Fusion-positive Rhabdomyosarcoma Research Articles

Dependence of PAX3-FOXO1 chromatin occupancy on ETS1 at important disease-promoting genes exposes new targetable vulnerability in Fusion-Positive Rhabdomyosarcoma

AbstractRhabdomyosarcoma (RMS), a malignancy of impaired myogenic differentiation, is the most common soft tissue pediatric cancer. PAX3-FOXO1 oncofusions drive the majority of the clinically more aggressive fusion-positive rhabdomyosarcoma (FP-RMS). Recent studies have established an epigenetic basis for PAX3-FOXO1-driven oncogenic processes. However, details of PAX3-FOXO1 epigenetic mechanisms, including interactions with, and dependence on, other chromatin and transcription factors, are incompletely understood. We previously identified a novel disease-promoting epigenetic axis in RMS, involving the histone demethylase KDM3A and the ETS1 transcription factor, and demonstrated that this epigenetic axis interfaces with PAX3-FOXO1 both phenotypically and transcriptomically, including co-regulation of biological processes and genes important to FP-RMS progression. In this study, we demonstrate that KDM3A and ETS1 colocalize with PAX3-FOXO1 to enhancers of important disease-promoting genes in FP-RMS, including FGF8, IL4R, and MEST, as well as PODXL, which we define herein as a new FP-RMS-promoting gene. We show that ETS1, which is induced by both PAX3-FOXO1 and KDM3A, exists in complex with PAX3-FOXO1, and augments PAX3-FOXO1 chromatin occupancy. We further show that the PAX3-FOXO1/ETS1 complex can be disrupted by the clinically relevant small molecule inhibitor YK-4-279. YK-4-279 displaces PAX3-FOXO1 from chromatin and interferes with PAX3-FOXO1-dependent gene regulation, resulting in potent inhibition of growth and invasive properties in FP-RMS, along with downregulation of FGF8, IL4R, MEST and PODXL expression. We additionally show that, in some FP-RMS, KDM3A also increases PAX3-FOXO1 levels. Together, our studies illuminate mechanisms of action of the KDM3A/ETS1 regulatory module, and reveal novel targetable mechanisms of PAX3-FOXO1 chromatin complex regulation, in FP-RMS.

Plasma-derived extracellular vesicles miR-335–5p as potential diagnostic biomarkers for fusion-positive rhabdomyosarcoma

BackgroundRhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, with embryonal (ERMS) and alveolar (ARMS) representing the two most common histological subtypes. ARMS shows poor prognosis, being often metastatic at diagnosis. Thus, the discovery of novel biomarkers predictive of tumor aggressiveness represents one of the most important challenges to overcome and may help the development of tailored therapies. In the last years, miRNAs carried in extracellular vesicles (EVs), small vesicles of endocytic origin, have emerged as ideal candidate biomarkers due to their stability in plasma and their tissue specificity.MethodsEVs miRNAs were isolated from plasma of 21 patients affected by RMS and 13 healthy childrens (HC). We performed a miRNA profile using the Serum/Plasma Focus microRNA PCR panels (Qiagen), and RT-qPCR for validation analysis. Statistically significant (p < 0.05) miRNAs were obtained by ANOVA test.ResultsWe identified nine EVs miRNAs (miR-483-5p, miR-132-3p, miR-766-3p, miR-454-3p miR-197-3p, miR-335-3p, miR-17-5p, miR-486-5p and miR-484) highly upregulated in RMS patients compared to HCs. Interestingly, 4 miRNAs (miR-335-5p, miR-17-5p, miR-486-5p and miR-484) were significantly upregulated in ARMS samples compared to ERMS. In the validation analysis performed in a larger group of patients only three miRNAs (miR-483-5p, miR-335-5p and miR-484) were differentially significantly expressed in RMS patients compared to HC. Among these, mir-335-5p was significant also when compared ARMS to ERMS patients. MiR-335-5p was upregulated in RMS tumor tissues respect to normal tissues (p = 0.00202) and upregulated significantly between ARMS and ERMS (p = 0.04). Furthermore, the miRNA expression correlated with the Intergroup Rhabdomyosarcoma Study (IRS) grouping system, (p = 0.0234), and survival (OS, p = 0.044; PFS, p = 0.025). By performing in situ hybridization, we observed that miR-335-5p signal was exclusively in the cytoplasm of cancer cells.ConclusionWe identified miR-335-5p as significantly upregulated in plasma derived EVs and tumor tissue of patients affected by ARMS. Its expression correlates to stage and survival in patients. Future studies are needed to validate miR-335-5p as prognostic biomarker and to deeply elucidate its biological role.

New dual inducible cellular model to investigate temporal control of oncogenic cooperating genes

The study of cooperating genes in cancer can lead to mechanistic understanding and identifying potential therapeutic targets. To facilitate these types of studies, we developed a new dual-inducible system utilizing the tetracycline- and cumate-inducible systems driving HES3 and the PAX3::FOXO1 fusion-oncogene, respectively, as cooperating genes from fusion-positive rhabdomyosarcoma. With this model, we can independently induce expression of either HES3 or PAX3::FOXO1, as well as simultaneously induce expression of both genes. This new model will allow us to further investigate the cooperation between HES3 and PAX3::FOXO1 including the temporal requirements for genetic cooperation. Functionally, we show that dual-induction of PAX3::FOXO1 and HES3 modifies sphere formation in a HEK293T-based system. More broadly, this lentiviral dual-inducible system can be adapted for any cooperating genes (overexpression or knockdown), allowing for independent, simultaneous, or temporally controlled gene expression.

Single cell transcriptomic profiling identifies tumor-acquired and therapy-resistant cell states in pediatric rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric tumor that resembles undifferentiated muscle cells; yet the extent to which cell state heterogeneity is shared with human development has not been described. Using single-cell/nucleus RNA sequencing from patient tumors, patient-derived xenografts, primary in vitro cultures, and cell lines, we identify four dominant muscle-lineage cell states: progenitor, proliferative, differentiated, and ground cells. We stratify these RMS cells/nuclei along the continuum of human muscle development and show that they share expression patterns with fetal/embryonal myogenic precursors rather than postnatal satellite cells. Fusion-negative RMS (FN-RMS) have a discrete stem cell hierarchy that recapitulates fetal muscle development and contain therapy-resistant FN-RMS progenitors that share transcriptomic similarity with bipotent skeletal mesenchymal cells. Fusion-positive RMS have tumor-acquired cells states, including a neuronal cell state, that are not found in myogenic development. This work identifies previously underappreciated cell state heterogeneity including unique treatment-resistant and tumor-acquired cell states that differ across RMS subtypes.

Rhabdomyosarcoma fusion oncoprotein initially pioneers a neural signature in vivo.

Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer molecularly characterized by arrested myogenesis. The defining genetic driver, PAX3::FOXO1, functions as a chimeric gain-of-function transcription factor. An incomplete understanding of PAX3::FOXO1's in vivo epigenetic mechanisms has hindered therapeutic development. Here, we establish a PAX3::FOXO1 zebrafish injection model and semi-automated ChIP-seq normalization strategy to evaluate how PAX3::FOXO1 initially interfaces with chromatin in a developmental context. We investigated PAX3::FOXO1's recognition of chromatin and subsequent transcriptional consequences. We find that PAX3::FOXO1 interacts with inaccessible chromatin through partial/homeobox motif recognition consistent with pioneering activity. However, PAX3::FOXO1-genome binding through a composite paired-box/homeobox motif alters chromatin accessibility and redistributes H3K27ac to activate neural transcriptional programs. We uncover neural signatures that are highly representative of clinical rhabdomyosarcoma gene expression programs that are enriched following chemotherapy. Overall, we identify partial/homeobox motif recognition as a new mode for PAX3::FOXO1 pioneer function and identify neural signatures as a potentially critical PAX3::FOXO1 tumor initiation event.

Targeted immunotherapy and nanomedicine for rhabdomyosarcoma: The way of the future.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. Histology separates two main subtypes: embryonal RMS (eRMS; 60%-70%) and alveolar RMS (aRMS; 20%-30%). The aggressive aRMS carry one of two characteristic chromosomal translocations that result in the expression of a PAX3::FOXO1 or PAX7::FOXO1 fusion transcription factor; therefore, aRMS are now classified as fusion-positive (FP) RMS. Embryonal RMS have a better prognosis and are clinically indistinguishable from fusion-negative (FN) RMS. Next to histology and molecular characteristics, RMS risk groupings are now available defining low risk tumors with excellent outcomes and advanced stage disease with poor prognosis, with an overall survival of about only 20% despite intensified multimodal treatment. Therefore, development of novel effective targeted strategies to increase survival and to decrease long-term side effects is urgently needed. Recently, immunotherapies and nanomedicine have been emerging for potent and effective tumor treatments with minimal side effects, raising hopes for effective and safe cures for RMS patients. This review aims to describe the most relevant preclinical and clinical studies in immunotherapy and targeted nanomedicine performed so far in RMS and to provide an insight in future developments.

Effect of oncolytic herpes simplex virus on fusion-positive rhabdomyosarcoma to radiotherapy.

10063 Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite multimodal interventions including surgery, chemotherapy, and radiotherapy, nearly a third of patients with localized disease will experience tumor recurrence. Patients with tumors harboring an oncogenic FOXO1 fusion are at greater risk of relapse and are most in need of novel therapies. Oncolytic viruses are being explored as treatments for pediatric tumors, and data suggest that oncolytic viruses may enhance responses to chemoradiotherapy. M002 is a genetically engineered oncolytic herpes simplex virus (oHSV) that exhibits aneurovirulence in normal cells. M002 selectively replicates in malignant cells, eliminating these cells through cellular burst during the lytic cycle. In the present study, we explored the efficacy of the M002 virus alone and in combination with low-dose ionizing radiotherapy in a human patient-derived xenograft (PDX) model of fusion-positive RMS (FP-RMS). Methods: A human FP-RMS PDX was established and maintained by injection into the quadriceps femoris muscle of athymic nude mice under Institutional Animal Care and Use Committee guidelines (IACUC-09363). Tumors were dissociated and cells were treated with increasing multiplicity of infection (MOI) of either the oHSV, R3659 or its genetically engineered variant, M002. A colorimetric assay evaluated the cytotoxicity of oHSV treatments on the RMS PDX cells after 24, 48, and 72 hours of infection. In separate experiments, cells treated with vehicle (control) or infected with M002 were exposed to irradiation (2 Gy), and viability was determined at 24, 48, 72, and 96 hours. Results: Treatment with M002 or its parent construct, R3659, significantly reduced RMS PDX cell viability in a dose-dependent manner at all time points. M002 showed slightly higher cytotoxicity than R3659 as measured by lethal dose 50% (LD50, 37±1.25 vs 44±0.95 PFU/cell, M002 vs R3659, at 24 hours, p=0.02). When sub-lethal doses of M002 were combined with low-dose irradiation, RMS PDX cell viability was significantly decreased compared to cells treated with either M002 or irradiation alone (26% decrease at 10 PFU/cell). Conclusions: Our preliminary data highlight that M002 exhibits cytotoxicity in a PDX model of FP-RMS. Treatment with M002 further increased the cytotoxicity of ionizing radiation compared to individual treatments, suggesting that oHSV sensitized the cells to radiotherapy. Future studies in vivo may provide more pre-clinical evidence for the radio-sensitizing and therapeutic potential of M002 in FP-RMS.

Abstract LB042: A potent FGFR4-targeted antibody-drug conjugate therapy for patients with rhabdomyosarcoma and other cancers expressing FGFR4

Abstract Background Rhabdomyosarcoma (RMS) is the most common pediatric sarcoma, representing 3-4% of childhood and adolescent cancers. Although multimodal therapies have significantly improved the overall survival of patients with localized disease, 5-year overall survival for patients with relapsed and metastatic disease has remained dismal. Therefore, new therapies are desperately needed for this deadly disease. Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the cytotoxic potency of chemotherapy drugs to selectively deliver potent anticancer agents to tumor cells while sparing healthy tissues. FGFR4 is a cell-surface receptor tyrosine kinase highly expressed in RMS and other human cancers such as hepatocellular carcinoma (HCC) and some breast cancers. It is expressed at much lower levels in normal tissues, making it a promising immune target. We hypothesize that an FGFR4-targeted ADC would have potent activity against RMS and other cancers expressing FGFR4 with minimal toxicity toward normal tissues. Methods We developed high-affinity binders to human FGFR4 and determined their internalization ability in RMS cancer cell lines. Our top candidate 3A11, a murine monoclonal antibody, was conjugated to monomethyl auristatin E (MMAE), a potent cytotoxic agent, using a valine-citrulline-p-aminocarbamate linker (VC-PABC), to generate an FGFR4-targeted ADC (3A11-vc-MMAE). We tested the in-vitro efficacy of 3A11-vc-MMAE against multiple cell lines of RMS, triple-negative breast cancer, HCC, and FGFR4 negative controls using a live-cell analysis system Incucyte. Western blots were performed to validate 3A11-vc-MMAE’s mechanism of action. We finally tested the in vivo efficacy of 3A11-vc-MMAE in subcutaneous xenograft models of a fusion-positive RMS (RH4) and a fusion-negative RMS with an FGFR4 V550L activating mutation (RMS559). Results We showed that 3A11 was internalized by FGFR4-expressing cell lines and trafficked to lysosomes. Cell viability assays demonstrated that 3A11-vc-MMAE selectively killed FGFR4-expressing cells with minimal effect on low or non-expression cells. Western blot confirmed that 3A11-vc-MMAE induced apoptosis specifically in FGFR4-expressing RMS cell lines. Mice treated with one cycle of 3A11-vc-MMAE (3mg/kg twice a week for 2 weeks) demonstrated a significantly increased survival rate of 30% for the RH4 model with a median survival of 43 days for 3A11-vc-MMAE and 23 days for control, vehicle-treated mice (p-value = 0.0038), and 70% for the RMS559 model, with a median survival not reached for of 3A11-vc-MMAE treated compared with 23 days for control mice (p-value &amp;lt; 0.0001). Of note, tumors were eradicated in 4 out of 10 mice bearing RMS559 xenografts. Conclusions and Future Directions Our results demonstrate that the ADC 3A11-vc-MMAE provides a potent and specific precision immunotherapy for rhabdomyosarcoma and other cancers with a high expression of FGFR4, which warrants further development for clinical translation. Citation Format: Meijie Tian, Katrina Jia, Jerry T. Wu, Jun S. Wei, Adam T. Cheuk, Zhongmei Zhang, Eleanor G. Pope, Boris Shor, L. Nathan Tumey, Javed Khan. A potent FGFR4-targeted antibody-drug conjugate therapy for patients with rhabdomyosarcoma and other cancers expressing FGFR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB042.

Germline Genetic Testing and Survival Outcomes Among Children With Rhabdomyosarcoma

Determining the impact of germline cancer-predisposition variants (CPVs) on outcomes could inform novel approaches to testing and treating children with rhabdomyosarcoma. To assess whether CPVs are associated with outcome among children with rhabdomyosarcoma. In this cohort study, data were obtained for individuals, aged 0.01-23.23 years, newly diagnosed with rhabdomyosarcoma who were treated across 171 Children's Oncology Group sites from March 15, 1999, to December 8, 2017. Data analysis was performed from June 16, 2021, to May 15, 2023. The presence of a CPV in 24 rhabdomyosarcoma-associated cancer-predisposition genes (CPGs) or an expanded set of 63 autosomal-dominant CPGs. Overall survival (OS) and event-free survival (EFS) were the main outcomes, using the Kaplan-Meier estimator to assess survival probabilities and the Cox proportional hazards regression model to adjust for clinical covariates. Analyses were stratified by tumor histology and the fusion status of PAX3 or PAX7 to the FOXO1 gene. In this study of 580 individuals with rhabdomyosarcoma, the median patient age was 5.9 years (range, 0.01-23.23 years), and the male-to-female ratio was 1.5 to 1 (351 [60.5%] male). For patients with CPVs in rhabdomyosarcoma-associated CPGs, EFS was 48.4% compared with 57.8% for patients without a CPV (P = .10), and OS was 53.7% compared with 65.3% for patients without a CPV (P = .06). After adjustment, patients with CPVs had significantly worse OS (adjusted hazard ratio [AHR], 2.49 [95% CI, 1.39-4.45]; P = .002), and the outcomes were not better among patients with embryonal histology (EFS: AHR, 2.25 [95% CI, 1.25-4.06]; P = .007]; OS: AHR, 2.83 [95% CI, 1.47-5.43]; P = .002]). These associations were not due to the development of a second malignant neoplasm, and importantly, patients with fusion-negative rhabdomyosarcoma who harbored a CPV had similarly inferior outcomes as patients with fusion-positive rhabdomyosarcoma without CPVs (EFS: AHR, 1.35 [95% CI, 0.71-2.59]; P = .37; OS: AHR, 1.71 [95% CI, 0.84-3.47]; P = .14). There were no significant differences in outcome by CPV status of the 63 CPG set. This cohort study identified a group of patients with embryonal rhabdomyosarcoma who had a particularly poor outcome. Other important clinical findings included that individuals with TP53 had poor outcomes independent of second malignant neoplasms and that patients with fusion-negative rhabdomyosarcoma who harbored a CPV had outcomes comparable to patients with fusion-positive rhabdomyosarcoma. These findings suggest that germline CPV testing may aid in clinical prognosis and should be considered in prospective risk-based clinical trials.

Abstract 1090: GPC2 directed CAR T cells are efficacious against fusion-positive and fusion-negative rhabdomyosarcomas

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive pediatric solid tumor with poor survival and new targeted therapies are needed. Aims: Using an integrative proteogenomic quantitative approach, we sought to identify candidate immunotherapeutic targets then validate top targets in RMS preclinical models. Methods: We first performed plasma membrane enrichment followed by mass spectrometry (MS) to define the surfaceome of 7 fusion-positive (FP) and 14 fusion-negative (FN) RMS patient-derived xenograft models. Surface proteins were scored by IMMUNOTAR, our custom pipeline which extracts and integrates quantitative features from diverse proteogenomic and annotation datasets to provide a score for each protein, using mean-average-precision for score optimization. Cell surface expression of IMMUNOTAR-prioritized proteins was validated using flow cytometry, focusing on targets with immunotherapies in development. CAR T cells targeting the lead protein were engineered and studied in RMS preclinical models. Results: A total of 847 and 882 high confidence cell surface proteins were detected in FP and FN RMS surfaceome, respectively, with 94% of proteins overlapping the phenotypes. IMMUNOTAR ranked anaplastic lymphoma kinase (ALK), a putative immunotherapeutic target in FP RMS, as the top FP target. Glypican 2 (GPC2), a differentially expressed cell surface proteoglycan identified in neuroblastoma and other neural-derived tumors, targetable with CAR T cells and antibody-drug conjugates [Bosse et al. Cancer Cell 2017], ranked as the top protein in FN and 5th protein in FP RMS. Overall, GPC2 was abundantly expressed in 16 of 21 RMS models profiled. Flow cytometry revealed expression of GPC2 comparable to neuroblastoma in 5 of 8 RMS cells lines. Additionally, both FP and FN GPC2+ RMS cell lines (FN: RH36, SMS-CTR; FP: RH41, RH30) activated GPC2 CAR T cells with either a CD28 or a 4-1BB costimulatory domain, inducing release of IFN-gamma and IL-2 upon co-incubation across different E:T ratios as quantified by ELISA. Further, GPC2 CAR T cells induced potent RMS cell cytotoxicity compared to control CD19 CAR T cells with near 100% specific cytotoxicity at 1:1 E:T ratio. In a pilot study, GPC2 CAR T cells with 4-1BB costimulatory domains induced significant tumor regression (p=0.03) and prolonged progression-free survival (p=0.01) compared to CD19 CAR T cells in an established murine RH41 flank xenograft model. Conclusion: In summary, we probed the RMS surfaceome using MS and identified candidate proteins amenable for immunotherapeutic targeting using our IMMUNOTAR algorithm. We validated GPC2 as a candidate target in FP and FN RMS and are corroborating our findings in additional pre-clinical RMS models. Demonstration of GPC2 CAR efficacy in these models could lead to inclusion of RMS in our ongoing phase 1 clinical trial (NCT05650749) in a planned expansion phase. Citation Format: Rawan Shraim, Patrick M. Schuerch, Anna Maria Giudice, Karina L. Conkrite, Amber K. Weiner, Alexander B. Radaoui, Brian Mooney, Raushan T. Kurmasheva, Peter J. Houghton, Gregg B. Morin, Yael B. Mosse, Poul H. Sorensen, Benjamin A. Garcia, Ahmet Sacan, John M. Maris, Kristopher R. Bosse, Sharon J. Diskin. GPC2 directed CAR T cells are efficacious against fusion-positive and fusion-negative rhabdomyosarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1090.

Abstract 138: UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma

Abstract PAX3-FOXO1 positive rhabdomyosarcoma (RMS) is an aggressive pediatric sarcoma with a terrible prognosis. The resultant fusion acts as a chimeric transcription factor, in part remodeling chromatin to inhibit terminal differentiation and maintain cancer cell proliferation. As this oncoprotein is not directly druggable, we have used genetic screens to identify dependencies downstream of PAX3-FOXO1 to improve our understanding of pathogenesis and develop new therapeutic targets. We performed pooled CRISPRi screening in an isogenic system of a patient-derived RMS cell line with or without knockdown of PAX3-FOXO1. Loss of the ubiquitin hydroxylase UCHL5 led to selective depletion of PAX3-FOXO1 positive Rh30 cells compared to PAX3-FOXO1 knockdown cells, suggesting that the fusion creates UCHL5 dependence. We validated this finding in competition growth assays in additional fusion positive (FP) and fusion negative (FN) RMS cell lines, and observed UCHL5 dependency only in the former. By contrast, UCHL5 was dispensable for cell growth and survival in FN RMS cell lines. These data were recapitulated in the large DepMap dataset, which also identified the top genetic co-dependencies of UCHL5 as INO80-complex (INO80c) members. We therefore investigated whether the fusion selective effects of UCHL5 were mediated by the INO80c, of which UCHL5 is an N-terminal member. We observed that knockdown of another N-terminal component of INO80c, NFRKB, phenocopies the fusion-selective effects of UCHL5 loss. Conversely, the catalytic ATPase INO80 was pan-essential, suggesting that only N-terminal subunits of INO80c regulate PAX3-FOXO1 specific functions. INO80c plays an essential role in mitigating DNA replication stress. Indeed, loss of UCHL5 led to DNA damage as measured by γH2AX and slowing of replication forks as measured by fiber assays in FP, but not FN RMS. Unexpectedly, acute degradation of PAX3-FOXO1 also slowed DNA replication fork speed, suggesting a genetic model in which UCHL5 and PAX3-FOXO1 cooperate to resolve replication stress in FP RMS. Finally, in vivo experiments using cell line xenografts confirms that loss of UCHL5 slows the growth of FP RMS, but not FN RMS. These findings suggest that loss of UCHL5 induces replication stress in a fusion-specific manner, and offers both an opportunity to study the mechanisms by which the PAX3-FOXO1 oncoprotein mitigates replication stress, and to develop UCHL5 inhibitors as precision therapies for FP RMS patients. Citation Format: Amit J. Sabnis, Pushpendra K. Sahu, Yue Pan. UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 138.

Abstract 706: Advancing radiotherapy strategies for high-risk rhabdomyosarcoma: Unveiling BCL-xL inhibition as a potent sensitizer to radiotherapy

Abstract Pediatric soft-tissue sarcomas are a group of solid tumors that account for about 7% of childhood cancer cases. Rhabdomyosarcoma (RMS), the most common subtype, further stratifies into PAX3 or PAX7 and FOXO1 fusion-negative (FN) or fusion-positive (FP) RMS. FP RMS is the higher risk subtype and is more aggressive, metastatic, and less responsive to treatment. Front-line treatments for RMS FP patients have had limited progress in the recent years and existing treatments such as radiotherapy lacks tumor cell selectivity, resulting in toxic side effects. To address this, we conducted a high-throughput drug screen to identify radiosensitizers to enhance efficacy of RMS radiotherapy. Using three patient-derived FP RMS tumoroids, we screened a comprehensive drug library with and without radiation, comprising 528 drugs at varying stages of pre-clinical and clinical trials. A validation screen of the top 29 drugs was conducted on a larger cohort of samples including eight patient-derived FP RMS tumoroids and three non-cancer models. The screening revealed that drugs targeting the anti-apoptotic protein B-cell lymphoma-extra-large (BCL-xL) exhibited both potent radiosensitization and cytotoxicity selectively for FP RMS. Notably, the sensitivity to BCL-xL inhibitors correlated with elevated BCL2L1 expression and PAX3 fusion status. Additionally, drugs targeting polo-like kinase 1 (PLK1) and other cell cycle-related genes exhibited robust radioprotective effects across both RMS and non-cancer models. Our findings propose the therapeutic potential of BCL-xL inhibition, either as a standalone treatment or in combination with radiotherapy. In parallel, we show the protective potential of PLK1 inhibition to mitigate radiation toxicity. In the long term, we aspire to integrate the identified radiosensitizing and radioprotecting compounds with existing treatment regiments for RMS, maximizing precision and efficacy in RMS radiotherapy. Citation Format: Sammy Se Whee Park, Nona Struyf, Tom Erkers, Sören Lehmann, Michael T. Meister, Jarno Drost, Frank C. Holstege, Nikolas Herold, Päivi Östling, Jakob Stenman, Olli Kallioniemi, Jan-Inge Henter, Brinton Seashore-Ludlow, Kasper Karlsson. Advancing radiotherapy strategies for high-risk rhabdomyosarcoma: Unveiling BCL-xL inhibition as a potent sensitizer to radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 706.

Abstract 1089: Endogenous HiBiT-tagging of PAX3-FOXO1 reveals downregulation of the fusion oncogene by CDK inhibitors and has synergy with vincristine

Abstract Background: Oncogenic fusion genes are attractive therapeutic targets due to their tumor-specific expression and driver roles in cancers. PAX3-FOXO1 (P3F) is the dominant oncogenic driver of fusion-positive rhabdomyosarcoma (FP-RMS) with no current targeted therapy. We developed methods to directly measure endogenous P3F protein levels amenable to high-throughput drug screens. Method: HiBiT tag, an 11 amino acid peptide of NanoLuc luciferase, was inserted into the endogenous P3F using CRISPR in FP-RMS cell lines RH4 and SCMC. Western blot was used for HiBiT tag validation. RNA-seq and ChIP-seq were used to assess transcriptomics and DNA binding of HiBiT-tagged P3F (P3F-HiBiT). High-throughput drug screen using Nano-Glo luciferase assay was performed using the Mechanism Interrogation PlatE (MIPE 5.0) drug library, a 2,480 drug library with known mechanisms of action. CellTiter-Glo was used to monitor cell viability. Mouse xenograft models of FP-RMS were used to investigate in vivo efficacy. Results: We validated HiBiT tagging of P3F by Western. Both P3F-HiBiT and unmodified P3F activated the same gene sets in fibroblasts by RNA-seq Gene Set Enrichment Analysis (GSEA). ChIP-seq using HiBiT antibody for P3F-HiBiT matched the genomic locations from ChIP-seq with P3F antibody in RH4 and SCMC. Using a cutoff value of &amp;gt; 90 (Area Under the Curve (AUC) of CellTiter-Glo minus AUC of Nano-Glo), in both RH4 and SCMC, we identified 182 compounds which downregulate P3F before cell death. Filtering for drugs with ≥ 3 hits for the same target identified 14 drug classes that suppressed P3F including HDAC inhibitors (3), BRD4 inhibitors (3), and CDK inhibitors (8). FP-RMS was sensitive to CDK1/2, CDK4/6, CDK9, and multi-CDK inhibitors. A multi-CDK inhibitor TG02, currently in human trials, downregulated P3F and RNA-seq GSEA showed marked suppression of P3F targets. TG02 also significantly delayed tumor progression in mouse xenograft model of FP-RMS without weight loss. Interestingly, the commonly used chemotherapeutic Vincristine (VCR) also downregulated P3F in vitro. TG02 with VCR showed synergy by Loewe analysis. In vivo, testing showed significant delay in tumor progression by the combination compared to TG02 or VCR alone. Of note, tumor RNA-seq GSEA following treatment with TG02, or VCR alone, and in combination significantly downregulated P3F targets. Conclusion And Future Directions: By HiBiT tagging the fusion oncogene P3F, we identified 182 drugs that suppress P3F levels of which TG02 was a top hit. TG02 showed in vivo efficacy indicating that FP-RMS is susceptible to CDK inhibition. We also found synergy between TG02 and VCR, resulting in a significant delay in FP-RMS tumor progression compared to single agents. Interestingly, we found that VCR alone can downregulate P3F and its targets. Combination therapy of TG02 with VCR shows promise for clinical translation in FP-RMS. Citation Format: Yong Yean Kim, Katrina Jia, Mehal Churiwal, Teresa Hawley, Silvia Pomella, Christine Evans, Raj Chari, David Milewski, Ranuka Sinniah, Young Song, Hsien-Chao Chou, Xinyu Wen, Craig Thomas, Michele Ceribelli, Jun Wei, Robert Hawley, Javed Khan. Endogenous HiBiT-tagging of PAX3-FOXO1 reveals downregulation of the fusion oncogene by CDK inhibitors and has synergy with vincristine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1089.

Abstract 1092: RUNX2 inhibition alters PAX3::FOXO1 driven fusion-positive rhabdomyosarcoma cell state and growth

Abstract Investigation: Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric cancer of skeletal muscle lineage. While the incidence of RMS is ~4.5 patients per million individuals aged &amp;lt;20 years, 5-year survival for high-risk children is &amp;lt;30%, and &amp;lt;8% when metastatic. The PAX3::FOXO1 (P3F) fusion gene, resulting from t(2:13), is the most prominent fusion oncogene that drives FP-RMS. P3F is a fusion transcription factor and supports FP-RMS by keeping it in a persistent myoblastic state. We found P3F functions in part by suppressing MST kinase activity in the Hippo tumor suppressor pathway, a developmental pathway involved in cell fate and organ size. To investigate how this may rewire gene expression to support FP-RMS, we performed ATAC-seq analysis of a myoblast-based model of FP-RMS in which MST was further inhibited and found the RUNX2 gene locus was more accessible. Since RUNX2 is a pioneer transcription factor known to interact with the “WW” domains of Hippo pathway members to support mesenchymal cell fate, we hypothesize that RUNX2 maintains these relationships to support FP-RMS cell state and survival. Methods &amp; Results: Inquiry of in silico databases and previously published literature unveiled that sarcomas including RMS have a high dependency on RUNX2 and that P3F directly binds a RUNX2 enhancer. Interrogation of a proximity labeling proteomics study from our collaborators in the Counter lab found that RUNX2 was part of the protein interactome of 7/7 FP-RMS fusions including P3F. Additionally, Co-IP experiments confirmed that there is a direct protein-protein relationship between RUNX2, P3F, and TAZ. Next, we performed in vitro genetic loss of function (LOF) of RUNX2 using RNAi, which showed that suppression of RUNX2 caused cell death, growth inhibition, increased TAZ expression, and induction of both myogenic and osteogenic markers. We also performed complementary pharmacologic LOF using two available small molecules, AI-14-91 (Bushweller lab) in vitro, and CADD522 (commercially available) in vitro and in vivo. We found that these partially phenocopied the genetic LOF and that CADD522 reduces tumor volume and growth. Conclusions: RUNX2 is a vulnerability in RMS based on in silico, in vitro, and in vivo investigation, and may act to prevent osteogenic and myogenic differentiation via protein-protein relationships in the Hippo pathway. The next step will be assessing mechanistic relationships between the Hippo pathway and RUNX2, as well as the genome wide changes upon RUNX2 LOF by performing ChIP-seq, RNA-seq, and scRNA-seq experiments. Citation Format: Elizabeth A. Mendes, Audrey L. Chambers, Samantha J. Weitzel, Michael Deel, Aanandi Munshi, Christopher M. Counter, Seth Zimmerman, John Bushweller, Corinne M. Linardic. RUNX2 inhibition alters PAX3::FOXO1 driven fusion-positive rhabdomyosarcoma cell state and growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1092.

Abstract 4739: Targeting resistance to BET inhibitors in Fusion-Positive Rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most frequent pediatric soft tissue sarcoma. Fusion-positive (FP-)RMS expressing the oncogenic chimeric transcription factor (TF) PAX3-FOXO1 (P3F) is at high risk of recurrence. FP-RMS cells show survival dependency on P3F. Despite P3F, as a TF, is considered undruggable, we and others demonstrated that P3F levels/functions can be epigenetically modulated. The BET protein and epigenetic reader BRD4 binds super-enhancers (SEs) to foster oncogenic transcription in cancer. We showed that P3F rewires the RMS enhancer landscape by recruiting BRD4 on oncogenic SEs. Accordingly, JQ1, a BET inhibitor (BETi) shuts down P3F functions halting tumor growth in vitro and in vivo. Therefore, BRD4 inhibition in FP-RMS results in a tumor subtype-specific vulnerability. Nonetheless, as for other targeted therapies, cancer cells can acquire resistance to BETi, thus, we investigated whether FP-RMS cells could develop BETi resistance. Therefore, we established resistant cells from two sensitive FP-RMS cell lines chronically exposed to escalating doses of JQ1, as a model of naturally acquired resistance. Our data show that the two established FP-RMS cell lines, acquired resistance by increasing JQ1 IC50 over the sensitive cells both in 2D and 3D settings. Compared to the sensitive ones, both the resistant cell lines show cross-resistance to other monovalent and bivalent BETi and degraders. Resistant cells also maintain the ability to form colonies in a clonogenic assay and to invade in a 3D Matrigel assay in the presence of JQ1. Moreover, conversely to sensitive cells, JQ1 treatment is unable to down-regulate P3F, MYCN and BCL2 (P3F target) levels in resistant cells. Furthermore, integration of transcriptomic and proteomic data reveals differentially modulated subset of genes/proteins among which MYCN and P3F targets, suggesting a restored activity of the TFs circuitry in resistant cells. Accordingly, MYCN silencing completely halts growth of resistant cells. In line, MYCN protein levels are reduced by JQ1 in a dose-dependent manner in sensitive but not in resistant cells and its half-life is 3 folds longer in resistant than in sensitive cells. Being MYCN protein stability affected by specific post-translational modifications among which the phosphorylation at Ser62 by ERKs, we evaluated the expression of phosphorylated MYCN and the activated form of ERK (pERK) and found both of them increased in resistant cells. In agreement, resistant cells are more sensitive to MEK-ERKs pathway inhibition, showing a lower IC50 of Trametinib (MEKi) compared to sensitive cells. Moreover, Trametinib and JQ1 co-treatment downregulates MYCN protein levels in resistant but not in sensitive cells, suggesting that ERKs overactivation could participate to BETi acquired resistance by stabilizing MYCN, thus representing an acquired vulnerability. The study has received funding from Ministero della Salute to SP. Citation Format: Matteo Cassandri, Erika Ferraro, Lucrezia D'Archivio, Francesca Aiello, Hsien-Chao Chou, Young K. Song, Berkley E. Gryder, Jun S. Wei, Simone Sidoli, Robert G. Hawley, Rossella Rota, Franco Locatelli, Javed Khan, Silvia Pomella. Targeting resistance to BET inhibitors in Fusion-Positive Rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4739.

Abstract 134: ZEB2 as a therapeutic target in fusion-positive rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is a highly aggressive childhood soft tissue cancer with skeletal muscle histogenesis. It is classified at the molecular level into fusion-negative (FN) and fusion-positive (FP) subtypes, with FP subtypes being defined by their signature oncofusions between the transcription factors PAX3 (or PAX7) and a variety of 3’ partners. Patients with FP-RMS have a five-year survival of &amp;lt30%, and current treatments remain ineffective against the driving oncofusions. To address this challenge, we are interested in identifying proteins that interact with the oncofusions as alternate therapeutic targets, and conducted an unbiased proximity labeling screen for seven of the known RMS oncofusions. This approach revealed that ZEB2 is a common member of the interactome for all seven FP-RMS subtypes. ZEB2 is a DNA-binding transcriptional repressor that interacts with SMAD proteins and is known to be important for coordinating muscle and nerve development during embryogenesis. ZEB2 has been identified as an oncogene in various cancer types such as leukemia, glioblastoma, lung, and breast cancers. Further analysis of ZEB2's expression in FP-RMS cell lines, compared to skeletal muscle and other cancer cell lines, showed significantly higher levels of expression. In silico analyses of genomic CRISPR and RNAi screens in DepMap suggested a strong dependency of FP-RMS on ZEB2, with ZEB2 being one of the top four differential CRISPR dependencies in RMS cell lines compared to all other cancer cell lines. To investigate the functional consequences of ZEB2 suppression, we employed a loss-of-function approach using RNAi (siRNA and shRNA) in human FP-RMS cells. Our experiments confirmed the knockdown of ZEB2 through qRT-PCR and immunoblotting. Interestingly, the extent of ZEB2 knockdown determined whether FP-RMS cells underwent apoptosis or differentiation, potentially towards a neurodifferentiation pathway. We hypothesize that ZEB2 holds potential as a therapeutic target for FP-RMS patients. We aim to elucidate ZEB2's phenotypic role and mechanism of action in FP-RMS tumorigenesis, and assess its value as a therapeutic target in vivo. Our long-term goals for this project are to contribute to the development of innovative therapies that can mitigate the oncogenic activity driven by oncofusions in FP-RMS. Citation Format: Assil Fahs, Aanandi Munshi, Leen Barbar, Seth Zimmerman, Chris Counter, Corinne Linardic. ZEB2 as a therapeutic target in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 134.

Abstract 1723: Targeting SMARCA1 in rhabdomyosarcoma: Potential therapeutic implications

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue in pediatric sarcoma, and studies demonstrate that RMS arises from skeletal muscle precursor cells. RMS, genetically divided into two subtypes: PAX-FOXO1 fusion positive RMS (FP-RMS), which is driven by chromosomal translocation involving PAX3 or PAX7 genes with FOXO1 and PAX-FOXO1 fusion negative RMS (FN-RMS), which is marked by mutations in RAS isoforms and some other genes. 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 have been classified as both oncogenes and tumor suppressors. The ISWI family protein, SMARCA1 has been implicated in tumorigenesis for several cancer types. ISWI complexes regulate cell differentiation and proliferation in other cell systems, but their impact on 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 employed RNA-seq, ATAC-seq, and CUT&amp;RUN to study the impact of SMARCA1 in RMS cells. Furthermore, phenotypic experiments were performed to determine the influence of SMARCA1 on differentiation and proliferation. Results: SMARCA1 is expressed at a high level in RMS tissues but not in muscle tissues. Our findings show that SMARCA1 is modestly upregulated during differentiation in normal myogenesis. Upon treatment with trametinib, FP-RMS cell lines with SMARCA1 knockout exhibited a dramatic decrease in cell confluency and viability. Intriguingly, the control FP-RMS cell lines demonstrated increased SMARCA1 protein levels in response to trametinib, which could suggest a resistance mechanism to the treatment. Trametinib also affected SMARCA1 mRNA expression in FN-RMS cells. Additionally, it was discovered that the elimination of SMARCA1 not only resulted in a reduction in cell migration but also led to a failure of these cells to develop spheres, unlike the control cells, where the migration gap healed in the usual manner and the spheres were observed. These findings may align with our hypothesis that SMARCA1 regulates chromatin accessibility, potentially impacting adhesion properties and signaling pathways. Analysis of gene clusters in RMS transcriptomic data indicated that SMARCA1 co-expresses with a RAS effector, RALA. SMARCA1 is also clustered with HDAC2. In addition, SMARCA1 interacted with HDAC2 in RMS cells, as determined by co-immunoprecipitation experiments. Conclusions: Our findings suggest that SMARCA1 may influence RMS tumor growth. This study might contribute to a deeper understanding of SMARCA1's role in RMS and support its credentialing as a novel therapeutic avenue. Citation Format: Ashwaq Aljabri, Katie Hebron, Yuliya Kriga, Juan Manuel Caravaca, Aiysha Althobaiti, Stacey Stauffer, Angela Kim, Lauren Stoak, Morgan Porter, Jyoti Shetty, Bao Tran, Matthew Geisler, Judith Davie, Marielle Yohe. Targeting SMARCA1 in rhabdomyosarcoma: Potential therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1723.

Abstract 2861: Rhabdomyosarcoma fusion-oncogene alters the chromatin landscape to initially drive a neural signature in vivo

Abstract Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer lacking curative therapies, and outcomes for children with this disease have not improved in decades. The chimeric transcription factor PAX3::FOXO1 is the most common and lethal driver of fusion-positive rhabdomyosarcoma, and it consists of the DNA binding domains of PAX3 fused to the transactivation domain of FOXO1. Despite its well-established essentiality for tumorigenesis, how PAX3::FOXO1 initially generates a tumorigenic cell state has been challenging to study due to the lack of in vivo models expressing the fusion-oncogene at accessible early developmental time points. Here, we developed a novel zebrafish mRNA injection model that ubiquitously expresses PAX3::FOXO1 during gastrulation representing all three germ layers. Using this high-throughput model, we used ChIP-seq to identify initial in vivo PAX3::FOXO1 binding sites and targets. PAX3::FOXO1 has pioneering activity, where it can utilize partial-motif recognition to bind to inaccessible chromatin and alter chromatin structure. With biochemical and 2D chromatin sequencing approaches in our model, we find evidence for in vivo PAX3::FOXO1 pioneering activity. PAX3::FOXO1 consists of two DNA binding domains, a paired and homeobox domain, and we demonstrate a new mode of pioneering activity driven by its homeobox domain given a strong enrichment of homeobox-related motifs at PAX3::FOXO1 binding sites. PAX3::FOXO1 activity in developing zebrafish embryos results in an arrested development phenotype and transcriptional signatures enriched for pathways such as an inhibition of segmentation and myogenic development. Critically, we find that PAX3::FOXO1 directly activates neural-related gene targets by increasing chromatin accessibility and re-distribution of the active histone mark H3K27Ac to these loci. These discoveries are particularly striking because rhabdomyosarcoma is traditionally associated with skeletal muscle characteristics. With our findings, we hypothesize neural gene activation is a critical mechanism for fusion-positive rhabdomyosarcoma tumor initiation and could be a critical pathway across various stages of rhabdomyosarcoma tumorigenesis. This hypothesis agrees with our previous discovery of a novel PAX3::FOXO1 cooperating neural transcription factor, HES3, which we find is directly activated by PAX3::FOXO1 in our mRNA injection model. Our long-term goal is to utilize this versatile model to functionally evaluate cooperation between PAX3::FOXO1 and both canonical rhabdomyosarcoma and neural transcription factors. Our approach will employ a cross-species comparative analysis across this injection model, our zebrafish tumor models, other model systems, and patient data. This work will identify conserved targets and mechanisms of PAX3::FOXO1 tumorigenesis that could become new therapeutic vulnerabilities. Citation Format: Jack Kucinski, Cenny Taslim, Alexi Tallan, Matthew Cannon, Katherine Silvius, Benjamin Stanton, Genevieve Kendall. Rhabdomyosarcoma fusion-oncogene alters the chromatin landscape to initially drive a neural signature in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2861.

KDM3B inhibitors disrupt the oncogenic activity of PAX3-FOXO1 in fusion-positive rhabdomyosarcoma

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.

PAX3-FOXO1 dictates myogenic reprogramming and rhabdomyosarcoma identity in endothelial progenitors

Fusion-positive rhabdomyosarcoma (FP-RMS) driven by the expression of the PAX3-FOXO1 (P3F) fusion oncoprotein is an aggressive subtype of pediatric rhabdomyosarcoma. FP-RMS histologically resembles developing muscle yet occurs throughout the body in areas devoid of skeletal muscle highlighting that FP-RMS is not derived from an exclusively myogenic cell of origin. Here we demonstrate that P3F reprograms mouse and human endothelial progenitors to FP-RMS. We show that P3F expression in aP2-Cre expressing cells reprograms endothelial progenitors to functional myogenic stem cells capable of regenerating injured muscle fibers. Further, we describe a FP-RMS mouse model driven by P3F expression and Cdkn2a loss in endothelial cells. Additionally, we show that P3F expression in TP53-null human iPSCs blocks endothelial-directed differentiation and guides cells to become myogenic cells that form FP-RMS tumors in immunocompromised mice. Together these findings demonstrate that FP-RMS can originate from aberrant development of non-myogenic cells driven by P3F.