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

Abstract

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.