Abstract 6193: TWIST2 promotes tumor progression and represses myogenic differentiation in rhabdomyosarcoma

Abstract

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma of myoblast origin. RMS tumors are unable to terminally differentiate into syncytial muscle despite elevated expression of the muscle master regulators, MyoD and Myf5. Fusion-positive RMS (FP-RMS) and fusion-negative RMS (FN-RMS) have emerged as the two major RMS subtypes. The pathogenesis of FP-RMS is commonly defined by the balanced translocation of chromosomes 2 and 3 resulting in the misexpression of a chimeric PAX3/7-FOXO1 oncoprotein that derails normal differentiation pathways. However, the molecular basis of FN-RMS is poorly understood as single nucleotide variations in genes like KRAS, NRAS, and TP53 have limited recurrence (less than 10% of all patient tumors). In contrast, DNA copy number variations are highly recurrent and the genes they encode represent vulnerabilities in FN-RMS. Strikingly, we discovered that 72% of FN-RMS patient tumors harbor copy number amplifications at loci of TWIST1, TWIST2, or both, resulting in the upregulation of TWIST gene expression. We demonstrated that depletion of TWIST2 using a doxycycline inducible shRNA system caused cell cycle arrest and increased myogenic differentiation in xenograft models of FN-RMS. To interrogate whether TWIST2 can directly regulate the expression of these genes, we performed RNA-seq and ChIP-seq for TWIST2, H3K27Ac, and MYOD in TWIST2 knockdown and control FN-RMS cells. Utilizing this genome-wide data of chromatin binding, we comprehensively demonstrated that TWIST2 competes with MYOD preferentially at a subset of enhancers required for myogenic differentiation, causing repression of these loci. Interestingly, while MYOD binding is gained at myogenic loci in the absence of TWIST2, binding of MYOD is not lost at growth-promoting, oncogenic enhancers despite a decrease in their associated gene expression. This suggests that TWIST2 interacts with a different slew of transcription factors or chromatin regulators to activate gene expression. To interrogate this possibility, we are characterizing the TWIST2 protein interactome in FN-RMS cells using BioID as well as motifs enriched at TWIST2-regulated peaks. By studying TWIST2-associated pathways that affect RMS growth and differentiation, we see an opportunity to unlock novel therapeutic strategies for RMS. Citation Format: Akansha Shah, Lei Guo, Priscilla Jaichander, Kenian Chen, Karla Cano Hernandez, Lin Xu, Rhonda Bassel-Duby, Eric Olson, Ning Liu. TWIST2 promotes tumor progression and represses myogenic differentiation in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6193.