Abstract 143: Molecular profiling of tumor-initiation events in rhabdomyosarcoma uncovers mitochondrial vulnerabilities

Abstract Background: Alveolar Rhabdomyosarcoma (ARMS) is a very aggressive soft tissue tumor affecting children and adolescents. It is found that pediatric patients with RMS harboring chimeric PAX fusion transcription factors (TFs) exhibit a greater incidence of tumor relapse, metastasis, and poor survival outcome, thereby underscoring the urgent need to develop effective therapies to treat this subtype of childhood cancer. Methods: We generated a novel ARMS model by stable transduction of myogenic progenitor cells with a cassette encoding the exact PAX3/7-FOXO1 fusion protein found in ARMS tumors. We analyzed genome-wide expression levels with RNA-seq and utilized Cut&Run to map the binding sites of PAX3/7-FOXO1, active histone marks, enhancers, and super-enhancers in our novel model. Hi-C and promoter capture HiC (pCHiC) were also performed to generate 3D chromatin conformation maps in cells expressing the oncogenic TFs. We compared these data with analogous datasets from orthotopic patient-derived xenografts (O-PDXs) of PAX3/7-FOXO1 fusion-positive ARMS tumors from St. Jude and established ARMS cell lines to validate whether our model system recapitulates ARMS tumors. CRISPRi was performed to silence key targets in two representative ARMS cell lines and proliferation was scored after silencing. Next, we found that ARMS tumors displayed anomalies in mitochondrial mass and numbers, metabolic parameters, and membrane potential by performing TEM, Seahorse assays, and FACS-based TMRM and mitotracker Red dye uptake in a panel of ARMS cells and PDX samples. We performed cell viability and colony forming assays in ARMS cells treated with single drugs and combinations of drugs targeting FGFR4 and mitochondrial metabolism. Results: Our data provide evidence to support muscle progenitors as potential cells-of-origin for ARMS. We showed that rewiring of chromatin architecture mediated by PAX3/7-FOXO1 TFs is evident across established cell lines, primary tumors as well as O-PDXs. By integrating PAX3/7-FOXO1 binding, RNA-seq, and pCHiC data, we identified key promoter-enhancer (P-En) interactions that are erased or established de novo at multiple loci of master TFs like MYOD, MYOG and MYCN. We also identified key regulators of proliferation, including FGFR4, and mitochondrial function that are essential for tumor growth and invasiveness. Our data indicated aberrant and enhanced mitochondrial metabolism in ARMS as well as selective sensitivity to mitochondrial inhibitors in RMS cells. Administration of these inhibitors preferentially inhibited cell viability and clonogenicity in ARMS, and prompted a pre-clinical phase II study to test these inhibitors at St. Jude Research Hospital. Conclusion: We present a novel treatment strategy leveraging the mitochondrial dependencies of fusion positive PAX-driven tumors which can be exploited for precision therapy in high-risk ARMS. Citation Format: Bhargab Kalita, Gerard Martinez-Cebrian, Justina McEvoy, Brittney Gordon, Kaley Blankenship, Asa Karlstorm, Elizabeth Stewart, Michael Dyer, Brian Dynlacht. Molecular profiling of tumor-initiation events in rhabdomyosarcoma uncovers mitochondrial vulnerabilities [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 143.