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Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and phenocopies a muscle precursor that fails to undergo terminal differentiation. The alveolar subtype (ARMS) has the poorest prognosis and represents the greatest unmet medical need for RMS. Emerging evidence supports the role of epigenetic dysregulation in RMS. Here we show that SMARCA4/BRG1, an ATP-dependent chromatin remodeling enzyme of the SWI/SNF complex, is prominently expressed in primary tumors from ARMS patients and cell cultures. Our validation studies for a CRISPR screen of 400 epigenetic targets identified SMARCA4 as a unique factor for long-term (but not short-term) tumor cell survival in ARMS. A SMARCA4/SMARCA2 protein degrader (ACBI-1) demonstrated similar long-term tumor cell dependence in vitro and in vivo. These results credential SMARCA4 as a tumor cell dependency factor and a therapeutic target in ARMS.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma occurring in children and carries a dismal prognosis when metastatic disease is detected. Our previous work has suggested the cytokine receptor IL4Rα may play a role in contributing to metastasis in the alveolar subtype of rhabdomyosarcoma (aRMS), and thus could present a therapeutic target. The IL4 signaling axis has been characterized in various adult cancers as well; however, pediatric trials often follow similar adult trials and the role of the IL4Rα receptor has not been explored in the context of a mediator of metastasis in adult disease. Here, we demonstrate that the impact of IL4Rα blockade in an orthotopic allograft model of aRMS is not mediated by a macrophage response. We further examine the effect of IL4 blockade in adult colon, breast, and prostate cancers and find that inhibition of IL4Rα signaling modulates in vitro cell viability of HCT-116 colon carcinoma cells; however, this finding did not translate to an autocrine-related in vivo difference in tumor burden or lung metastasis. Our results suggest that if humanized IL4 mouse host strains are not available (or not ideal due to the need for immunosuppressing the host innate immune response for xenograft systems), then genetically-engineered mice and mouse allograft studies may be the best indicator of therapeutic targeting efficacy.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood with a propensity to metastasize. Current treatment for patients with RMS includes conventional systemic chemotherapy, radiation therapy, and surgical resection; nevertheless, little to no improvement in long term survival has been achieved in decades—underlining the need for target discovery and new therapeutic approaches to targeting tumor cells or the tumor microenvironment. To evaluate cross-species sarcoma extracellular matrix production, we have used murine models which feature knowledge of the myogenic cell-of-origin. With focus on the RMS/undifferentiated pleomorphic sarcoma (UPS) continuum, we have constructed tissue microarrays of 48 murine and four human sarcomas to analyze expression of seven different collagens, fibrillins, and collagen-modifying proteins, with cross-correlation to RNA deep sequencing. We have uncovered that RMS produces increased expression of type XVIII collagen alpha 1 (COL18A1), which is clinically associated with decreased long-term survival. We have also identified significantly increased RNA expression of COL4A1, FBN2, PLOD1, and PLOD2 in human RMS relative to normal skeletal muscle. These results complement recent studies investigating whether soft tissue sarcomas utilize collagens, fibrillins, and collagen-modifying enzymes to alter the structural integrity of surrounding host extracellular matrix/collagen quaternary structure resulting in improved ability to improve the ability to invade regionally and metastasize, for which therapeutic targeting is possible.

// Narendra Bharathy 1 , Matthew N. Svalina 1 , Teagan P. Settelmeyer 1 , Megan M. Cleary 1 , Noah E. Berlow 1 , Susan D. Airhart 2 , Sunny Xiang 2 , James Keck 2 , James B. Hayden 3 , Jack F. Shern 4, 5 , Atiya Mansoor 6 , Melvin Lathara 7 , Ganapati Srinivasa 7 , David M. Langenau 8, 9 and Charles Keller 1 1 Children’s Cancer Therapy Development Institute, Beaverton, OR 97005, USA 2 The Jackson Laboratory, Sacramento, CA 95838, USA 3 Department of Orthopedics and Rehabilitation, Oregon Health & Science University, Portland, OR 97239, USA 4 Genetics Branch, Oncogenomics Section, Center for Cancer Research, National Institutes of Health, Bethesda, MD 20892, USA 5 Pediatric Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, MD 20892, USA 6 Department of Pathology, Oregon Health & Science University, Portland, OR 97239, USA 7 Omics Data Automation, Beaverton, OR 97005, USA 8 Molecular Pathology, Cancer Center, and Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA 9 Harvard Stem Cell Institute, Cambridge, MA 02129, USA Correspondence to: Narendra Bharathy, email: naren@cc-tdi.org Charles Keller, email: charles@cc-tdi.org Keywords: rhabdomyosarcoma, preclinical testing, patient-derived xenograft, GSK3β, myodifferentiation Received: December 07, 2016 Accepted: June 01, 2017 Published: June 16, 2017 ABSTRACT Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. RMS often arise from myogenic precursors and displays a poorly differentiated skeletal muscle phenotype most closely resembling regenerating muscle. GSK3β is a ubiquitously expressed serine-threonine kinase capable of repressing the terminal myogenic differentiation program in cardiac and skeletal muscle. Recent unbiased chemical screening efforts have prioritized GSK3β inhibitors as inducers of myodifferentiation in RMS, suggesting efficacy as single agents in suppressing growth and promoting self-renewal in zebrafish transgenic embryonal RMS (eRMS) models in vivo . In this study, we tested the irreversible GSK3β-inhibitor, tideglusib for in vivo efficacy in patient-derived xenograft models of both alveolar rhabdomyosarcoma (aRMS) and eRMS. Tideglusib had effective on-target pharmacodynamic efficacy, but as a single agent had no effect on tumor progression or myodifferentiation. These results suggest that as monotherapy, GSK3β inhibitors may not be a viable treatment for aRMS or eRMS.

Alveolar rhabdomyosarcoma (aRMS) is a pediatric soft tissue cancer commonly associated with a chromosomal translocation that leads to the expression of a Pax3:Foxo1 or Pax7:Foxo1 fusion protein, the developmental underpinnings of which may give clues to its therapeutic approaches. In aRMS, the NFκB–YY1–miR-29 regulatory circuit is dysregulated, resulting in repression of miR-29 and loss of the associated tumor suppressor activity. To further elucidate the role of NFκB in aRMS, we first tested 55 unique sarcoma cell lines and primary cell cultures in a large-scale chemical screen targeting diverse molecular pathways. We found that pharmacological inhibition of NFκB activity resulted in decreased cell proliferation of many of the aRMS tumor cultures. Surprisingly, mice that were orthotopically allografted with aRMS tumor cells exhibited no difference in tumor growth when administered an NFκB inhibitor, compared to control. Furthermore, inhibition of NFκB by genetically ablating its activating kinase inhibitor, IKKβ, by conditional deletion in a mouse model harboring the Pax3:Foxo1 chimeric oncogene failed to abrogate spontaneous tumor growth. Genetically engineered mice with conditionally deleted IKKβ exhibited a paradoxical decrease in tumor latency compared with those with active NFκB. However, using a synthetic-lethal approach, primary cell cultures derived from tumors with inactivated NFκB showed sensitivity to the BCL-2 inhibitor navitoclax. When used in combination with an NFκB inhibitor, navitoclax was synergistic in decreasing the growth of both human and IKKβ wild-type mouse aRMS cells, indicating that inactivation of NFκB alone may not be sufficient for reducing tumor growth, but, when combined with another targeted therapeutic, may be clinically beneficial., Summary: In a genetically engineered mouse model of aRMS, disrupting the NFκB pathway facilitated tumor initiation, suggesting it is a modifier of the disease rather than the driver.

Alveolar and embryonal rhabdomyosarcoma (RMS) are soft-tissue cancers that affect children, adolescents, and young adults. Sometimes referred to as muscle cancer, RMS is a cancer of muscle and non-muscle origin that phenocopies incompletely differentiated myoblasts or activated satellite (muscle stem) cells. Interestingly, embryonal RMS (ERMS) has been observed to undergo terminal myogenic differentiation in response to stress induced by chemotherapy and radiation therapy4, 9, 24. Given the propensity of rhabdomyosarcoma to differentiation, in this report we explore the use of differentiation therapy combining MEK inhibitor (MEKi) cobimetinib and chemotherapy as a strategy to halt RMS growth. We evaluated a representative panel of RMS cell lines with cobimetinib and chemotherapy in two dosing schedules that mimic clinical use followed by cell growth evaluation and high content analysis (differentiation) assays. We uncovered that cobimetinib does not have significant additive or synergistic effects on cell differentiation or cell growth with chemotherapy in RMS and can have unanticipated antagonistic effects; specifically, pre-exposure of cobimetinib to cells can decrease the effectiveness of chemotherapy-mediated cell growth inhibition in vitro. Although differentiation-therapy is still a potential viable strategy in RMS, our data do not support MEKi/chemotherapy co-treatment in this context.

The childhood muscle cancer rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. In the last 40 years, outcomes for low and intermediate risk patients have improved; however, high risk patients with metastatic disease still have poor overall survival. Differentiation therapy for RMS has been considered a potential clinical approach to halting tumor progression by inducing the terminal myogenic differentiation program, and thus reducing the need for cytotoxic chemotherapy. Both the NOTCH and MEK pathway have been shown to play varying roles in inducing differentiation in RMS cells. Here, we tested several different RMS cell lines harboring varying genetic abnormalities with the MEK inhibitor trametinib alone, and in combination with γ-secretase inhibitors and found no significant effect on cell viability when used together.