ERMS Cell Research Articles

Indole Family and Neomycin Sulfate: Inductors of Differentiation in C2C12 and RD cell Lines

Rhabdomyosarcoma (RMS) is a highly aggressive tumor primarily affecting the pediatric population, that generally originates from a failure in the embryonic differentiation of myogenic precursor cells. Standard treatment involves surgery, chemotherapy and radiation therapy, with a poor prognosis, especially when it spreads to other parts of the body, highlighting the need for new treatment approaches. In this study, cellular differentiation was investigated as a potential therapeutic approach for an embryonal subtype of RMS (eRMS) employing a human eRMS cell line, RD; and comparing it with a normal murine myoblast cell line, C2C12. We observed that not only a serum free or a low serum concentration in the cultured medium induce morphological changes towards differentiations, but also staurosporine, bisindolylmaleimide, and neomycin sulfate (direct or indirect protein kinase inhibitors) induces differentiation in RD and C2C12 cell lines, suggesting a crucial role of PKC, PLC and its signaling pathways in a proliferation/differentiation switch. These findings underscore the importance of exploring cellular differentiation as a therapeutic strategy against RMS and highlight the need for further research into the underlying mechanisms of kinase inhibitor-induced cellular differentiation as a potential cancer treatment.

Abstract LB_A23: The role of SMARCA1 in Rhabdomyosarcoma and skeletal muscle differentiation

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue of pediatric sarcoma, and studies demonstrate that RMS arises from skeletal muscle precursor cells. RMS, genetically and histologically, is divided into two subtypes: PAX-FOXO1 fusion positive (alveolar) RMS (aRMS), which is driven by chromosomal translocation involving PAX3 or PAX7 genes with FOXO1 and PAX-FOXO1 fusion negative (embryonal) RMS (eRMS), which is marked by mutations in RAS isoforms and some genes such as TP53, PIK3CA, CTNNB1 and FGFR4. Chromatin was one of the earliest identified targets for cancer therapy. Several chromatin remodeling proteins are associated with cancer progression processes such as proliferation, differentiation, apoptosis, and tumorigenesis. Components of chromatin remodeling complexes, such as the imitation switch (ISWI) complex, have been classified as both oncogenes and tumor suppressors. The ISWI family protein, SMARCA1 (also known as SNF2L), has been implicated in tumorigenesis for several cancer types. ISWI complexes regulate cell differentiation and proliferation in other cell systems, but their impact in myogenesis is not well understood. In this study, we will characterize the function of SMARCA1 in RMS cells and skeletal muscle. We hypothesize that SMARCA1 acts to modulate chromatin accessibility, and drive RMS tumorigenic growth. Methods: We will use RNA-seq, ATAC-seq, and CUT&RUN to study the impact of SMARCA1 deletion in RMS cells. Furthermore, phenotypic experiments will be performed to determine the influence of SMARCA1 on differentiation. Results: SMARCA1 is expressed at a high level in RMS tissues but not in muscle tissues. Furthermore, our findings show that SMARCA1 is modestly upregulated during the differentiation in normal myogenesis. Curiously, eRMS cell lines show no change in the protein level of SMARCA1 when treated with a MEK inhibitor, trametinib. Analysis of gene clusters in RMS transcriptomic data of indicated that SMARCA1 co-expresses with a RAS effector, RALA. SMARCA1 also clustered with HDAC2. In addition, SMARCA1 interacted with HDAC2 in RMS cells as determined by co-immunoprecipitation experiments. Conclusions: This study will deepen our understanding of how SMARCA1 impacts RMS differentiation and proliferation and may credential SMARCA1 as a novel therapeutic target in rhabdomyosarcoma. Citation Format: Ashwaq K Aljabri, Katie E Hebron, Yuliya Kriga, Juan Manuel Caravaca, Aiysha Althobaiti, Alex Emmons, Stacey Stauffer, Angela Kim, Lauren Stoak, Morgan Porter, Jyoti Shetty, Peter Fitzgerald, Bao Tran, Matthew Geisler, Judith K Davie, Marielle E Yohe. The role of SMARCA1 in Rhabdomyosarcoma and skeletal muscle differentiation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A23.

Expression of oncogenic HRAS in human Rh28 and RMS-YM rhabdomyosarcoma cells leads to oncogene-induced senescence

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. The two predominant histologic variants of RMS, embryonal and alveolar rhabdomyosarcoma (eRMS and aRMS, respectively), carry very different prognoses. While eRMS is associated with an intermediate prognosis, the 5-year survival rate of aRMS is less than 30%. The RMS subtypes are also different at the molecular level—eRMS frequently has multiple genetic alterations, including mutations in RAS and TP53, whereas aRMS often has chromosomal translocations resulting in PAX3-FOXO1 or PAX7-FOXO1 fusions, but otherwise has a “quiet” genome. Interestingly, mutations in RAS are rarely found in aRMS. In this study, we explored the role of oncogenic RAS in aRMS. We found that while ectopic oncogenic HRAS expression was tolerated in the human RAS-driven eRMS cell line RD, it was detrimental to cell growth and proliferation in the human aRMS cell line Rh28. Growth inhibition was mediated by oncogene-induced senescence and associated with increased RB pathway activity and expression of the cyclin-dependent kinase inhibitors p16 and p21. Unexpectedly, the human eRMS cell line RMS-YM, a RAS wild-type eRMS cell line, also exhibited growth inhibition in response to oncogenic HRAS in a manner similar to aRMS Rh28 cells. This work suggests that oncogenic RAS is expressed in a context-dependent manner in RMS and may provide insight into the differential origins and therapeutic opportunities for RMS subtypes.

Abstract A05: RAS signaling promotes ERMS cell viability via sustaining TAZ expression and protein stability

Abstract Rhabdomyosarcoma (RMS) is a pediatric cancer characterized by skeletal muscle histogenesis, with the embryonal histologic variant (ERMS) being the most common. Mutations of the RAS pathway are found in 40-60% of ERMS patient specimens, and as in other human cancers the RAS protein is currently not druggable, and inhibition of downstream RAS effector pathways fails due to compensating signaling. Thus, there is an urgent need to understand the dysregulated signaling in RAS-driven ERMS (rERMS). Hippo signaling was originally identified for its role in controlling organ size and tissue homeostasis, but it is now known to also have a critical role in cancer initiation and progression. In ERMS patients, higher YAP1 gene expression correlates with a poor prognosis, and in vitro and in vivo studies using ERMS cell lines and mouse models also show that YAP1 supports ERMS cell and tumor growth. Whether and how the YAP1 paralog WWTR1 (TAZ) contributes to rERMS tumorigenesis remains understudied. Our initial studies examining ERK suppression (genetic inhibition via shRNA or pharmacologic inhibition via trametinib) showed that this slowed rERMS cell growth and proliferation, and induced rERMS cell differentiation. However, we also found CTGF and CYR61 (downstream targets of YAP1/TAZ) significantly repressed upon trametinib treatment. While YAP1 appeared unaffected, trametinib treatment significantly suppressed TAZ at both the mRNA and protein levels in a dose-dependent manner. Suppression of TAZ with shRNA decreased rERMS cell growth and proliferation, suggesting that sustained TAZ expression is crucial for rERMS cell viability. Interestingly, there was no change in the activity of MST and LATS. Our next focus will be on the mechanism of ERK-mediated TAZ expression, with a potential role for glycogen synthase kinase 3 beta as a mediator of TAZ degradation. In summary, these studies identify a new role for RAS signaling in promoting rERMS cell survival via TAZ expression. Citation Format: Liz Yi-Tzu Lin, Corinne Linardic. RAS signaling promotes ERMS cell viability via sustaining TAZ expression and protein stability [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A05.

C-Myb regulates tumorigenic potential of embryonal rhabdomyosarcoma cells

Rhabdomyosarcomas (RMS) are a heterogeneous group of mesodermal tumors, the most common sub-types are embryonal (eRMS) and alveolar (aRMS) rhabdomyosarcoma. Immunohistochemical analysis revealed c-Myb expression in both eRMS and aRMS. c-Myb has been reported to be often associated with malignant human cancers. We therefore investigated the c-Myb role in RMS using cellular models of RMS. Specific suppression of c-Myb by a lentiviral vector expressing doxycycline (Dox)-inducible c-Myb shRNA inhibited proliferation, colony formation, and migration of the eRMS cell line (RD), but not of the aRMS cell line (RH30). Upon c-Myb knockdown in eRMS cells, cells accumulated in G0/G1 phase, the invasive behaviour of cells was repressed, and elevated levels of myosin heavy chain, marker of muscle differentiation, was detected. Next, we used an RD-based xenograft model to investigate the role of c-Myb in eRMS tumorigenesis in vivo. We found that Dox administration did not result in efficient suppression of c-Myb in growing tumors. However, when c-Myb-deficient RD cells were implanted into SCID mice, we observed inefficient tumor grafting and attenuation of tumor growth during the initial stages of tumor expansion. The presented study suggests that c-Myb could be a therapeutic target in embryonal rhabdomyosarcoma assuming that its expression is ablated.

The histone deacetylase inhibitor Suberoylanilide Hydroxamic Acid (SAHA) as a therapeutic agent in rhabdomyosarcoma

ABSTRACT Rhabdomyosarcoma (RMS) is an aggressive childhood sarcoma with two distinct subtypes, embryonal (ERMS) and alveolar (ARMS) histologies. More effective treatment is needed to improve outcomes, beyond conventional cytotoxic chemotherapy. The pan-histone deacetylase inhibitor, Suberoylanilide Hydroxamic Acid (SAHA), has shown promising efficacy in limited preclinical studies. We used a panel of human ERMS and ARMS cell lines and xenografts to evaluate the effects of SAHA as a therapeutic agent in both RMS subtypes. SAHA decreased cell viability by inhibiting S-phase progression in all cell lines tested, and induced apoptosis in all but one cell line. Molecularly, SAHA-treated cells showed activation of a DNA damage response, induction of the cell cycle inhibitors p21Cip1 and p27Kip1 and downregulation of Cyclin D1. In a subset of RMS cell lines, SAHA promoted features of cellular senescence and myogenic differentiation. Interestingly, SAHA treatment profoundly decreased protein levels of the driver fusion oncoprotein PAX3-FOXO1 in ARMS cells at a post-translational level. In vivo, SAHA-treated xenografts showed increased histone acetylation and induction of a DNA damage response, along with variable upregulation of p21Cip1 and p27Kip1. However, while the ARMS Rh41 xenograft tumor growth was significantly inhibited, there was no significant inhibition of the ERMS tumor xenograft RD. Thus, our work shows that, while SAHA is effective against ERMS and ARMS tumor cells in vitro, it has divergent in vivo effects . Together with the observed effects on the PAX3-FOXO1 fusion protein, these data suggest SAHA as a possible therapeutic agent for clinical testing in patients with fusion protein-positive RMS.

PO-369 KDM3A/Ets1/MCAM axis in rhabdomyosarcoma and osteosarcoma

IntroductionRhabdomyosarcoma (RMS) and Osteosarcoma (OS) are the most common primary malignant tumours of soft tissue and bones, respectively, affecting children, adolescents and young adults. RMS can be divided into two major histopathological and molecular subtypes: driver oncofusion (PAX3/7-FOXO1) positive, alveolar (ARMS), and oncofusion-negative embryonal (ERMS). Oncofusion positive ARMS carries a worse prognosis, in part due to higher propensity for metastasis. OS is also an aggressive disease with high propensity for metastasis. Patients with metastatic RMS and OS have few therapeutic options, including currently no effective targeted therapies, and typically face poor outcomes. Our laboratory has previously identified a novel, potentially druggable, tumour and metastasis-promotional, axis in Ewing Sarcoma, another aggressive bone and soft tissue cancer of childhood, involving the histone demethylase KDM3A, the Ets1 transcription factor, and the cell adhesion protein MCAM. We have additionally observed KDM3A to be overexpressed in RMS, and in a subset of OS. The aim of this study was to examine whether the KDM3A/Ets1/MCAM axis is intact in RMS and OS, and what impact it has on the biology of these diseases.Material and methodsRMS and OS cell lines were analysed for KDM3A, Ets1 and MCAM expression, and selected cell lines were subjected to stable shRNA-mediated KDM3A, Ets1 and MCAM knockdown (KD). Phenotypic effects of knockdown were analysed by clonogenic and transendothelial invasion assays. Preliminary tumour xenograft studies were also performed.Results and discussionsKDM3A is uniformly overexpressed in ERMS and ARMS cell lines and a subset of OS cell lines. KDM3A KD inhibits colony formation and transendothelial invasion in ERMS and ARMS cells and a high-expressing OS cell line. In preliminary studies KDM3A KD also inhibits ARMS tumorigenesis in an orthotopic model. Strikingly, similar to our prior studies in Ewing Sarcoma, a KDM3A/Ets1/MCAM regulatory axis is intact in RMS and OS, and MCAM KD exerts similar phenotypic effects to those achieved with KDM3A KD.ConclusionOur studies indicate that a KDM3A/Ets1/MCAM molecular axis is intact in RMS and OS, and regulates growth and invasive properties. Together with our previous studies in Ewing Sarcoma, these findings suggest that this may be an important tumour and metastasis promotional axis in all three common paediatric sarcomas, despite distinct cellular origins of these cancers.

Targeting Anaplastic Lymphoma Kinase (ALK) in Rhabdomyosarcoma (RMS) with the Second-Generation ALK Inhibitor Ceritinib.

BackgroundThe receptor tyrosine kinase (RTK) anaplastic lymphoma kinase (ALK) has been implicated in the tumorigenesis of rhabdomyosarcoma (RMS). However, the exact role of ALK in RMS is debatable and remains to be elucidated.ObjectiveTo determine the in vitro and in vivo effects and mechanism of action of the second-generation ALK inhibitor ceritinib on RMS cell growth.MethodsEffects of ceritinib on cell proliferation, wound healing, cell cycle, and RTK signaling were determined in alveolar and embryonal rhabdomyosarcoma (ARMS, ERMS). In addition, possible synergistic effects of combined treatment with ceritinib and the Abl/Src family kinase inhibitor dasatinib were determined.ResultsCeritinib treatment led to decreased cell proliferation, cell cycle arrest, apoptosis, and decreased in vivo tumor growth for the ARMS subtype. ERMS cell lines were less affected and showed no cell cycle arrest or apoptosis. Both subtypes lacked intrinsic ALK phosphorylation, and ceritinib was shown to affect the IGF1R signaling pathway. High levels of phosphorylated Src (Tyr416) were present following ceritinib treatment, making combined treatment with a Src inhibitor a potential treatment option. Combined treatment of ceritinib and dasatinib showed synergistic effects in both ERMS and ARMS cell lines.ConclusionThis study shows that monotherapy with an ALK inhibitor, such as ceritinib, in RMS, has no effect on ALK signaling. However, the synergistic effects of ceritinib and dasatinib are promising, most probably due to targeting of IGF1R and Src. Electronic supplementary materialThe online version of this article (10.1007/s11523-017-0528-z) contains supplementary material, which is available to authorized users.

Abstract 5830: A novel Notch-YAP circuit drives stemness and tumorigenesis in embryonal rhabdomyosarcoma

Abstract Introduction: Rhabdomyosarcoma (RMS), a tumor characterized by skeletal muscle features, is the most common soft tissue sarcoma in children and adolescents. The standard of care treatment has not changed in over 40 years, resulting in a stagnant survival rate of less than 30% for high risk groups. Previous studies have identified the developmental pathways Notch and YAP as potent oncogenic signals in the embryonal subtype of RMS (eRMS). Understanding the signaling between Notch and YAP in cancers driven by these pathways, such as eRMS, will be crucial to designing rational combination therapies. Experimental Procedures: For in vitro assays, we utilized 3D sphere cell culture methods that permit the study of stem cell behavior. To analyze signaling, we performed genetic gain and loss-of-function (dox-inducible shRNAs and constitutively active constructs), and pharmacologic inhibition. Target validation was assessed by qPCR and immunoblot, and mechanism was determined by ChIP and limiting dilution assays. For in vivo experiments, we performed subcutaneous xenografts in immunocompromised mice. Immunohistochemistry was used to analyze protein expression in human and murine tumors. Statistical analysis was performed to assess significant differences. Results: We have discovered a novel Notch-YAP circuit that drives cancer cell stemness and tumorigenesis. In eRMS 3D spheres, Notch transcriptionally upregulates YAP and increases its activity, while YAP transcriptionally upregulates the Notch ligands JAG1 and DLL1 and the core Notch transcription factor RBPJ to provide positive feedback. In vivo, YAP suppression decreases Notch signaling, stemness, and proliferation. This circuit boosts expression of the stem cell gene SOX2, which is functionally required for eRMS cell stemness. Inhibiting one node of the circuit, for example through Notch pharmacologic blockade, is not sufficient to shut down the circuit, as stemness and cell growth can be rescued by ectopic YAP. Rather, dual inhibition of Notch and YAP is necessary to silence the circuit’s signaling and sphere growth. Importantly, nuclear Notch and YAP protein expression correlate in eRMS patient tumor samples, suggesting this circuit is intact in human eRMS tumors. Conclusions: While unidirectional signaling between these two pathways has been reported in metazoan development and rarely in human malignancy, this is the first finding of an intact bidirectional circuit between Notch and YAP that supports cancer cell stemness and tumorigenesis. This study provides mechanistic insight into the long-standing question of how eRMS cancer stem cells maintain plasticity. Additionally, while this signaling is identified here in RMS, it could be applicable to many cancer types that are Notch and YAP-dependent. Lastly, this work provides evidence and rationale for research into combination therapies targeting this Notch-YAP signaling circuit. Citation Format: Katherine K. Slemmons, Lisa E.S. Crose, Stefan Riedel, Manuela Sushnitha, Brian Belyea, Corinne M. Linardic. A novel Notch-YAP circuit drives stemness and tumorigenesis in embryonal rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5830. doi:10.1158/1538-7445.AM2017-5830

Abstract 4996: Targeting FGFR4 with monoclonal antibodies as therapeutic agents for the treatment of rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with two major subtypes, embryonal (ERMS) and alveolar (ARMS), and current treatment modalities have yielded event free 5-year survival in only 30% of the patients with high-risk disease. Therefore, there is a need for novel strategies to identify and validate clinically relevant targets and therapeutics for the treatment of RMS. The fibroblast growth factor receptor 4 (FGFR4) is a very attractive therapeutic target because: 1) the FGFR4 gene is over expressed in RMS, 2) it is directly transcriptionally induced by the PAX3-FOXO1 fusion oncogene found in ARMS, 3) it is crucial for survival, proliferation, metastasis and drug resistance, 4) activating mutations in the kinase domain lead to aggressive growth and poor survival in patients with ERMS and 5) genetic or pharmacologic inhibition of FGFR4 signaling inhibited tumor growth in vitro and in vivo. Most normal human tissues do not express FGFR4 protein as determined by immunohistochemistry and electrochemiluminesence assay. Based these, we hypothesize that monoclonal antibodies (mAbs) targeting FGFR4 and their derivatives can serve as potential therapeutic agents for the treatment of RMS. We have developed a total of 15 mAbs against human FGFR4 protein from rabbit and mouse (by hybridoma technology), and from human immunoglobulin libraries (by recombinant DNA technology). These mAbs specifically react with human FGFR4 protein in an ELISA and not to the other members of the FGFR family. Flow cytometer analysis demonstrated that they specifically bound to cell surface FGFR4 protein in RMS cell lines although the FGFR4 expression levels was variable in both ARMS and ERMS cell lines. Furthermore, cell surface FGFR4 mediated internalization of the bound antibody upon incubation at 37oC for as little as 30 min and maximum internalization was observed at 2 h. Two of the mouse mAbs tested were able to mediate specific cytotoxicity in FGFR4-expressing cell lines in the presence of an anti-mouse secondary antibody conjugated to cytotoxic drugs (2oADCs), e.g. monomethyl auristatin F (MMAF) or a duocarmycin derivative (DMDM). Dose titration of the anti-FGFR4 mAbs revealed maximum killing at 100 pM indicating their robust activity in vitro, and both ARMS and ERMS cell lines were susceptible to the mAb-mediated cytotoxicity. Ongoing investigations include generation of FGFR4-targeting direct ADCs and evaluation of their efficacy in vivo. Together, these observations support the contention that anti-FGFR4 mAb can be used as a vehicle to deliver a cytotoxic payload in the form of small molecule drugs and toxins. Thus, FGFR-ADCs may serve as potential therapeutic agents for the treatment of patients with RMS, as well other FGFR4-positive cancers including breast, liver, prostate and lung. Citation Format: Sivasubramanian Baskar. Targeting FGFR4 with monoclonal antibodies as therapeutic agents for the treatment of rhabdomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4996.

Abstract 2452: Exosomes as mediators of paracrine signalling that promote invasive behavior in rhabdomyosarcoma

Abstract Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells. Tumor exosomes contain intact and functional protein, mRNA and miRNA that may alter the cellular environment to favor tumor growth. Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, with two distinct subtypes, alveolar (ARMS) and embryonal (ERMS) histology. ARMS tumors are characterized in the majority of cases by a fusion oncoprotein PAX3-FOXO1, thought to be an initiating factor in tumorigenesis. To date, no studies have been performed to investigate the possible role or content of exosomes in paracrine signaling in RMS. In the present study, we isolated and characterized exosomes secreted from RMS cells, using 3 ERMS (fusion oncoprotein negative) and 2 ARMS (fusion oncoprotein positive) cell lines. RNA analysis showed that small RNA were enriched in RMS-derived exosomes, therefore we focused on evaluating the miRNA content. Array expression analysis showed that exosome miRNA clustered together well, and to a higher extent than cellular miRNA, in ARMS cell lines. Similarly, in the 2 ERMS cell lines that had mutant p53, exosome miRNA clustered together and the pattern of expression was very different from the corresponding cellular miRNA. ARMS and ERMS exosome-enriched miRNA were different, and only 2 miRNA were found to be common among both; putative targets are implicated in cancer and inflammation. Functionally, using in vitro assays, we found that both ERMS- and ARMS-derived exosomes significantly increased the cellular migration and invasion of normal human fibroblasts, and had a positive effect on viability and proliferation of both fibroblasts and RMS cells. In vivo, Matrigel plug assay showed that RMS-derived exosomes had a positive effect on the migration and invasion of stromal cells, suggesting a positive role on angiogenesis. We conclude that RMS-derived exosomes can exert specific paracrine effects on recipient cells, enhancing cell viability as well as invasive properties of fibroblasts and stromal cells, and therefore is likely to play a role in promoting RMS angiogenesis and metastasis. Ongoing studies are aimed at directly assessing the role of RMS-derived exosomes in clinical angiogenesis and metastasis, using in vivo murine models, in addition to dissecting the role of specific enriched miRNA on recipient cell biology. Citation Format: Sandra E. Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Raya Saab. Exosomes as mediators of paracrine signalling that promote invasive behavior in rhabdomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2452.

Abstract 4733: Notch signaling increases the number of relapse-driving tumor propagating cells in embryonal rhabdomyosarcoma

Abstract Embryonal rhabdomyosarcoma (ERMS) is a devastating pediatric muscle cancer with extremely poor prognosis at relapse. Work from our group has identified the tumor-propagating cell (TPC) in a transgenic zebrafish model of kRASG12D-induced ERMS that is responsible for driving continued tumor growth and relapse. The TPC is molecularly similar to an activated muscle satellite-cell and expresses myf5, c-met, and m-cadherin. Building on these observations, we have identified the Notch pathway as a potent enhancer of ERMS self-renewal and TPC number. Specifically, bulk tumor limiting dilution cell transplantation experiments revealed that TPCs are increased 10-fold in kRASG12D expressing ERMS that co-express activated intracellular Notch1 (ICN1). This increase in TPC number is partly the result of ICN1 expressing ERMS exhibiting a 3-fold expansion of relapse-driving myf5-GFP+/mylz2-mCherry-negative ERMS-cell population. Unexpectedly, cell transplantation experiments revealed that Notch pathway activation also conferred tumor-propagating ability to the myf5-GFP+/mylz2-mCherry+ mid-differentiated ERMS cells - a population of cells previously shown to lack self-renewal capacity. Single cell engraftment studies uncovered that NOTCH activation caused cells to oscillate between the TPC and mid-differentiated ERMS molecular states that was not observed in kRASG12D-expressing ERMS, suggesting that Notch has important roles in both self-renewal and cell state transitions. Next, we validated our findings in human ERMS where NOTCH1 is highly expressed both in tumors and ERMS-cell lines. We show important roles for NOTCH in regulating self-renewal and differentiation in human ERMS. Specifically, human ERMS cells that expressed activated NOTCH1 had elevated sphere-colony formation, a surrogate for self-renewal in vitro. By contrast, shRNA knockdown of NOTCH1 resulted in decreased sphere-colony formation and robust terminal differentiation of ERMS cells into late-stage, myosin-expressing myoblasts. Moreover, we identified that NOTCH1 directly activated SNAI1 expression and was required for both efficient sphere formation and differentiation in ERMS cell lines. SNAI1 is commonly over-expressed in human ERMS and its expression is positively correlated with NOTCH1. Taken together, our data indicate that Notch signaling is an important modifier of human ERMS acting to regulate both TPC self-renewal and differentiation. Notch and/or SNAl1 pathway inhibition may have potential benefit for a subset of patients with relapsed ERMS. Citation Format: Myron Ignatius, Riadh Lobbardi, Madeline Hayes, Eleanor Chen, Karin McCarthy, G. Petur Nielsen, Brian Beleyea, Corinne Linardic, Javed Khan, Charles Keller, David M. Langenau. Notch signaling increases the number of relapse-driving tumor propagating cells in embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4733. doi:10.1158/1538-7445.AM2015-4733

Abstract A14: Canonical WNT/β-catenin pathway activation suppresses embryonal rhabdomyosarcoma growth and self-renewal

Abstract Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress TPC function, a large-scale chemical screen comprising of ~40,000 compounds including 47% FDA-approved drugs was completed, identifying GSK3-inhibitors as potent suppressors of ERMS growth through inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT pathway, recombinant WNT3A and a stabilized β-catenin enhanced differentiation and reduced self-renewal in human ERMS cell lines. Moreover, treatment of tumor-bearing zebrafish with a GSK3 inhibitor activated the WNT/β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. As canonical WNT signaling is essential for transition from muscle stem cell proliferation to myogenic differentiation during regeneration, our findings suggest that the same developmental pathways that regulate muscle stem cell self-renewal also contribute to tumorigenesis in ERMS. GSK3 inhibitors are being assessed for their efficacy in inhibiting growth as well as inducing differentiation of human ERMS in xenograft mouse models. Our work has identified the vital role of canonical WNT pathway in regulating differentiation and self-renewal of ERMS and demonstrated the effective application of small molecule inhibitors to target differentiation of TPCs in ERMS. Citation Format: Eleanor Chen, Michael DeRan, Katherine Brooke Grandinetti, Myron Ignatius, Ryan Clagg, Karin McCarthy, Riadh Lobbardi, Xu Wu, David Langenau. Canonical WNT/β-catenin pathway activation suppresses embryonal rhabdomyosarcoma growth and self-renewal. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A14.

Abstract A47: Self-renewing cellular compartments in embryonal rhabdomyosarcoma are modulated by hedgehog signaling

Abstract Rhabdomyosarcoma (RMS) is a heterogeneous group of malignancies with features of impaired skeletal muscle differentiation and represents the majority of soft tissue cancers in the pediatric age group. Current treatment regimens show clinical benefit but this has reached a plateau. Since there is significant risk of adverse side effects and secondary tumors later in life, identifying and targeting sub-populations with higher tumorigenic potential and self renewal capacity would not only decrease tumor burden but also lower the probability of relapse. Using in vitro self renewal assays we could identify self-renewing cellular compartments within embryonal RMS (eRMS) cell lines. These compartments showed upregulation of several components of the sonic hedgehog pathway. Here, we now show using both genetic loss- and gain-of-function experiments and clinically relevant small molecule modulators that Hedgehog signalling is important for determining the self-renewal, tumor initiation capability both in vitro as well as in vivo. In addition, the hedgehog pathway can also influence chemoresistance of eRMS cells and altered the differentiation status of the cells by directly modulating the expression of various stem cell associated transcription factors. The pathway was found to be heterogeneously activated within eRMS patient samples in a comprehensive tissue microarray analysis. Importantly, the presence of Hedgehog-active cells identified using effector transcription factors could negatively predict patient survival in a retrospective analysis of this patient cohort. This work elucidates both a novel and central role played by the Hedgehog pathway in eRMS and underscores the importance of studying tumor heterogeneity in pediatric cancer. Citation Format: Sampoorna Satheesha, Amandine Bovay, Gabriele Manzella, Peter Bode, Simone Feuchtgruber, Ewa Koscielniak, Reka Belle, Elisa Casanova, Nurhak Dogan, Beat W. Schäfer. Self-renewing cellular compartments in embryonal rhabdomyosarcoma are modulated by hedgehog signaling. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A47.

Abstract A67: Canonical WNT/β;-catenin pathway activation suppresses embryonal rhabdomyosarcoma growth and self-renewal

Abstract Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress TPC function, a large-scale chemical screen comprising of ∼40,000 compounds including 47% FDA-approved drugs was completed, identifying GSK3-inhibitors as potent suppressors of ERMS growth through inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT pathway, recombinant WNT3A and a stabilized β-catenin enhanced differentiation and reduced self-renewal in human ERMS cell lines. Moreover, treatment of tumor-bearing zebrafish with a GSK3 inhibitor activated the WNT/β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. As canonical WNT signaling is essential for transition from muscle stem cell proliferation to myogenic differentiation during regeneration, our findings suggest that the same developmental pathways that regulate muscle stem cell self-renewal also contribute to tumorigenesis in ERMS. GSK3 inhibitors are being assessed for their efficacy in inhibiting growth as well as inducing differentiation of human ERMS in xenograft mouse models. Our work has identified the vital role of canonical WNT pathway in regulating differentiation and self-renewal of ERMS and demonstrated the effective application of small molecule inhibitors to target differentiation of TPCs in ERMS. Citation Format: Eleanor Chen, Michael DeRan, Katherine Brooke Grandinetti, Myron Ignatius, Ryan Clagg, Karin McCarthy, Riadh Lobbardi, Xu Wu, David Langenau. Canonical WNT/β;-catenin pathway activation suppresses embryonal rhabdomyosarcoma growth and self-renewal. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A67.

Abstract LB-200: Potent antitumor activity of MLN0128, an investigational selective mTORC1/2 kinase inhibitor, in preclinical models of rhabdomyosarcoma

Abstract Phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) axis regulates growth and homeostasis, and is often dysregulated in rhabdomyosarcoma. Agents that target mammalian target of rapamycin complex 1 (mTORC1), one of the two multi-protein complexes comprising the serine-threonine kinase mTOR, have been disappointing in clinical trials, and novel therapeutics are urgently needed to treat patients with relapsed, refractory, or metastatic alveolar (ARMS) and embryonal (ERMS) rhabdomyosarcoma. It is hypothesized that modest clinical results with mTORC1 only inhibitors may be attributed to the lack of inhibition of the second mTOR complex, mTORC2, and its downstream effectors, as well as release of mTOR-mediated negative feedback of the pathway that leads to activation of receptor tyrosine kinases and feedback activation of AKT. In the present preclinical study, MLN0128, a selective and highly potent inhibitor of both mTORC1 and mTORC2, was tested for its antitumor activity in vitro and in a mouse xenograft model. Data suggest that the anti-proliferative effect of MLN-0128 was superior to rapamycin, a selective mTORC1 inhibitor, in a broad panel of ERMS and ARMS cell lines where it was found to be highly effective at low concentrations from 5 to 175 nanomolar. Western immunoblot analysis of treated cells showed inhibition of phosphorylation of mTORC1 and mTORC2 targets including phosphorylated S6 ribosomal protein (pS6), phospho-p70-S6K, phospho-AKT-S473, phospho-AKT-T308, and phospho-4E-BP1. Apoptosis in both ERMS and ARMS cell lines was confirmed by the presence of cleaved poly ADP ribose. Additionally, MLN0128 was found to have superior anti-tumor effect in a xenograft model of rhabdomyosarcoma without significant toxicity when compared with rapamycin. These pre-clinical studies support further research of MLN0128 as a potential therapeutic approach for patients with ARMS and ERMS. Citation Format: Emily Slotkin, Parag Patwardhan, Shyamprasad Vasudeva Deraje, Gary Schwartz, Tap William. Potent antitumor activity of MLN0128, an investigational selective mTORC1/2 kinase inhibitor, in preclinical models of rhabdomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-200. doi:10.1158/1538-7445.AM2014-LB-200

RAGE signaling deficiency in rhabdomyosarcoma cells causes upregulation of PAX7 and uncontrolled proliferation

Embryonal rhabdomyosarcomas (ERMSs) show elevated levels of PAX7, a transcription factor that marks quiescent adult muscle stem (satellite) cells and is important for proliferation and survival of activated satellite cells and whose timely repression is required for myogenic differentiation. However, the mechanism of PAX7 accumulation in ERMSs and whether high PAX7 causes uncontrolled proliferation in ERMS remains to be elucidated. The receptor for advanced glycation end-products (RAGE, encoded by AGER) transduces a myogenic and anti-proliferative signal in myoblasts, and stable transfection of the ERMS cell line TE671, which does not express RAGE, with AGER results in reduced proliferation and formation of tumor masses in vivo, and enhanced apoptosis and myogenic differentiation. Herein, we show that RAGE expression is low or absent in human ERMSs. We also show that in ERMS cells (1) PAX7 accumulates owing to absent or low RAGE signaling; (2) elevated PAX7 levels reduce RAGE expression and levels of MyoD and myogenin, muscle-specific transcription factors required for myoblast proliferation arrest and differentiation, respectively; (3) PAX7 supports myoblast proliferation by reducing the levels of MyoD, primarily by promoting its degradation; and (4), when ectopically expressed in ERMS cells, that RAGE upregulates myogenin which upregulates MyoD and downregulates PAX7, with consequent inhibition of proliferation and stimulation of differentiation. Thus, failure to express RAGE and, hence, MyoD and myogenin above a critical level in ERMS cells might result in deregulated PAX7 expression leading to uncontrolled proliferation and, potentially, to rhabdomyosarcomagenesis.

Distinct cellular origin and genetic requirement of Hedgehog-Gli in postnatal rhabdomyosarcoma genesis.

Dysregulation of the Hedgehog (Hh)-Gli signaling pathway is implicated in a variety of human cancers, including basal cell carcinoma (BCC), medulloblastoma (MB) and embryonal rhabdhomyosarcoma (eRMS), three principle tumors associated with human Gorlin syndrome. However, the cells of origin of these tumors, including eRMS, remain poorly understood. In this study, we explore the cell populations that give rise to Hh-related tumors by specifically activating Smoothened (Smo) in both Hh-producing and -responsive cell lineages in postnatal mice. Interestingly, we find that unlike BCC and MB, eRMS originates from the stem/progenitor populations that do not normally receive active Hh signaling. Furthermore, we find that the myogenic lineage in postnatal mice is largely Hh quiescent and that Pax7-expressing muscle satellite cells are not able to give rise to eRMS upon Smo or Gli1/2 overactivation in vivo, suggesting that Hh-induced skeletal muscle eRMS arises from Hh/Gli quiescent non-myogenic cells. In addition, using the Gli1 null allele and a Gli3 repressor allele, we reveal a specific genetic requirement for Gli proteins in Hh-induced eRMS formation and provide molecular evidence for the involvement of Sox4/11 in eRMS cell survival and differentiation.

Abstract 2648: Self-renewing cellular compartments in embryonal rhabdomyosarcoma are modulated by Hedgehog signaling.

Abstract Rhabdomyosarcoma (RMS) is a heterogeneous group of malignancies with features of impaired skeletal muscle differentiation and represents the majority of the soft tissue cancers in the pediatric age group. Current treatment regimens offer some clinical respite but at a significant risk of averse side effects and secondary tumors later in life. Identifying and targeting sub-populations with higher tumorigenic potential and self renewal capacity not only decreases tumor burden but also lowers the probability of relapse. Using the sphere formation assay we could select for self-renewing cells from the heterogeneous monolayer cultures of embryonal RMS (eRMS) cell lines, RD and Rh36, which had multipotent differentiation ability in vitro and greater tumorigenic potential in vivo. These cells also showed increased expression of embryonic stem cell genes. Interestingly, quantitative gene expression analysis revealed increased expression of various components of the Hedgehog pathway, a developmental pathway important in eRMS biology. Inhibition or activation of the pathway in vitro by small molecule agents led to increased or decreased self renewing ability respectively. Furthermore we observed that eRMS adherent monolayer cultures contained a sub-population of GLI1-positive cells which could be modulated by pathway manipulation. Importantly, inhibition of the hedgehog pathway in vitro led to significantly delayed tumor initiation in vivo. Ongoing experiments aim at confirming these results on the genetic level. We postulate that the Hedgehog pathway sustains a primitive cellular compartment necessary for tumor initiation within embryonal RMS cell lines. Citation Format: Sampoorna Satheesha, Nurhak Dogan, Elisa A. Casanova, Dagmar Walter, Beat W. Schäfer. Self-renewing cellular compartments in embryonal rhabdomyosarcoma are modulated by Hedgehog signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2648. doi:10.1158/1538-7445.AM2013-2648

Abstract 3813: Uniparental disomy is associated with embryonal rhabdomyosarcoma in Costello Syndrome and nonsyndromic patients.

Abstract Embryonal rhabdomyosarcoma (ERMS) is the most prevalent pediatric soft tissue tumor and is characterized at the molecular level by loss of heterozygosity (LOH) at 11p15.5. We aimed to study ERMS in the context of Costello syndrome (CS), a rare syndromic predisposition to ERMS. Because CS arises from a typically paternal germline mutation in the proto-oncogene HRAS, it is considered a rasopathy. CS results in failure to thrive, intellectual disabilities, short stature, coarse facial features, skeletal abnormalities, congenital heart disease, and a predisposition for cancer, most commonly ERMS. We molecularly characterized seven ERMS tumors from six unrelated CS patients, carrying paternally derived HRAS p.G12S (5) or p.G12A (2) mutations. LOH was evaluated in all CS ERMS tumors by microarray and/or using short tandem repeat (STR) markers along both arms of chromosome 11. One patient developed a second primary ERMS tumor showing the ERMS hallmark LOH limited to 11p15.5. The six other CS ERMS tumors displayed paternal uniparental disomy (UPD) of the entire chromosome 11. Paternal UPD was also observed in a pure ERMS cell line, established using fresh tumor sample from one of the patients with a p.G12A germline mutation. This represents, to our knowledge, the first CS ERMS cell line. Additional cell lines derived from non CS patients were used in this study and included two ERMS (RD and RH36), one Alveolar RMS (RH30), and one mixed A/ERMS (RH18). RH36 cells carried an HRAS mutation at position 61 (p.Q61K). Unexpectedly, both ERMS cell lines, RD and RH36, displayed a complete LOH for chromosome 11. The ARMS RH30 cells carried no mutation in HRAS or LOH. RH18 cells derived from an A/ERMS had no HRAS mutation and showed LOH on both, the p and q arm of chromosome 11, except at locus D11S1338. Cytogenetic analysis with fluorescence in situ hybridization (FISH) on three CS tumors revealed multiple copies of chromosome 11 leading to trisomies and tetrasomies in the tumor and cultured cells. Gene expression revealed loss of imprinting in the growth regulating genes H19, IGF2, and CDKN1C on chromosome 11 not only in CS tumors, but also in nonsyndromic ERMS. Citation Format: Katherine M. Robbins, Deborah L. Stabley, Katrina Conard, David J. Wu, Hunter Stitik, Edward A. Kolb, Andrew Napper, Robert W. Mason, Karen W. Gripp, Katia Sol-Church. Uniparental disomy is associated with embryonal rhabdomyosarcoma in Costello Syndrome and nonsyndromic patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3813. doi:10.1158/1538-7445.AM2013-3813