Fusion-positive Rhabdomyosarcoma Research Articles

An Integrative Morphologic and Molecular Approach for Diagnosis and Subclassification of Rhabdomyosarcoma.

Rhabdomyosarcoma, the most common soft tissue sarcoma of children, is currently classified into the following 4 subtypes: embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, spindle cell/sclerosing rhabdomyosarcoma, and pleomorphic rhabdomyosarcoma, based on recent molecular genetic knowledge and morphologic features. To highlight the most recent advances of molecular genetic alterations, and to familiarize pathologists with most recent genotype and phenotype correlation in rhabdomyosarcoma. Data were derived from the World Health Organization Classification of Soft Tissue and Bone Tumors, fifth edition, recently published literature (PubMed), and clinical practice experience. Current classification has been significantly impacted by genotype and phenotype correlation, especially with PAX-FOXO1 fusion-positive rhabdomyosarcoma versus fusion-negative rhabdomyosarcoma, and with the emergence of 3 distinct new subtypes of spindle cell/sclerosing rhabdomyosarcoma. Although all rhabdomyosarcomas were considered a single diagnostic entity in the past, they are now considered to be a group of histologically similar but biologically diverse entities because their clinical behavior and underlying molecular alterations dramatically differ. This review outlines recent molecular genetic developments, corresponding morphologic features, and current challenges faced by pathologists in daily practice.

BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor.

Genetic Characterization, Current Model Systems and Prognostic Stratification in PAX Fusion-Negative vs. PAX Fusion-Positive Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents and accounts for approximately 2% of soft tissue sarcomas in adults. It is subcategorized into distinct subtypes based on histological features and fusion status (PAX-FOXO1/VGLL2/NCOA2). Despite advances in our understanding of the pathobiological and molecular landscape of RMS, the prognosis of these tumors has not significantly improved in recent years. Developing a better understanding of genetic abnormalities and risk stratification beyond the fusion status are crucial to developing better therapeutic strategies. Herein, we aim to highlight the genetic pathways/abnormalities involved, specifically in fusion-negative RMS, assess the currently available model systems to study RMS pathogenesis, and discuss available prognostic factors as well as their importance for risk stratification to achieve optimal therapeutic management.

High Frequency of Tumor Propagating Cells in Fusion-Positive Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Fusion-positive RMS (FPRMS), expressing the PAX3/7-FOXO1, has a worse prognosis compared to the more common fusion-negative RMS (FNRMS). Although several studies reported hierarchical organization for FNRMS with the identification of cancer stem cells, the cellular organization of FPRMS is not yet clear. In this study we investigated the expression of key stem cell markers, developed a sphere assay, and investigated the seven most common FPRMS cell lines for subpopulations of tumor propagating cancer stem-like cells, also called cancer stem cells (CSCs). Moreover, loss- and gain-of-functions of the stem cell genes SOX2, OCT4, and NANOG were investigated in the same cells. Single-cell clonal analysis was performed in vitro as well as in vivo. We found that no stable CSC subpopulation could be enriched in FPRMS. Unlike depletion of PAX3-FOXO1, neither overexpression nor siRNA-mediated downregulation of SOX2, OCT4, and NANOG affected physiology of RMS cells. Every single subclone-derived cell clone initiated tumor growth in mice, despite displaying considerable heterogeneity in gene expression. FPRMS appears to contain a high frequency of tumor propagating stem-like cells, which could explain their higher propensity for metastasis and relapse. Their dependency on PAX3-FOXO1 activity reinforces the importance of the fusion protein as the key therapeutic target.

Serine hydroxymethyltransferase 2 expression promotes tumorigenesis in rhabdomyosarcoma with 12q13-q14 amplification.

The 12q13-q14 chromosomal region is recurrently amplified in 25% of fusion-positive (FP) rhabdomyosarcoma (RMS) cases and is associated with a poor prognosis. To identify amplified oncogenes in FP RMS, we compared the size, gene composition, and expression of 12q13-q14 amplicons in FP RMS with those of other cancer categories (glioblastoma multiforme, lung adenocarcinoma, and liposarcoma) in which 12q13-q14 amplification frequently occurs. We uncovered a 0.2 Mb region that is commonly amplified across these cancers and includes CDK4 and 6 other genes that are overexpressed in amplicon-positive samples. Additionally, we identified a 0.5 Mb segment that is only recurrently amplified in FP RMS and includes 4 genes that are overexpressed in amplicon-positive RMS. Among these genes, only serine hydroxymethyltransferase 2 (SHMT2) was overexpressed at the protein level in an amplicon-positive RMS cell line. SHMT2 knockdown in amplicon-positive RMS cells suppressed growth, transformation, and tumorigenesis, whereas overexpression in amplicon-negative RMS cells promoted these phenotypes. High SHMT2 expression reduced sensitivity of FP RMS cells to SHIN1, a direct SHMT2 inhibitor, but sensitized cells to pemetrexed, an inhibitor of the folate cycle. In conclusion, our study demonstrates that SHMT2 contributes to tumorigenesis in FP RMS and that SHMT2 amplification predicts differential response to drugs targeting this metabolic pathway.

Abstract 47: Identification of first-in-class KDM3B inhibitors that suppress PAX3-FOXO1 oncogene activity in fusion positive rhabdomyosarcoma

Abstract Background: PAX3/7-FOXO1 fusion gene is the major oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), a highly aggressive soft tissue sarcoma of childhood. The chimeric gene results from either t(2;13) or t(1;13) translocation and has been shown to drive FP-RMS carcinogenesis by activation of super enhancer driven transcription. Thus, this fusion gene represents a unique vulnerability in FP-RMS which can be targeted by small molecules. MATERIALS AND METHODS: Using luciferase assays simultaneously monitoring PAX3-FOXO1 super enhancer and general transcription activity in RMS cells, 62,643 compounds were screened for selective inhibitors of PAX3-FOXO1 activity. RNA-seq was performed on FP-RMS cell lines treated with the top hits and gene set enrichment analysis (GSEA) was performed. To characterize the functions of fusion gene inhibitors, histone lysine demethylase (KDM) enzyme inhibition assay and in vitro proliferation assay was performed. Western analysis was used to assess expression level of MYOG, PARP, and PAX3-FOXO1 in RMS cells after treatment with inhibitors. NMR techniques WaterLOGSY and CPMG were used to determine direct binding of compound to KDM3B. In vivo FP-RMS xenograft mouse model was used to determine tumor suppression by PAX3-FOXO1 inhibitors. RESULTS: PAX3-FOXO1 selective cell-based assay identified 64 compounds that inhibited PAX3-FOXO1 activity without general inhibition of transcription or cell death at 24 hours. Compound PFI-63 was identified as the top hit, and its analog PFI-90 was identified using a chemical similarity screen based on PFI-63. RNA-seq analysis on RMS cells treated with PFI-63 and PFI-90 indicated these compounds inhibit KDMs, resulting in apoptosis and myogenic differentiation while PAX3-FOXO1 downstream gene sets were repressed. Activation of apoptosis and myogenic differentiation was validated by Western analysis for PARP cleavage and increased expression of MYOG respectively. In vitro enzymatic inhibition assay confirmed activity against KDMs with highest specificity for KDM3B. Western analysis for methylation status of histone 3 lysines at positions K27, K4, and K9 showed increased histone 3 methylation at K4 and K9 sites after treatment. Furthermore, WaterLOGSY and CPMG NMR techniques showed direct binding of PFI-90 to KDM3B. Finally, PFI-90 delayed tumor progression in a FP-RMS xenograft mouse model. CONCLUSION: We identified two KDM3B selective inhibitors which directly bind to KDM3B and disrupt PAX3-FOXO1 downstream targets in FP-RMS. PFI-63 and PFI-90 induce apoptosis and cell differentiation resulting in delayed tumor progression in vivo. Thus, we describe here novel inhibitors of KDM3B that inhibit PAX3-FOXO1 action. These novel compounds represent a potential new therapy for FP-RMS. Citation Format: Yong Yean Kim, Robert G. Hawley, Berkley Gryder, Silvia Pomella, Joshua Kowalczyk, Joseph Barchi, Young Song, Javed Khan. Identification of first-in-class KDM3B inhibitors that suppress PAX3-FOXO1 oncogene activity in fusion positive rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 47.

Abstract 3048: Surfaceome profiling identifies the immune checkpoint molecule B7-H3 as a key player of immune-evasion in rhabdomyosarcoma

Abstract Introduction: Pediatric rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children, with nearly 20% of children presenting with locally aggressive and/or metastatic disease. Despite aggressive chemotherapy and radiotherapy, the 5-year overall survival rate is still less than 40%. In addition, resistance to chemotherapy remains the major cause of cancer-related mortality. Therefore, there is an unmet need to develop more effective and tolerable therapeutic strategies for the treatment of RMS. Our overall objective is to leverage transcriptomic and proteomic profiling methods to identify RMS-specific surface proteins targetable with immune-based therapies. Methods: To identify therapeutic targets, we performed cell-surface capture and mass spectrometry on fusion-positive (FP), fusion-negative (FN) RMS and normal skeletal muscle. Transcriptomic datasets of RMS tumor specimens and normal tissue were employed to identify high-confidence RMS-specific cell-surface proteins. Expression of immune checkpoint molecules was confirmed by flow cytometry, western blot and PCR. Paraffin-embedded tissue sections were used to determine protein expression and prognostic value in RMS patients. We developed an in vitro T-cell killing assay to assess the functional role of immune checkpoint molecules on the antitumor immune response. Results: Surfaceome profiling of RMS cells revealed unique protein signatures specific for each tumor type and normal tissue. We have identified 5061 cell-surface proteins in RMS and 31.6% of proteins were correctly located at the plasma membrane. By integrating proteomic and transcriptomic data, we have identified 30 and 38 proteins significantly overexpressed in fusion-negative and fusion-positive RMS respectively compared to normal muscle. We have identified the immune checkpoint molecule B7-H3 as cell-surface protein overexpressed in both RMS subtypes. Strong expression of B7-H3 was also observed in patient's tumor specimens. Genetic deletion of B7-H3 gene in RMS cells was associated with higher antitumor immune response in tumor cells co-cultured with CD8+-T cells. No difference was observed in tumor cell proliferation and radioresistance. Conclusion: Through integrated transcriptomic and proteomic profiling, we have discovered B7-H3 as a key regulator of tumor immune-evasion in RMS. B7-H3 is a suitable immunotherapy target for the treatment of RMS. Further studies are warranted to elucidate underlying mechanisms of B7-H3 function in RMS. Citation Format: Roxane R. Lavoie, Yohan Kim, Mohamed E. Ahmed, Doris Phelps, Emily Baer, Cristine Charlesworth, Benjamin Madden, Liguo Wang, Haidong Dong, Peter Houghton, John Cheville, Patricio Gargollo, Candace Granberg, Fabrice Lucien. Surfaceome profiling identifies the immune checkpoint molecule B7-H3 as a key player of immune-evasion in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3048.

Fenretinide Acts as Potent Radiosensitizer for Treatment of Rhabdomyosarcoma Cells.

Fusion-positive rhabdomyosarcoma (FP-RMS) is a highly aggressive childhood malignancy which is mainly treated by conventional chemotherapy, surgery and radiation therapy. Since radiotherapy is associated with a high burden of late side effects in pediatric patients, addition of radiosensitizers would be beneficial. Here, we thought to assess the role of fenretinide, a potential agent for FP-RMS treatment, as radiosensitizer. Survival of human FP-RMS cells was assessed after combination therapy with fenretinide and ionizing radiation (IR) by cell viability and clonogenicity assays. Indeed, this was found to significantly reduce cell viability compared to single treatments. Mechanistically, this was accompanied by enhanced production of reactive oxygen species, initiation of cell cycle arrest and induction of apoptosis. Interestingly, the combination treatment also triggered a new form of dynamin-dependent macropinocytosis, which was previously described in fenretinide-only treated cells. Our data suggest that fenretinide acts in combination with IR to induce cell death in FP-RMS cells and therefore might represent a novel radiosensitizer for the treatment of this disease.

Romidepsin (FK228) fails in counteracting the transformed phenotype of rhabdomyosarcoma cells but efficiently radiosensitizes, in vitro and in vivo, the alveolar phenotype subtype

Purpose Herein we describe the in vitro and in vivo activity of FK228 (Romidepsin), an inhibitor of class I HDACs, in counteracting and radiosensitizing embryonal (ERMS, fusion-negative) and alveolar (ARMS, fusion-positive) rhabdomyosarcoma (RMS). Methods RH30 (ARMS, fusion-positive) and RD (ERMS, fusion-negative) cell lines and human multipotent mesenchymal stromal cells (HMSC) were used. Flow cytometry analysis, RT-qPCR, western blotting and enzymatic assays were performed. Irradiation was delivered by using an x-6 MV photon linear accelerator. FK228 (1.2 mg/kg) in vivo activity, combined or not with radiation therapy (2 Gy), was assessed in murine xenografts. Results Compared to HMSC, RMS expressed low levels of class I HDACs. In vitro, FK228, as single agents, reversibly downregulated class I HDACs expression and activity and induced oxidative stress, DNA damage and a concomitant growth arrest associated with PARP-1-mediated transient non-apoptotic cell death. Surviving cells upregulated the expression of cyclin A, B, D1, p27, Myc and activated PI3K/Akt/mTOR and MAPK signaling, known to be differently involved in cancer chemoresistance. Interestingly, while no radiosensitizing effects were detected, in vitro or in vivo, on RD cells, FK228 markedly radiosensitized RH30 cells by impairing antioxidant and DSBs repair pathways in vitro. Further, FK228 when combined with RT in vivo significantly reduced tumor mass in mouse RH30 xenografts. Conclusion FK228 did not show antitumor activity as a single agent whilst its combination with RT resulted in radiosensitization of fusion-positive RMS cells, thus representing a possible strategy for the treatment of the most aggressive RMS subtype.

Epigenetic regulator BMI1 promotes alveolar rhabdomyosarcoma proliferation and constitutes a novel therapeutic target.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma. There are two main subtypes of RMS, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma. ARMS typically encompasses fusion‐positive rhabdomyosarcoma, which expresses either PAX3‐FOXO1 or PAX7‐FOXO1 fusion proteins. There are no targeted therapies for ARMS; however, recent studies have begun to illustrate the cooperation between epigenetic proteins and the PAX3‐FOXO1 fusion, indicating that epigenetic proteins may serve as targets in ARMS. Here, we investigate the contribution of BMI1, given the established role of this epigenetic regulator in sustaining aggression in cancer. We determined that BMI1 is expressed across ARMS tumors, patient‐derived xenografts, and cell lines. We depleted BMI1 using RNAi and inhibitors (PTC‐209 and PTC‐028) and found that this leads to a decrease in cell growth/increase in apoptosis in vitro, and delays tumor growth in vivo. Our data suggest that BMI1 inhibition activates the Hippo pathway via phosphorylation of LATS1/2 and subsequent reduction in YAP levels and YAP/TAZ target genes. These results identify BMI1 as a potential therapeutic vulnerability in ARMS and warrant further investigation of BMI1 in ARMS and other sarcomas.

FOXF1 is required for the oncogenic properties of PAX3-FOXO1 in rhabdomyosarcoma.

The PAX3-FOXO1 fusion protein is the key oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue malignancy with a particularly poor prognosis. Identifying key downstream targets of PAX3-FOXO1 will provide new therapeutic opportunities for treatment of FP-RMS. Herein, we demonstrate that Forkhead Box F1 (FOXF1) transcription factor is uniquely expressed in FP-RMS and is required for FP-RMS tumorigenesis. The PAX3-FOXO1 directly binds to FOXF1 enhancers and induces FOXF1 gene expression. CRISPR/Cas9 mediated inactivation of either FOXF1 coding sequence or FOXF1 enhancers suppresses FP-RMS tumorigenesis even in the presence of PAX3-FOXO1 oncogene. Knockdown or genetic knockout of FOXF1 induces myogenic differentiation in PAX3-FOXO1-positive FP-RMS. Over-expression of FOXF1 decreases myogenic differentiation in primary human myoblasts. In FP-RMS tumor cells, FOXF1 protein binds chromatin near enhancers associated with FP-RMS gene signature. FOXF1 cooperates with PAX3-FOXO1 and E-box transcription factors MYOD1 and MYOG to regulate FP-RMS-specific gene expression. Altogether, FOXF1 functions downstream of PAX3-FOXO1 to promote FP-RMS tumorigenesis.

Abstract PO-009: Disrupting chromatin architecture: The NuRD subunit and ATPase CHD4 as a new therapeutic target in pediatric sarcoma

Abstract Cancer-specific chromosomal aberrations producing chimeric fusion genes are recurrently found in pediatric sarcomas. Fusion positive rhabdomyosarcoma (FP-RMS) and Ewing sarcoma (ES) are two rare but lethal pediatric malignancies driven by such chromosomal translocations. PAX3-FOXO1 and EWS-FLI1 are the most common products of the fusion genes found in FP-RMS and ES, respectively, and they are commonly perceived as the founding genetic abnormality driving the development of these malignancies by changing gene expression. Since direct targeting of transcription factors is still very challenging, acting on the activity of these oncogenic transcription factors at the chromatin level presents a robust alternative for targeted therapy. The Nucleosome Remodeling and Deacetylase (NuRD) complex subunit CHD4 has been previously identified as an interactor of both PAX3-FOXO1 and EWS-FLI1. Hence, we decided here to further characterize the role of this chromatin remodeler in the regulation of fusion protein-mediated gene expression in both FP-RMS and ES. Our NuRD-centered CRISPR/Cas9 screen demonstrated that both these malignancies are especially dependent on CHD4 amongst all other complex members. In fact, CHD4 silencing in both tumors through shRNA knockdown or CRISPR knockout drastically reduces tumor cell proliferation and induces cell death. In vivo, CHD4 knockdown also impaired tumour growth in both FP-RMS and ES. Mechanistically, our RNA-seq assays demonstrated that silencing of the nucleosome remodeller CHD4 alters gene expression in both tumours and our ChIP-seq experiments show that CHD4 binding sites are highly enriched for the binding motif of PAX3-FOXO1 in FP-RMS and EWS-FLI1 in ES. In FP-RMS, we observed that CHD4 particularly regulates super-enhancer accessibility creating a chromatin architecture permissive to the binding of PAX3-FOXO1 and allowing the expression of the fusion gene signature. Similar studies in ES to further investigate CHD4 as a regulator of gene expression are currently ongoing. Finally, our analysis of genome-wide cancer dependency databases identified CHD4 as general novel cancer vulnerability amongst NuRD subunits and other SNF2-like ATPases. In summary, we have unravelled the prominent role of CHD4 in regulation of super-enhancer driven gene expression in FP-RMS and exposed this chromatin remodeler as novel potential drug target for pediatric sarcoma therapy. Our work has motivated us to establish several collaborations with computational and biophysics experts and we are now currently working to identify the first CHD4 specific small molecule inhibitor. Citation Format: Joana G. Marques, Berkley Gryder, Blaz Pavlovic, Yeonjoo Chung, Quy Ngo, Marco Wachtel, Javed Khan, Beat Schäfer. Disrupting chromatin architecture: The NuRD subunit and ATPase CHD4 as a new therapeutic target in pediatric sarcoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-009.

Survival outcomes of patients with localized FOXO1 fusion-positive rhabdomyosarcoma treated on recent clinical trials: A report from the Soft Tissue Sarcoma Committee of the Children's Oncology Group.

The objective of this analysis was to evaluate the clinical factors influencing survival outcomes in patients with localized (clinical group I-III), FOXO1 fusion-positive rhabdomyosarcoma (RMS). Patients with confirmed FOXO1 fusion-positive RMS who were enrolled on 3 completed clinical trials for localized RMS were included in the analytic cohort. Outcomes were analyzed using the Kaplan-Meier method to estimate event-free survival (EFS) and overall survival (OS), and the curves were compared using the log-rank test. A Cox proportional hazards regression model was used to perform multivariate analysis of prognostic factors that were significant in the univariate analysis. The estimated 4-year EFS and OS of 269 patients with localized, FOXO1 fusion-positive RMS was 53% (95% CI, 47%-59%) and 69% (95% CI, 63%-74%), respectively. Univariate analysis revealed that several known favorable clinical characteristics, including age at diagnosis between 1 and 9 years, complete surgical resection, tumor size ≤5 cm, favorable tumor site, absence of lymph node involvement, confinement to the anatomic site of origin, and PAX7-FOXO1 fusion, were associated with improved outcomes. Multivariate analysis identified older age (≥10 years) and large tumor size (>5 cm) as independent, adverse prognostic factors for EFS within this population, and patients who had both adverse features experienced substantially inferior outcomes. Patients with localized, FOXO1 fusion-positive RMS can be further risk stratified based on clinical features at diagnosis, and older patients with large primary tumors have the poorest prognosis.

Abstract 4175: Identification of novel inhibitors of the PAX3-FOXO1 fusion oncogene in rhabdomyosarcoma

Abstract Background: PAX3/7-FOXO1 fusion gene is the major oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), a highly aggressive soft tissue sarcoma of childhood. The chimeric gene results from either t(2;13) or t(1;13) translocation and has been shown to drive FP-RMS carcinogenesis by the activation of superenhancer driven transcription. Thus, this fusion gene represents a unique vulnerability in FP-RMS which can be targeted by small molecules. MATERIALS AND METHODS: Novel luciferase assays were developed which simultaneously monitor PAX3-FOXO1 super enhancer and general transcription activity. Using this assay, 62,643 compounds were screened for selective inhibitors of PAX3-FOXO1 activity. RNA-seq was performed on FP-RMS cell lines treated with the top hits and gene set enrichment analysis (GSEA) was performed to assess the molecular effects of the inhibitors. CRISPR-Cas9 lethal screen was performed with and without the top hit molecule to identify synthetic lethal combination to help elucidate the mechanism of action. Western analysis was performed for MYOG, PARP, and PAX3-FOXO1. In vitro direct enzyme inhibition analysis was performed. RESULTS: PAX3-FOXO1 selective cell-based assay identified 63 compounds that inhibited PAX3-FOXO1 activity without general inhibition of transcription or cell death at 24 hours. Compound PFI-63 was identified as the top hit. RNA-seq and genome wide synthetic lethality studies indicated the compound inhibits histone demethylases. Also, RNA-seq showed activation of apoptosis and myogenic differentiation pathways while PAX3-FOXO1 gene sets were repressed. Activation of apoptosis and myogenic differentiation was validated by Western analysis for PARP cleavage and increase expression of MYOG respectively. CRISPR-Cas9 screen validated the targeting of PAX3-FOXO1 gene sets by PFI-63. In vitro enzymatic inhibition assay confirmed activity of PFI-63 against KDM's with highest specificity to KDM3B. Western analysis for methylation status of histone 3 lysines at positions K27, K4, and K9 showed increase after treatment with PFI-63. However, due to poor solubility of PFI-63, in vivo validation was lacking. Thus, we performed a similarity search of PFI-63 and screened additional compounds leading to the discovery of PFI-90. PFI-90 showed a superior inhibition of KDM family of proteins with highest inhibition of KDM3B via in vitro enzymatic assay. PFI-90 had significantly improved solubility allowing for in vivo administration of drug. CONCLUSION: We identified two KDM3B selective inhibitors which has activity against FP-RMS in vitro. PFI-63 and PFI-90 disrupts the PAX3-FOXO1 downstream effects and elicits apoptosis and differentiation. Pre-clinical validation by in-vivo experiments is planned. Thus we describe here a novel inhibitor of KDM3B that results in epigenetic inhibition of PAX3-FOXO1 activity representing a potential new therapy for FP-RMS. Citation Format: Yong Yean Kim, Robert Hawley, Berkley Gryder, Silvia Pomella, Josh Kowalczyk, Ranu Sinniah, Young Song, Javed Khan. Identification of novel inhibitors of the PAX3-FOXO1 fusion oncogene in rhabdomyosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4175.

Abstract 3882: YAP1 and WWTR1 (TAZ) positively regulate PAX3-FOXO1 transcriptional programming in fusion-positive rhabdomyosarcoma

Abstract Although more than 20 years have passed since the discovery that fusion-positive rhabdomyosarcoma (FP-RMS) is driven by the chimeric fusion oncogene PAX3-FOXO1 (P3F), therapeutically tractable components of the P3F tumorigenic program have yet to be uncovered and survival rates remain dismal (5-yr overall survival <50%). YAP1 and TAZ, transcriptional co-activators of the Hippo pathway, are potent oncogenes known to mediate transcriptional addiction in malignancy. Here, we demonstrate that YAP1/TAZ complex with P3F and positively regulate P3F transcriptional activity in FP-RMS. Immunohistochemical (IHC) staining of human tissue microarrays were used to identify the relative abundance of YAP1 and TAZ in FP-RMS tumor samples. Functional interactions of YAP1/TAZ and P3F were investigated using P3F reporters, immunoblots, and co-immunoprecipitation (co-IP); while co-IP-coupled mass spectrometry (IP-MS) was used to identify the YAP1/TAZ/P3F interactome. Experiments utilized gain- and loss-of-function vectors expressing control, wild-type YAP1/TAZ, constitutively active YAP1/TAZ (S89A/S127A), or shRNA knockdown. To identify P3F-mediated genes and pathways that are YAP1/TAZ-dependent, we used RNA-Seq and quantitative proteomics via tandem mass tag labeling. We demonstrate via IHC that YAP1/TAZ are highly abundant in FP-RMS, and through functional assays that YAP1/TAZ regulate many FP-RMS cancer phenotypes. Mechanistically, an interaction between YAP/TAZ and P3F was demonstrated via co-IPs for endogenous as well as epitope-tagged proteins. This was confirmed with IP-MS, which also revealed that YAP/TAZ and P3F share an enrichment for co-immunoprecipitated proteins involved in DNA binding and transcriptional regulation. IP-MS also demonstrated that chromatin remodeling complex proteins were enriched with TAZ immunoprecipitation. YAP1/TAZ functionally augment P3F transcriptional activity in reporter assays as well as expression of candidate P3F target genes. An unbiased approach using RNA-Seq and quantitative proteomics demonstrate that YAP1/TAZ positively regulate differential expression of P3F target genes, as well as proteins involved in cell cycle progression and pan-cancer related proteins that are typically up- or down-regulated across multiple malignancy types.In conclusion, we identify a novel complex between YAP1/TAZ and P3F, show YAP1/TAZ are positive regulators of P3F transcriptional activity, and identify the YAP1/TAZ axis as a vulnerability for P3F-transcriptional reprogramming in FP-RMS. Citation Format: Tooba Rashid, Breanne A. Burgess, Corinne M. Linardic, Michael D. Deel. YAP1 and WWTR1 (TAZ) positively regulate PAX3-FOXO1 transcriptional programming in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3882.

NuRD subunit CHD4 regulates super-enhancer accessibility in rhabdomyosarcoma and represents a general tumor dependency.

The NuRD complex subunit CHD4 is essential for fusion-positive rhabdomyosarcoma (FP-RMS) survival, but the mechanisms underlying this dependency are not understood. Here, a NuRD-specific CRISPR screen demonstrates that FP-RMS is particularly sensitive to CHD4 amongst the NuRD members. Mechanistically, NuRD complex containing CHD4 localizes to super-enhancers where CHD4 generates a chromatin architecture permissive for the binding of the tumor driver and fusion protein PAX3-FOXO1, allowing downstream transcription of its oncogenic program. Moreover, CHD4 depletion removes HDAC2 from the chromatin, leading to an increase and spread of histone acetylation, and prevents the positioning of RNA Polymerase 2 at promoters impeding transcription initiation. Strikingly, analysis of genome-wide cancer dependency databases identifies CHD4 as a general cancer vulnerability. Our findings describe CHD4, a classically defined repressor, as positive regulator of transcription and super-enhancer accessibility as well as establish this remodeler as an unexpected broad tumor susceptibility and promising drug target for cancer therapy.

Abstract A08: Loss of noncanonical Hippo signaling in fusion-positive alveolar rhabdomyosarcoma increases invasiveness and a dedifferentiated phenotype associated with metastasis

Abstract A hallmark of alveolar rhabdomyosarcoma (ARMS) is the presence of a chromosomal translocation encoding the PAX3-FOXO1 fusion oncogene (FP-ARMS). Patients presenting with FP-ARMS represent the subset of rhabdomyosarcoma patients with the worst prognosis, and when metastatic, survival for these children and adolescents is less than 10%. Nevertheless, standard of care has not changed in the clinic in decades due to a lack of novel effective therapeutics, and as a result, efforts are ongoing to develop better models of this disease in vivo and identify new and more predictive biomarkers. Recently, our group identified a role for Hippo/MST signaling in a genetically engineered mouse model (GEMM) of FP-ARMS that demonstrates Stk3F/F;Stk4F/F; Pax3PF/PF;Cdkn2aF/F;Myf6ICN/+ ARMS tumor-derived cells are less differentiated and more invasive in vitro as compared to Pax3PF/PF;Cdkn2aF/F; Myf6ICN/+ counterparts. Preliminary in vivo studies show that FP-ARMS GEMM tumor-derived cells form secondary metastases in both the lungs and livers of recipient animals, suggesting a new utility of the model for studies of metastatic potential. Studies of bone marrow metastasis, a lethal disease progression in PF-ARMS patients, are ongoing. Further, canonical signaling down the kinase cascade from MST1/2 to YAP1 is largely unchanged in this model, implicating instead noncanonical Hippo signaling and a potential role for MOB1, a Hippo pathway modulator implicated in mitotic exit and expressed highly in the bone marrow niche. Together, these data present an opportunity to interrogate the biology driving the predisposition of PF-RMS patients to develop metastases, and a foundation to identify novel preclinical targets. Citation Format: Kristianne M. Oristian, Lisa E.S. Crose, Napasorn Kuprasertkul, Rex C. Bentley, Yi-Tzu Lin, Nerissa Williams, David G. Kirsch, Corinne M. Linardic. Loss of noncanonical Hippo signaling in fusion-positive alveolar rhabdomyosarcoma increases invasiveness and a dedifferentiated phenotype associated with metastasis [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 A08.

Abstract B70: Clonal evolution of chemotherapy-resistant rhabdomyosarcoma via multifocal genomic analysis of pretreatment and treatment-resistant autopsy specimens

Abstract Objective: Outcomes for patients with rhabdomyosarcoma (RMS) who have relapsed or refractory disease remain poor. Multiple large-scale tumor genomic profiling efforts have been undertaken; however, little is known about its spatial intratumoral heterogeneity (ITH) and temporal clonal evolutionary processes. Methods: To address this issue, we performed 80x whole-genome sequencing of 23 metastases and 2 pretreatment samples from two patients with lethal rhabdomyosarcoma. Multiregion and spatially separated metastases were available through our research autopsy donation program. RA 17-1 had fusion-positive alveolar RMS, while RA 17-10 had Li-Fraumeni syndrome and anaplastic embryonal RMS. Results: We observed on average 65 and 98 coding single-nucleotide variants (SNV) per sample in RA 17-1 and RA 17-10, respectively. RA 17-1 was found to have the canonical PAX03-FOX01 fusion along with MYC-N amplification, which was truncal to all samples. A somatic TP53 mutation in combination with TP53 loss of heterozygosity (LOH) was present in all metastases sequenced at autopsy. A TP53 mutation was not identified in the pretreatment biopsy sample selected for sequencing; however, TP53 immunohistochemistry demonstrated the presence of a TP53 mutation in at least 2 out of 6 pretreatment biopsy cores. In RA 17-10 each sample exhibited numerous copy number alterations and complex structural variants, including LOH of TP53, a TSC2 mutation with LOH, and MYC amplification, which were truncal to all samples. PTCH1 amplification was private to all relapse specimens in RA 17-10. RA 17-10 had more structural and copy number variations per sample than RA 17-1. Phylogenies were derived based on SNVs and demonstrated a branched evolutionary pattern for each patient. When comparing related samples’ mutational signatures, several dynamics were apparent, including the predominance of signatures 3 and 8 (DNA double-strand break repair pathways) in metastatic samples, consistent with the fact that both patients harbored TP53 mutations. Conclusion: Defining important elements of ITH within metastases remains a goal for genomic study in pediatrics, especially in patients with germline predisposition syndromes. Our findings demonstrate the dynamic nature of genomic instability processes, highlighting the importance of longitudinal sampling at the time of recurrence to define treatment effect and the changing mutational landscape in these tumors. The case of RA 17-1 demonstrates the limitations of targeted sequencing on a single sample at diagnosis, as the subclonal TP53 mutation was not originally identified on the clinical assay. TP53 mutations in fusion-positive RMS have rarely been described and likely contributed to this patient’s refractory disease course. Both RA 17-1 and RA 17-10 had more structural variants per sample then what has previously been reported for fusion-positive and fusion-negative RMS, respectively, likely highlighting the contribution of the TP53 mutation to genomic instability in each patient. Citation Format: Michael D. Kinnaman, Alvin Makohon-Moore, Nancy Bouvier, Dominik Glodzik, Max Levine, Ellie Papaemmanuil, Filemon Dela Cruz, Leonard Wexler, Andrew Kung, Christine Iacobuzio-Donahue. Clonal evolution of chemotherapy-resistant rhabdomyosarcoma via multifocal genomic analysis of pretreatment and treatment-resistant autopsy specimens [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B70.

Abstract A47: BMI1 constitutes a novel therapeutic vulnerability in fusion-positive rhabdomyosarcoma

Abstract Background: Despite intense efforts within pediatric oncology, novel, effective therapy for alveolar rhabdomyosarcoma, known as fusion-positive rhabdomyosarcoma (FP RMS) given the PAX-FOXO1 fusions characteristic of the disease, remains unrealized. Like many pediatric tumors, FP RMS displays a quiet genomic landscape, when focusing on the coding genome. However, the epigenome plays key roles in shaping tumor aggression, and previous studies have demonstrated that FP RMS is specifically enriched for methylation of Polycomb target genes, suggesting that Polycomb complexes may be deregulated and could constitute novel therapeutic targets. We hypothesized that BMI1, a key member of the Polycomb family and a tractable therapeutic target, represents a novel therapeutic vulnerability in FP RMS. Methods: We analyzed RNA and protein expression in FP RMS cell lines, patient-derived xenografts (PDXs), and human tumor specimens. We used genetic and pharmacologic approaches to manipulate BMI1 in FP RMS cells and measured effects on proliferation, cell cycle, apoptosis, and signal transduction. To examine the effect of in vivo inhibition of BMI1, we utilized xenograft models of FP RMS. Results: We examined RNA-Seq tumor datasets and tumor microarrays and demonstrated that BMI1 is robustly expressed in FP RMS tumors, PDXs, and cell line models. Next, in 2 cell line models, we depleted BMI-1 using shRNAs and siRNAs, and found that this led to striking (~70%) decreases in cell growth secondary to both G1/S phase arrest and apoptosis. Given these findings, we asked whether small-molecule inhibitors of BMI-1 mediated similar effects. We treated 4 independent FP RMS cell line models with both PTC-209 (first-generation inhibitor) and PTC-028 (orally available second-generation inhibitor with higher potency). Both compounds inhibited BMI-1 function and greatly reduced cell proliferation in FP rhabdomyosarcoma cell line models. Similar to genetically mediated depletion, pharmacologic inhibition of BMI1 led to G1/S phase arrest and apoptosis, as demonstrated by Annexin V staining and PARP cleavage. Finally, in a xenograft model of an aggressive FP RMS, PTC-028 treatment decreased tumor growth (p=0.0005) and significantly prolonged survival by 17 days (p=0.0002). Importantly, treatment was well tolerated without evidence of toxicity or weight loss. Conclusions: BMI1 is robustly expressed in FP RMS and both genetic and pharmacologic inhibition of BMI1 lead to striking decreases in cell proliferation, with concomitant cell cycle arrest and apoptosis. BMI1 inhibition significantly decreases tumor growth, prolongs survival, and is well tolerated. Currently, we are further investigating combining BMI1 inhibition with standard chemotherapy and novel agents, as well as defining molecular mechanisms by which BMI1 exerts molecular functions in FP RMS. Targeting BMI-1 could provide a novel therapeutic option for patients with FP RMS, with potential broader implications for additional aggressive sarcomas. Citation Format: Cara E. Shields, Selma M. Cuya, Sarah Chappell, Komal Rathi, Shiv Patel, Sindhu Potlapalli, Robert W. Schnepp. BMI1 constitutes a novel therapeutic vulnerability in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A47.

Abstract B12: PAX3 translocations co-opt super enhancers and intrinsically disordered fusion partners in rhabdomyosarcoma

Abstract Chromosomal translocations drive many types of childhood cancer. Fusion-positive rhabdomyosarcoma (FP-RMS) tumors are found most frequently with a fusion between PAX3 and FOXO1, but less frequent translocation partners have been discovered, including INO80D and NCOA1. We hypothesized that all FP-RMS translocations are selecting simultaneously for (1) enhancers active in a myoblast-like epigenome and (2) protein partners with high-levels of intrinsic disorder to give PAX3 enhanced transcriptional strength. Analysis of primary tumors revealed these diverse PAX3 fusions recapitulate a near-identical transcriptome, suggesting uniform underlying molecular mechanisms. ChIP-seq evidence from cell lines and primary tumors suggested that in all FP-RMS tumors, large super-enhancer (SE) elements were present near each chosen translocation partner (distal to FOXO1, INO80D, or NCOA1). Using tools to determine the 3-D folding of chromatin (3C, 4C-seq, and HiChIP), we discovered an extensive network of hijacked FOXO1 enhancers and a SE that physically interact together and with the PAX3 promoter, only in PAX3-FOXO1 positive cells. Furthermore, pooled CRISPR tiling of cis-regulatory elements revealed special dependence on the FOXO1 SE, or certain CTCF boundary elements that facilitate enhancer interactions. ChIP-seq paired to short-term CRISPR experiments shows PAX3-FOXO1 transcription depends on an extended network of related enhancers distal to FOXO1. We find these enhancers are unique to early myoblast stages of differentiation and are bound by myogenic TFs in RMS, suggesting miswiring of normal myogenic enhancer logic. While many SE-driven genes exist in FP-RMS, most are never selected for translocation partners. We found that FOXO1, INO80D, and NCOA1 are all highly disordered proteins, a theme they hold in common despite having no amino acid sequence homology. Initial evidence suggests that PAX3-FOXO1 enables the formation of phase condensates in the nucleus that recruit high-levels of transcriptional machinery such as BRD4. Ongoing work is exploring the mechanistic chemical determinants of these interactions, and how phase condensates are manipulated by drugs that block PAX3-FOXO1’s transcriptional output. Together our studies are illuminating new paradigms for understanding how fusion transcription factors drive cancer. Citation Format: Berkley E. Gryder, Marco Wachtel, Winston Ewert, Kenneth Chang, Osama El Demerdash, Young Song, Beat W Schäfer, Christopher R. Vakoc, Javed Khan. PAX3 translocations co-opt super enhancers and intrinsically disordered fusion partners in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B12.