Trophoblast cell surface antigen 2 (Trop-2) is a glycoprotein that spans the epithelial membrane surface and plays a role in cell self-renewal, proliferation, and transformation.1,2 Encoded by the TACSTD2 gene, Trop-2 is a 35-kDa protein composed of a large extracellular domain, a single transmembrane domain, and a short intracellular tail that is the functionally dominant part of the protein.1-4

Under physiological conditions, Trop-2 plays an essential role in embryonic development, placental tissue formation, embryo implantation, stem cell proliferation, and organ development.2 A low basal expression level of Trop-2 is found on the surface of multiple normal epithelial tissues, including skin and oral mucosa.1,3 Trop-2 can promote tumor growth and its overexpression is common in many types of malignant epithelial tumors.1,2,4

Expression of Trop-2 is regulated by several pro-oncogenic transcription factors (eg, CREB1, nuclear factor [NF]B, and HOXA10) via positive feedback relationships.2 Trop-2 expression may be upregulated because of the inactivation of several transcription factors (eg, HNF4A, TP63/TP53L, ERG, HNF1A/TCF-1, and FOXP3).1,2 Overexpression of Trop-2 accelerates the cancer cell cycle and drives cancer growth. Knocking out the TACSTD2 gene disturbs the proliferation of tumor cells, further validating the role of Trop-2 in tumorigenesis.1

Trop-2 was first elucidated as a transducer of intracellular calcium signals; however, it is now known to function in a variety of cell signaling pathways associated with tumorigenesis (Figure 1).1,2,4 Expression of Trop-2, as a calcium signal transducer, causes calcium to be mobilized from internal stores. Increased intracellular calcium levels activate MAPK, which in turn increases levels of phosphorylated ERK1 and ERK2.2,4 ERK1 and ERK2 are important mediators of cell cycle progression, angiogenesis, cell proliferation, cell invasion, and metastasis.2,4 Intracellular calcium also activates the NF-B pathway, which is involved in stimulation of cell growth, and the RAF pathway, which is essential for the upregulation of FOXM1, one of the most commonly overexpressed genes in human solid tumors.2

In addition to stimulating calcium release and MAPK signaling, Trop-2 is involved in several other pro-oncogenic signaling pathways, leading to tumor cell growth and proliferation. Activation of cyclin E and D further promotes cell cycle progression.4Alteration of the Notch, Hedgehog, and Wnt pathways may discourage appropriate stem cell proliferation and differentiation.2,4 Trop-2 signaling also appears to be dependent on -catenin.5 Direct interaction between -catenin and the intracellular domain of Trop-2, through -catenin signaling, enhances stem celllike properties (eg, self-renewal and transformation) of cancer cells.5 Attenuation of IGF-1 receptor signaling by Trop-2 encourages cancer growth and malignancy, particularly in lung cancers.2

Trop-2 is inextricably linked to cancer progression and metastasis because of its role as a key regulator of the hallmarks of cancer, including cell growth, proliferation, migration, invasion, and survival.4 A variety of human epithelial cancer cells are characterized by Trop-2 overexpression, including breast, lung, urothelial, gastric, colorectal, pancreatic, prostatic, cervical, head and neck, and ovarian carcinomas.2,3 In an analysis of 702 tissue samples from patients with breast cancer, Trop-2 expression was detected via immunohistochemistry (IHC) across a wide range of breast cancer subtypes.6 Trop-2 expression is substantially higher in hormone receptorpositive/HER2-negative (HR+/HER2-) disease and triple-negative breast cancer (TNBC) compared with other breast cancer subtypes, including HER2-positive disease.7

Trop-2 overexpression is also common in nonsmall cell lung cancer (NSCLC).8 Using IHC on tissues collected from the tumors of 68 patients with NSCLC, Trop-2 expression was significantly higher in NSCLC tissues compared with matched healthy tissues (P < .05). Moreover, its overexpression was associated with worse tumor, node, metastasis stage (P = .012), lymph node metastasis (P = .038), and histologic grade (P = .013).9

Bladder cancer, the most common urothelial cancer, is also marked by elevated Trop-2 expression.10,11 In a study of 102 transitional cell bladder cancer samples, IHC staining for Trop-2 demonstrated increased Trop-2 expression compared with noncancerous samples, and this expression pattern was significantly associated with worsened tumor grade (P = .001), stage (P < .0 01), and bladder cancer recurrence (P = .0 3).11

Molecular markers that influence the biological progress of tumors often serve as important prognostic indicators. Overexpression of Trop-2 has been associated with more aggressive disease, poorer overall survival (OS), and worse disease-free survival in patients with solid tumors.4 A meta-analysis conducted in 2016 explored the association of Trop-2 expression and prognosis in patients with a variety of solid tumors (N = 2569). Results from the study showed that high Trop-2 expression negatively affected OS (hazard ratio, 1.896; 95% CI, 1.599-2.247; P < .001) and disease-free survival (pooled hazard ratio, 2.336; 95% CI, 1.596-3.419; P < .0 01).12

Specific to breast cancers, increased Trop-2 mRNA is a strong predictor of lymph node involvement, distant metastasis, and poor OS.13,14 Trop-2 is expressed across all breast cancer subtypes; however, overexpression appears more common in aggressive disease subtypes, including HR+/HER2- disease and TNBC.7

Trop-2 overexpression is also associated with poor outcomes in patients with urothelial cancer. In an analysis of 102 tissue samples collected from patients with noninvasive bladder cancer, Trop-2 expression was higher in samples from patients who experienced disease recurrence compared with those who did not have recurrent disease (P = .0 3). Additionally, patients with Trop-2 overexpression had significantly lower rates of recurrence-free survival (P = .0 01).11 In a separate study, high Trop-2 expression analyzed by IHC was strongly correlated with bladder cancer severity and worsened disease prognosis, with particularly strong Trop-2 expression in muscle-invasive bladder cancer tissues compared with normal bladder tissues (P < .0 01).15

Taken together, the data indicate that Trop-2 is a potentially valuable therapeutic target, given the connection between its overexpression and poor prognosis in various solid tumors.4,15 Its value as a prognostic indicator and potential target for therapeutic development is particularly evident in advanced cancers that have limited or few treatment options available, such as TNBC and metastatic urothelial cancers.

Metastatic TNBC

TNBC is an aggressive form of invasive breast cancer that accounts for 15% to 20% of all breast cancers and a disproportionate number of deaths due to breast cancer.16-18 Its prevalence is particularly high in premenopausal women and those of African American and Hispanic descents.17,19 TNBC is characterized by a lack of estrogen and progesterone receptors and a low expression of HER2; therefore, TNBC cannot be effectively treated with standard hormone-based therapies and HER2-targeted agents.16, 20Although chemotherapy has shown promising results in early TNBC, the majority of patients relapse and progress to metastatic TNBC within the first 3 to 5 years after initial treatment.18 The treatment of metastatic TNBC remains a clinical challenge, as no standard-of-care chemotherapy exists for previously treated patients.17,18 There is an urgent unmet need for effective treatment options in patients with metastatic TNBC.18

Metastatic Urothelial Cancer

In the United States, an estimated 81,400 new cases of urothelial cancer will be diagnosed in 2020, and approximately 18,000 Americans will die from the disease.21 The majority of urothelial cancers arise in the bladder, and established risk factors for bladder cancer include older age, male gender, Caucasian race, family history, and smoking.22,23 Muscle-invasive and meta-static urothelial cancers represent 25% of urothelial carcinoma cases and are characterized by substantially worse prognostic outcomes.23,24 Current chemotherapeutic options for metastatic disease offer a modest median OS of 15 months and a 5-year survival of less than 5%.23,24 Long-term survival is infrequent, and newer treatment modalities that target distinct molecular biomarkers are warranted.24,25

As Trop-2 is a clinically relevant cell surface antigen among several solid tumor types, its overexpression on cancer cells makes it an ideal candidate for targeting by specific therapies.26 One targeted approach involves the use of antibody-drug conjugates (ADCs), a technology that has revolutionized the approach to cancer chemo-therapy over the past 2 decades.26

An ADC is designed to contain 3 components: a monoclonal antibody (mAb), a cytotoxic drug called a payload, and a linker that connects the mAb to the cytotoxin. The mAb binds specifically to its tumor-associated antigen (eg, Trop-2), thereby delivering the cytotoxin to the surface of the tumor cell. Once bound, the ADC is internalized through receptor-mediated endocytosis. Lysosomal degradation of the ADC ensues, facilitating the release of the cytotoxin and enabling it to bind to its intracellular target and induce apoptotic cell death (Figure 2).26,27 The targeted nature of ADCs allows potent therapy to be delivered to the cancer cell itself, limiting systemic exposure. The result is fewer adverse effects (AEs), a wider therapeutic window, and reduced exposure of the drug to efflux mechanisms that can increase drug resistance.26,27

Sacituzumab govitecan-hziy is the only FDA-approved Trop-2targeted ADC, and several other agents are under preclinical and clinical development.28

Sacituzumab govitecan-hziy is an ADC that binds to Trop-2 and delivers a potent cytotoxic drug into tumor cells.29,30 The FDA recently granted it accelerated approval for the treatment of metastatic TNBC, and it has also received fast track designation for metastatic urothelial carcinoma, NSCLC, and small cell lung cancer.28,30-32

The composition of sacituzumab govitecan-hziy has been optimized to effectively target tumors expressing Trop-2. A humanized monoclonal antibody (hRS7) binds to Trop-2 and delivers govitecan (SN-38) to the cell surface. SN-38 is the active metabolite of irinotecan and functions as a DNA topoisomerase I inhibitor. A hydrolysable CL2a linker covalently binds SN-38 to h R S 7.30 When released intracellularly, SN-38 causes double-stranded DNA breaks that lead to apoptosis.29 Additionally, the hydrolysable linker allows a portion of the SN-38 payload to be released into the tumor microenvironment, leading adjacent tumor cells to be killed via a bystander effect.31,32

Sacituzumab govitecan-hziy delivers SN-38 in its most active nonglucuronidated form. Because of its moderate toxicity profile, SN-38 is conjugated to hRS7 at a high drug-to-antibody ratio of up to 8 SN-38 molecules per antibody, allowing for greater drug delivery than systemic irinotecan can achieve.29,32 Irinotecan causes grade 3 to 4 diarrhea in approximately one-third of patients, whereas the lower toxicity of SN-38 may confer an improved therapeutic index.29,30 This high level of drug delivery may overcome the ability of Trop-2expressing tumors to repair DNA breaks.30

On April 22, 2020, sacituzumab govitecan-hziy received accelerated approval from the FDA for the treatment of adult patients with metastatic TNBC who have received at least 2 prior therapies for metastatic disease.28 Approval was based on findings of the phase 1/2, single-arm, multicenter IM-T-IMMU-132-01 trial (NCT01631552), in which sacituzumab govitecan-hziy produced durable responses in a subset of patients with heavily pretreated metastatic TNBC.31,33

The IM-T-IMMU-132-01 trial enrolled 108 patients with metastatic TNBC who had received at least 2 prior treatments for metastatic disease. In the study population, the median number of prior systemic therapies in the metastatic setting was 3, and the majority of patients received prior taxanes (98%) and anthracyclines (86%) in the neoadjuvant or metastatic setting. The median age of study patients was 55 years (range 31-80); 99% were female, and 76% were Caucasian.31Brain metastases were present in 23% of patients, and visceral metastases were present in 77% of patients; these included metastases in the lung/pleura (57%), the liver (42%), and other visceral organs (adrenal glands, pancreas, and kidney; 7%).31

Patients received sacituzumab govitecan-hziy 10 mg/kg administered intravenously on days 1 and 8 of 21-day cycles. Treatment continued until disease progression or unacceptable toxicity. The primary efficacy end point was objective response rate (ORR) assessed according to RECIST 1.1 tumor criteria. The secondary efficacy end points included time to response, duration of response, clinical benefit rate (defined as a complete or partial response or stable disease for 6 months), progression-free survival (PFS), and OS.31

After a median follow-up duration of 9.7 months, an objective response occurred in 36 of 108 patients (ORR, 33.3%; 95% CI, 24.6%-43.1%), including a complete response in 3 patients.31 The median time to response was 2 months (range 1.6-13.5). The median response duration was 7.7 months (95% CI, 4.9-10.8), with 55.6% of patients responding at 6 months and 16.7% of patients still responding at 12 months.31,34 An independent central review of the data found a similar ORR and median response duration (34.3% and 9.1 months, respectively).31 The clinical benefit rate, including stable disease for at least 6 months, was 45.4%. Median PFS was 5.5 months; the estimated probability of PFS at 6 and 12 months was 41.9% and 15.1%, respectively. Median OS was 13 months (95% CI, 11.2-13.7); the estimated probability of survival at 6 and 12 months was 78.5% and 51.3%, respectively.31

The most common AEs of any grade were nausea (67%), neutropenia (64%), diarrhea (62%, predominantly grade 1), and fatigue (55%). Of grade 3 or 4 AEs, the most common were neutropenia, decreased white cell count, and anemia (occurring in 42%, 11%, and 11% of patients, respectively).31 Serious AEs occurred in 32% of patients, with the most common being febrile neutropenia (7%), vomiting (6%), nausea (4%), diarrhea (3%), and dyspnea (3%). Occurrence of AEs led to treatment interruption in 44% of patients, dose reductions in 34%, and discontinuation of treatment in 3%.31,34

IM-T-IMMU-132-01 Trial HR+/HER2- Subpopulation Analysis

Treatment with sacituzumab govitecan-hziy showed encouraging results in a prespecified subpopulation of patients with histologically confirmed HR+/HER2- metastatic breast cancer from the IM-T-IMMU-132-01 trial.32

A total of 54 patients with histologically confirmed HR+/HER2- metastatic breast cancer were enrolled. Eligible patients had received at least 1 line of hormone-based therapy and at least 1 prior chemotherapy in the metastatic setting. The median age of enrollees was 54 years (range, 33-79); aside from required prior hormone-based therapy, previous chemotherapies included a taxane (85%), an anthracycline (67%), capecitabine (65%), a CDK4/6 inhibitor (61%), an mTOR inhibitor (44%), and an immune checkpoint inhibitor (1.9%). After a washout period of at least 2 weeks since prior treatment, sacituzumab govitecan-hziy was dosed at 10 mg/kg via intravenous infusion on days 1 and 8 of 21-day cycles.32

The primary efficacy end point was ORR. Of the 54 patients enrolled, 17 patients achieved partial responses during a median follow-up duration of 11.5 months (ORR, 31.5%; 95% CI, 19.5%-45.6%). In the key secondary outcomes, patients experienced a median PFS of 5.5 months (95% CI, 3.6-7.6) and a median OS of 12.0 months (95% CI, 9.0-18.2). The median time to response was 2.1 months (95% CI, 1.4-7.8), and median duration of response was 8.7 months (95% CI, 3.7-12.7). Of the 17 responders, 4 achieved a response lasting more than 12 months (24%). The clinical benefit rate was 44.4% (95% CI, 30.9%-58.6%), with 7 patients showing stable disease for at least 6 months.32

Safety analyses showed a manageable AE profile for sacituzumab govitecan-hziy. There were no reports of cardiac toxicity or severe peripheral neuropathy. The most common grade 3 or higher treatment-related AE was neutropenia, which occurred in 50% of patients. The incidence of diarrhea was 46% and was mild overall. Grade 3 diarrhea was reported in 4 patients, with no reports of grade 4.32 Serious AEs occurred in 2 patients, who experienced febrile neutropenia and 1 case each of neutropenia, viral pneumonia, sepsis, diarrhea, nausea, vomiting, dehydration, and acute respiratory failure.32

ESMO 2020 Data: ASCENTTrial in Metastatic TNBC (NCT02574455)

Final results of the international, multicenter, open-label ASCENT trial (NCT02574455) were presented at the European Society for Medical Oncology (ESMO) Virtual Congress 2020. ASCENT was the first phase 3 study of an ADC to show improvement in PFS and OS compared with standard-of-care chemotherapy in patients with previously treated metastatic TNBC.35,36

A total of 529 patients with metastatic TNBC were randomized 1:1 to receive either sacituzumab govitecan-hziy or physicians choice of single-agent chemotherapy (capecitabine, eribulin, vinorelbine, or gemcitabine). The dose of sacituzumab govitecan-hziy was 10 mg/kg intravenously on days 1 and 8 of 21-day cycles. All patients had histologically or cytologically confirmed TNBC refractory to or relapsed after at least 2 prior chemotherapies including a taxane. The median age of the study population was 54 years, and the median number of prior chemotherapies received was 4.

In the primary end point, sacituzumab govitecan-hziy significantly improved median PFS (hazard ratio, 0.41; P< .0001) compared with chemotherapy. The sacituzumab govitecan-hziy treatment group achieved a median PFS of 5.6 months compared with 1.7 months in the chemotherapy treatment group. Compared with chemotherapy, sacituzumab govitecan-hziy treatment also significantly improved key secondary end points of OS (12.1 vs 6.7 months; hazard ratio, 0.48; P < .0001) and ORR (35% vs 5%; P < .0001).35,36

The most common treatment-related grade 3 or higher AEs with sacituzumab govitecan-hziy compared with chemotherapy were neutropenia (51% vs 33%, respectively), diarrhea (10.5% vs < 1.0%), anemia (8% vs 5%), and febrile neutropenia (6% vs 2%). No treatment-related deaths were reported, and no cases of neuropathy or interstitial lung disease greater than grade 3 occurred with sacituzumab govitecan-hziy.35

ESMO 2020 Data: Sacituzumab Govitecan-hziy in Combination with Talazoparib for Patients with Metastatic TNBC (NCT04039230)

At the ESMO Virtual Congress 2020, investigators presented the trial design, objectives, and status of a phase 1/2, open-label study that will investigate the efficacy and safety of sacituzumab govitecan-hziy in combination with the PARP inhibitor talazoparib for patients with metastatic TNBC.37 PARP is involved in repairing damaged DNA and is required for clearance of Trop-2 cleavage complexes; thus, PARP inhibitors may be complementary therapeutic partners with sacituzumab govitecan-hziy.37, 38

This study will include a dose escalation in phase 1b followed by a dose expansion in phase 2. Patients will receive sacituzumab govitecan-hziy on days 1 and 8 of 21-day cycles and talazoparib daily on days 15 to 21 of each cycle.38 The primary objective of phase 1b is to assess the dose-limiting toxicity rate and maximum tolerated dose of sacituzumab govitecan-hziy when given in combination with talazoparib. From these data, investigators will determine the recommended phase 2 dose. During phase 2, investigators will assess the ORR, PFS, OS, and clinical benefit rate. As of August 30, 2020, the trial was undergoing active recruitment, and a total of 20 patients were enrolled.37, 38

ESMO 2020 Data: Sacituzumab Govitecan-hziy for Breast Cancer Brain Metastases (NCT03995706)

SN-38, the cytotoxic payload delivered by sacituzumab govitecan-hziy, crosses the blood-brain barrier and is often included in central nervous system (CNS) cancer regimens.39 Investigators hypothesized that sacituzumab govitecan-hziy would yield therapeutically relevant SN-38 concentrations within the CNS of patients under-going craniotomy for breast cancer brain metastases or recurrent glioblastoma.39,40

In this single-center, nonrandomized, phase 0 study (NCT03995706), patients receive a single 10-mg/kg intravenous dose of sacituzumab govitecan-hziy the day prior to craniotomy and then resume therapy (on days 1 and 8 of 21-day cycles) after recovery. To date, 14 patients have been treated. For patients with recurrent glioblastoma (n = 7), the mean SN-38 concentration was 420 nM; for patients with breast cancer brain metastases (n = 7), the mean SN-38 concentration was 626 nM. Among those patients with residual measurable disease, 2 partial intracranial responses have been observed in each group after 12 weeks of treatment (ORR, 28% and 50% for glioblastoma and breast cancer brain metastases, respectively). As of September 2020, recruitment for this trial was ongoing.39,40

The metastatic urothelial cancer cohort of the IM-T-IMMU-132-01 trial reported encouraging activity with sacituzumab govitecan-hziy monotherapy (ORR, 31%; median PFS, 7.3 months; and median OS, 18.9 months).41Sacituzumab govitecan-hziy has FDA fast track desig-nation for metastatic urothelial cancer and is currently under further investigation in the phase 2 TROPHY U-01 trial (NCT03547973) and the upcoming phase 3 TROPiCS-04 trial (NCT04527991).42-45

Final data for cohort 1 and the trial design for cohort 3 were presented at the ESMO Virtual Congress 2020 for the pivotal phase 2, open-label, multicohort TROPHY U-01trial. The TROPHY U-01trial is investigating the safety and efficacy of sacituzumab govitecan-hziy in patients with heavily pretreated metastatic urothelial cancer across several cohorts. The study population across the TROPHY U-01 trial includes patients with disease progression despite treatment with platinum (PLT)-based chemotherapy, checkpoint inhibitors, or both. For all cohorts, the primary efficacy end point is ORR, and key secondary end points include PFS, OS, duration of response, and safety analyses.44,45

Cohort 1

Cohort 1 included a total of 113 patients who were treated with sacituzumab govitecan-hziy. The study population included patients who experienced disease progression after both PLT-based chemotherapy and checkpoint inhibitor therapy.44 Overall, patients in cohort 1 were previously treated with a median of 3 therapies and were a median of 66 years of age. In the results presented at ESMO 2020, a total of 31 patients had achieved an objective response (ORR, 27%; 95% CI, 19%-37%), of which 6 were complete responses and 25 were partial responses. The median duration of response was 5.9 months (95% CI, 4.7-8.6); median PFS and OS were 5.4 months (95% CI, 3.5-6.9) and 10.5 months (95% CI, 8.2-12.3), respectively. Sacituzumab govitecan-hziy demonstrated manageable toxicity. Key grade 3 or higher AEs were neutropenia (35%), anemia (14%), febrile neutropenia (10%), and diarrhea (10%).44

Cohort 3

As of March 2020, cohort 3 had started enrollment and is ongoing. The study plans to enroll a total of 61 patients with metastatic urothelial cancer who are nave to checkpoint inhibitor agents and have experienced disease progression or recurrence after PLT-based chemotherapy.45 As checkpoint inhibitors are the stan-dard-of-care therapy for patients who have failed on PLT-based chemotherapy, this study will investigate combination therapy with sacituzumab govitecan-hziy and the checkpoint inhibitor pembrolizumab. Exclusion criteria include active autoimmune disease or a history of interstitial lung disease, given the coadministration of pembrolizumab. A 10-patient lead-in cohort will determine standard the recommended phase 2 dose of sacituzumab govitecan-hziy (given on days 1 and 8 of 21-day cycles), to be given along with pembrolizumab 200 mg on day 1 of each cycle. The primary end point of ORR and secondary end points of PFS, OS, clinical benefit rate, duration of response, and safety will be assessed.45

The phase 3, global, open-label TROPiCS-04trial aims to enroll 482 patients to investigate the efficacy and safety of sacituzumab govitecan-hziy in patients with metastatic or locally advanced unresectable urothelial cancer who have progressed despite prior therapy with PLT-based chemotherapy and a PD-1 or PD-L1 checkpoint inhibitor. Sacituzumab govitecan-hziy will be compared with physicians choice of chemotherapy (paclitaxel, docetaxel, or vinflunine). The primary outcome measure will be OS; secondary outcomes will include PFS, ORR, safety, and quality of life. As of August 2020, the trial was not yet recruiting patients.43

Evaluation of novel and existing ADCs has revealed that success is not based on the use of any one particular cytotoxic compound or conjugate platform. Factors such as the consistency and level of target-antigen expression, tumor progression, and specific properties of the cancer and stage of disease also play important roles.46 Several additional Trop-2targeted ADCs are currently being investigated in solid tumors (Table).33,36,37,40,42,43,47-51

DS-1062a is a Trop-2directed ADC that contains the cytotoxic compound DXd, a derivative of exatecan that acts as a DNA topoisomerase I inhibitor.52 It is currently being investigated for the treatment of advanced NSCLC in an ongoing phase 1, multicenter, open-label study (NC T 03 401385).48

The study involves a dose-escalation phase and a dose-expansion phase. Dose-limiting toxicity, maximum tolerated dose, and AEs will be explored in both phases.47 Eligible patients have experienced disease progression or recurrence despite previous treatments, have measurable disease per RECIST 1.1 criteria, and are able to provide a sufficient tumor tissue sample for Trop-2 measurement. Patients with multiple primary malignancies or untreated brain metastases are ineligible for the study.48

As of November 2018, a total of 22 patients had been treated with 1 of 3 escalating doses of DS-1062a. Nearly 82% of patients experienced at least 1 treatment-emergent AE, with fatigue being the most common complaint. Fatigue was the only reported grade 3 or higher AE and was reported by 1 patient. Of 18 tumor-evaluable patients, 1 showed a partial response and 8 showed stable disease. Maximum-tolerated dose has not been achieved, and investigators will continue to monitor for safety and disease progression.47, 48

RN927C

RN927C, also known as PF-06664178, is an ADC composed of a Trop-2directed antibody conjugated with the cytotoxic microtubule inhibitor PF-06380101. Release of PF-06380101 leads to mitotic arrest, apoptosis, and cell death.3 Preclinical studies demonstrated the ability of RN927C to induce cell death among various tumor cell lines, including those from the skin, lung, head and neck, breast, ovary, and colon.3

RN927C was investigated in a phase 1, open-label, nonrandomized dose-escalation study (NCT02122146)of patients with advanced or metastatic solid tumors that were unresponsive to current therapies or for whom no standard therapy was available. The primary objective of the study was to determine the maximum tolerated dose and recommended phase 2 dose. Secondary outcomes included safety and preliminary evidence of antitumor activity. A total of 31 patients were enrolled and received treatment with escalating doses of RN927C. Stable disease was noted in 11 patients (39%), but no partial or complete responses were seen. Doses of 3.6 mg/kg, 4.2 mg/kg, and 4.8 mg/kg were considered intolerable, primarily because of skin reactions and development of neutropenia. The next-lower dose of 2.4 mg/kg was well tolerated, but the study was terminated early because of minimal anti-tumor activity and excessive toxicities.50

BAT8003

BAT8003 is an ADC composed of a Trop-2directed antibody conjugated to a potent cytotoxic maytansine derivative. The ADC has been optimized to facilitate site-specific conjugation, which allows for a more controllable drug-antibody ratio. In addition, a fucosylation of the Fc region of the antibody enhances its antibody-dependent cell-mediated cytotoxicity effect. In preclinical xenograft and primate models, BAT8003 demonstrated strong inhibition of tumor growth at doses of 5 mg/kg and 15 mg/kg, with a highest nonseverely toxic dose of 20 mg/kg given once every 3 weeks.51, 53

Given the promising preclinical data, a phase 1 dose-escalation study (NCT03884517) is currently investigating the safety, tolerability, and pharmacokinetics of BAT8003 in patients with advanced epithelial cancer who are either ineligible for standard therapy or have disease refractory to standard therapy.Eligible patients will receive escalating doses of BAT8003 (0.2-10.0 mg/kg) on day 1 of each 21-day cycle. The study will be divided into 3 periods: (1) the first 21-day cycle, which will examine the safety of a single BAT8003 administration, observe for dose-limiting toxicities, and establish preliminary pharmacokinetic parameters; (2) cycles 2 through 8, which will examine safety, immunogenicity, and preliminary efficacy of escalating doses of BAT8003; and (3) an expansion period, which could include an additional 10 to 30 cases to further assess safety and efficacy once a safe and effective dose has been established. As of the last update on March 21, 2019, the trial was actively recruiting patients.51

Trop-2 has established itself as a clinically meaningful biomarker among several types of solid malignancies. Its ability to promote self-renewal, proliferation, and cell invasion makes it an ideal candidate for targeted anti-tumor therapies, including ADCs.

Sacituzumab govitecan-hziy is the first Trop-2directed ADC to receive FDA approval for the treatment of metastatic TNBC. In the pivotal IM-T-IMMU-132-01 trial, sacituzumab govitecan-hziy showed encouraging results in patients with multiple difficult-to-treat solid tumor types, including TNBC, HR+/HER2- metastatic breast cancer, and metastatic urothelial cancer.31,32,41 Sacituzumab govitecan-hziy and other Trop-2directed ADCs represent a novel strategy to improve outcomes among these populations of patients with few therapeutic options. Data from additional trials of sacituzumab govitecan-hziy were presented at the ESMO Virtual Congress 2020. In the ASCENT trial, sacituzumab govitecan improved response rates and survival outcomes in patients with metastatic TNBC compared with standard-of-care therapy.35 Data from a cohort of patients with metastatic urothelial cancer in the TROPHY U-01 trial indicated positive survival impacts with manageable toxicity.44 Additional trials of sacituzumab-govitecan-hziy (as monotherapy or in combination with PARP inhibitors or checkpoint inhibitors) are under way in patients with metastatic TNBC, breast cancer brain metastases, and metastatic or locally advanced urothelial cancer.37,40,43,45 Other Trop-2directed ADCs are under investigation in NSCLC and advanced epithelial cancers.47, 51

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Role of Trop-2 as an Actionable Biomarker in Solid Tumors - OncLive

Cardiac Stem Cells Comments Off on Role of Trop-2 as an Actionable Biomarker in Solid Tumors – OncLive | October 29th, 2020

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Segmentation by Product Type

Segmentation by End User

The stem cell-derived cells market is categorized based on product type and end user. Based on product type, the stem cell-derived cells are classified into two major types stem cell-derived cell kits and accessories. Among these stem cell-derived cell kits, stem cell-derived hepatocytes kits are the most preferred stem cell-derived cells product type. On the basis of product type, stem cell-derived cardiomyocytes kits segment is projected to expand its growth at a significant CAGR over the forecast years on the account of more demand from the end use segments. However, the stem cell-derived definitive endoderm cell kits segment is projected to remain the second most lucrative revenue share segment in stem cell-derived cells market. Biotechnology and pharmaceutical companies followed by research and academic institutions is expected to register substantial revenue growth rate during the forecast period.

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North America and Europe cumulatively are projected to remain most lucrative regions and register significant market revenue share in global stem cell-derived cells market due to the increased patient pool in the regions with increasing adoption for stem cell based therapies. The launch of new stem cell-derived cells kits and accessories on FDA approval for the U.S. market allows North America to capture significant revenue share in stem cell-derived cells market. Asian countries due to strong funding in research and development are entirely focused on production of stem cell-derived cells thereby aiding South Asian and East Asian countries to grow at a robust CAGR over the forecast period.

Some of the major key manufacturers involved in global stem cell-derived cells market are Takara Bio Inc., Viacyte, Inc. and others.

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The Stem Cell-Derived Cells market to be in conjunction to growth from 2020 to 2030 - PRnews Leader

IPS Cell Therapy Comments Off on The Stem Cell-Derived Cells market to be in conjunction to growth from 2020 to 2030 – PRnews Leader | October 29th, 2020

The global animal stem cell therapy market is growing at rapid pace on the back of increased research and development activities in the healthcare sector. Stem cells are widely utilized for the replacement of neurons, which are damaged due to various health issues such as Parkinsons disease, stroke, Alzheimers disease, and spinal cord injury.

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Stem cell therapy is gaining popularity on the back of increased technological advancements in worldwide healthcare sector. This technique is increasingly used for the treatment of numerous diseases and health disorders in animals as well. In recent years, there is remarkable increase in cases of different diseases in animals across the globe. This situation is resulted in growing utilization of animal stem cell therapy. As a result, the global animal stem cell therapy market is foreseen to gain prominent amount of money in the form of revenues in the forthcoming years.

Players Focus on Mergers and Acquisitions to Maintain Leading Market Position

The global animal stem cell therapy market experiences presence of many enterprises in it. As a result, the nature of this market is fairly fragmented. At the same time, the competitive landscape of the market for animal stem cell therapy is intense. Players operating in this market are using various organic as well as inorganic strategies to maintain their leading market position. One of the trending strategies used by vendors working in the global animal stem cell therapy market is mergers and acquisitions.

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Several stakeholders in the animal stem cell therapy market are seen investing heavily in research and development activities. This move is helping them in achieving advancement in products quality. Apart from this, many companies are increasing engagement in collaborations, partnerships, joint ventures, and new product launches. All these activities are indicative of rapid expansion of the animal stem cell therapy market in the forthcoming years.

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Spinal Cord Stem Cells Comments Off on Tag: Animal Stem Cell Therapy Market – TMR Research Blog | October 29th, 2020

Central nervous system comprises brain and spinal cord, and is responsible for integration of sensory information. Brain is the largest and one of the most complex organs in the human body. It is made up of 100 billion nerves that communicate with 100 trillion synapses. It is responsible for the thought and movement produced by the body. Spinal cord is connected to a section of brain known as brain stem and runs through the spinal canal. The brain processes and interprets sensory information sent from the spinal cord. Brain and spinal cord serve as the primary processing centers for the entire nervous system, and control the working of the body. Neuroprosthetics improves or replaces the function of the central nervous system. Neuroprosthetics, also known as neural prosthetics, are devices implanted in the body that stimulate the function of an organ or organ system that has failed due to disease or injury. It is a brain-computer interface device used to detect and translate neural activity into command sequences for prostheses. Its primary aim is to restore functionality in patients suffering from loss of motor control such as spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis, and stroke. The major types of neuroprosthetics include sensory implants, motor prosthetics, and cognitive prosthetics. Motor prosthetics support the autonomous system and assist in the regulation or stimulation of affected motor functions.

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Similarly, cognitive prosthetics restore the function of brain tissue loss in conditions such as paralysis, Parkinsons disease, traumatic brain injury, and speech deficit. Sensory implants pass information into the bodys sensory areas such as sight or hearing, and it is further classified as auditory (cochlear implant), visual, and spinal cord stimulator. Some key functions of neuroprosthetics include providing hearing, seeing, feeling abilities, pain relief, and restoring damaged brain cells. Cochlear implant is among the most popular neuroprosthetics. In addition, auditory brain stem implant is also a neuroprosthetic meant to improve hearing damage.

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North America dominates the global market for neuroprosthetics due to the rising incidence of neurological diseases and growth in geriatric population in the region. Asia is expected to display a high growth rate in the next five years in the global neuroprosthetics market, with China and India being the fastest growing markets in the Asia-Pacific region. Among the key driving forces for the neuroprosthetics market in developing countries are the large pool of patients, increasing awareness about the disease, improving healthcare infrastructure, and rising government funding in the region.

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Increasing prevalence of neurological diseases such as traumatic brain injury, stroke and Parkinsons disease, rise in geriatric population, increase in healthcare expenditure, growing awareness about healthcare, rapid progression of technology, and increasing number of initiatives by various governments and government associations are some key factors driving growth of the global neuroprosthetics market. However, factors such as high cost of devices, reimbursement issues, and adverse effects pose a major restraint to the growth of the global neuroprosthetics market.

Innovative self-charging neural implants that eliminate the need for high risk and costly surgery to replace the discharge battery and controlling machinery with thoughts would help to develop opportunities for the growth of the global neuroprosthetics market. The major companies operating in the global neuroprosthetics market are Boston Scientific Corporation, Cochlear Limited, Medtronic, Inc., Cyberonics, Inc., NDI Medical LLC, NeuroPace, Inc., Nervo Corp., Retina Implant AG, St. Jude Medical, and Sonova Group.

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The Neuroprosthetics market to grow in the wake of incorporation of the latest technology - PRnews Leader

Spinal Cord Stem Cells Comments Off on The Neuroprosthetics market to grow in the wake of incorporation of the latest technology – PRnews Leader | October 29th, 2020

An 11-year-old girl is in urgent need of a bone marrow transplant after being diagnosed with a rare, life-threatening condition.

Arya Lloyd, who was born in the Leicester Royal Infirmary, started to complain of abdominal pain and aches in her back and ribs during the summer.

Her father, Geraint said "she had always been fit and healthy" and did well in sports. So it was a shock to him and his wife, Arya's mother Brundha, when she was diagnosed with aplastic anaemia in late July this year.

Aplastic anaemia, also known as bone marrow failure, is a rare disease affecting the blood whereby the bone marrow and stem cells do not produce enough blood cells.

Arya then went on to have two bone marrow biopsies after her diagnosis.

"Her blood count continued to drop and that's why it is so important to get a donor match," Geraint said.

But finding a match will be a challenge. Arya, whose mother is of Indian heritage and father is Caucasian, will wait longer to find a suitable match due to her dual heritage.

Currently, those of Asian, Black or mixed ethnic background have a 20 per cent chance of finding the best possible match from an unrelated donor, compared with the 70 per cent chance of finding a match for Caucasian patients.

Arya and her parents, who a currently live in Cambridge are "staying positive" while Arya undergoes immunosuppressive treatment at St Mary's Hospital in Paddington.

Geraint told LeicestershireLive: She has been really brave and she just gets on with it. I wish it was me rather than her going through all this but she's optimistic."

The 11-year-old schoolgirl is expected to be discharged from the hospital this week and has spent her time keeping in touch with friends and catching up with homework when she can.

It has now been 22 days since she was admitted to hospital where she has been able to stay with her mother. Due to Covid-19 restrictions, the pair have had to stay in an isolated room and unable to see Geraint who has kept in touch through video calls.

"It's been really difficult, our whole world has been turned upside-down but we need to be optimistic and find a match," Geraint said.

The family is now urging people to come forward and join the bone marrow donor register.

"It's very urgent and so important that particularly people of Indian and mixed heritage join the register as they are hugely underrepresented," Geraint said.

So far, no suitable match has been found for Arya and it will take several months to determine the effects of the treatment she is currently having.

Following the immunosuppressive treatment, Arya will be infection-prone and have to be careful to avoid any trauma or injury due to her low blood count. This also leaves her in the category of people who are at higher risk from Covid-19.

While Arya and her family continue to adjust and stay positive, they need your help.

Joining the bone marrow donor register is simple and can be done from home by ordering a swab kit that is then sent back and analysed.

You can find out more about Arya's story and how to join the register at http://www.dkms.org.uk/en/arya.

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11-year-old urgently needs a bone marrow transplant after being diagnosed with life-threatening condition - Leicestershire Live

Bone Marrow Stem Cells Comments Off on 11-year-old urgently needs a bone marrow transplant after being diagnosed with life-threatening condition – Leicestershire Live | October 29th, 2020

Ongoing studies of Cynata Therapeutics Cymerus stem cell products are beginning to reveal a wide range of commercial possibilities for the ASX-listed companys cutting edge biotechnology that it is looking to apply to a multitude of ailments from the treatment of osteoarthritis and heart attacks through to COVID-19.

In its most advanced trials to date, Cynata will soon embark on a Phase 3 trial of its CYP-004 product, the companys mesenchymal stem cell or MSC product developed to treat osteoarthritis. The 448 person trial is being sponsored by The University of Sydney and will be funded by a project grant from the Australian Government National Health and Medical Research Council.

The company is also progressing on multiple other fronts developing a range of Cymerus MSC therapeutics with the CYP-001 product being another lead candidate. CYP-001 is being developed to treat acute graft-versus-host disease, or GVHD an affliction suffered by bone marrow transplant recipients. GVHD can develop from donated bone marrow that does not take well to a recipients body which triggers an immune response, attacking the host.

Presently, GVHD is treated with steroid therapy however sufferers tend to have a very low survival rate, with less than 20 per cent of patients living for more than two years and few alternate treatment pathways are available.

This looks set to change following Phase 1 trial of Cynatas CYP-001 product on a cohort of patients which saw the survival rate of sufferers of GVHD triple to 60 per cent over a two-year period. The company is now moving CYP-001 into Phase 2 testing and towards commercialisation with partner and shareholder, Fujifilm Corporation.

The matchup with the Japanese-based multi-national is already paying dividends with Cynata receiving an upfront US$3 million payment with further staged payments and royalties to follow in a licensing deal potentially worth more than US$50 million in the longer term.

Stem cells are the building blocks of the human body - essentially the cells from which all other cells are derived and under the right conditions, they can divide to produce more cells sometimes known as Daughter cells. These Daughter cells can become new stem cells or more specialised cells such as blood, bone or even the cells that make up brain or heart tissue.

When appropriately manipulated, stem cells have the potential to treat a range of diseases and aid in the healing and recovery of patients suffering both disease and trauma.

There are a limited number of sources of stem cells - embryonic stem cells, perinatal stem cells and adult stem cells.

Embryonic stem cells are thought to be the most useful and versatile but only harvestable in very small quantities. Perinatal stem cells found in amniotic fluid and umbilical cord blood are also only harvestable in limited quantities although their potential is yet to be fully understood.

Adult stem cells, found in bone marrow or fat, were previously thought to be only useful in producing a limited range of specialised cells with multiple donors required to generate practical amounts of therapeutical medicines.

However, ongoing research shows that by utilising a form of genetic reprogramming, mature cells can be re-programmed to behave like embryonic stem cells. These manipulated cells are called induced pluripotent stem cells, or iPSCs which is where Cynatas Cymerus technology comes into the picture.

Cynatas proprietary Cymerus technology uses iPSCs and a precursor cell called a mesenchymoangioblast to manufacture MSC therapies at a commercial scale without the need for multiple donors. This is where the Cymerus platform diverges from similar therapies, doing away with the need for multiple donors and overcoming a bottleneck in the generation of its product.

Other Cynata MSC products in development include a therapy to assist in the treatment and recovery of heart attacks, which is also showing promise according to the company. Another Cynata product undergoing pre-clinical trials with potential application in the treatment of lung disease is idiopathic pulmonary fibrosis, or IPF. Cynatas research in lung diseases has an unexpected spin-off in that its MSCs may assist in a patients recovery of COVID-19 according to the company. This application is being pursued in a clinical trial in COVID-19 patients presently being conducted in NSW.

These latest results with Cymerus MSCs add to the large body of evidence on the potency of these cells and their potential utility in treating a wide range of devastating diseases. IPF represents an enormous unmet medical need, as existing treatment options have only modest effects on disease progression and survival rates.

Cynatas is now modelling potential MSC therapies to treat various other afflictions too including critical limb ischemia, asthma, sepsis, cytokine release syndrome and diabetic wounds.

In the world of biotechnology, you really only have to produce one winner to attract a longing stare from the big biotechs who can swallow you whole with their massive cheque books with a range of targets and opportunities in its armoury that look to be developing well, dont be surprised if Cynata eventually disappears under the giant footprint of one of the big biotechs.

Is your ASX listed company doing something interesting? Contact: matt.birney@wanews.com.au

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Cynata looking to revolutionise stem cell therapy - The West Australian

Bone Marrow Stem Cells Comments Off on Cynata looking to revolutionise stem cell therapy – The West Australian | October 29th, 2020

Cell Harvesting Systems: Introduction

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Cell Harvesting Systems Market: Increasing demand for stem cell transplantation along with stem cell-based therapy to drive the market - BioSpace

Bone Marrow Stem Cells Comments Off on Cell Harvesting Systems Market: Increasing demand for stem cell transplantation along with stem cell-based therapy to drive the market – BioSpace | October 29th, 2020

WASHINGTON, Oct. 26, 2020 /PRNewswire/ --Despite the increasing use and promise of hematopoietic cell transplantation (HCT) as curative therapy for children with cancer and other life-threatening diseases, new research suggests that children transplanted for cancer are more likely to die from treatment-related complications if they live in poorer neighborhoods. The study, published today in the journal Blood, also found that having Medicaid versus private insurance, another marker of poverty, was associated with a higher chance of dying. Researchers say the results underscore the need to better understand and mitigate the effects of poverty and other social determinants of health on pediatric cancer care.

Hematopoietic cell transplantation, also called stem cell or bone marrow transplantation, is a treatment option for patients with blood cancers such as leukemia or lymphoma, as well as certain non-malignant conditions such as sickle cell disease or immunodeficiencies. It is only accessible at some medical centers. Together with radiation therapy or chemotherapy, HCT is designed to increase the chance of eliminating the cancerous or abnormal blood cells, and of restoring normal blood cell production.

The data revealed that children under the age of 18 with cancer who live in communities with high poverty rates had a 34% greater risk of treatment-related mortality following HCT compared with children in low-poverty areas. Even after adjusting for a child's disease and transplant-related factors, the data revealed children on Medicaid had a 23% greater risk of dying from any cause within five years of undergoing HCT and a 28% greater risk of treatment-related mortality when compared to children with private insurance.

"Our study shows that even after children with cancer have successfully accessed this high-resource treatment at specialized medical centers, those who are exposed to poverty are still at higher risk of dying of complications after treatment and of dying overall," said lead author Kira Bona, MD, MPH, Attending Physician, Dana-Farber/Boston Children's Cancer and Blood Disorders Center. "Simply providing the highest quality complex medical care to children who are vulnerable from a social perspective is inadequate if our goal is to cure every child with cancer."

One in five children in the U.S. lives in a household with an income below the federal poverty level. While previous studies have shown an association between household poverty and poorer outcomes in HCT procedures generally, there are limited data on how poverty influences the success of HCT in children specifically.

Dr. Bona and her team sought to fill this gap by reviewing outcomes data for pediatric allogeneic transplant recipients from the Center for International Blood and Marrow Transplant Research Database, the largest available repository of HCT outcomes. The researchers looked at two cohorts of patients: 2,053 children with malignant disease and 1,696 children with non-malignant disease, who underwent a first HCT between 2006 and 2015. Neighborhood poverty exposure was defined according to U.S. Census definitions as living within a ZIP code in which 20% or more of the residents live below 100% of the Federal Poverty Level. They also stratified patients by type of insurance and used Medicaid as a proxy measure for household level poverty. The researchers looked at pediatric patients' overall survival defined as the time from HCT until death from any cause, as well as relapse, transplant-related mortality, acute and chronic graft-versus-host disease, and infection in the first 100 days following HCT.

Interestingly, neighborhood poverty or having Medicaid insurance did not seem to affect outcomes, including overall survival, relapse, or infection, among children transplanted for non-malignant diseases such as sickle cell disease. Dr. Bona said the study does not explain why this might be and more research is needed; however, it is possible that physicians and families of children with non-malignant conditions who face social health challenges may elect to avoid intensive HCT procedures.

One study limitation is its reliance on proxy measures of household poverty (ZIP code and Medicaid insurance) that do not provide insight into specific aspects of an individual child's socioeconomic exposures and the home environment in which they live that may interfere with their ability to navigate the health care system. Dr. Bona says researchers and clinicians have historically not considered social determinants of health as being as important as biological variables in specialized cancer care and so have not collected data on these factors as part of research. She says this is a missed opportunity.

"We as a field need to recognize that non-biological variables such as your exposure to poverty and other social determinants of health matter just as much as many of the biological variables we pay close attention to when thinking about outcomes for children, and these variables must be collected systematically for research if we want to optimize the care and outcomes of the children we serve," Dr. Bona said.

If future studies could collect more nuanced measures of poverty such as household material hardship (e.g., food insecurity, access to heat and electricity, housing insecurity, transportation insecurity) or language barriers, targeted interventions in the form of assistance programs could potentially help mitigate social hardships and improve the overall care of children with cancer.

Blood(www.bloodjournal.org), the most cited peer-reviewed publication in the field of hematology, is available weekly in print and online. Blood is a journal of the American Society of Hematology (ASH) (www.hematology.org).

SOURCE American Society of Hematology/Blood Journal

http://www.bloodjournal.org

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Study: Poverty Linked to Higher Risk of Death Among Children with Cancer Undergoing Stem Cell Transplantation - PRNewswire

Bone Marrow Stem Cells Comments Off on Study: Poverty Linked to Higher Risk of Death Among Children with Cancer Undergoing Stem Cell Transplantation – PRNewswire | October 29th, 2020

The lymphoid organs purpose is to provide immunity for the body. This second article in a six-part series explains the primary and secondary lymphoid organs and their clinical significance and structure. It comes with a self-assessment enabling you to test your knowledge after reading it

This article is the second in a six-part series about the lymphatic system. It discusses the role of the lymphoid organs, which is to develop and provide immunity for the body. The primary lymphoid organs are the red bone marrow, in which blood and immune cells are produced, and the thymus, where T-lymphocytes mature. The lymph nodes and spleen are the major secondary lymphoid organs; they filter out pathogens and maintain the population of mature lymphocytes.

Citation: Nigam Y, Knight J (2020) The lymphatic system 2: structure and function of the lymphoid organs. Nursing Times [online]; 116: 11, 44-48.

Authors: Yamni Nigam is professor in biomedical science; John Knight is associate professor in biomedical science; both at the College of Human and Health Sciences, Swansea University.

This article discusses the major lymphoid organs and their role in developing and providing immunity for the body. The lymphoid organs include the red bone marrow, thymus, spleen and clusters of lymph nodes (Fig 1). They have many functional roles in the body, most notably:

The red bone marrow and thymus are considered to be primary lymphoid organs, because the majority of immune cells originate in them.

Bone marrow is a soft, gelatinous tissue present in the central cavity of long bones such as the femur and humerus. Blood cells and immune cells arise from the bone marrow; they develop from immature stem cells (haemocytoblasts), which follow distinct developmental pathways to become either erythrocytes, leucocytes or platelets. Stem cells rapidly multiply to make billions of blood cells each day; this process is known as haematopoiesis and is outlined in Fig 2.

To ensure there is a continuous production and differentiation of blood cells to replace those lost to function or age, haematopoietic stem cells are present through adulthood. In the embryo, blood cells are initially made in the yolk sac but, as development of the embryo proceeds, this function is taken over by the spleen, lymph nodes and liver. Later in gestation, the bone marrow takes over most haematopoietic functions so that, at birth, the whole skeleton is filled with red bone marrow.

Red bone marrow produces all erythrocytes, leucocytes and platelets. Haematopoietic stem cells in the bone marrow follow either the myeloid or lymphoid lineages to create distinct blood cells (Fig2); these include myeloid progenitor cells (monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells and platelets), and lymphoid progenitor cells (T-lymphocytes, B-lymphocytes and natural killer cells).

Some lymphoid cells (lymphocytes) begin life in the red bone marrow and become fully formed in the lymphatic organs, including the thymus, spleen and lymph nodes. As puberty is reached and growth slows down, physiological conversion occurs, changing red bone marrow to yellow bone marrow. This entire process is completed by the age of 25years, when red bone marrow distribution shows its adult pattern in the bones.

The pattern is characterised by:

However, under particular conditions, such as severe blood loss or fever, the yellow marrow may revert back to red marrow (Malkiewicz and Dziedzic 2012).

Any disease or disorder that poses a threat to the bone marrow can affect many body systems, especially if it prevents stem cells from turning into essential cells. Those known to damage the marrows productive ability and destroy stem cells include:

A growing number of diseases can be treated with a bone marrow transplant or haematopoietic stem cell transfer; this is often achieved by harvesting suitable donor stem cells from the posterior iliac crests of the hip bone, where the concentration of red bone marrow is highest.

The thymus gland is a bi-lobed, pinkish-grey organ located just above the heart in the mediastinum, where it rests below the sternum (breastbone). Structurally, the thymus resembles a small bow tie, which gradually atrophies (shrinks) with age. In pre-pubescents, the thymus is a relatively large and very active organ that, typically, weighs around 40g, but in a middle-aged adult it may have shrunk sufficiently to be difficult to locate. By 20 years of age, the thymus is 50% smaller than it was at birth, and by 60years of age it has shrunk to a sixth of its original size (Bilder, 2016); this is called thymic involution

Each of the two lobes of the thymus is surrounded by a capsule, within which are numerous small lobules typically measuring 2-3mm in width which are held together by loose connective tissue. Each lobule consists of follicles that are composed of a framework of thyomsin-secreting epithelial cells and a population of T-lymphocytes; these cells are commonly referred to as T-cells (the T denotes their origin as mature cells from the thymus). Lobules have two distinct areas:

In addition to being a major lymphoid organ, the thymus is also recognised as part of the endocrine system because it secretes a family of hormones collectively referred to as thymosin; this is a group of several structurally related hormones secreted by the thymic epithelial cells. These hormones are essential for normal immune function and many members of the thymosin family are used therapeutically to treat cancers, infections and diseases such as multiple sclerosis (Severa et al, 2019).

T-cells originate as haematopoietic stem cells from the red bone marrow (Fig2). A population of these haematopoietic stem cells infiltrate the thymus, dividing further within the cortical regions of the lobules then migrating into the medullary regions to mature into active T-cells; this process of T-cell maturation is controlled by the hormone thymosin. A proportion of these mature T-cells continually migrate from the thymus into the blood and other lymphoid organs (spleen and lymph nodes), where they play a major role in the bodys specific immune responses (which will be discussed in detail in part 3 of this series). The importance of these cells is apparent in patients who have depleted T-cell populations, such as those infected with HIV.

One of the most important functions of the thymus is programming T-cells to recognise self antigens through a process called thymic education. This process allows mature T-cells to distinguish foreign, and therefore potentially pathogenic, material from antigens that belong to the body. It has been demonstrated that removal of the thymus may lead to an increase in autoimmune diseases, as this ability to recognise self is diminished (Sherer et al, 1999).

Diseases of the thymus include thymic cancer and myasthenia gravis (MG). MG occurs when the thymus produces antibodies that block or destroy the muscle-receptor sites, causing the muscles to become weak and easily tired. It most commonly affects muscles that control the eyes and eyelids, resulting in droopy eyelids and difficulty making facial expressions; chewing, swallowing and speaking also become difficult. MG can affect people of any age, but typically starts in women aged <40years and men aged >60years.

In most cases of either MG or thymic cancer, thymectomy is recommended. Patients who have had a thymectomy may develop an immunodeficiency known as Good syndrome, which increases their susceptibility to bacterial, fungal and viral opportunistic pathogens; this condition is, however, relatively rare.

The spleen and lymph nodes are two major secondary lymphoid organs that play key roles in:

When foreign antigens reach these organs, they initiate lymphocyte activation and subsequent clonal expansion and maturation of these important white blood cells. Mature lymphocytes can then leave the secondary organs to enter the circulation, or travel to other areas, and target foreign antigens.

The spleen is the largest lymphoid organ. Situated in the upper left hypochondriac region of the abdominal cavity, between the diaphragm and the fundus of the stomach, it primarily functions as a filter for the blood, bringing it into close contact with scavenging phagocytes (white blood cells in the spleen that will seek out and eat any pathogens in the blood) and lymphocytes.

Due to its extensive vascularisation, the spleen is a dark-purplish oval-shaped organ; in adults it is approximately 12cm long, 7cm wide and weighs around 150g. However, the size of the spleen can vary with circumstance: it diminishes in starvation, after heavy exercise and following severe haemorrhage (Gujar et al, 2017), and recent investigations indicate an increase in size in well-fed individuals and during the ingestion of food (Garnitschnig et al, 2020).

The spleen (Fig3) is enclosed in a dense, fibro-elastic capsule that protrudes into the organ as trabeculae; these trabeculae constitute the organs framework. Blood enters the spleen from the splenic artery and leaves via the splenic vein, both of which are at the hilum; the splenic vein eventually becomes a tributary of the hepatic portal vein.

The spleen is made up of two regions:

White pulp is a mass of germinal centres of dividing B-lymphocytes (B-cells), surrounded by T-cells and accessory cells, including macrophages and dendritic cells; these cells are arranged as lymphatic nodules around branches of the splenic artery. As blood flows into the spleen via the splenic artery, it enters smaller, central arteries of the white pulp, eventually reaching the red pulp. The red pulp is a spongy tissue, accounting for 75% of the splenic volume (Pivkin et al, 2016); it consists of blood-filled venous sinuses and splenic cords.

Splenic cords are made up of red and white blood cells and plasma cells (antibody-producing B-cells); therefore, the red pulp primarily functions as a filtration system for the blood, whereas the white pulp is where adaptive T- and B-cell responses are mounted. The colour of the white pulp is derived from the closely packed lymphocytes and the red pulps colour is due to high numbers of erythrocytes (Stewart and McKenzie, 2002).

The spleen has three major functions:

The spleens main immunological function is to remove micro-organisms from circulation. The lymphatic nodules are arranged as sleeves around the blood vessels, bringing blood into the spleen. Within the white pulp are splenic nodules called Malpighian corpuscles, which are rich in B-cells, so this portion of lymphoid tissue is quick to respond to foreign antigenic stimulation by producing antibodies. The walls of the meshwork of sinuses in the red pulp also contain phagocytes that engulf foreign particles and cell debris, effectively filtering and removing them from circulation.

In the spleens destruction of old and senescent red blood cells, they are digested by phagocytic macrophages in the red pulp. The haemoglobin is then split apart into haem and globin. The globin is broken down into its constituent amino acids, which can be utilised in the synthesis of a new protein. Haem consists of an iron atom surrounded four non-iron (pyrrole) rings.

The iron is removed and transported to be stored as ferritin, then reused to make new haemoglobin in the red bone marrow; macrophages convert the pyrrole rings into the green pigment biliverdin and then into the yellow pigment bilirubin. Both are transported to the liver bound to plasma albumin. Bilirubin, the more toxic pigment, is conjugated in the liver to form a less toxic compound, which is excreted in bile.

The red pulp partly serves to store a large reserve of the bodys platelets up to a third of the total platelet supply. In some animals particularly athletic mammals such as horses, greyhounds and foxes the spleen is also an important reservoir of blood, which is released into circulation during times of stress to improve aerobic performance. In humans, however, the spleen contributes only a small percentage of blood cells into active circulation under physiological stress; the total stored blood volume is believed to be only 200-250ml (Bakovic et al, 2005). The capsule of the spleen may contract following haemorrhage, releasing this reserve into circulation in the body.

The spleen also plays a minor role in haematopoiesis: usually occuring in foetuses of up to five months gestation, erythrocytes, along with the bone marrow, are produced by the spleen.

As the spleen is the largest collection of lymphoid tissue in the body, infections that cause white blood cell proliferation and antigenic stimulation may cause germinal centres in the organ to expand, resulting in its enlargement (splenomegaly). This happens in many diseases for example, malaria, cirrhosis and leukaemia. The spleen is not usually palpable, but an enlarged spleen is palpable during deep inspiration. Enlargement may also be caused by any obstruction in blood flow, for example in the hepatic portal vein.

The anatomical position of the spleen coincides with the left tenth rib. Given its proximity to the abdominal wall, it is one of the most commonly injured organs in blunt abdominal trauma. The spleen is a fragile organ and, due to its highly vascularised nature, any injury causing rupture will rapidly lead to severe intraperitoneal haemorrhage; death may result due to massive blood loss and shock.

A moderate splenic injury may be managed conservatively, but an extensively burst or ruptured spleen may be treated by complete and prompt removal (splenectomy). However, current data supports successful non-operative management of many traumatic splenic injuries, with the intention of reducing the need for complete removal (Armstrong et al, 2019).

Patients being treated for certain malignant diseases may also require a partial or total splenectomy and, although other structures such as the bone marrow and liver can take over some of the functions that are usually carried out by the spleen, such patients may be at increased risk of infection. With an overwhelming post-splenectomy infection, there is also an increased risk of sepsis, which is associated with significant morbidity and mortality. Infection is usually with encapsulated pathogens, including Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis. Clinical guidelines to help reduce the risk of infection advocate education about infection prevention, vaccination and antibiotic prophylaxis (Arnott et al, 2018).

Swollen lymph nodes and a fever are sure signs that the body is mounting an effective immune response against an offending pathogen

Lymph nodes vary in size and shape, but are typically bean-shaped structures found clustered at specific locations throughout the body. Although their size varies, each node has a characteristic internal structure (Fig4).

The central portions of the lymph node are essential to its function; here, there are large numbers of fixed macrophages, which phagocytose foreign material such as bacteria on contact, and populations of B- and T-cells. Lymph nodes are crucial to most antibody-mediated immune responses: when the phagocytic macrophages trap pathogenic material, that material is presented to the lymphocytes so antibodies can be generated.

Each lymph node is supplied by one or more afferent lymphatic vessels, which deliver crude, unmodified lymph directly from neighbouring tissues. A healthy, fully functioning node removes the majority of pathogens from the lymph before the fluid leaves via one or more efferent lymphatic vessels. In addition to its lymphatic supply, each lymph node is supplied with blood via a small artery; the artery delivers a variety of leucocytes, which populate the inner regions of the node.

When infection is present, the lymph nodes become increasingly metabolically active and their oxygen requirements increase. A small vein carries deoxygenated blood away from each node and returns it to the major veins. In times of infection, this venous blood may carry a variety of chemical messengers (cytokines) that are produced by the resident leucocytes in the nodes. These cytokines act as general warning signals, alerting the body to the potential threat and activating a variety of specific immune reactions.

The structure of a lymph node is not unlike that of the spleen. Each lymph node is divided into several regions:

During infection, antibody-producing B-cells begin to proliferate in the germinal centres, causing the affected lymph nodes to enlarge and become palpable and tender. Some of the cytokines released are pyrogenic (meaning they cause fever) and act directly on the thermoregulatory centre in the hypothalamus to increase body temperature. As the majority of human pathogens divide optimally at around 37C, this increase in body temperature serves to slow down bacterial replication, allowing the infection to be dealt with more efficiently by the immune system. Swollen lymph nodes and a fever are both sure signs that the body is mounting an effective immune response against the offending pathogen; this will be discussed in more detail in part 3 of this series.

Other types of lymphatic tissue also exist. Mucosa-associated lymphoid tissue (MALT) is positioned to protect the respiratory and gastrointestinal tracts from invasion by microbes. The following are made up of MALT:

The tonsils are aggregates of lymphatic tissue strategically located to prevent foreign material and pathogens from entering the body. The palatine tonsils are in the pharynx, the lingual tonsils in the oral cavity and the pharyngeal tonsils (adenoids) are at the back of the nasal cavity; as a result of this, the tonsils themselves are at high risk of infection and inflammation (tonsillitis). This will also be discussed further in part 3.

Armstrong RA et al (2019) Successful non-operative management of haemodynamically unstable traumatic splenic injuries: 4-year case series in a UK major trauma centre. European Journal of Trauma and Emergency Surgery; 45: 5, 933-938.

Arnott A et al (2018) A registry for patients with asplenia/hyposplenism reduces the risk of infections with encapsulated organisms. Clinical Infectious Diseases; 67: 4, 557-561.

Bakovi D et al (2005) Effect of human splenic contraction on variation in circulating blood cell counts. Clinical and Experimental Pharmacology and Physiology; 32: 11, 944-951.

Bilder G (2016) Human Biological Aggin: From Macromolecules to Organ Systems. Wiley.

Garnitschnig L et al (2020) Postprandial dynamics of splenic volume in healthy volunteers. Physiological Reports; 8: 2, e14319.

Gujar S et al (2017) A cadaveric study of human spleen and its clinical significance. National Journal of Clinical Anatomy; 6: 1, 35-41.

Makiewicz A, Dziedzic M (2012) Bone marrow reconversion: imaging of physiological changes in bone marrow. Polish Journal of Radiology; 77: 4, 45-50.

Pivkin IV et al (2016) Biomechanics of red blood cells in human spleen and consequences for physiology and disease. Proceedings of the National Academy of Sciences of the United States of America; 113: 28, 7804-7809.

Severa M et al (2019) Thymosins in multiple sclerosis and its experimental models: moving from basic to clinical application. Multiple Sclerosis and Related Disorders; 27: 52-60.

Sherer Y et al (1999) The dual relationship between thymectomy and autoimmunity: the kaleidoscope of autoimmune disease. In: Paul S (ed) Autoimmune Reactions. Contemporary Immunology. Totowa, NJ: Humana Press.

Stewart IB, McKenzie DC (2002) The human spleen during physiological stress. Sports Medicine; 32: 6, 361-369.

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The lymphatic system 2: structure and function of the lymphoid organs - Nursing Times

Bone Marrow Stem Cells Comments Off on The lymphatic system 2: structure and function of the lymphoid organs – Nursing Times | October 29th, 2020

Mirror photo by Patrick Waksmunski / Johnathan Dodson, an intensive care unit nurse practitioner who treats COVID-19 patients at UPMC Altoona, recently met the woman who donated the stem cells that helped him overcome leukemia.

A few weeks ago, nurse practitioner and former leukemia patient Johnathan Dodson interrupted a reporters phone interview to give his two young sons a hug and a kiss before they went to sleep.

The interview concerned the Claysburg natives recent appearance as a healthcare hero on Jimmy Kimmel Live, because Dodson treats COVID-19 patients at UPMC Altoona.

The segment also featured Dodsons surprise virtual meeting on the show with his own healthcare hero: the Texas woman who donated the stem cells that enabled Dodson to survive past his early 20s via a transplant.

Theyre here because of her, Dodson, 36, said of the little boys hed just sent off to bed.

In the interaction that followed the on-screen introduction to his donor, Dodson tried to explain his feelings about what the woman had done: how it hadnt been limited to saving his life, but had also kept his parents, siblings and friends from losing him and had spread out to allow for the establishment of his own family, including those kids, Chase, now 7, and Karter, now 4.

I dont think she realized the ripple effects, Dodson said.

He had long thought about a first encounter with Shannon Weishuhn of Rowlett, Texas.

I had kind of prepared this thank-you speech in my head, he said.

(But) how do you thank someone who saved your life? Dodson asked.

For Weishuhn, also a nurse, the donation was an ancient memory, Dodson said, based on an off-screen conversation he had with her, which included a virtual meeting with his family.

She had no idea of the butterfly effect that her action had on his world, he said, speaking of the idea that small occurrences can have big consequences. Thats the message I was trying to convey, he said.

Almost didnt make it

Dodson almost didnt make it to the transplant.

But in the process of getting through his difficulties with leukemia, he found his calling.

He was diagnosed initially in 2003.

He went through chemotherapy to wipe out my immune system, which also wiped out the cancer cells, he said.

The idea was to do an immune system reset, with the hope that the cancer cells wouldnt grow back, he said.

He went into remission, but relapsed at the beginning of 2004, he said.

So he underwent chemotherapy again.

He relapsed again.

The third time he got chemo was in preparation for the transplant.

He nearly died multiple times, and at one point, his survival chances shrunk to about 3 percent, Dodson said.

The cancer had broken into his spine and his brain, he said.

Only a handful of prior cases had been treated successfully when that had happened, he said.

There were three options a shunt in his head and more chemotherapy, spinal taps with chemo or hospice at home, he said.

His parents knew he didnt want a shunt in his head, so that was out of the question, Dodson said.

His parents asked the doctors what theyd do if he was their son, and they recommended hospice, he said.

But a nurse stepped in and said you need to give him a chance, arguing that his survival from two previous crises should merit another try, Dodson said.

Thats when my parents switched and opted for treatment, Dodson said. That sealed the deal.

Once the decision was made, there was talk about sending him to Texas, the only place where the contemplated treatment had been done successfully, he said.

Dodson nixed that.

If I was going to die, I was going to die here, he said.

The reason Im here today

By that time, the nurses who took care of him at West Penn Hospital, now part of Allegheny Health Network, had almost become family, he said.

They along with his donor are the reason Im here today, he said.

The nurses are also the reason hes a nurse himself.

The transplant, however, didnt suddenly make things all better.

He had a really rough go (afterwards), said Dr. John Lister, chief of the division of hematology and cellular therapy of Allegheny Health Network Cancer Institute and a member of Dodsons transplant team.

Caring for patients after leukemia transplants is as challenging as anything in medicine, said Lister, who is a descendant of Joseph Lister, a pioneer in antiseptic surgery.

Its challenging because the blood stem cells harvested from the donors blood, when injected into the recipient, create a new white-blood-cell immune system that attacks the recipients diseased white-blood-cell immune system, Lister indicated.

It can be fatal, he said. And extremely debilitating.

Doctors deal with it by giving powerful immunosuppressant medications, he said.

The direction of attack the donor material attacking the recipients is the opposite of the direction of attack with transplants of organs like kidneys, Lister said.

After those other transplants, the recipients immune system attacks the donor organ, he said.

Dodson was kept alive due to the intensive efforts of many people, Lister said.

Eventually, the initial reaction dies down, Lister said.

Hes totally normal at this point, Lister said of Dodson. I would say hes cured.

The donor matched Dodson in certain key genes that make the immune system work, Lister said.

The harvesting of donor stem cells occurs after the donor is given a growth factor that causes those stem cells to leave the bone marrow and enter the bloodstream, Lister said.

Blood stem cells can become any of the three types of blood cells, given the right conditions.

When injected into the recipient, they home to the marrow where theyre needed, according to Lister.

There they divide and repopulate, he said.

Anyone willing to make a bone marrow or stem cell donation can go to bethematch.org.

Its free to register, Dodson said. More ethnically diverse donors are needed, he added.

Last year, the web site helped facilitate 6,425 transplants, Dodson said.

You could change someones life forever, he said.

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UPMC nurse practitioner hailed 'healthcare hero' on live TV - Altoona Mirror

Bone Marrow Stem Cells Comments Off on UPMC nurse practitioner hailed ‘healthcare hero’ on live TV – Altoona Mirror | October 29th, 2020

The research report on Mesenchymal Stem Cells Market gives thorough insights regarding various key trends that shape the industry expansion with regards to regional perspective and competitive spectrum. Furthermore, the document mentions the challenges and potential restrains along with latent opportunities which may positively impact the market outlook in existing and untapped business spaces. Moreover, it presents the case studies, including the ones related to COVID-19 pandemic, to convey better understanding of the industry to all the interested parties.

The recent market trend of increasingly using Mesenchymal Stem Cells for understanding the development of a disease extensively fuel the growth of this market in the coming years. Another trend that will aid the growth of the global Mesenchymal Stem Cells market is the escalating demand for personalized medicine. Extensive investments are being made by various organizations, pharmaceutical companies, and governments for the research and development of drugs, and this is another trend that is benefiting the growth of the global Mesenchymal Stem Cells market. This is because Mesenchymal Stem Cells techniques enable researchers to compare Mesenchymal Stem Cells changes between disease samples and normal samples. Public health can thus be analyzed as the changes in Mesenchymal Stem Cells are influenced by internal biological system and environment directly.

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The report covers extensive analysis of the key market players in the market, along with their business overview, expansion plans, and strategies. The key players studied in the report include: Advanced Cell Technology Incorporated, Stem cell technologies Inc., Stemedica Cell Technologies, Inc., Cyagen Biosciences Inc., EMD Millipore Corporation, ScienCell Research Laboratories., Cytori Therapeutics Inc., Cell Applications, Inc., Axol Bioscience Ltd., Aastrom Biosciences, BrainStorm Cell Therapeutics., R&D Systems, Inc., Genlantis, Inc., Celprogen, Inc..

Mesenchymal Stem Cells Market Segmentation:

In market segmentation by types of Mesenchymal Stem Cells, the report covers-

Bone MarrowUmbilical Cord BloodPeripheral BloodLung TissueSynovial TissuesAmniotic FluidsAdipose Tissues

In market segmentation by applications of the Mesenchymal Stem Cells, the report covers the following uses-

InjuriesDrug DiscoveryCardiovascular InfractionOthers

Regional Analysis for Mesenchymal Stem Cells Market-:

1) North America- (United States, Canada)

2) Europe- (Germany, France, UK, Italy, Russia, Spain, Netherlands, Switzerland, Belgium)

3) Asia Pacific- (China, Japan, Korea, India, Australia, Indonesia, Thailand, Philippines, Vietnam)

4) Middle East & Africa- (Turkey, Saudi Arabia, United Arab Emirates, South Africa, Israel, Egypt, Nigeria)

5) Latin America- (Brazil, Mexico, Argentina, Colombia, Chile, Peru)

The report provides insights on the following pointers :

Market Penetration: Comprehensive information on the product portfolios of the top players in the Supply Chain Analytics market.

Product Development/Innovation: Detailed insights on the upcoming technologies, R&D activities, and product launches in the market

Competitive Assessment: In-depth assessment of the market strategies, geographic and business segments of the leading players in the market

Market Development: Comprehensive information about emerging markets. This report analyzes the market for various segments across geographies

Market Diversification: Exhaustive information about new products, untapped geographies, recent developments, and investments in the Supply Chain Analytics market

NOTE: Our analysis involves the study of the market taking into consideration the impact of the COVID-19 pandemic. Please get in touch with us to get your hands on an exhaustive coverage of the impact of the current situation on the market. Our expert team of analysts will provide as per report customized to your requirement.

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Table of Content

Chapter 1 Mesenchymal Stem Cells Introduction and Market Overview

Chapter 2 Executive Summary

Chapter 3 Industry Chain Analysis

Chapter 4 Global Mesenchymal Stem Cells Market, by Type

Chapter 5 Mesenchymal Stem Cells Market, by Application

Chapter 6 Global Mesenchymal Stem Cells Market Analysis by Regions

Chapter 7 North America Mesenchymal Stem Cells Market Analysis by Countries

Chapter 8 Europe Mesenchymal Stem Cells Market Analysis by Countries

Chapter 9 Asia Pacific Mesenchymal Stem Cells Market Analysis by Countries

Chapter 10 Middle East and Africa Mesenchymal Stem Cells Market Analysis by Countries

Chapter 11 South America Mesenchymal Stem Cells Market Analysis by Countries

Chapter 12 Competitive Landscape

Chapter 13 Industry Outlook

Chapter 14 Global Mesenchymal Stem Cells Market Forecast

Chapter 15 New Project Feasibility Analysis

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Big Market Research has a range of research reports from various domains across the world. Our database of reports of various market categories and sub-categories would help to find the exact report you may be looking for.

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Mesenchymal Stem Cells Market Augmented Expansion to Be Registered by 2020-2025 - Eurowire

Bone Marrow Stem Cells Comments Off on Mesenchymal Stem Cells Market Augmented Expansion to Be Registered by 2020-2025 – Eurowire | October 29th, 2020

Catalent Biologics has agreed to manufactureNurOwn, the cell-based therapy by BrainStorm Cell Therapeuticsbeing evaluated in a soon-to-conclude pivotal trial as a possible treatment of amyotrophic lateral sclerosis (ALS).

With this agreement, Catalent will produce NurOwn under current Good Manufacturing Practices standards set to ensure that batches of a medicine are produced with consistent high quality at its new 32,000-square-foot cell therapy manufacturing facility in Houston.

We are proud to have a partner in Catalent whose excellence in manufacturing quality therapies will support commercial supply of NurOwn, Chaim Lebovits, BrainStorms CEO, said in a press release.

With NurOwn, a patients mesenchymal stem cellsare collected from the bone marrow and treated in the lab to produce proteins called neurotrophic factors (NTFs), compounds that promote nervous tissue growth and survival. (Mesenchymal stem cells, orMSCs, are stem cells that can differentiate into a variety of other cell types.)

The modified cells called MSC-NTF cells are then injected into the patients spinal cord, where their NTFs are expected to promote the growth and survival of nerve cells, which are damaged over the course of ALS.

Using a patients own cells as a therapy minimizes the risk of an immune reaction, as might occur with cells from a donor.

The U.S. Food and Drug Administration has given NurOwn bothfast track and orphan drugdesignations to support and speed its development for ALS. The medicine also received orphan drug designation from the European Medicines Agency.

We know that ALS patients are in urgent need of a new treatment option. If NurOwn is successful in the current clinical trials, this agreement will be integral to ensuring rapid access for patients, Lebovits added.

NurOwn showed an ability to slow progression in people with fast-progressing disease in a Phase 2 trial (NCT02017912). This led Brainstorm to open a Phase 3 trial (NCT03280056)to confirm those findings in a larger group of ALS patients.

The trial, taking place at six sites in the U.S., enrolled 200 patients and randomly assigned them to either NurOwn or a placebo, given in three intrathecal (into the spinal cord) injections at two-month intervals.

Researchers are evaluating NurOwns effectiveness using therevised amyotrophic lateral sclerosis functional rating scale(ALSFRS-R), which assesses such daily life abilities as swallowing, speaking, dressing and washing oneself, climbing stairs, and turning over in bed.

Its primary goal is to determine whether NurOwn outperforms a placebo at reducing the rate of decline in ALSFRS-R scores over six months. A change of 1.25 points or more in ALSFRS-R scores each month, compared to scores recorded prior to treatment, defines a responder.

Other trial goals include safety, the number of patients whose disease has not progressed, total ALSFRS-R decline, and overall survival. Samples of blood and cerebrospinal fluid will also be collected to evaluate biomarkers, like neurotrophic factors and immune molecules, in response to the treatment.

BrainStorm expects to deliver top-line results this year; the study is set to fully conclude in December.

Should results be positive and NurOwn be approved for clinical use, BrainStorm and Catalent will consider extending their partnership to allow for commercial manufacturing of NurOwn at the Houston facility.

Forest Ray received his PhD in systems biology from Columbia University, where he developed tools to match drug side effects to other diseases. He has since worked as a journalist and science writer, covering topics from rare diseases to the intersection between environmental science and social justice. He currently lives in Long Beach, California.

Total Posts: 45

Ins holds a PhD in Biomedical Sciences from the University of Lisbon, Portugal, where she specialized in blood vessel biology, blood stem cells, and cancer. Before that, she studied Cell and Molecular Biology at Universidade Nova de Lisboa and worked as a research fellow at Faculdade de Cincias e Tecnologias and Instituto Gulbenkian de Cincia. Ins currently works as a Managing Science Editor, striving to deliver the latest scientific advances to patient communities in a clear and accurate manner.

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Catalent to Produce BrainStorm's NurOwn Cell Therapy for ALS - ALS News Today

Bone Marrow Stem Cells Comments Off on Catalent to Produce BrainStorm’s NurOwn Cell Therapy for ALS – ALS News Today | October 29th, 2020

GAITHERSBURG, Md., Oct. 27, 2020 (GLOBE NEWSWIRE) -- NexImmune, a clinical-stage biotechnology company developing unique non-genetically-engineered T cell immunotherapies, announced today that it has signed a research initiative related to its AIM nanoparticle technology with City of Hope, a world-renowned independent research and treatment center for cancer, diabetes and other life-threatening diseases.

City of Hope is a participating clinical site in the ongoing Phase 1/2 study of NEXI-001. The cancer center will leverage both patient samples from the ongoing NexImmune Phase 1/2 clinical study of NEXI-001 in acute myeloid leukemia (AML) patients with relapsed disease after allogeneic stem cell transplantation and the centers tumor repository bank of primary leukemia samples, one of the largest collections in the world, to drive the research.

NEXI-001 is a cellular product candidate that contains populations of naturally occurring CD8+ T cells directed against multiple antigen targets for AML, and it is the first clinical product generated by the Companys AIM nanoparticle technology.

NexImmune has developed a unique and versatile technology platform that lends itself very effectively to important areas of ongoing research in the field of AML, said Guido Marcucci, M.D., Chair and Professor with City of Hopes Department of Hematologic Malignancies Translational Science. Our collective goal is to translate future research findings into new, more effective T cell immunotherapies to the benefit of these very difficult to treat patients.

A key objective of the research will focus on the identification of new antigen targets that are expressed on both leukemic blasts as well as leukemic stem cells, and those which represent survival proteins to both. Once identified, these antigen targets will be loaded on NexImmune AIM-nanoparticles to expand antigen-specific CD8+ T cells, and evaluated in pre-clinical models for anti-tumor potency, tumor-specific killing, and response durability.

In addition, the research initiative will aim to further understand different mechanisms of tumor escape, such as tumor antigen and human leukocyte antigen (HLA) downregulation due to immune pressure.

Research between NexImmune and City of Hope will inform a scientific understanding of how the immune system can address certain tumor escape mechanisms to more effectively fight aggressive cancers like AML, and how this might be accomplished with NexImmunes AIM technology and T cell products, said Monzr Al Malki, M.D., Director of City of Hopes Unrelated Donor BMT Program and Haploidentical Transplant Program and an Associate Clinical Professor with Department of Hematology and Hematopoietic Cell Transplantation. Based on our current clinical experience with this technology, were excited to learn what more this research will tell us.

City of Hope is a world-class clinical research institution that has built one of the largest banks of leukemia samples in the world, said Han Myint, M.D., NexImmune Chief Medical Officer. The depth of expertise that Drs. Marcucci, Al Malki and their team bring to this research initiative will help NexImmune continue to develop innovative products that can help patients with AML and other hard-to-treat cancers.

City of Hope is a leader inbone marrow transplantation. More than 16,000 stem cell and bone marrow transplants have been performed at City of Hope, and more than 700 are performed annually. City of Hopes BMT program is the only one in the nation that has had one-year survival above the expected rate for 15 consecutive years, based on analysis by the Center for International Blood and Marrow Transplant Research.

About NexImmuneNexImmune is a clinical-stage biotechnology company developing unique approaches to T cell immunotherapies based on its proprietary Artificial Immune Modulation (AIM) technology. The AIM technology is designed to generate a targeted T cell-mediated immune response and is initially being developed as a cell therapy for the treatment of hematologic cancers. AIM nanoparticles (AIM-np) act as synthetic dendritic cells to deliver immune-specific signals to targeted T cells and can direct the activation or suppression of cell-mediated immunity. In cancer, AIM-expanded T cells have demonstrated best-in-class anti-tumor properties as characterized by in vitro analysis, including a unique combination of anti-tumor potency, antigen target-specific killing, and long-term T cell persistence. The modular design of the AIM platform enables rapid expansion across multiple therapeutic areas, with both cell therapy and injectable products.

NexImmunes two lead T cell therapy programs, NEXI-001 and NEXI-002, are in Phase 1/2 clinical trials for the treatment of relapsed AML after allogeneic stem cell transplantation and multiple myeloma refractory to >3 prior lines of therapy, respectively. The Companys pipeline also has additional preclinical programs, including cell therapy and injectable product candidates, for the treatment of oncology, autoimmune disorders, and infectious diseases.

For more information, visit http://www.neximmune.com.

Media Contact:Mike BeyerSam Brown Inc. Healthcare Communications312-961-2502mikebeyer@sambrown.com

Investor Contact:Chad RubinSolebury Trout+1-646-378-2947crubin@soleburytrout.com

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NexImmune Establishes Research Initiative with City of Hope to Focus on Novel Immunotherapeutic Approaches to Acute Myeloid Leukemia - GlobeNewswire

Bone Marrow Stem Cells Comments Off on NexImmune Establishes Research Initiative with City of Hope to Focus on Novel Immunotherapeutic Approaches to Acute Myeloid Leukemia – GlobeNewswire | October 29th, 2020

After tackling two major research challenges, the founders of Be Biopharma are ready to announce their official launch along with a $52 million funding round. They are intent upon usingthe bodys B cells to treat a range of diseases.

The Series A round was led by Atlas Venture and RA Capital Management. Joining in were Longwood, Alta Partners and Takeda Ventures.

We have an ambitious plan to be the company that knows how to make B cells and mirror them precisely and make them at scale, Aleks Radovic-Moreno, Be Biopharmas president and director, said in a phone interview.

B cells, which play a leading role in the bodys immune response, can be taken out, genetically programmed to help fight specific diseases and then put back in the body. The cells also can come from healthy donors.

The challenges involved being able to efficiently edit the cells and then make them in sufficient quantities, Radovic-Moreno said. Those are the two big problems we have overcome.

Founded this year, Be Biopharma is looking to hire people in research, engineering and manufacturing, as well as a full-time leadership team, said Radovic-Moreno, an entrepreneur in residence at Longwood Fund, a co-founder and investor in Be Biopharma. The startups CEO is David Steinberg, a general partner at Boston-based Longwood.

From there, the company hopes to begin developing therapeutics for use in people. Cancers and autoimmune diseases are potential targets, as are monogenic diseases like cystic fibrosis. B cells, for example, could replace the need for invasive bone marrow transplants, Radovic-Moreno said.

If we can do that, it would change the lives of so many people. So, were trying to move that as fast as humanly possible, said Radovic-Moreno, who declined to offer a specific timeline.

The path for B cells could be relatively quick, he said, based on the experience of T cells and stem cells, he said. B cell therapies, though, are expected to be safer and less toxic than those involving T cells.

Be Biopharma is drawing on research undertaken at the Seattle Childrens Research Institute by Dr. David Rawlings and Richard James. They are among the co-founders of the new company.

B cells play a key role in combatting diseases by catalyzing humoral immunity the arm of the immune system that manufactures large quantities of proteins to neutralize disease-causing pathogens and manipulate immune cell behavior, Rawlings, director of the Center for Immunity and Immunotherapies at the Seattle institute, said in a statement. Today, this powerful part of the immune system is only passively and/or indirectly addressed therapeutically. Our ambition is to advance the field by building a new class of engineered B cell medicines that will provide direct control over the power of humoral immunity and help transform the prognosis for patients who currently have limited treatment options.

Picture: Feodora Chiosea, Getty Images

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Startup focused on B-cell therapies launched with $52M in Series A - MedCity News

Bone Marrow Stem Cells Comments Off on Startup focused on B-cell therapies launched with $52M in Series A – MedCity News | October 29th, 2020

What is Adipose Tissue Derived Stem Cell Therapy?

Adipose tissue derived stem cells (ADSCs) are stem cells originated from adipocytes. ADSCs have characteristics similar to bone marrow mesenchymal stem cells. Thus Adipose-derived stem cells substitute for bone marrow as a source of stem cells. Different varieties of manual and automatic stem cell separation procedures are used to separate adipose stem cells (ASCs) from adipose tissue. Flow cytometry can be utilized to isolate ADSCs from other stem cells within a cell solution. Currently, adipose derived stem cells (ADSCs) are generally used in the generation of regenerative medicine due to its anti-inflammatory, anti-apoptotic, and immunomodulatory properties.

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The research provides answers to the following key questions:

A new market study report by The Insight Partners on the Adipose Tissue Derived Stem Cell Therapy Market has been released with reliable information and accurate forecasts for a better understanding of the current and future market scenarios. The report offers an in-depth analysis of the global market, including qualitative and quantitative insights, historical data, and estimated projections about the market size and share in the forecast period. The forecasts mentioned in the report have been acquired by using proven research assumptions and methodologies. Hence, this research study serves as an important depository of the information for every market landscape. The report is segmented on the basis of types, end-users, applications, and regional markets. Some of the key players in the study are AlloCure, Inc, Antria, Inc., Celgene Corporation, Cellleris SA, Corestem, Inc., Cytori Therapeutics, LLC, Intrexon, Inc., Mesoblast Ltd., Pluristem Therapeutics, Inc., Tissue Genesis, Inc. etc.

Market Insights:

The Adipose Tissue-derived Stem Cell Therapy Market is growing due to increasing use of regenerative medicine in disease treatment and increasing private and public funding for stem cell therapy. However, high cost associated with stem cell processing hampers growth of this market.

An Overview of the Impact of COVID-19 on this Market:

Due to the pandemic, we have included a special section on the Impact of COVID 19 on the Adipose Tissue Derived Stem Cell Therapy Market which would mention How the Covid-19 is Affecting the Adipose Tissue Derived Stem Cell Therapy Industry, Market Trends and Potential Opportunities in the COVID-19 Landscape, Covid-19 Impact on Key Regions and Proposal for Adipose Tissue Derived Stem Cell Therapy Players to fight Covid-19 Impact.

Adipose Tissue Derived Stem Cell Therapy Market: Regional analysis includes:

The Adipose Tissue Derived Stem Cell Therapy Market segments and Market Data Break Down are illuminated below:By Cell Type (Autologous Stem Cells, Allogeneic Stem Cells);

Product (Cell Line, Culture Media);

Disease (Cancer, Obesity, Wounds and Injuries, Musculoskeletal Diseases, Cardiovascular Diseases, Others);

End User (Hospitals and Trauma Centers, Cell banks and Tissue Banks, Research Laboratories and Academic Institutes, Others)

The study conducts SWOT analysis to evaluate strengths and weaknesses of the key players in the Adipose Tissue Derived Stem Cell Therapy market. Further, the report conducts an intricate examination of drivers and restraints operating in the market. The report also evaluates the trends observed in the parent market, along with the macro-economic indicators, prevailing factors, and market appeal with regard to different segments. The report predicts the influence of different industry aspects on the Adipose Tissue Derived Stem Cell Therapy market segments and regions.

This report strategically examines the micro-markets and sheds light on the impact of technology upgrades on the performance of the Adipose Tissue Derived Stem Cell Therapy market.

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Adipose Tissue Derived Stem Cell Therapy Market to Set Phenomenal Growth in Key Regions by 2027 | AlloCure, Inc, Antria, Inc., Cellleris SA, Tissue...

Bone Marrow Stem Cells Comments Off on Adipose Tissue Derived Stem Cell Therapy Market to Set Phenomenal Growth in Key Regions by 2027 | AlloCure, Inc, Antria, Inc., Cellleris SA, Tissue… | October 29th, 2020

June 27, 2020

Following promising phase 1 testing, Mayo Clinic is launching phase 2 of a randomized clinical trial of stem cell treatment for patients with severe spinal cord injury. The clinical trial, known as CELLTOP, involves intrathecal injections of autologous adipose-derived stem cells.

"The field of spinal cord injury has seen advances in recent years, but nothing in the way of a significant paradigm shift. We currently rely on supportive care. Our hope is to alter the course of care for these patients in ways that improve their lives," says Mohamad Bydon, M.D., a neurosurgeon at Mayo Clinic in Rochester, Minnesota.

The first participant in the phase 1 trial was a superresponder who, after stem cell therapy, saw significant improvements in the function of his upper and lower extremities.

"Not every patient who receives stem cell treatment is going to be a superresponder. Among the 10 participants in our phase 1 study, we had some nonresponders and moderate responders," Dr. Bydon says. "One objective in our future studies is to delineate the optimal treatment protocols and understand why patients respond differently."

In CELLTOP phase 2, 40 patients will be randomized to receive stem cell treatment or best medical management. Patients randomized to the medical management arm will eventually cross over to the stem cell arm.

Study participants must be age 18 or older and have experienced traumatic spinal cord injury within the past year. The spinal cord injuries must be American Spinal Injury Association (ASIA) grade A or B.

The initial participant in CELLTOP phase 1 sustained a C3-4 ASIA grade A spinal cord injury. As described in the February 2020 issue of Mayo Clinic Proceedings, the neurological examination at the time of the injury revealed complete loss of motor and sensory function below the level of injury.

After undergoing urgent posterior cervical decompression and fusion, as well as physical and occupational therapy, the patient demonstrated improvement in motor and sensory function. But that progress plateaued six months after the injury.

Stem cells were injected nearly a year after his injury and several months after his improvement had plateaued. Clinical signs of efficacy in both motor and sensory function were observed at three, six, 12 and 18 months following the stem cell injection.

"Our patient also reported a strong improvement with his grip and pinch strength, as well as range of motion for shoulder flexion and abduction," Dr. Bydon says.

Spinal cord injury has a complex pathophysiology. After the primary injury, microenvironmental changes inhibit axonal regeneration. Stem cells can potentially provide trophic support to the injured spinal cord microenvironment by modulating the inflammatory response, increasing vascularization and suppressing cystic change.

"In the phase 2 study, we will begin to learn the characteristics of individuals who respond to the therapy in terms of their age, severity of injury and time since injury," says Anthony J. Windebank, M.D., a neurologist at Mayo's campus in Minnesota and director of the Regenerative Neurobiology Laboratory. "We will also use biomarker studies to learn about the characteristics of responders' cells. The next phase would be studying how we can modify everyone's cells to make them more like the cells of responders."

CELLTOP illustrates Mayo Clinic's commitment to regenerative medicine therapies for neurological care. "Our findings to date will be encouraging to patients with spinal cord injuries," Dr. Bydon says. "We are hopeful about the potential of stem cell therapy to become part of treatment algorithms that improve physical function for patients with these devastating injuries."

Bydon M, et al. CELLTOP clinical trial: First report from a phase I trial of autologous adipose tissue-derived mesenchymal stem cells in the treatment of paralysis due to traumatic spinal cord injury. Mayo Clinic Proceedings. 2020;95:406.

Regenerative Neurobiology Laboratory: Anthony J. Windebank. Mayo Clinic.

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Stem cell treatment after spinal cord injury: The next ...

Spinal Cord Stem Cells Comments Off on Stem cell treatment after spinal cord injury: The next … | October 27th, 2020

At least six international studies have reported T cell reactivity against SARS-CoV-2 in 20% to 50% of people with no known exposure to the virus. Experts suggest doing three tests simultaneously: RTPCR, antibody test, and a T-cell assay, which will give a picture to policymakers if the State or the country has achieved herd immunity against SARS-CoV-2.

A type of white blood cell, T cells are part of the immune system and develop from stem cells in the bone marrow. They help protect the body from infection. Also called T lymphocyte and thymocyte.

For latest updates on coronavirus outbreak, click here

T-cell mediated immunity can be acquired due to previous exposure to other beta coronaviruses which cause the common cold. Knowing a threshold for herd immunity can allow the government to focus on that section of the population who do not have immunity. But they also caution that very few basic science labs in the country like NIMHANS, IISc, or the National Centre for Biological Sciences can do T cell assays in their labs as it is cumbersome and expensive.

Assessing how much of the population has IgG (non-neutralising antibodies), the current active Covid case burden, and T-cell induced protection, simultaneously will give a clear picture of the health of the population, with respect to Covid-19.

"Currently, we do not know when the pandemic will end. If we know how much of the population is immune, it is easier to decide how much of our resources should be allocated to fight Covid, the economy, etc. If done at the state or the sub-state level, we can understand which region needs more resources," said Dr Giridhar Babu, epidemiologist, and member of the State Covid-19 technical advisory committee (TAC).

In the serosurvey undertaken in Karnataka whose results are yet to be announced, with samples from all the eight zones of Bengaluru included, unlike the serosurveys of Delhi, Mumbai, Pune, and Punjab, Karnataka are supposed to have done all three: RTPCR, antibody, and antigen tests simultaneously in the statewide survey.

Coronavirus India update: State-wise total number of confirmed cases, deaths on October 23

Dr V Ravi, Senior Professor and Head, Neurovirology, NIMHANS, and member of State Covid-19 TAC, told DH, "T cell response assay is very cumbersome and complicated to do. Peripheral blood has to be drawn, lymphocytes separated, culture them, stimulate them with antigens, and then take a readout. It is expensive and resource-intensive. Basic science institutes like IISc, NCBS, National Institute of Immunology, ISER, some of them may have the capacity for doing it, but not the medical college laboratories."

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Covid-19: Has Karnataka achieved herd immunity? Simultaneous triple tests will give true picture - Deccan Herald

Bone Marrow Stem Cells Comments Off on Covid-19: Has Karnataka achieved herd immunity? Simultaneous triple tests will give true picture – Deccan Herald | October 24th, 2020

INTRODUCTION

Cleidocranial dysplasia (CCD) is a hereditary disease characterized by incomplete closure of the fontanelle, abnormal clavicle, short stature, and skeletal dysplasia. It has been reported that there are multiple Runx2 mutations in human CCD syndrome (1, 2). Mature osteoblasts defect and bone mineralization disorders were observed in Runx2-deficient mice. The Runx2-heterozygous mice show similar phenotypes to the CCD syndrome (24). RUNX2 triggers mesenchymal stem cells (MSCs) to differentiate into osteoblasts (3, 5). According to the skeletal pathology studies in humans and mice, it is important to accurately regulate Runx2 activity during bone formation and bone remodeling (6, 7). However, the molecular regulation of Runx2 activity remains to be further studied.

The evolutionarily conserved Hippo pathway is essential for tissue growth, organ size control, and cancer development (811). Numerous evidences revealed the important roles of Hippo components in regulating bone development and bone remodeling. YAP, the essential downstream effector of Hippo pathway, regulates multiple steps of chondrocyte differentiation during skeletal development and bone repair (12). YAP also promotes osteogenesis and suppresses adipogenic differentiation by regulating -catenin signaling (13). VGLL4, a member of the Vestigial-like family, acts as a transcriptional repressor of YAP-TEADs in the Hippo pathway (14). Our previous work found that VGLL4 suppressed lung cancer and gastric cancer progression by directly competing with YAP to bind TEADs through its two TDU (Tondu) domains (9, 15). We also found that VGLL4 played a critical role in heart valve development by regulating heart valve remodeling, maturation, and homeostasis (16). Moreover, our team found that VGLL4 regulated muscle regeneration in YAP-dependent manner at the proliferation stage and YAP-independent manner at the differentiation stage (17). Our previous studies suggest that VGLL4 plays an important role to regulate cell differentiation in multiple organs. However, the function of VGLL4 in skeletal formation and bone remodeling is unknown.

Here, we reveal the function of VGLL4 in osteoblast differentiation and bone development. Our in vivo data show that global knockout of Vgll4 results in a wide variety of skeletal defects similar to Runx2 heterozygote mice. Our in vitro studies reveal that VGLL4 deficiency strongly inhibits osteoblast differentiation. We further demonstrate that TEADs can bind to RUNX2, thereby inhibiting the transcriptional activity of RUNX2 independent of YAP binding. VGLL4 could relieve the inhibitory function of TEADs by breaking its interaction with RUNX2. In addition, deletion of VGLL4 in MSCs shows similar skeletal defects with the global Vgll4-deficient mice. Further studies show that knocking down TEADs or overexpressing RUNX2 in VGLL4-deficient osteoblasts reverses the inhibition of osteoblast differentiation.

To study the function of VGLL4 in bone, we first measured -galactosidase activity in Vgll4LacZ/+ mice (16). -Galactosidase activity was enriched in trabecular bones, cortical bones, cranial suture, and calvaria cultures (fig. S1, A to C). Furthermore, in bone marrow MSCs (BMSCs), Vgll4LacZ/+ mice displayed -galactosidase activity in osteoblast-like cells (fig. S1D). During osteoblast differentiation in vitro, osteoblast marker genes such as alkaline phosphatase (Alp) and Sp7 transcription factor (Osterix) were increased and peaked at day 7. Vgll4 showed similar trend in this process at both mRNA and protein levels (Fig. 1A and fig. S1, E and F). To further clarify the important role of VGLL4 in bone development, we used a Vgll4Vgll4-eGFP/+ reporter mouse line in which VGLL4enhanced green fluorescent protein (eGFP) fusion protein expression is under the control of the endogenous VGLL4 promoter, and GFP staining reflects VGLL4 expression pattern in skeletal tissues (16). GFP staining was performed at embryonic day 18.5, week 1, week 2, and week 4 stages. The results indicated that the VGLL4 expression level was increased during bone development (fig. S1G). In addition, VGLL4 was enriched in trabecular bones, cortical bones, chondrocytes, cranial suture, and calvaria (fig. S1, G and K to M). We then observed the colocalization of VGLL4-eGFP with markers of MSCs (CD105), osteoblasts [osteocalcin (OCN)], and chondrocytes [collagen 2a1 (Col2a1)] in long bone and calvaria (fig. S1, H to M). Next, we analyzed VGLL4 expression pattern during osteoblast development in vivo (fig. S1N), which was similar to Alp and Osterix expression patterns in mouse BMSCs of different ages. Together, both in vivo and in vitro data suggest that VGLL4 may play roles in osteoblast differentiation and bone development.

(A) Immunoblotting showed the expression profile of VGLL4 during osteoblast differentiation in C57BL/6J mouse BMSCs. Samples were collected at 0, 1, 4, 7, and 10 days after differentiation. (B) Skeletons of WT and Vgll4/ mice at postnatal day 1 (P1) were double-stained by Alizarin red/Alcian blue (n = 5). Scale bar, 5 mm. (C) Quantification of body length in (B). (D) Skull preparations from control and Vgll4/ mouse newborns were double-stained with Alizarin red and Alcian blue at P1. -QCT images of skulls were taken from control and Vgll4/ mice at P4. Scale bar, 5 mm. (E) Quantification of skull defect area in (D). (F) Clavicle preparations from control and Vgll4/ mouse newborns were double-stained with Alizarin red and Alcian blue at P1 and quantification of clavicle length. Scale bar, 5 mm. (G) Alp staining and Alizarin red staining of calvarial cells from WT and Vgll4/ mice after cultured in osteogenic medium. Scale bar, 3 mm. (H) Relative mRNA levels were quantified by RT-PCR. (I) Hematoxylin and eosin (H&E) staining of femur from WT and Vgll4/ mice at embryonic day 16.5. Scale bar, 125 m. (J) In situ hybridization for Col11 immunostaining. Scale bar, 125 m. In (C), (E), (F), and (H), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001, ns, no significance; unpaired Students t test. Photo credit: Jinlong Suo, State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai.

To investigate the potential function of VGLL4 in bone, we next analyzed the phenotype of Vgll4 knockout (Vgll4/) mice (16). The newborn Vgll4 knockout mice were significantly smaller and underweight compared with their control littermates (Fig. 1, B and C, and fig. S2, A and B). In particular, the membranous ossification of the skull was impaired in Vgll4/ newborns compared with the control littermates (Fig. 1, D and E). Furthermore, Vgll4 knockout mice developed a marked dwarfism phenotype with short legs and short clavicles (Fig. 1, C and F). To assess the role of VGLL4 in osteoblast differentiation, calvarial cells from Vgll4/ mice and wild-type (WT) mice were cultured in osteogenic medium. The activity of Alp in the Vgll4 deletion group was significantly reduced at the seventh day of differentiation (Fig. 1G, top) and was markedly weakened over a 14-day culture period as revealed by Alizarin red S staining (Fig. 1G, bottom). The declined osteogenesis in Vgll4 knockout cells was confirmed by the decreased expression of a series of osteogenic marker genes (Fig. 1H), including Alp, Osterix, and collagen type1 1 (Col11). In addition, in Vgll4/ mice, bone development was severely impaired with remarkable decrease in bone length and almost a complete loss of bone ossification (Fig. 1I). Consistently, immunohistochemical analysis of bone tissue sections from embryos at embryonic day 14.5 further confirmed the defects of bone formation and impaired osteoblast differentiation in Vgll4/ mice (Fig. 1J). Together, our study suggests that VGLL4 is likely to regulate MSC fate by enhancing osteoblast differentiation.

Given that the smaller size of mice is often caused by dysplasia, we also paid attention to the development of cartilage after Vgll4 deletion. As we expected, cartilage development was delayed in Vgll4-deficient mice determined by Safranin O (SO) staining (fig. S2C). Immunohistochemical analysis of collagen X (Col X) further confirmed the delay of cartilage development in Vgll4/ mice (fig. S2D). However, additional experiments would be required to determine the regulatory mechanism behind the observed chondrodysplasia. Although dwarfism was observed and trabecular bones were significantly reduced in the adult Vgll4/ mice, no significant cartilage disorder was observed by SO staining (fig. S2E). In adults, bone is undergoing continuous bone remodeling, which involves bone formation by osteoblasts and bone resorption by osteoclasts. We speculated that Vgll4 deletion might lead to decreased osteoclast activity. To distinguish this possibility, we performed histological analysis by tartrate-resistant acid phosphatase (TRAP) staining to detect osteoclast activity. The results showed that osteoclast activity was comparable between Vgll4/ mice and their control littermates (fig. S2F). Together, our results suggest that the phenotypes observed in Vgll4/ mice are mainly due to the defect of osteoblast activity.

To further explore the role of Vgll4 in the commitment of MSCs to the fate of osteoblasts, we generated Prx1-cre; Vgll4floxp/floxp mice (hereafter Vgll4prx1 mice) (fig. S3A). Prx1-Cre activity is mainly restricted to limbs and craniofacial mesenchyme cells (18, 19). Western blot analysis confirmed that VGLL4 was knocked out in BMSCs (fig. S3B). Vgll4prx1 mice survived normally after birth and had normal fertility. However, Vgll4prx1 mice exhibited marked dwarfism that was independent of sex (Fig. 2, A and B, and fig. S3C), which was similar to the phenotype of Vgll4/ mice. In particular, the membranous ossification of the skull and clavicle was also impaired in Vgll4prx1 mouse newborns compared with control littermates (Fig. 2, C to E). To assess the role of VGLL4 in osteoblast differentiation, BMSCs from Vgll4prx1 and Vgll4fl/fl mice were cultured in osteogenic medium. Markedly decreased ALP activity and mineralization were observed in Vgll4prx1 mice (Fig. 2, F and G). The declined osteogenesis in Vgll4 knockout osteoblasts was also proved by the decreased expression of a series of osteogenic marker genes, including Alp, Osterix, and Col1a1 (Fig. 2H). Normal Runx2 expression was detected in Vgll4prx1 mice (Fig. 2H). To further verify the role of VGLL4 in osteoblast differentiation, BMSCs from Vgll4fl/fl mice were infected with GFP and Cre recombinase (Cre) lentivirus and then cultured in osteogenic medium. Vgll4fl/fl BMSCs infected with Cre lentivirus showed markedly decreased ALP activity and mineralization (fig. S4A). Reduced VGLL4 expression by Cre lentivirus was confirmed by reverse transcription polymerase chain reaction (RT-PCR) (fig. S4B). The declined osteogenesis was also proved by the decreased expression of a series of osteogenic marker genes, including Alp, Osterix, and Col1a1 (fig. S4B).

(A) Skeletons of Vgll4fl/fl and Vgll4prx1 mice at P1 were double-stained by Alizarin red and Alcian blue. Scale bar, 5 mm. (B) Quantification of body length in (A) (n = 6). (C) Skull and clavicle preparation from Vgll4fl/fl and Vgll4prx1 mouse newborns were double-stained with Alizarin red and Alcian blue at P1. Scale bars, 5 mm. (D) Quantification of the defect area of skulls in (C) (n = 6). (E) Quantification of clavicle length in (C) (n = 6). (F) Alp staining and Alizarin red staining of BMSCs from Vgll4fl/fl and Vgll4prx1 mice after cultured in osteogenic medium. Scale bars, 3 mm. (G) Alp activity was measured by phosphatase substrate assay. (H) Relative mRNA levels were quantified by RT-PCR. (I) 3D -QCT images of trabecular bone (top) and cortical bone (bottom) of distal femurs. (J to N) -QCT analysis for trabecular bone volume per tissue volume (BV/TV, Tb) (J), trabecular number (Tb.N/mm) (K), trabecular thickness (Tb.Th/mm) (L), trabecular separation (Tb.Sp/mm) (M), and cortical bone thickness (Cor.Th/mm) (N). (O) Representative images of von Kossa staining of 12-week-old Vgll4fl/fl and Vgll4prx1 mice. Scale bar, 500 m. (P) Representative images of calcein and Alizarin red S labeling of proximal tibia. Scale bar, 50 m. (Q) Quantification of MAR. (R and S) ELISA analysis of serum PINP (ng ml1) and CTX-1 (ng ml1) from 10-week-old Vgll4fl/fl and Vgll4prx1 mice (n = 5). In (B), (D), (E), (G), (H), (J) to (N), and (Q) to (S), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test. Photo credit: Jinlong Suo, State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai.

We next performed PCNA (proliferating cell nuclear antigen) staining and MTT assay to detect whether VGLL4 influences cell proliferation during bone development. No significant differences were found after VGLL4 deletion (fig. S5, A to C). We also did not detect significant changes of proliferation-related genes and YAP downstream genes (fig. S5, D and E). We next performed TUNEL (terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labeling) staining to detect whether VGLL4 influences cell apoptosis. In addition, no significant differences were found after VGLL4 deletion (fig. S5, F and G).

To further determine the function of VGLL4 in skeletal system, we did micro-quantitative computed tomography (-QCT) analysis to compare the changes in bone-related elements in the long bones of Vgll4prx1 mice and control littermates. We found that the 3-month-old Vgll4prx1 mice showed decreased bone mass per tissue volume (BV/TV) relative to age-matched control littermates (Fig. 2, I and J). Further analysis showed a reduction in trabecular number (Tb.N) of Vgll4prx1 mice compared to control mice (Fig. 2K), which was accompanied by a decrease in trabecular thickness (Tb.Th) and an increase in trabecular separation (Tb.Sp) compared to control mice (Fig. 2, L and M). Vgll4prx1 mice also showed decreased cortical bone thickness (Cor.Th) relative to the Vgll4fl/fl mice (Fig. 2N). The von Kossa staining showed reduced bone mineral deposition in 3-month-old Vgll4prx1 mice (Fig. 2O). The mineral apposition rate (MAR) was also decreased in Vgll4prx1 mice compared with control littermates by fluorescent double labeling of the mineralizing front (Fig. 2, P and Q). Consistent with the decreased bone mass in Vgll4prx1 mice, the enzyme-linked immunosorbent assay (ELISA) assay of N-terminal propeptide of type I procollagen (PINP), a marker of bone formation, revealed a reduced bone formation rate in Vgll4prx1 mice (Fig. 2R). However, the ELISA assay of C-terminal telopeptide of collagen type 1 (CTX-1), a marker of bone resorption, showed that the bone resorption rate of Vgll4prx1 mice did not change significantly (Fig. 2S). Collectively, Vgll4 conditional knockout mice mimicked the main phenotypes of the global Vgll4 knockout mice, further indicating that VGLL4 specifically regulates bone mass by promoting osteoblast differentiation.

To further determine whether the abnormal osteogenesis in Vgll4prx1 mice was caused by a primary defect in osteoblast development, we generated an osteoblast-specific Osx-cre; Vgll4floxp/floxp mice (hereafter Vgll4Osx mice) by crossing Vgll4fl/fl mice with Osx-Cre mice, a line in which Cre expression is primarily restricted to osteoblast precursors (fig. S6A) (6, 20). Vgll4Osx mice survived normally after birth and had normal fertility, but exhibited marked dwarfism in comparison with Osx-Cre mice (fig. S6, B and C), which was similar to the phenotypes of Vgll4/ and Vgll4prx1 mice. In addition, the membranous ossification of the skull and clavicle was also impaired in Vgll4Osx mice compared with control littermates (fig. S6C). -QCT analysis further confirmed the osteogenic phenotype of Vgll4Osx mice (fig. S6, D to J). Hence, the Vgll4Osx mice summarized the defects observed in the Vgll4prx1 mice, thus supporting the conclusion that VGLL4 is necessary for the differentiation and function of committed osteoblast precursors.

We next worked to figure out the mechanism how VGLL4 controls bone mass and osteoblast differentiation. The pygmy and cranial closure disorders in Vgll4/ mice were similar to that of Runx2-heterozygous mice. We therefore examined the potential interaction between VGLL4 and RUNX2. However, coimmunoprecipitation experiments did not show the interaction between VGLL4 and RUNX2 (Fig. 3A). Previous studies showed that VGLL4 could compete with YAP for binding to TEADs (9). The TEAD family contains four highly homologous proteins (8), which is involved in the regulation of myoblast differentiation and muscle regeneration (21). We determined whether the binding of VGLL4 with RUNX2 requires TEADs. Coimmunoprecipitation experiments showed that RUNX2 and TEAD14 had almost equivalent interactions (Fig. 3B). Next, we investigated whether TEADs control the transcriptional activity of Runx2. We used the 6xOSE2-luciferase reporter system that is specifically activated by RUNX2 to verify the role of TEADs (22). We performed dual-luciferase reporter assay with 6xOSE2-luciferase and Renilla in C3H10T1/2 cells, and the results showed that TEAD14 significantly inhibited the activation of 6xOSE2-luciferase induced by RUNX2 (Fig. 3C). Consistently, knockdown of TEADs by small interfering RNAs (siRNAs) markedly enhanced both basic and RUNX2-induced 6xOSE2-luciferase activity (fig. S8A). TEAD family is highly conserved, which consists of an N-terminal TEA domain and a C-terminal YAP-binding domain (YBD) (Fig. 3D) (23). Glutathione S-transferase (GST) pull-down assay revealed the direct interaction between RUNX2 and TEAD4 (Fig. 3E). Moreover, both TEA and YBD domains of TEAD4 could bind to RUNX2 (Fig. 3, F and G).

(A) Coimmunoprecipitation experiments of RUNX2 and VGLL4 in HEK-293T cells. The arrow indicated IgG heavy chain. (B) Coimmunoprecipitation experiments of RUNX2 and TEAD14 in HEK-293T cells. The arrow indicated IgG heavy chain. (C) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2 and TEAD14. Data were calculated from three independent replicates. (D) Schematic illustration of the domain organization for TEAD4, TEAD4-Nt, and TEAD4-Ct. (E) GST pull-down (PD) analysis between purified GST-RUNX2 and HIS-SUMO-TEAD4 proteins. (F) GST pull-down analysis between purified GST-RUNX2 and HIS-SUMO-TEAD4-TEA proteins. (G) Lysates from HEK-293T cells with Flag and Flag-RUNX2 expressions were incubated with recombinant GST-TEAD4-YBD protein. GST pull-down assay showed the binding between RUNX2 and TEAD4-YBD. (H) Cells isolated from WT mice were infected with TEAD lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (I) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (J) Relative mRNA levels of Alp, Col11, and Osterix were quantified by RT-PCR. (K) Cells isolated from WT mice were infected with TEAD shRNA lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (L) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (M) Relative mRNA levels of Runx2, Alp, Col11, and Osterix were quantified by RT-PCR. (N) Relative mRNA levels of Tead1-4 were quantified by RT-PCR. In (C), (I), (J), and (L) to (N), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test.

To determine whether overexpression of TEAD14 affects osteoblast differentiation, BMSCs from WT mice were infected with TEAD14 lentivirus and then cultured in osteogenic medium. The activities of ALP in TEAD14 overexpression groups were significantly reduced at the seventh day of differentiation [Fig. 3, H (top) and I] and were significantly weakened by Alizarin red S staining over a 14-day culture period (Fig. 3H, bottom). The declined osteogenesis in TEAD14 overexpression cells was confirmed again by the decreased expression of a series of osteogenic marker genes, including Alp, Col11, and Osterix (Fig. 3J). Next, we blocked the total activities of TEAD14 by short hairpin RNA (shRNA) lentiviral infection (Fig. 3N). The activity of Alp in TEAD14 knockdown group was significantly increased [Fig. 3, K (top) and L]. Over a 14-day culture period, osteogenic differentiation was significantly enhanced by Alizarin red S staining (Fig. 3K, bottom). The enhanced osteogenesis in TEAD14 knockdown cells was further confirmed by elevated expression of a series of osteogenic marker genes, including Alp, Col11, and Osterix (Fig. 3M). These results suggest that TEAD14 act as repressors of RUNX2 to inhibit osteoblast differentiation.

To investigate the mechanistic role of VGLL4 in inhibiting osteoblast differentiation, we then verified whether VGLL4 could affect the interaction between TEADs and RUNX2. We found that VGLL4 reduced the interaction between RUNX2 and TEADs (Fig. 4A). To further illustrate the relationship between RUNX2/TEADs/VGLL4, we checked the interaction between RUNX2 and TEADs in the BMSC of Vgll4fl/fl mice treated with GFP or Cre lentivirus. We found that the interaction between RUNX2 and TEADs was enhanced in Cre-treated cells (Fig. 4B). We noticed that there were conserved binding sites of RUNX2 (5-AACCAC-3) and TEAD (5-CATTCC-3) in the promoter regions of Alpi, Osx, and Col1a1, which are three target genes of RUNX2 (17, 24). We performed TEAD4 and RUNX2 chromatin immunoprecipitation (ChIP) assays in BMSCs. The results indicated that both TEAD4 and RUNX2 bound on Alp, Osx, and Col1a1 promoters (fig. S7, A to I). VGLL4 was a transcriptional cofactor, which could not bind DNA directly. We have demonstrated that VGLL4 promoted RUNX2 activity by competing for its binding to TEADs. Consistently, VGLL4 partially blocked TEADs-repressed transcriptional activity of RUNX2 (Fig. 4C). However, overexpression of VGLL4 in TEADs knockdown cells showed no marked change on RUNX2-induced 6xOSE2-luciferase activity compared with TEAD knockdown (fig. S8B). We then asked whether loss of VGLL4-induced disorders of osteoblast differentiation is related to TEADs. We knocked down TEADs by lentiviral infection in Vgll4-deficient BMSCs and then induced these cells for osteogenic differentiation. The differentiation disorders caused by VGLL4 deletion were restored after TEAD knockdown (Fig. 4, D to F). These data supported that VGLL4 released the inhibition of TEADs on RUNX2, thereby promoting osteoblast differentiation.

(A) Coimmunoprecipitation experiments of RUNX2, TEADs, and VGLL4 in HEK-293T cells. The arrow indicated IgG heavy chain. (B) Coimmunoprecipitation experiments of RUNX2 and TEADs in BMSCs cells of Vgll4fl/fl mice treated with GFP and Cre lentivirus. (C) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, TEADs, and VGLL4. (D) Cells isolated from Vgll4fl/fl and Vgll4prx1 mice were infected with GFP and TEAD shRNA lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (E) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (F) Relative mRNA levels of Vgll4, Runx2, Alp, Col11, and Osterix were quantified by RT-PCR. In (B), (D), and (E), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test.

YAP, the key transcription cofactor in the Hippo pathway, has been widely reported in regulating bone development and bone mass (12, 13). VGLL4, a previously identified YAP antagonist, directly competes with YAP for binding to TEADs (9). Therefore, we suspected that the inhibition of RUNX2 transcriptional activity caused by VGLL4 deletion might be dependent on YAP. To this end, we validated the role of YAP by 6xOSE2-luciferase reporter system. The data showed that YAP promoted RUNX2 activity in a dose-dependent manner (Fig. 5A). Moreover, TEAD4 significantly inhibited 6xOSE2-luciferase activity induced by YAP (Fig. 5B). TEAD4Y429H, a mutation that impairs the interaction between TEAD4 and YAP/TAZ (Fig. 5C) (25), did not promote 3xSd-luciferase activity induced by YAP (Fig. 5D). We found that both TEAD and TEAD4Y429H could interact with RUNX2 (Fig. 5E), and both TEAD4 and TEAD4Y429H could inhibit the activity of RUNX2 in a dose-dependent manner (Fig. 5, F and G). Restoring the expression of both TEAD4 and TEAD4Y429H could reverse the increased osteoblast differentiation in TEAD knockdown BMSCs (Fig. 5, H and I). Furthermore, overexpression of TEAD1 could further inhibit osteogenic differentiation of BMSCs after YAP knockdown (Fig. 5J). Together, these data suggest that the inhibition of RUNX2 activity by TEADs is independent of YAP binding.

(A) Effects of YAP on Runx2-activated 6xOSE2-luciferase activity in C3H10T1/2 cells. (B) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, YAP, and TEAD4. (C) Schematic illustration of TEAD4 and TEAD4Y429H mutation. (D) 3xSd-luciferase activity was determined in HEK-293T cells cotransfected with YAP, TEAD4, and TEAD4Y429H. (E) Coimmunoprecipitation experiments of RUNX2, TEAD4, and TEAD4Y429H in HEK-293T cells. The arrow indicated IgG heavy chain. (F) Effects of TEAD4 on RUNX2-activated 6xOSE2-luciferase activity in C3H10T1/2 cells. (G) Effects of TEAD4Y429H on RUNX2-activated 6xOSE2-luciferase activity in C3H10T1/2 cells. (H) Cells isolated from WT mice were infected with GFP or TEAD shRNAs, TEAD4, or TEAD4Y429H lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (I) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (J) Relative mRNA levels of Runx2, Alp, Col11, Osterix, Tead1, and Yap were quantified by RT-PCR. In (A), (B), (D), (F), (G), (I), and (J), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test.

We next examined how VGLL4 breaks the interaction between RUNX2 and TEADs. It has been reported that VGLL4 relies on its own two TDU domains to interact with TEADs (9), and VGLL4 HF4A mutation can disrupt the interaction between VGLL4 and TEADs (15). We hypothesized that VGLL4 competes with RUNX2 for TEAD1 binding depending on its TDU domain. On the basis of these previous studies, we performed coimmunoprecipitation experiments and found that VGLL4 HF4A abolished the interaction between VGLL4 and TEAD1 but did not affect the interaction between TEAD1 and RUNX2 (Fig. 6A). VGLL4 partially rescued the inhibition of RUNX2 transcriptional activity by TEAD1; however, VGLL4 HF4A lost this function (Fig. 6B). We then overexpressed TEAD1 by lentivirus infection in primary calvarial cells and found that the transcriptional level of Alp was significantly inhibited. This inhibition was released by overexpressing VGLL4 but not VGLL4 HF4A (Fig. 6C). To further verify the specific regulation of RUNX2 activity by VGLL4, we performed a coimmunoprecipitation experiment with low and high doses of VGLL4 and VGLL4 HF4A. The results showed that the TEAD1-RUNX2 interaction was gradually repressed along with an increasing dose of VGLL4 but not VGLL4 HF4A (Fig. 6D). Similarly, the inhibition of RUNX2 transcriptional activity by TEAD1 was gradually released with an increasing dose of VGLL4 but not VGLL4 HF4A (Fig. 6E). Super-TDU, a peptide mimicking VGLL4, could also reduce the interaction between purified RUNX2 and TEAD4 proteins (Fig. 6F). Thus, these findings suggest that VGLL4 TDU domain competes with RUNX2 for TEADs binding to release RUNX2 transcriptional activity.

(A) Coimmunoprecipitation experiments of RUNX2, TEAD1, VGLL4, and VGLL4 HF4A in HEK-293T cells. The arrow indicated IgG heavy chain. (B) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, VGLL4, VGLL4 HF4A, and TEAD1 (n = 3). (C) RT-PCR analysis of Alp expression in calvarial cells. Cells isolated from WT mice were infected with GFP, TEAD1, VGLL4, or VGLL4 HF4A lentivirus. (D) Coimmunoprecipitation experiments of RUNX2, TEAD1, and an increasing amount of VGLL4 or VGLL4 HF4A in HEK-293T cells. The arrow indicated IgG heavy chain. (E) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, TEAD1, and an increasing amount of VGLL4 or VGLL4 HF4A. (F) Competitive GST pull-down assay to detect the effect of VGLL4 Super-TDU on the interaction between RUNX2 and TEAD4. (G) Cells isolated from Vgll4fl/fl and Vgll4prx1 mice were infected with GFP and RUNX2 lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (H) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (I) Relative mRNA levels of Vgll4, Runx2, Alp, Col11, and Osterix were quantified by RT-PCR. (J) Schematic model of VGLL4/TEADs/RUNX2 in regulating osteogenic differentiation. In (B), (C), (E), (H), and (I), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test.

Furthermore, we overexpressed RUNX2 by lentivirus infection in Vgll4 knockout BMSCs during osteogenic differentiation, and we found that RUNX2 could significantly restore the osteogenic differentiation disorder caused by Vgll4 deletion (Fig. 6, G to I). Together, these data suggest a genetic interaction between VGLL4/TEADs/RUNX2 and provide evidences that RUNX2 overexpression rescues osteogenic differentiation disorders caused by VGLL4 deletion.

Collectively, our study demonstrates the important roles of VGLL4 in osteoblast differentiation, bone development, and bone homeostasis. In the early stage of osteoblast differentiation, TEADs interact with RUNX2 to inhibit its transcriptional activity in a YAP bindingindependent manner. During differentiation progress, VGLL4 expression gradually increases to dissociate the interaction between TEADs and RUNX2, thereby releasing the inhibition of RUNX2 transcriptional activity by TEADs and promoting osteoblasts differentiation (Fig. 6J).

Accumulating evidences have suggested that the Hippo pathway plays key roles in regulating organ size and tissue homeostasis (8, 10). However, the transcription factors TEADs have not been reported in skeletal development and bone-related diseases. VGLL4 functions as a new tumor suppressor gene, which has been reported to negatively regulate the YAP-TEADs transcriptional complex. Our previous studies show that VGLL4 plays important roles in many tissue homeostasis and organ development, such as heart and muscle (16, 17). In this study, we provide evidences to show that VGLL4 can break TEADs-mediated transcriptional inhibition of RUNX2 to promote osteoblast differentiation and bone development independent of YAP binding.

Overall, our studies establish the Vgll4-specific knockout mouse model in the skeletal system. We show that VGLL4 deletion in MSCs leads to abnormal osteogenic differentiation with delayed skull closure and reduced bone mass. Our data also reveal that VGLL4 deletion leads to chondrodysplasia. Recent researches identified that chondrocytes have the ability to transdifferentiate into osteoblasts (2628), suggesting the possibility that loss of VGLL4 might reduce or delay the pool of chondrocytes that differentiate into osteoblasts. We identify that VGLL4 regulates the RUNX2-TEADs transcriptional complex to control osteoblast differentiation and bone development. TEADs can bind to RUNX2 and inhibit its transcriptional activity in a YAP bindingindependent manner. Recent studies pointed out that reciprocal stabilization of ABL and TAZ regulates osteoblastogenesis through transcription factor RUNX2 (29); however, we found that TEAD4-Y429H, a mutation at the binding site of TAZ and TEAD (25, 30, 31), can still significantly inhibit the activity of RUNX2. Therefore, we consider that the way TEAD regulates RUNX2 may not depend on TAZ regulation. Further research found that VGLL4, but not VGLL4 HF4A, can alleviate the inhibition by influencing the binding between RUNX2 and TEADs. It is possible that VGLL4 might influence the structure organization of the RUNX2-TEAD complex to some extent. Structural information may be required to answer this question and may provide more insights into the mechanism of VGLL4 in osteogenic differentiation.

Previous studies showed that mutations in RUNX2 cause CCD and Runx2+/ mice show a CCD-like phenotype. However, many patients with CCD do not have RUNX2 mutations. Our study may provide clues to the pathogenesis of these patients. A significant reduction of bone mass was observed in the adult mice, suggesting that VGLL4 and TEADs might be drug targets for treatment of cranial closure disorders and osteoporosis. In addition, further investigation of the clinical correlation of VGLL4 and cleidocranial dysplasia in a larger cohort will provide more accurate information for bone research. Our work also provides clues to researchers who are studying the roles of VGLL4 in tumors or other diseases. RUNX2 is highly expressed in breast and prostate cancer cells. RUNX2 contributes to tumor growth in bone and the accompanying osteolytic diseases (32). The regulation of RUNX2 transcriptional activity by TEADs and VGLL4 is likely to play essential roles in tumor, bone metastasis, and osteolytic diseases. Our work may provide clues to researchers who are studying the role of VGLL4 in bone tumors.

We demonstrate that TEADs are involved in regulating osteoblast differentiation by overexpressing and knocking down the TEAD family in vitro. However, the exact roles of TEADs in vivo need to be further confirmed by generation of TEAD1/2/3/4 conditional knockout mice. In the follow-up work, we will continue to study the mechanism of TEADs in skeletal development and bone diseases. Overall, although there are still some shortcomings, our work has greatly contributed to understand the TEADs regulation of RUNX2 activity.

Our work defines the role of VGLL4 in regulating osteoblast differentiation and bone development, and identifies that TEADs function as repressors of RUNX2 to inhibit osteoblast differentiation. We propose a model that VGLL4 dissociates the combination between TEADs and RUNX2. It is not clear whether VGLL4 is also involved in regulating other transcription factors or signaling pathways in the process of osteoblast differentiation and bone development. If that is the case, how to achieve cooperation will be another interesting issue worthy of further study.

Vgll4Lacz/+ mice, Vgll4 knockout (Vgll4/) mice, Vgll4Vgll4-eGFP/+ mice, and Vgll4 conditional knockout (Vgll4fl/fl) mice were generated as previously described (16, 17), and Vgll4fl/fl mice were crossed with the Prx1-Cre and Osx-Cre strain to generate Vgll4prx1 and Vgll4Osx mice. All mice analyzed were maintained on the C57BL/6 background. All mice were monitored in a specific pathogenfree environment and treated in strict accordance with protocols approved by the Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

The following antibodies were used: anti-Osterix antibody (1:1000; Santa Cruz Biotechnology, SC133871), anti-RUNX2 antibodies (1:1000; Santa Cruz Biotechnology, SC-390351 and SC-10758), anti-Flag antibody (1:5000; Sigma-Aldrich, F-3165), anti-HA (hemagglutinin) antibody (1:2000; Santa Cruz Biotechnology, SC-7392), anti-HA antibody (1:1000; Sangon Biotech, D110004), anti-MYC antibody (1:1000; ABclonal Technology, AE010), anti-PCNA antibody (1:1000; Santa Cruz Biotechnology, SC-56), rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology, SC-2027), mouse IgG (Sigma-Aldrich, I5381), anti-VGLL4 antibody (1:1000; ABclonal, A18248), anti-TEAD1 antibody (1:1000; ABclonal, A6768), anti-TEAD2 antibody (1:1000; ABclonal, A15594), anti-TEAD3 antibody (1:1000; ABclonal, A7454), anti-TEAD4 antibody (1:1000; Abcam, ab58310), and antipan-TEAD (1:1000; Cell Signaling Technology, 13295).

Cells were cultured at 37C in humidified incubators containing an atmosphere of 5% CO2. Human embryonic kidney (HEK)293T cells were maintained in Dulbeccos Modified Eagle Medium (DMEM) (Corning, Corning, NY) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco) solution. C3H10T1/2 cells were maintained in -minimum essential medium (-MEM) (Corning, Corning, NY) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) solution. To induce differentiation of BMSC into osteoblasts, cells were cultured in -MEM containing 10% FBS, l-ascorbic acid (50 g/ml), and -glycerophosphate (1080 mg/ml). The osteoblast differentiation level assay was performed following a previously published method (33). To quantitate Alp activity, cells incubated with Alamar Blue to calculate cell numbers and then incubated with phosphatase substrate (Sigma-Aldrich, St. Louis, MO) dissolved in 6.5 mM Na2CO3, 18.5 mM NaHCO3, and 2 mM MgCl2 after washing by phosphate-buffered saline (PBS). Alp activity was then read with a luminometer (Envision). Bone nodule formation was stained with Alizarin red S solution (1 mg/ml; pH 5.5) after 14 days of induction.

We collected femurs and tibias from mice and flushed out the bone marrow cells with 10% FBS in PBS. All nuclear cells were seeded (2 106 cells per dish) in 100-mm culture dishes (Corning) and incubated at 37C under 5% CO2 conditions. After 48 hours, nonadherent cells were washed by PBS and adherent cells were cultured in -MEM (Corning, Corning, NY) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) solution for an additional 5 days. Mouse BMSCs in passage one were used in this study.

Total RNA was isolated from cells with TRIzol reagent (T9424, Sigma-Aldrich), and first-strand complementary DNA (cDNA) was synthesized from 0.5 g of total RNA using the PrimeScript RT Reagent Kit (PR037A, TaKaRa). The real-time RT-PCR was performed with the Bio-Rad CFX96 System. Gene expression analysis from RT-PCR was quantified relative to Hprt.

C3H10T1/2 cells were seeded overnight at 1 105 cells per well into a 12-well plate and transfected by PEI (polyethylenimine linear) with a luciferase reporter plasmid along with various expression constructs, as indicated. All wells were supplemented with control empty expression vector plasmids to keep the total amount of DNA constant. At 36 to 48 hours after transfection, the cells were harvested and subjected to dual-luciferase reporter assays according to the manufacturers protocol (Promega).

293T cells were seeded at 1 107 cells per 10-cm dish and cultured overnight. At 36 to 48 hours after transfection with PEI, cells were harvested and washed with cold PBS following experimental treatments. Then, cells were lysed with EBC buffer [50 mM tris (pH 7.5), 120 mM NaCl, and 0.5% NP-40] containing protease inhibitor cocktail (1:100; MedChem Express, HY-K0010). After ultrasonication, lysates were subjected to immunoprecipitation with anti-Flag antibodies (M2, Sigma-Aldrich) at 4C overnight, followed by washing in lysis buffer, SDSpolyacrylamide gel electrophoresis (PAGE), and immunoblotting with the indicated antibody.

RUNX2 and TEAD4-YBD were cloned into pGEX-4T-1-GST vector and expressed in Escherichia coli BL21 (DE3) cells. TEAD4 and TEAD4-TEA were cloned into HT-pET-28a-HIS-SUMO vector and expressed in E. coli BL21 (DE3) cells. The two TDU domains of VGLL4 were cloned into HT-pET-28a-MBP vector and expressed in E. coli BL21 (DE3) cells. VGLL4 Super-TDU was designed as previously described (15). GST, HIS-SUMO, and MBP-fused proteins were purified by affinity chromatography as previously described (17). The input and output samples were loaded to SDS-PAGE and detected by Western blotting.

CalceinAlizarin red S labeling measuring bone formation rate was performed as previously described (33).

Preparation of skeletal tissue and -QCT analysis were performed as previously described (34). The mouse femurs isolated from age- and sex-matched mice were skinned and fixed in 70% ethanol. Scanning was performed with the -QCT SkyScan 1176 System (Bruker Biospin). The mouse femurs were scanned at a 9-m resolution for quantitative analysis. Three-dimensional (3D) images were reconstructed using a fixed threshold.

ChIP experiments were carried out in BMSCs according to a standard protocol. The cell lysate was sonicated for 20 min (30 s on, 30 s off), and chromatin was divided into fragments ranging mainly from 200 to 500 base pairs in length. Immunoprecipitation was then performed using antibodies against TEAD4 (Abcam, ab58310), RUNX2 (Santa Cruz Biotechnology, SC-10758), and normal IgG. The DNA immunoprecipitated by the antibodies was detected by RT-PCR. The primers used were as follows: Alp-OSE2-ChIP-qPCR-F (5-GTCTCCTGCCTGTGTTTCCACAGTG-3), Alp-OSE2-ChIP-qPCR-R (5-GAAGACGCCTGCTCTGTGGACTAGAG-3), Alp-TBS-ChIP-qPCR-F (5-CCTTGCATGTAAATGGTGGACATGG-3), Alp-TBS-ChIP-qPCR-R (5-TATCATAGTCACTGAGCACTCTCTTGCG-3), Osx-OSE2-ChIP-qPCR-F (5-TTAACTGCCAAGCCATCGCTCAAG-3), Osx-OSE2-ChIP-qPCR-R (5-CCTCTATGTGTGTATGTGTGTTTACCAAACATC-3), Osx-TBS-ChIP-qPCR-F (5-ATGCCAAGAGATCCCTCATTAGGGAC-3), Osx-TBS-ChIP-qPCR-R (5-AGCTTGGTGAGCACAGCAAAGACAC-3), Col1a1-TBS/OSE2-Chip-qPCR-F (5-CTCAGCCTCAGAGCTGTTATTTATTAGAAAGG-3), and Col1a1-TBS/OSE2-Chip-qPCR-R (5-TTAATCTGATTAGAACCTATCAGCTAAGCAGATG-3). TBS indicated TEAD binding sites.

Mouse TEAD1, TEAD2, TEAD3, and TEAD4 siRNAs and the control siRNA were synthesized from Shanghai Gene Pharma Co. Ltd., Shanghai, China. siRNA oligonucleotides were transfected in C3H10T1/2 by Lipofectamine RNAiMAX (Invitrogen) following the manufacturers instructions. Two pairs of siRNAs were used to perform experiments.

Hematoxylin and eosin stain and immunohistochemistry were performed as previously described (7). Tissue sections were used for TRAP staining according to the standard protocol. Tissues were fixed in 4% paraformaldehyde for 48 hours and incubated in 15% DEPC (diethyl pyrocarbonate)EDTA (pH 7.8) for decalcification. Then, specimens were embedded in paraffin and sectioned at 7 m. Immunofluorescence was performed as previously described (33). Sections were blocked in PBS with 10% horse serum and 0.1% Triton for 1 hour and then stained overnight with anti-PCNA antibody (SC-56). Donkey anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes, A21206) was used as secondary antibodies. DAPI (4,6-diamidino-2-phenylindole) (Sigma-Aldrich, D8417) was used for counterstaining. Slides were mounted with anti-fluorescence mounting medium (Dako, S3023), and images were acquired with a Leica SP5 and SP8 confocal microscope. For embryonic mice, 5-mm tissue sections were used for immunohistochemistry staining, DIG-labeled in situ hybridization (Roche), and immunohistochemical staining (Dako).

TUNEL staining for apoptosis testing was performed as provided by Promega (G3250).

MTT assay for cell viability was performed as provided by Thermo Fisher Scientific.

We determined serum concentrations of PINP using the Mouse PINP EIA Kit (YX-160930M) according to the instructions provided. In addition, we determined serum concentrations of CTX-1 using the Mouse CTX-1 EIA Kit (YX-032033M) according to the instructions provided.

Tissue sections were used for SO staining according to the standard protocol. After paraffin sections were dewaxed into water, they were acidified with 1% acetic acid for 10 s and then fast green for 2 min, acidified with 1% acetic acid for 10 s, stained with SO for 3 min and 95% ethanol for 5 s, and dried and sealed with neutral glue.

Statistical analysis was performed by unpaired, two-tailed Students t test for comparison between two groups using GraphPad Prism Software. A P value of less than 0.05 was considered statistically significant.

Acknowledgments: We thank A. McMahon (Harvard University, Boston, MA) for providing the Prx1-Cre mouse line. We thank the cell biology core facility and the animal core facility of Shanghai Institute of Biochemistry and Cell Biology for assistance. Funding: This work was supported by the National Natural Science Foundation of China (nos. 81725010, 31625017, 81672119, and 31530043), National Key Research and Development Program of China (2017YFA0103601 and 2019YFA0802001), Strategic Priority Research Program of Chinese Academy of Sciences (XDB19000000), Shanghai Leading Talents Program, Science and Technology Commission of Shanghai Municipality (19ZR1466300), and Youth Innovation Promotion Association CAS (2018004). Author contributions: Z.W., L.Z., and W.Z. conceived and supervised the study. J.S. conceived and designed the study, performed the experiments, analyzed the data, and wrote the manuscript. X.F. made the constructs, performed the in vitro pull-down assay and ChIP experiments, analyzed the data, and revised the manuscript. L.Z. and Z.W. provided genetic strains of mice. J.S. and Z.W. bred and analyzed Vgll4/ mice. J.L. and J.W. cultured the cells and made the constructs. W.Z., L.Z., X.F., and Z.W. edited the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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VGLL4 promotes osteoblast differentiation by antagonizing TEADs-inhibited Runx2 transcription - Science Advances

Bone Marrow Stem Cells Comments Off on VGLL4 promotes osteoblast differentiation by antagonizing TEADs-inhibited Runx2 transcription – Science Advances | October 24th, 2020

INTRODUCTION

Ewing sarcoma (EwS) is an aggressive, poorly differentiated, human tumor characterized by a chromosomal translocation involving a member of the FET family of genes (FUS, EWSR1 and TAF15) and a member of the ETS family of transcription factors, with the EWSR1-FLI1 gene fusion the most common one (1). EwS genomes present low mutation rates with FET-ETS rearrangements as the dominant genetic aberration in the majority of tumors (2). Notably, the cell of origin of EwS is still a controversial field, although human mesenchymal stem cells (hMSCs) and human neural crest stem cells are the most accepted (35).

The EWSR1-FLI1 fusion protein, which contains the transcriptional activation and RNA binding domains of EWSR1 and the DNA binding domain of FLI1, is the main driver of tumorigenesis (3, 6). The resulting fusion oncoprotein has the ability to act as an aberrant transcription factor, leading to gene activation and repression for a well-described set of genes (3, 7). A decade ago, EWSR1-FLI1 was found to bind preferentially to DNA sites containing GGAA microsatellite repeats (8, 9). Recent studies have reported that binding of EWSR1-FLI1 multimers to GGAA repeats acts as a pioneer factor and induces the formation of de novo active enhancers by recruiting the acetyl transferases CBP/p300, E2F3, and the BRG1/BRM-associated factor chromatin remodeling complex (1012). On the other hand, it was hypothesized that monomeric EWSR1-FLI1 inhibits transcription at enhancers by displacing endogenous ETS transcription factors from GGAA motifs (10). Therefore, the mechanisms by which EWSR1-FLI1 acts as either a gene activator or repressor depend on both DNA sequence and cofactors.

Several proteins from the Polycomb group (PcG) have previously been implicated in EwS tumorigenesis. PcG was first described in Drosophila melanogaster as a key regulator of Hox genes expression. PcG proteins not only prevent differentiation by repressing lineage-specific genes but also mark bivalent chromatin regions for subsequent activation. EZH2 (the enzymatic subunit of PRC2) methylates histone H3 at lysine 27 (H3K27me3), while RING1B (the enzymatic subunit of PRC1) ubiquitinates H2A at lysine 119 (H2Aub), both considered repressive histone marks (13).

The canonical PRC1 complex (defined by the presence of four subunits, comprising one variant each of PCGF, PHC, CBX, and RING1) has mostly been associated with maintaining gene repression. However, increasing evidence indicates that PRC1 complexes containing RING1B have the potential for transcription activation, via their catalytic-independent association with UTX, an H3K27me3 demethylase, and p300 acetyltransferase (14, 15). With respect to EwS, it was recently shown that EZH2 blocks endothelial and neuroectodermal differentiation (16), BMI1 promotes tumorigenicity (17), and RING1B represses the nuclear factor B pathway (18). The molecular mechanisms behind the contribution of PcG to EwS have not been addressed. Notably, the GGAA repeats are significantly decorated with H3K27me3 in H1 human embryonic cell lines and human umbilical vein endothelial cells (HUVECs) (19). This is in stark contrast with the lack of H3K27me3 mark at EWSR1-FLI1 binding sites in EwS cells (10, 11), thus suggesting a different role of PcG in EwS. Last, comparison between malignant and nonmalignant tissues revealed a misregulation of PcG target genes in EwS (20). Together, these findings suggest a potential role of the PcG during the early steps of EwS pathogenesis. Here, we report that RING1B and EWSR1-FLI1 interact and colocalize at the same genomic loci. Notably, we find that RING1B is present at promoters and enhancers of actively transcribed EWSR1-FLI1 target genes. Furthermore, we demonstrate that modulation of RING1B interferes with EWSR1-FLI1 recruitment and with the expression of EWSR1-FLI1 targets, thus unveiling an interdependent cooperation between both proteins.

Human pediatric MSCs (hpMSCs) have been proposed as a plausible cell of origin for EwS (21). Nevertheless primary human endothelial HUVECs share high similarity in gene expression profiles with EwS cells (22). Thus, to investigate the potential contribution of epigenetic alteration in the initiation of EwS, we analyzed the role of epigenetic marks in these models and compared to established EwS cell lines. We first analyzed the levels of H3K27me3 and H3K4me3 in the human EwS-derived cell line A673 at several bona fide direct targets of EWSR1-FLI1 (table S1) by chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR). Promoter of genes that are transcriptionally activated by EWSR1-FLI1, such as FCGRT, NR0B1, CACNB2, EZH2, IGF1, NKX2-2, and HOXD11, was enriched for the H3K4me3 mark, and lacked the H3K27me3 mark, in agreement with previous data (8, 20, 23, 24) (fig. S1A). On the other hand, transcriptionally repressed genes, such as KCNA5 (25), were enriched for H3K27me3. We next compared the levels of H3K27me3 and H3K4me3 at the same loci in HUVECs and in hpMSCs. In an apparently reversed situation to the A673 EwS cell line, analysis of those promoters presented strong enrichment for H3K27me3 but not for H3K4me3 (fig. S1B). Accordingly, infection of HUVECs with the EWSR1-FLI1 oncogene (Fig. 1A) not only led to the activation of these targets (FCGRT, NR0B1, CACNB2, EZH2, IGF1, NKX2-2, and HOXD11) (Fig. 1B) but also decreased the levels of H3K27me3 (Fig. 1C). This demonstrates that, although H3K27me3 is not present at oncogene binding regions in EwS cell lines such as A673, these regions are repressed by PcG before oncogene expression.

(A) Western blot showing ectopic expression of EWSR1-FLI1 upon infection of HUVECs with an empty pLIV vector or EWSR1-FLI1pLIV. (B) RT-qPCR determination of relative mRNA expression of EWSR1-FLI1 target genes upon infection of HUVECs with an empty pLIV vector or EWSR1-FLI1pLIV. Values are normalized to TBP. (C) H3K27me3 ChIP-qPCR at EWSR1-FLI1 target gene promoters in HUVECs infected with an empty pLIV vector or EWSR1-FLI1pLIV. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input with subtracted immunoglobulin G (IgG). (D) Bar plots of chromatin state relative frequencies in the whole genome [background (BG)] and in published EWSR1-FLI1 binding sites (FLI1) for three selected cell lines. Genome segmentations were extracted from the Epigenome Roadmap Consortium. (E) Heatmap with percentages of each chromatin state in the whole genome (BG) as compared to the frequency within published EWSR1-FLI1 binding regions for indicated cell lines by grouping in 8 similar chromatin states the initial classification containing 15 (quiescent segments were excluded). Bold format indicates enrichments greater than 10%. Enrichment scores were calculated as the difference between the value in EWSR1-FLI1 and the value at the whole genome, normalized by the value at the whole genome. (F) Cell proliferation expressed as cell number in 293T, A673, SK-ES1, and A4573 cells transiently transfected with small interfering RNA (siRNA) against a control (siCTRL) or two different RING1B sequences (siRING1B#1 and #2). Error bars in (B), (C), and (F) indicate SD of three biological independent experiments. Statistical significance in (D) and (F) is as follows: ***P < 0.001 and *P < 0.05.

To explore the chromatin and transcriptional states of EWSR1-FLI1 binding sites (10), we measured the frequency of each chromatin state at these regions (26) and compared to the corresponding value obtained for the whole genome in several cell lines [HUVECs, H1, and H9 human embryonic cells, H1-derived MSCs, bone marrow (BM)derived MSCs, and adipose-derived MSCs]. This analysis indicated that EWSR1-FLI1 binding sites are overrepresented in chromatin states associated with zinc finger genes and repeats (ZNF/repeats) and active promoters (Fig. 1D and table S1). In cells with MSC origin (such as H1, adipose, and BM-derived cell lines), EWSR1-FLI1 binding sites are overrepresented in PcG weak repressed state, which represents flanking regions of H3K27me3 peaks summit (Fig. 1D and fig. S1, C and D). Similar results were obtained when we grouped chromatin states of similar categories (Fig. 1E). This suggests that EWSR1-FLI1 occupies flanking regions of H3K27me3 summit peaks in hMSC, which are considered to be the potential cell of origin for EwS.

Data from our group have revealed that the PRC1 subunit RING1B, is highly overexpressed in EwS primary tumors (18). We thus assessed whether RING1B modulates the growth rate of EwS cells as has been reported for other PcG subunits, such as EZH2 and BMI1 (16, 17). RING1B depletion caused a reduction in cell viability in the A673, SK-ES1, and, with a lesser extent, in A4573 EwS cell lines but not in the control cell line 293T (Fig. 1F and fig. S1E), suggesting that RING1B represents an epigenetic vulnerability for EwS cells.

Chan et al. (27) recently proposed that RING1B might play a role in modulating enhancer activity. Together with its role in promoter regulation, EWSR1-FLI1 has been recently reported to generate de novo enhancers (10). This led us to postulate whether EWSR1-FLI1 and RING1B might cooperate during EwS tumorigenesis. We first aimed to define the genome-wide localization of RING1B and its repressive histone mark H2Aub in the A673 cell line by chromatin immunoprecipitation sequencing (ChIP-seq). In two independent experiments, we identified 2573 and 3945 peaks of RING1B, and 26424 and 10269 peaks of H2Aub. Using differential binding analysis (DiffBind), which allows for the identification of statistically common peaks (28), we found 2459 RING1B and 5392 H2Aub significant peaks between duplicates (P < 0.05, fig. S2A), corresponding to 1264 target genes and 3013 target genes, respectively (table S2). Genomic distribution of peaks showed that RING1B is more abundant in intergenic regions, whereas H2Aub is mainly located in promoters (Fig. 2A). Moreover, 38% of RING1B peaks were found at intergenic regions with respect to 21.5% of H2Aub peaks, and 29.2% of RING1B peaks were in promoters with respect to 40.5% of H2Aub peaks, further supporting the potential role of RING1B at enhancers. We then categorized peaks for RING1B, H2Aub, and EWSR1-FLI1 in active or poised enhancers, and in active or poised promoters, based on H3K27me3, H3K4me3, H3K27ac, and H3K4me1 (29). To complement the above data, we performed a ChIP-seq analysis using a different antibody directed against FLI1 (fig. S2B and table S2). We found that an important fraction of RING1B peaks (35%) and EWSR1-FLI1 (46%) are located at transcriptionally active enhancers and promoters of A673 cells (Fig. 2B, left). On the other hand, as expected, 35% of RING1B peaks and 37% of H2Aub peaks showed a preference for transcriptionally repressed regulatory regions (Fig. 2B, left). We then intersected the list of genes associated to RING1B and H2Aub peaks with published data of EWSR1-FLI1 target genes in A673 cells, producing a common set of 162 genes (fig. S2C and table S3). Comparing this set with 386 genes containing only RING1B and H2Aub or the group of 324 EWSR1-FLI1/RING1B genes without H2Aub confirmed that the presence of EWSR1-FLI1 correlated with higher level of transcription (P < 1016; fig. S2D, left). Functional analysis of the common gene set of 324 EWSR1-FLI1/RING1B genes (table S3) returned Gene Ontology (GO) categories related to chondrocyte and neuronal differentiation (fig. S2D, right). EWSR1-FLI1/RING1B/H2Aub genes were also enriched in neuronal differentiation category, while the RING1B/H2Aub genes were related to general transcription. These data suggest that RING1B is a positive regulator of a specific set of genes implicated in EwS and that this activity is independent of its canonical repressive mark.

(A) Pie chart showing genomic distribution of RING1B and H2Aub peaks relative to functional categories including promoter (2.5 kb from TSS), gene body (intragenic region not overlapping with promoter), and intergenic (rest of the genome). (B) Boxplot depicting percentage of regulatory elements (active/bivalent enhancers and promoters) in each described group. (C) Venn diagram depicting the overlap between RING1B and EWSR1-FLI1 in A673 cells at the peak level. (D) Aggregated plot showing the average ChIP-seq signal of RING1B and EWSR1-FLI1 at EWSR1-FLI1 binding sites. (E) Aggregated plots showing the average ChIP-seq signal of H3K27ac, H2Aub, and H3K27me3 in the three sets of RING1B and EWSR1-FLI1 peaks. (F) Heatmap showing RING1B, EWSR1-FLI1, H3K27ac, H2Aub, and H3K27me3 ChIP-seq signals segregating in the three sets of RING1B and EWSR1-FLI1 peaks. Top MEME motif for every group is shown. (G) University of California Santa Cruz (UCSC) genome browser ChIP-seq signal tracks for RING1B, EWSR1-FLI1, H2Aub, H3K27ac, H3K4me3, and H3K27me3 at NKX2-2, CCND1, VRK1, and CAV1 gene promoters and intergenic enhancer regions. Gray boxes represent EWSR1-FLI1 and RING1B colocalization and ES super-enhancers (SEnh; as shown at VRK1 and CAV1/2).

To fully understand the association of RING1B with transcriptional activation in EwS, we intersected EWSR1-FLI1 peaks with those of RING1B and obtained 955 common regions (Fig. 2C). Notably, intersection between H2Aub and RING1B peaks returned only 589 common peaks. Among the 955 overlapping EWSR1-FLI1/RING1B peaks, we inspected for genes containing an enhancer within 100 kb and obtained 1276 genes, of which 235 (18%) were reported to be regulated by EwS super-enhancers (table S4) (11). The common targets of RING1B and EWSR1-FLI1 sites were found within active enhancers, while the majority of RING1B peaks not overlapping with EWSR1-FLI1 were located in transcriptionally repressed regulatory elements (Fig. 2B, right). The distribution of RING1B peaks was centered on EWSR1-FLI1 binding sites (Fig. 2D), suggesting that their binding occurs at the same loci. We next assessed the distribution of H3K27ac, H2Aub, and H3K27me3 in genomic regions occupied by EWSR1-FLI1, RING1B, or shared (Fig. 2E). Common peaks were decorated with H3K27ac, lacking H2Aub (Fig. 2, E and F), and presented narrow RING1B peaks located in intergenic or intronic regions (fig. S2E, right). These data suggest that common sites likely represent enhancers. Known EWSR1-FLI1 target genes such as NKX2-2, CCND1, VRK1, or CAV1 presented an intergenic peak of RING1B, which overlaps with defined super-enhancers in the case of VRK1 and CAV1 (Fig. 2G). Intronic enhancers such as JARID2 or MYOM2 (fig. S2G) constitute the majority of the 162 common RING1B, EWSR1-FLI1, and H2Aub genes (53% of sites, fig. S2C). On the other hand, RING1B-specific peaks were associated with H3K27me3 and H2Aub (Fig. 2, E and F) and presented a broader distribution [e.g., HNF1B and TAL1 (fig. S2H)] mainly located within promoter or gene body regions (fig. S2E, left). The bivalent marks H3K4me3 and H3K27me3 decorated 63% of the 932 downstream genes associated to RING1B-specific peaks (P < 10300, table S4) (29). RING1Btranscription start sites (TSS) do not overlap with EWSR1-FLI1 and are decorated with H2K27me3 and H2Aub, while RING1B-distal sites overlap with EWSR1-FLI1 and with H3K27ac (fig. S2F).

Last, de novo motif analysis revealed that EWSR1-FLI1specific sites contained predominantly (P < 10282) one single occurrence of the canonical ETS motif GGAA (Fig. 2F). When EWSR1-FLI1 was associated with RING1B, we observed a significant enrichment for multimeric GGAA repeats (P < 101072) (10). Furthermore, RING1B-sepecific sites were enriched for CG sequence, as previously reported (P < 10176) (30). Together, we identified two major types of RING1B peaks in EwS: a prominent group with narrow peaks that colocalizes with EWSR1-FLI1 at enhancers of actively transcribed genes and a second group with broader peaks located at promoters, where RING1B is associated with H2Aub.

To further characterize RING1B binding regions (table S4), we analyzed several EWSR1-FLI1 active promoters (CAV1, FCGRT, NR0B1, CACNB2, FEZF1, and KIAA1797) and enhancers (CCND1, IGF1, CAV2, JARID2, VRK1, and NKX2-2) by ChIP-qPCR. Both groups showed enrichment for RING1B, with stronger signals at enhancers (Fig. 3A). Known repressed targets of the oncogene (e.g., IGFBP3, TGFBR2, and LOX) also showed binding of RING1B. At these repressed promoters, RING1B was accompanied by its canonical repressive mark H2Aub (fig. S3A). We also validated the occupancy of RING1B in EWSR1-FLI1activated promoters (CAV1, FCGRT, NR0B1, and FEZF1) and enhancers (CCND1, CAV2, JARID2, and VRK1) in SK-ES1 cells (fig. S3B). Similar to A673 cells, H2Aub correlated with RING1B at promoters of repressed genes (IGFBP3, TGFBR2, and LOX) (fig. S3C). Last, we observed that the PRC1 and PRC2 subunits, BMI1 and EZH2, respectively, were present at repressed promoters but not in active enhancers (fig. S3, D and E), as well as in promoters with broad peaks of RING1B concomitant with H3K27me3 and H2Aub but no EWSR1-FLI1 (e.g., TAL1, IGF1R, and HNF1B) (fig. S3F). Furthermore, genome-wide analysis demonstrated that BMI1 and CBX7 (31) subunits of the PRC1 canonical complex colocalize with RING1B only at repressed regions (TAL1) as shown in Fig. 3B, while no detectable peaks are present at active enhancers where EWSR1-FLI1 is present (VRK1). Thus, while RING1B decorates EWSR1-FLI1activated promoters and enhancers, it also maintains its canonical role at several oncogene repressed regions, as well as in a subgroup of genes with no EWSR1-FLI1.

(A) RING1B ChIP-qPCR of EWSR1-FLI1 bound active promoters, repressed promoters, and active enhancers. Control regions indicate the absence of RING1B and EWSR1-FLI1 binding at these sites. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input. (B) UCSC genome browser ChIP-seq signal tracks for EWSR1-FLI1, RING1B, CBX7, BMI1, H2Aub, and H3K27me3 at TAL1 promoter and VRK1 enhancer. (C) Histogram depicting percentages of activated and repressed genes in A673 and SK-ES1 cells with stable RING1B knockdown seq#2 (shRING1B#2) versus control seq#2 (shCTRL#2), with P < 0.05 and an absolute fold change (FC) > 1.25 or 1.5. (D) Western blot showing RING1B, RING1A, H2Aub, and H3K27me3 in A673 and SK-ES1 cells with either shCTRL#2 or shRING1B#2. Lamin B and histone H4 are used as loading controls. (E) Venn diagram showing intersection between differentially activated or repressed genes for EWSR1-FLI1 and RING1B in A673 cells; P < 0.05. (F) RT-qPCR determination of mRNA expression of EWSR1-FLI1 target genes with active enhancers in shCTRL and shRING1B A673 cells (#1 and #2). Values are normalized to GAPDH. (G) Same analysis as in (F) for SK-ES1 cells. Error bars in (A), (F), and (G) indicate SD of four independent biological experiments and ***P < 0.001, **P < 0.01, and *P < 0.05.

To understand whether RING1B behaves as a canonical repressor and/or activator in EwS, we analyzed the expression changes after knocking down RING1B using two different sets of short hairpin RNA (shRNA, seq#1 and seq#2; fig. S4A). The data obtained showed that 71.94 and 63.85% of genes were down-regulated in the A673 and SK-ES1 cell lines, respectively (FC < -1.5, Fig. 3C). This confirms our finding that RING1B acts predominantly as an activator, despite its presence at several EWSR1-FLI1repressed targets. Furthermore, H2Aub levels remained unchanged after RING1B knockdown (Fig. 3D), while RING1A knockdown produces a notable decrease in H2Aub levels (fig. S4B). These data suggest that RING1B main function in EwS is uncoupled from its ubiquitin ligase activity toward H2A and that RING1A is the main histone H2A mono-ubiquitin ligase. To further elucidate to what extent RING1B cooperates with EWSR1-FLI1 in transcription regulation, we intersected differentially expressed genes in RING1B knockdown cells (absolute FC > 1.25) with those affected by EWSR1-FLI1 knockdown (absolute FC > 1.5) (10), obtaining an overlap of 1078 genes. After segregating these data into down- and up-regulated genes, we found that RING1B and EWSR1-FLI1activated 229 genes and repressed 162 genes (Fig. 3E and table S5). Among the 229 activated genes, we found several developmental genes, including SOX2, SIX3, LYAR, and KIT. GO analysis showed regulation of the potassium channel and mechanisms that control actin monomers and filaments as the main categories (fig. S4C), in agreement with previous publications (25, 32). Among the activated genes, SOX2 and KIT harbored RING1B and EWSR1-FLI1 peaks in intergenic and intronic enhancer regions, respectively (fig. S4E). TGFBR2, a gene repressed by both EWSR1-FLI1 and RING1B, also contained an intronic enhancer where both proteins colocalized. Notably, the expression of known targets of EWSR1-FLI1, such as NKX2-2 or IGF1 (fig. S4D), was just below our logFC cutoff value. Nonetheless, we confirmed by reverse transcription (RT)qPCR the changes in expression levels of selected repressed and activated genes cobound by EWSR1-FLI1 and RING1B. We noticed that RING1B knockdown causes a significant reduction in the expression levels of those genes where both EWSR1-FLI1 and RING1B were co-occupying enhancer regions (Fig. 3, F and G). The expression of CAV1, NKX2-2, SOX2, IGF1, JARID2, and VRK1 was affected in stronger manner upon EWSR1-FLI1 knockdown, indicating that some cofactors could remain when RING1B is depleted (fig. S4F). The effect of RING1B knockdown was less pronounced when both proteins were enriched at promoter regions of active genes (fig. S4, G and H, left). As expected, at those genes where EWSR1-FLI1 acts as a repressor, RING1B knockdown induces a promoter reactivation (fig. S4, G and H, right). Overall, these data indicate that RING1B and EWSR1-FLI1 cooperate in gene activation, at both the promoter and enhancer levels, while RING1B retains its canonical role at those targets repressed by the oncogene. Since a large number of EWSR1-FLI1 and RING1B cotargets were not altered by RING1B knockdown, we postulate compensatory mechanism(s) or additional cofactors involved in their regulation.

Wild-type EWSR1 interacts with RING1B in the VCaP prostate cancer cell line (33). We also confirmed this interaction in SK-ES1 cells (Fig. 4A). Since RING1B and EWSR1-FLI1 are enriched at transcriptionally active regions, we next aimed to investigate whether both proteins interact. Coimmunoprecipitation experiments in HeLa cells where EWSR1-FLI13xFlag was overexpressed (34) confirmed that indeed oncogene interacts with RING1B (Fig. 4, B and C). Analysis of published mass spectrometry data demonstrated that several SWI/SNF subunits interact with RING1B (33), further supporting an active role of RING1B in EwS gene regulation. Together, our results indicate that EWSR1-FLI1 and RING1B not only colocalize at the same genomic regions but also physically interact, mainly through the EWSR1 component of the fusion protein.

(A) Western blot showing endogenous coimmunoprecipitation of RING1B with EWSR1 in the SK-ES1 cell line. (B) Western blot showing overexpression of EWSR1-FLI1-3xFlag and RING1B levels in HeLa stably transfected cells upon induction with indicated doxycycline concentrations for 24 hours. Calnexin is used as loading control. (C) Coimmunoprecipitation of RING1B with EWSR1-FLI1-3xFlag under induction conditions (0.5 g/ml). Inputs in (A) and (C) contain 10% of immunoprecipitated material and IgG is used as control. (D) Western blot showing RING1B and EWSR1-FLI1 in cytoplasm, soluble, and bound chromatin fractions in shCTRL#1 or shRING1B#1 SK-ES1 cells. Histone H4 is used as a control of bound chromatin, and GAPDH as a control of cytoplasmic fraction. Blot quantification of the same ordered samples is depicted below. (E) ChIP-qPCR analysis of FLI1, RING1B, and H3K27ac at EWSR1-FLI1activated enhancers of NKX2-2, SOX2, or IGF1 genes in shCTRL#2 and shRING1B#2 A673 cells. ENC1 is used as negative control region. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input. Error bars indicate SD of three independent biological experiments. Statistical significance is as follows ***P < 0.001, **P < 0.01, and *P < 0.05. (F) Aggregated plot and boxplot showing the average ChIP-seq signal of RING1B and FLI1 peaks at RING1B and EWSR1-FLI1 binding sites, respectively, in shCTRL#2 and shRING1B#2 A673 cells. (G) UCSC genome browser ChIP-seq signal tracks for EWSR1-FLI1 and RING1B in shCTRL#2 and shRING1B#2 A673 cells at SOX2 and VRK1 enhancer regions.

Next, we analyzed whether RING1B depletion affects the EWSR1-FLI1 recruitment to chromatin. As expected, after knockdown, we observed a notable reduction of RING1B in the chromatin bound fraction (Fig. 4D). EWSR1-FLI1 was also evicted from chromatin bound and enriched in the soluble chromatin fraction (Fig. 4D). We then monitored the occupancy of EWSR1-FLI1, RING1B, and H3K27ac at several enhancers (e.g., SOX2, NKX2-2, and IGF1). The data in Fig. 4E showed that upon RING1B knockdown, enrichments at those enhancers decreased to control values [immunoglobulin G (IgG) or ENC1 region]. To assess the decrease of EWSR1-FLI1 recruitment genome-wide, we performed ChIP-seq analysis of RING1B and FLI1 in shCTRL and shRING1B A673 cells. The analysis indicated that upon RING1B depletion, EWSR1-FLI1 binding to chromatin was reduced (Fig. 4, F and G). In sum, we conclude that in EwS, RING1B exerts its main role as activator by promoting recruitment of EWSR1-FLI1 to enhancer regions.

RING1B stimulates tumor growth and metastasis in melanoma, leukemia, and breast cancers (14, 27). We observed a reduction in colony number when RING1B is depleted in the SK-ES1 cell line (fig. S5A). To gain functional insight into the cancer pathways potentially modulated by RING1B, we performed gene set enrichment analysis (GSEA) by comparing SK-ES1 shCTRL versus shRING1B cells. The top 10 most significant pathways included interferon-, epithelial-to-mesenchymal transition, hedgehog signaling, and angiogenesis, with a 0.25 Q value cutoff (fig. S5B). In EwS, disruption of angiogenic pathways has been described (4, 22). Further inspection of angiogenic gene list revealed that key genes such as PDGFA, FGFR1, SLCO2A1, CXCL6, and S100A4 were down-regulated upon RING1B depletion (fig. S5C).

To assess the relevance of RING1B in vivo, we generated xenografts by injecting SK-ES1 shCTRL or shRING1B cells (seq#1 and seq#2) subcutaneously into athymic nude mice. Cells with reduced RING1B levels showed delayed engraftment and slower tumor growth (Fig. 5A). At 21 days after injection, tumors derived from shRING1B cells were significantly smaller than those from control cells (fig. S5D). Notably, the median survival increases from 26 days for shCTRL cells to 30 days for shRING1B seq#1 and from 20 to 27 days for shRING1B seq#2 (Fig. 5B). Immunohistochemical analyses of tumors confirmed reduced levels of RING1B, while the ES marker CD99 remained essentially unchanged (Fig. 5C and fig. S5E). Furthermore, shCTRL tumors displayed higher proliferation rates than shRING1B, as shown by Ki-67 staining (Fig. 5C).

(A) Tumor volume curve in xenografts established by subcutaneous injection of shCTRL and shRING1B#1 (n = 9 and n = 10, respectively, above) or shRING1B#2 (n = 12 both groups, below) SK-ES1 cells in athymic nude mice. (B) Kaplan-Meier xenograft survival curves in shCTRL and shRING1B SK-ES1 cells (#1 and #2). (C) Immunohistochemistry staining of EWSR1-FLI1, CD99, and RING1B on sections of tumors excised from shCTRL#1 and shRING1B#1 SK-ES1 xenografts. Proliferation was analyzed by Ki67 immunohistochemistry; hematoxylin and eosin (H&E) was used as control. (D) Heatmap depicting fold changes in gene expression in six tumors excised from shCTRL#1 and shRING1B#1 SK-ES1 groups. (E) RT-qPCR levels of mRNA expression for RING1B and EWSR1-FLI1 in shCTRL#1 and shRING1B#1 SK-ES1derived tumors; ***P < 0.001. (F) RT-qPCR levels of mRNA expression for genes regulated by EWSR1-FLI1/RING1B enhancers (left) and angiogenic genes (right) in shCTRL#1 and shRING1B#1 SK-ES1 derived tumors; *P < 0.05.

To better characterize xenograft derived tumors, we performed RNA sequencing (RNA-seq) of a cohort of tumors (six for each group, Fig. 5D). GSEA analysis confirmed the enrichment of angiogenic genes in the shCTRL tumors (fig. S5, F and G). Since RING1B retains its repressive function at several promoters, we hypothesized that the delay in survival and in tumor growth upon RING1B knockdown could be related to up-regulation of tumor suppressor genes (TSG). GSEA applied to 983 genes from TSG database (https://bioinfo.uth.edu/TSGene), indicated that this gene list was enriched in shCTRL phenotype, suggesting that tumor growth and survival differences observed were not due to RING1B repression of TSG (fig. S5H). The NKX2-2, SOX2, and IGF1 genes are necessary for EwS tumor proliferation (21, 23, 35). In agreement, confirmed RING1B and EWSR1-FLI1 expression reduction (Fig. 5E) is associated to down-regulation of these genes in xenograft tumors (Fig. 5F, left), as we previously shown in EwS cells (Fig. 3, F and G). Furthermore, after RING1B knockdown, we also validated down-regulation of S100A4, SLCO2A1, and VEGFA, which are main activators of angiogenic signaling pathways (Fig. 5F, right). All these data highlight the role of RING1B as an activator in EwS tumorigenesis.

Several kinases (including AURKB, MEK1, and CK2) have been reported to modulate the activating transcriptional function of RING1B (14, 15, 36). To investigate which pathway(s) regulates RING1B at active enhancers in EwS, we analyzed the expression levels of these three kinases in a publicly available database (4) comprising a cohort of 27 tumor samples and BM-MSCs. While MEK1 and CK2 were not expressed in primary tumors with respect to BM-MSCs (control), 11 of 27 EwS tumors (40%) showed higher levels of AURKB compared to control (fig. S6A). EWSR1-FLI1 directly regulates the expression of AURKB (37), as also demonstrated by AURKB down-regulation in EwS cell lines upon oncogene knockdown (fig. S6A).

AZD1152 is a specific AURKB inhibitor, with a median inhibitory concentration (IC50) of 19 nM in EwS cell lines (38). Accordingly, we observed IC50 values of 5 and 6 nM in SK-ES1 and A4573 cells, respectively; in contrast, the IC50 for A673 was 5 M, and AZD1152 had no effect on the control cell line 293T (fig. S6B). EwS cells that survived to the treatment showed an atypical phenotype, suggesting enhanced differentiation (fig. S6C). Furthermore, viability of EwS cell lines was not affected by the inhibition of RING1B E3 ubiquitin ligase activity with PRT4165 (fig. S6B). To further elucidate the effect of AZD1152 in EwS, cell death was analyzed by Annexin V staining. A 72-hour AZD1152 treatment of A673, SK-ES1, and A4573 cells led to an increase in the early and late apoptosis populations as compared to 293T cells (Fig. 6A). Analysis of cleaved PARP levels further demonstrated that AZD1152 stimulated apoptotic pathways in EwS cell lines, with SK-ES1 being the most sensitive (Fig. 6B). It is worth noting that the levels of EWSR1-FLI1 were decreased after AZD1152 treatment in SK-ES1 and A4573, yet RING1B levels were unaffected (Fig. 6B and fig. S6, D and E, right). To understand how AURKB modulates RING1B in EwS, we analyzed H2Aub levels after AZD1152 treatment. We observed increased levels of H2Aub repressive mark after AURKB inhibition, suggesting that this kinase indeed inhibits the ubiquitin ligase activity of RING1B in EwS (Fig. 6C). Furthermore, in SK-ES1 and A4573 cells, the increase in ubiquitin ligase activity correlated with decreased expression of EWSR1-FLI1 targets co-occupied by RING1B, with more pronounced effect on those genes where both proteins colocalize at the enhancer region (Fig. 6D and fig. S6, D and E, left). For the A673 cell line, higher doses were required to reach oncogene target deregulation, as expected. Next, we reasoned that AURKB should be present at those regions where it inhibits RING1B activity. Using ChIP-qPCR, we demonstrated that AURKB is enriched in active enhancers (CAV2, driving CAV1 expression, and SOX2; Fig. 6E) and promoters (NR0B1; fig. S6F). Furthermore, EWSR1-FLI1 down-regulation could be explained by the presence of RING1B at the EWSR1 promoter, which indirectly decreases upon AZD1152 incubation (fig. S6G). Although part of AZD1152 cytotoxicity might be related to reduction of EWSR1-FLI1 availability, the data presented suggest that RING1B regulation of oncogene targets is susceptible to AURKB inhibition. The translational value of this potential targetable vulnerability is the matter of ongoing work.

(A) Annexin V staining of SK-ES1, A673, and A4573 cells after treatment with AZD1152 (20 nM). 293T cells were used as a control cell line. (B) Western blot analysis of cleaved poly(ADP-ribose) polymerase (cPARP), EWSR1-FLI1, RING1B, and AURKB after treatment with 10 or 20 nM AZD1152, in the A673, SK-ES1, A4573, and 293T cell lines. Tubulin was used as loading control. (C) Western blot analysis of H2Aub and H3S10phospho (H3S10ph) in the A673, SK-ES1, and A4573 cell lines treated with 5 or 20 nM AZD1152. Histone H4 was used as loading control. (D) RT-qPCR determination of mRNA expression of target genes with RING1B/EWSR1-FLI bound enhancers in SK-ES1 and A4573 cells after treatment with 20 nM AZD1152. RPL27 was used for normalization. DMSO, dimethyl sulfoxide. (E) AURKB ChIP-qPCR at CAV2 and SOX2 EWSR1-FLI1/RING1B enhancers (above) and control regions (below). The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input. Error bars in (D) and (E) indicate SD of three independent biological experiments. (F) Schematic representation illustrating the EWSR1-FLI1 recruitment by RING1B to repressed regions containing GGAA repeats. Once EWSR1-FLI1 has been recruited, additional cooperating factors such as AURKB might inhibit RING1B ubiquitin ligase activity, which, in turn, is able to participate in transcription activation.

Here, we investigated the genome-wide occupancy of RING1B in EwS. In agreement with previous data, we identified a set of regions bound by RING1B where it exerts its canonical repressive function. We also report that RING1B co-occupy together with EWSR1-FLI1 many intergenic and intronic regions decorated with H3K27ac. A strong enrichment in GGAA repeats has been described in regulatory elements where EWSR1-FLI1 binds producing active enhancers (10). The presence of GGAA repeats, as well as the H3K27ac association, indicates that cobinding of RING1B and EWSR1-FLI1 occurs in active enhancers. BMI1 or EZH2 was not found at these enhancer regions, suggesting a Polycomb-independent function for RING1B. Enhancers are key regulatory regions implicated in cell fate determination. Here, we unveiled that an aberrant transcription factor such as EWSR1-FLI1 relies on RING1B to activate enhancers, causing an altered gene expression profile, which favor cell transformation.

In accordance with RNA-seq data from melanoma and breast cancer, where a positive association of RING1B with transcription activation has been reported (14, 27), we observed in EwS cells a higher number of genes activated than repressed by RING1B. We found NKX2-2, SOX2, and IGF1 being direct targets down-regulated both in vivo and in vitro upon RING1B knockdown. In EwS, NKX2-2 and SOX2 are key players in tumorigenesis (21, 23), suggesting that modulation of their expression in vivo upon RING1B knockdown might contribute to decreased tumor volume and better survival, supporting an oncogenic role for RING1B.

Recent studies in hpMSCs have demonstrated that, before oncogene recruitment, H3K27me3 is enriched at regions where EWSR1-FLI1 could bind (39). In agreement with these data, we further demonstrate that upon EWSR1-FLI1 expression, those same regions loose H3K27me3 marks while becoming transcribed. Moreover, we report that enrichment in Polycomb repressed chromatin states is specific for H1-, adipose- and BM-derived MSCs, reinforcing hMSC as the putative cell of origin, which has already been described by other groups (4, 21). The existence of H3K27me3 repressed regions decorated only with PRC1 complex has already been described during differentiation of neural precursor cells, where RING1B and PCGF2 are retained while the PRC2 subunit Suz12 is not (40). In melanoma, CCND2 is marked with H3K27me3 before RING1B activation by phosphorylation (14). We have observed that GGAA repeats are differentially enriched in the binding motif analysis when RING1B is associated to chromatin with EWSR1-FLI1. In this scenario, given the interaction observed for RING1B and EWSR1-FLI1, it is tempting to speculate that RING1B targets EWSR1-FLI1 to specific sites. In line with this hypothesis, the reduced recruitment of EWSR1-FLI1 to chromatin (including enhancer regions, such as NKX2-2, SOX2, and IGF1) upon RING1B knockdown underlines the importance of RING1B in the initials steps of EwS tumorigenesis. Overall, our data suggest that RING1B is required for the recruitment of EWSR1-FLI1 to multimeric GGAA repeats (Fig. 6F).

We have demonstrated that RING1B is an essential partner of EWSR1-FLI1 triggering chromatin remodeling. Recent studies demonstrated the requirement of SWI/SNF, WDR5, and p300 acetyltransferase for EWSR1-FLI1induced transcription. Similarly, in synovial sarcoma, the SS18-SSX oncogenic fusion protein and the SWI/SNF complex colocalize at KDM2B-repressed target genes together with the noncanonical PRC1.1 complex to produce transcriptional active regions (41). Along the same lines, in leukemia, noncanonical PRC1.1 also targets active genes independently of H3K27me3 (42). Further mechanistic insights are needed to elucidate the contribution of PRC1.1 repressive complex in EwS, where somatic mutations in BCOR have been reported (1). The noncanonical PRC1.1 complex contains a DNA binding ZnF-CXXC domain able to target chromatin via KDM2B (43). ZNF/repeats chromatin state was statistically enriched in five of the six EwS cell lines analyzed.

Recently, different cell models have shown that the E3 ubiquitin ligase activity of RING1B is inactivated by phosphorylation (15, 36). Our results showing the recruitment of AURKB to enhancers are compatible with a model in which RING1B is unable to repress the newly formed ES enhancers, which were previously Polycomb-repressed regions. Once the oncogene binds to chromatin, RING1B would cooperate to induce transcription activation if its ubiquitin ligase activity is inhibited by phosphorylation (either directly or indirectly) (Fig. 6F). More studies are needed to clarify how oncogenic fusion proteins act as binding scaffolds to recruit a specific set of interactors to generate previously unknown functional units (such as neo-enhancers).

Inhibition of super-enhancers activity with BET inhibitors has emerged as a successful preclinical strategy in the fight against different pediatric cancers such as EwS, neuroblastoma, and rhabdomyosarcoma (4446). Inhibition of AURKB with AZD1152 increases H2Aub and decreases expression of key oncogene targets, thus suggesting that RING1B is essential for enhancer deregulation by EWSR1-FLI1. Nevertheless, as RING1B account for catalytic and noncatalytic dependencies (14), further investigation should address its clinical therapeutic implications. In agreement with our data, combined inhibition of AURKA and AURKB, as well as synergistic activity of AURKB with focal adhesion kinase inhibitors, has been described effective in EwS preclinical studies, although AURKB efficiency as single agent has not been proved (47, 48). In EwS cells, AZD1152 could affect the levels of RING1B, and this likely reverberates on the regulation of the oncogenes promoter since RING1B occupies the EWSR1 promoter (fig. S6, E and G).

In summary, we demonstrate the oncogenic dependency to high levels of RING1B in EwS. The data support a model in which RING1B plays a pivotal role for EWSR1-FLI1 recruitment to the multimeric DNA repeats. This, in turn, allows for transcriptional activation that defines the characteristic transcriptome of EwS. Given the role of RING1B in the activation of super-enhancers, which are critical elements for cell fate determination, we propose that the EwS cell of origin is predefined by high levels of RING1B.

The Ewings sarcoma cell lines A673, SK-ES1, and A4573, which carry the EWSR1-FLI1 translocation types I, II, and III, respectively, and the HEK293 cell line from human embryonic kidney infected with AgT from SV40 (293T), were cultured in RPMI 1640 media (Gibco) and supplemented with 10% fetal bovine serum, l-glutamine, and penicillin/streptomycin. Cells were cultured at 37C with 5% CO2. The A673 and SK-ES1 cell lines harboring shCTRL and shRING1B with seq#1 and seq#2 as well as A673 cell line with doxycycline inducible knockdown of EWSR1-FLI1 were previously described (11, 18). hpMSCs were isolated following published protocols (21). Ectopic expression of EWSR1-FLI1 3xFLAG C terminus in HeLa cells was induced with doxycycline (0.5 g/ml) (34).

All experiments performed with AZD1152 were incubated 72 hours, with the exception of RNA expression assays that were incubated 24 hours. For IC50 calculations, A673, SK-ES1, A4573, and 293T cell lines were seeded at 2000 cells per well in 96-well culture plates. AZD1152 and PRT4165 (Sigma-Aldrich) was added to complete growth medium; after 72 hours, cells were subjected to the ATPlite assay (PerkinElmer), and measurements were performed using a Tecan plate reader. Inhibitory concentrations were calculated using OriginPro 9.0 software.

EWSR1-FLI1 type 2 was amplified from a pSG5 vector with primers containing Bgl II and Hind III sequences (forward, 5-ggaggaaggAGATCTAATGGCGTCCACGG-3; reverse, 5-aagAAGCTTGTAGTAGCTGCCTAA-3). The PCR product was purified using an Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare Life Sciences). The product of the amplification was subcloned into the TOPO TA Cloning Kit for Sequencing following the manufacturers instructions. TOPO-EWSR1-FLI1 plasmid and the acceptor vector pEGFP-N1 were double digested with Bgl II and Hind III at 37C. The resulting EWSR1-FLI1 band was ligated into pEGFP-N1, and ligation product was then transformed into JM109 cells.

Target sequences for siRNA are described in table S6. Transfection of small duplexes (Sigma-Aldrich) was performed with Lipofectamine RNAiMAX and Optimem (Invitrogen), using 30 pmol when cells were 80% confluent; samples were collected after a 72-hour incubation. Transient transfections of GFP constructs or empty vector were done using FuGENE XP (Roche) with 1 to 2 g of plasmid when cells were 60% confluent; samples were collected after 48 hours. Both reagents were used according to the manufacturers recommendations.

Empty pLIV and EWSR1-FLI1pLIVexpressing lentiviruses were provided by N. Riggi (University Institute of Pathology Lausanne, Switzerland). Lentiviruses were produced in Lenti-X 293T packaging cells (Takara, Cultek) at a low passage number. For each plate, 7 g of the lentiviral plasmid, 5 g of the envelope plasmid (VSV-G), and 6 g of the packaging plasmid (PAX8) were prepared and introduced by calcium phosphate transfection, according to standard protocols. The supernatant containing lentiviruses was collected 48 hours after transfection. The HUVEC cell line was seeded at 3000 cells/cm2 and transduced with 3:1 of the lentiviral supernatant with fresh media containing Polybrene (Sigma-Aldrich) at 6 g/ml. Cells were selected with fresh growth media containing puromycin (0.3 g/ml) for 72 hours. A control dish without the transduction media was also selected with puromycin, to control for killing of nontransduced cells.

Histone extracts of cultured cells were isolated using the EpiQuick Histone Extraction kit (Epigentek) following the manufacturers instructions. Total cell extracts were prepared in IPH buffer [50 mM tris-HCl (pH 8), 150 mM NaCl, 5 mM EDTA, and 0.5% NP-40] with EDTA-free protease inhibitor cocktail (Roche). For protein, fractionation standard protocols were used. Histone or total protein extracts were quantified by Bradford assay. Immunoprecipitation was performed with total cellular extracts incubated at 4C overnight with primary antibody. After incubation of immunoprecipitated samples on protein A/G and agarose beads (Santa Cruz Biotech), 30 to 50 g of whole protein extracts or 5 g of histones was resolved by polyacrylamide gel electrophoresis. Western blotting was performed using standard protocols. Incubation with primary antibodies was done at 4C overnight and LI-COR secondary antibodies that are detectable by near-infrared fluorescence were used for detection (table S6). Blots were scanned with an Odyssey CLx Infrared Imaging System at medium intensities.

Treated cells were fixed in 70% ethanol, stained with 25 l of propidium iodide (PI) (1 mg/ml), and 25 l of ribonuclease (RNase) (10 mg/ml), and incubated 30 min at 37C. For Annexin V binding, the Alexa Fluor 488 fluorophore kit (Invitrogen) was used for apoptotic cell detection. After culture and treatment, cells were resuspended in annexin binding buffer with 5 l of Alexa Fluor 488 Annexin V and 1 l of PI working solution (100 g/ml). After 15 min, samples were run in Gallios multicolor flow cytometer (Beckman Coulter) set up with the 3-lasers, 10 colors standard configuration. Histograms and cytograms were further analyzed with FlowJo 10.2.

Total RNA was isolated and purified from collected cells using the RNeasy Mini Kit (Qiagen) according to the manufacturers protocol. After quantification using the NanoDrop software (Thermo Fisher Scientific), RT was performed. A 1-g aliquot of each RNA sample was converted to cDNA in a reaction catalyzed by a retrotranscriptase enzyme (M-MLV Reverse Transcriptase Promega). Random primers and RNase inhibitor (RNasin Plus RNase Inhibitor, Promega) were also added to the reaction. cDNA obtained was analyzed by qPCR using SYBR Green PCR Master Mix (ABI). cDNA was amplified with specific oligonucleotides (table S6). Each cDNA sample was run in triplicate, and its levels were analyzed using the 7500 Fast PCR instrument (Applied Biosystems). To compare between different conditions studied, relative quantification of each target was normalized to a housekeeping gene. Last, data were analyzed using the comparative 2-ct method.

Gene expression microarrays were performed at the Microarray Analysis Service, Hospital del Mar Medical Research Institute (IMIM, Barcelona). RNA samples were amplified, labeled according to a GeneChip WT PLUS Reagent kit, and hybridized to Human Gene 2.0 ST (Affymetrix) in a GeneChip Hybridization Oven 640. Washing and scanning were performed using the Expression Wash, Stain, and Scan Kit and the GeneChip System of Affymetrix (GeneChip Fluidics Station 450 and GeneChip Scanner 3000 7G). After quality control, raw data were background corrected, quantile-normalized, and summarized to a gene level using the robust multichip average; a total of 48,144 transcript clusters, excluding controls, were obtained, which roughly corresponds to genes and other RNAs, such as long intergenic noncoding RNAs and microRNAs. NetAffx 36 annotations, based on the human genome 19, were used to summarize data into transcript clusters and to annotate analyzed data. Linear Models for Microarray (limma), a moderated t statistics model, was used for detecting differentially expressed genes between the conditions. All data analyses were performed in R (version 3.4.3) with R/Bioconductor packages aroma.affymetrix, Biobase, affy, limma, genefilter, ggplots, and Vennerable. Genes with a P less than 0.05 were selected as significant.

Raw sequencing reads in the fastq files were mapped with STAR version 2.6.a (49). GENCODE release 29, based on the GRCh38 reference genome, and the corresponding GTF file were used. The table of counts was obtained with featureCounts function in the package subread, version 1.6.4. The differential gene expression analysis (DEG) was assessed with voom+limma in the limma package version 3.40.2 and using R version 3.6.0. Raw library size differences between samples were treated with the weighted trimmed mean method implemented in the edgeR package. Clustering method used is Ward.D2 with correlation distances and principal components analysis. For the differential expression analysis, read counts were converted to log2 counts per million, and the mean-variance relationship was modeled with precision weights using voom approach in limma package. Raw data are accessible at the NCBI Gene Expression Omnibus (GEO) accession code GSE131286.

Intersection of DEG for A673 shRING1B knockdown with those for A673 shEWSR1-FLI1 with accession number GSE61953 (10) was obtained by calculating a delta-score as described by the authors. Absolute FC > 1.25 and 1.5 for RING1B and EWSR1-FLI1 datasets were selected, respectively. Overlaps for positive and negative gene sets were obtained using Vennerable R package and BioVenn. Functional analysis of the intersection between RING1B and EWSR1-FLI1 gene lists was performed in Enrichr. Normalized enrichment scores on A673 and SK-ES1 shRING1B versus shCTRL were obtained with GSEA using the Hallmark gene set collection. GSEA was used to analyze enrichment on the list of 983 down-regulated TSG in tumor samples versus normal tissue from TSGene database (50) (https://bioinfo.uth.edu/TSGene/). Analysis of expression levels for AURKB, CSNK2A1, and MAP2K1 were performed using information from GEO2R GSE7007 for the probes 209464_at, 212075_s_at, and 202670_at, respectively.

Immunohistochemical analyses were performed following standard techniques. The antibodies used are given in table S6. Tumors were fixed in formalin and embedded in paraffin for subsequent processing. Consecutive, sections were deparaffinized, rehydrated, and heated with Epitope Retrieval Solution (pH 6.0) (Novocastra Laboratories). Reactions were developed with Novolink Polymer Detection System (Novocastra Laboratories). Immunoreactivity was visualized by diaminobenzidine, and nuclei were counterstained with hematoxylin. Tissue was then dehydrated with alcohol, permeated with xylene, and mounted with Permount organic mounting solution (Thermo Fisher Scientific). Images were evaluated by a pathologist to select regions of interest and analyzed with the Dotslide Microscope and Olympia Software (Olympus). Similar regions of every sample were selected from every section.

Cells were treated with 1% formaldehyde at room temperature for 10 min, and the cross-linking reaction was stop by adding 500 l glycine (1.25 M). Cells were resuspended in lysis buffer [0.1% SDS, 0.15 M NaCl, 1% Triton X-100, 1 mM EDTA, 20 mM tris (pH 8), and protease inhibitors (1 mg/ml)] and sonicated with Bioruptor Pico (Diagenode) for 10 cycles until chromatin was sheared to an average fragment length of 200 bp. After centrifugation, a small fraction of eluted chromatin was measured with Qubit. Starting with 30 g of sample, immunoprecipitation for each antibody was performed overnight (table S6); 50 l of Dynabeads Protein A (Invitrogen) was then added and incubated for 2 hours at 4C under rotation. Immunoprecipitates were washed once with TSE I [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM tris-HCl (pH 8), and 150 mM NaCl], TSE II [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM tris-HCl (pH 8), and 500 mM NaCl], and TSE III [0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, and 10 mM tris-HCl (pH 8)] and then twice with tris-EDTA buffer. Washed pellets were eluted with 120 l of a solution of 1% SDS and 0.1 M NaHCO3. Eluted pellets were decross-linked for 5 hours at 65C and purified on 50 l of tris-EDTA buffer with the QIAquick PCR Purification Kit (Qiagen). Differences in the DNA content at each binding region (sequences in table S6) from every immunoprecipitation assay were determined by real-time PCR using the ABI 7700 sequence detection system and SYBR Green master mix protocol (Applied Biosystems). Each immunoprecipitation was done in triplicate, and PCR assays were performed using fixed amounts of input and immunoprecipitated DNA. For every amplicon, standard curves to calculate efficiency and melting curves to confirm single amplicons were obtained. The reported data represent real-time PCR values normalized to input DNA and are expressed as percentage (%) of bound/input signal.

Libraries were prepared using the NEBNext Ultra DNA Library Prep from Illumina according to the manufacturers protocol. Briefly, 5 ng of input and ChIP-enriched DNA were subjected to end repair and addition of A bases to 3 ends, ligation of adapters, and USER excision. All purification steps were performed using AgenCourt AMPure XP beads (Qiagen). Library amplification was performed by PCR using NEBNext Multiplex Oligos from Illumina. Final libraries were analyzed using Agilent high sensitivity chip to estimate the quantity and to check size distribution and then were quantified by qPCR using the KAPA Library Quantification Kit (KapaBiosystems) before amplification with Illuminas cBot. Libraries were loaded onto the flow cell sequencer 1 50 on Illuminas HiSeq 2500.

ChIP-seq samples were mapped against the hg19 human genome assembly using BowTie with the option m 1 to discard those reads that could not be uniquely mapped to just one region. A second replicate of RING1B and H2Aub was sequenced to evaluate the statistical significance of the results. Model-based analysis of ChIP-seq (MACS) was run individually on each replicate with the default parameters but with the shift size adjusted to 100 bp to perform the peak calling against the corresponding control sample (51). DiffBind was initially run over the peaks reported by MACS for each pair of replicates of the same experiment to generate a consensus set of peaks (28). Next, DiffBind was run again over each pair of replicates of the same experiment, samples and inputs, to find the peaks from the consensus set that were significantly enriched in both replicates in comparison to the corresponding controls (categories, DBA_CONDITION; block, DBA_REPLICATE; and method, DBA_DESEQ2_BLOCK). DiffBind RING1B peaks with P < 0.05 and H2Aub peaks with P < 0.05 and false discovery rate < 0.00001 were selected for further analysis. The genome distribution of each set of peaks was calculated by counting the number of peaks fitted on each class of region according to RefSeq annotations. Promoter is the region between 2.5 kb upstream and 2.5 kb downstream of the TSS. Genic regions correspond to the rest of the gene (the part that is not classified as promoter), and the rest of the genome is considered to be intergenic. Peaks that overlapped with more than one genomic feature were proportionally counted the same number of times. Each set of target genes was retrieved by matching the ChIP-seq peaks in the region 2.5 kb upstream of the TSS until the end of the transcripts as annotated in RefSeq. Reports of functional enrichments of GO categories were generated using the EnrichR tool. Aggregated plots showing the average distribution of ChIP-seq reads around the summit of each peak were generated by counting the number of reads for each region and then averaging the values for the total number of mapped reads of each sample and the total number of peaks in the particular gene set. To perform the comparison between two sets of peaks, a minimum overlap of one nucleotide was necessary to consider one match. The heatmap displaying the density of ChIP-seq reads 5 kb around the summit of each peak set were generated by counting the number of reads in this region for each individual peak and normalizing this value with the total number of mapped reads of the sample. Peaks on each ChIP heatmap were ranked by the logarithm of the average number of reads in the same genomic region. On the other hand, we separated the single peaks of RING1B into distal and TSS (5 kb around one RefSeq gene) to generate the heatmap of ChIP-seq signal strength of RING1B, EWSR1-FLI1, H3K27me3, H2Aub, and H3K27ac over the two classes of RING1B peaks detected above (distal and TSS). To build our collection of enhancers and promoters, we reanalyzed published ChIP-seq samples of H3K4me1, H3K27ac, H3K27me3, and H3K4me3 in A673 cells (10). H3K27ac and H3K27me3 peaks were used to discriminate between active or repressed regulatory regions. Promoters were defined as ChIP peaks of H3K27 found up to 2.5 kb from the TSS of one gene and enhancers on intergenic areas outside promoters or within gene introns. H3K4me3 was required to be present in promoters but absent in enhancers. We defined four classes of regulatory elements: active enhancers (H3K27ac), active promoters (H3K27ac + H3K4me3), poised enhancers (H3K27me3), and bivalent promoters (H3K27me3 + H3K4me3). The MEME-ChIP tool was used to perform motif-finding analysis of the sequences bound by each factor. The UCSC genome browser was used to generate the screenshots of each group of experiments along the manuscript (52). Raw data, genome-wide profiles, and peaks of each ChIP-seq experiment are accessible at the NCBI GEO accession code GSE131286.

We have determined the composition of 3945 EWSR1-FLI1 biding sites in terms of 15 chromatin states from the segmentations generated by Epigenome Roadmap Consortium (GEO code: GSE61953) for six different cell types: HUVECs (E122), H1 (E003) and H9 ES cells (E008), H1-derived mesenchymal stem cells (E006), BM-derived MSCs (E026), and adipose-derived MSC (E025) (26). The statistical significance of the relative frequency of each stage at every cell type was assessed in comparison to the same value measured along the whole genome, using the Fishers exact test. The R package GenomicRanges from Bioconductor was used for calculations of compositions. Next, to generate the final heatmap, we have grouped certain states for semantic similarity (active TSS category includes active and flanking active TSS states; transcription includes flanking, strong, and weak states; enhancers account for both genic and intergenic; bivalent TSS include also flanking bivalent promoters and PcG repressed include both repressed and weak repressed). Thus, the relative frequencies of the new eight states were recalculated, while quiescent state was discarded from the analysis. Last, the enrichment percentage at a particular stage was calculated as the difference between the relative frequency at the EWSR1-FLI1 ChIP-seq sites minus the relative frequency at the whole genome normalized by the relative frequency at the whole genome again.

In vivo studies were performed after the approval of the Institutional Animal Research Ethics Committee. Athymic nude mice (Envigo) were injected subcutaneously with 4 106 cells for shCTRL#seq1 and shRING1B#seq1 and 2 106 for seq#2. shCTRL cells were resuspended in 200 l of Matrigel (Becton Dickinson) with phosphate-buffered saline and injected into both flanks (5 mice n = 10 for seq#1 and 6 mice, n = 12 for seq#2). The same procedure was performed for the SK-ES1 shRING1B cell line. Tumor growth was monitored three times a week by measuring tumor volume with a digital caliper. Mice were euthanized when tumors reached a size of 2.5 cm in any dimension. Survival curves were calculated using the Kaplan-Meier method and were compared with a log-rank test. At the end of the experiment, tumors were excised; half of each specimen was frozen in liquid nitrogen for RNA extraction, and the other was fixed in 10% formalin for immunohistochemistry experiments.

Acknowledgments: We thank N. Riggi for reagents and technical advice and M. Martnez-Balbs for technical advice and critical reading of the manuscript. We also thank G. Pascual-Pasto, S. Mateo, and M. Suol for technical advice, S. Perez-Jaume for statistical advice, and L. Nonell from the Microarray Analysis Service, Hospital del Mar Medical Research Institute (IMIM, Barcelona) for technical advice. Last, we are grateful to the Band of Parents at Hospital Sant Joan de Du for supporting the overall research activities of the developmental tumor laboratory, PCCB. Funding: S.S.-M. and the project were supported by the Spanish Association Against Cancer (AECC) consolidated groups grant (GCB13131578) consortium. The project also had the support from the Asociacion Pablo Ugarte (APU). E.F.-B. was supported by the Spanish government grant, Instituto de Salud Carlos III (PI16/00245) to J.M. The work in the Di Croce laboratory was supported by grants from the Spanish of Economy, Industry and Competitiveness (MEIC) (BFU2016-75008-P), and Fundacion Vencer El Cancer (VEC). Author contributions: S.S.-M., L.D.C., and J.M. designed the study, conducted experiments, and wrote the manuscript. J.M. supervised all the work. S.S.-M., E.F.-B., M.S.-J., P.T., C.B., E.P., L.H.-P., and D.J.G.-D. performed the experiments. E.B. and S.G. performed all the bioinformatic analysis. I.H.-M., O.M.T., A.M.C., C.L., and E.. provided expertise and feedback. All authors reviewed the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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RING1B recruits EWSR1-FLI1 and cooperates in the remodeling of chromatin necessary for Ewing sarcoma tumorigenesis - Science Advances

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Global cosmetic skin care market is set to witness a substantial CAGR of 5.5% in the forecast period of 2019- 2026. The report contains data of the base year 2018 and historic year 2017. Increasing self-consciousness among population and rising demand for anti- aging skin care products are the factor for the market growth.

Global Cosmetic Skin Care Market By Product (Anti-Aging Cosmetic Products, Skin Whitening Cosmetic Products, Sensitive Skin Care Products, Anti-Acne Products, Dry Skin Care Products, Warts Removal Products, Infant Skin Care Products, Anti-Scars Solution Products, Mole Removal Products, Multi Utility Products), Application (Flakiness Reduction, Stem Cells Protection against UV, Rehydrate the skins surface, Minimize wrinkles, Increase the viscosity of Aqueous, Others), Gender (Men, Women), Distribution Channel (Online, Departmental Stores and Convenience Stores, Pharmacies, Supermarket, Others), Geography (North America, Europe, Asia-Pacific, South America, Middle East and Africa) Industry Trends and Forecast to 2026

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Market Definition: Global Cosmetic Skin Care Market

Cosmetic skin care is a variety of products which are used to improve the skins appearance and alleviate skin conditions. It consists different products such as anti- aging cosmetic products, sensitive skin care products, anti- scar solution products, warts removal products, infant skin care products and other. They contain various ingredients which are beneficial for the skin such as phytochemicals, vitamins, essential oils, and other. Their main function is to make the skin healthy and repair the skin damages.

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Geographically, this report is segmented into several key regions, with sales, revenue, market share and growth Rate of industry in these regions, from 2020 to 2027, covering

Global cosmetic skin care market is highly fragmented and the major players have used various strategies such as new product launches, expansions, agreements, joint ventures, partnerships, acquisitions, and others to increase their footprints in this market. The report includes market shares of cosmetic skin care market for Global, Europe, North America, Asia-Pacific, South America and Middle East & Africa.

Few of the major competitors currently working in the global cosmetic skin care market are LOral, Unilever, New Avon Company, Este Lauder Companies, Espa, Kao Corporation, Johnson & Johnson Services, Inc., Procter & Gamble, Beiersdorf, THE BODY SHOP INTERNATIONAL LIMITED, Shiseido Co.,Ltd., Coty Inc., Bo International, A One Cosmetics Products, Lancme, Clinique Laboratories, llc., Galderma Laboratories, L.P., AVON Beauty Products India Pvt Ltd, Nutriglow Cosmetics Pvt. Ltd, Shree Cosmetics Ltd among others.

Cosmetic Skin Care Market: Key Questions Answered in Report

The research study on the Cosmetic Skin Care market offers inclusive insights about the growth of the market in the most comprehensible manner for a better understanding of users. Insights offered in the Cosmetic Skin Care market report answer some of the most prominent questions that assist the stakeholders in measuring all the emerging possibilities.

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Cosmetic Skin Care Market (Covid 19 Impact Analysis) Data Highlighting Major Vendors, Promising Regions, Anticipated Growth Forecast To 2027 - PRnews...

Skin Stem Cells Comments Off on Cosmetic Skin Care Market (Covid 19 Impact Analysis) Data Highlighting Major Vendors, Promising Regions, Anticipated Growth Forecast To 2027 – PRnews… | October 24th, 2020