Abstract 131: Utilizing human induced pluripotent stem cells (hiPSCs) as a model to study the genetic drivers of rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is a childhood soft tissue sarcoma composed of skeletal myoblast-like cells that lack the capacity to differentiate despite their high expression of terminal differentiation markers like myogenin. There are two main types of RMS: fusion-positive RMS (FP-RMS), which is associated with a chromosomal translocation resulting in an oncogenic fusion protein (PAX3-FOXO1), and the more common form, fusion-negative RMS (FN-RMS) which lacks this translocation and is instead thought to be driven by numerous other oncogenes. Our lab used a Bayesian-based computational algorithm to analyze NGS sequencing data from 290 RMS samples and identified 25 potential genetic drivers of RMS. We aim to use induced pluripotent stem cells as a new model to study how these drivers, including the PAX3-FOXO1 fusion gene, influence RMS formation and progression. To this aim, we have successfully made a doxycycline inducible lentiviral vector containing the full-length sequence of human PAX3-FOXO1 which we used to ectopically express PAX3-FOXO1 in two well-characterized iPSC lines. We subcutaneously injected these iPSCs into NOD/SCID mice and treated them with doxycycline to form teratomas, non-malignant tumors containing tissue from all three germ layers. We hypothesized that PAX3-FOXO1 induction in these differentiating teratomas, may have transforming capabilities and may deregulate transcriptional programs to form FP-RMS-like tumors. We have found that PAX3-FOXO1 overexpressing teratomas grew faster and histologically look more “immature” than their control counterparts but do not seem to give rise to rhabdo-like tumors. We want to further understand what other critical factors are necessary to drive RMS formation by focusing on one of our drivers of interest EZH2, a histone methyltransferase implicated as either an oncogene or a tumor suppressor in certain forms of cancer. We have data suggesting that EZH2-dependent histone methylation activity may act as a proto-oncogene with PAX3-FOXO1 to silence the expression of key tumor suppressor genes CDKN2A and CDKN2B in RMS. To this aim, we reasoned that iPSCs differentiated into myogenic progenitors, one of the potential cells of origin of RMS, will be a novel way to study the role of oncogenes like PAX3-FOXO1 and EZH2 in RMS. We have derived myogenic progenitors from iPSCs (iPSC-MPCs) that express key myogenic factors like PAX7 and MYOD and differentiate and fuse to form mature myofibers. We plan to next explore how ectopic expression of EZH2 and/or PAX3-FOXO1 alters gene expression and histone methylation and whether these alterations can drive RMS formation in these iPSC-MPCs. Using this model system of iPSC-MPCs to drive expression of these oncogenes has the potential to offer new and exciting ways to identify the most important genes in driving cancer formation hopefully providing insight into better targets for treatment of RMS. Citation Format: Celeste Romero, Yanbin Zheng, Lin Xu, Stephen Skapek. Utilizing human induced pluripotent stem cells (hiPSCs) as a model to study the genetic drivers of 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 (6Suppl): Abstract nr 131.