Abstract 4992: Miswired super enhancer logic driving childhood sarcoma

Abstract

Abstract Super enhancers (SEs) are regulatory regions with unusually large deposits of active histone marks, chromatin regulators and transcriptional coactivators. Chromosomal rearrangements allowing SEs to drive oncogene expression is an emerging mechanism in tumor biology. An aggressive myoblastic cancer of childhood, alveolar (fusion-positive) rhabdomyosarcoma (FP-RMS), universally possesses a chromosomal translocation, involving most commonly PAX3 and FOXO1, more rarely PAX7-FOXO1, and in exceptional cases novel PAX3-INO80D and PAX3-NCOA1 fusions. Patients with a PAX3-fusion frequently relapse and have low survival rates. PAX3 initiates specification of the muscle lineage, but is shut off during myogenic differentiation, which is in turn dominated by master regulators MYOD and finally MYOG. FP-RMS has the master regulators needed to trigger muscle differentiation, but are halted in an early myoblastic and thus more proliferative epigenetic state. We hypothesized that the translocations miswires regulation of the fusion oncoprotein in FP-RMS by hijacking SEs and creating new topologically associated domains (TADs) which allow for continued expression of PAX fusions, thus circumventing normal myogenic enhancer logic. Thus, we recently completed the first epigenetic landscape of FP-RMS and uncovered a strong dependence on SEs for tumor survival, with PAX3-FOXO1 being a chief determinant of SE formation in collaboration with MYOD and MYOG, and oncogene MYCN. Importantly, we discovered a key SE 300 kb distal of FOXO1 which was occupied by all four of these master regulators. Further, we found that PAX3-FOXO1 is driven by this novel translocated SE forming a key TAD structure which was necessary to directly influence PAX3 upon translocation, with CTCF analysis in FP-RMS cells confirming the predicted boundaries. The CTCF motif orientation was found to be antiparallel after the translocation event, permitting chromatin loop extrusion. We demonstrate these elements to physically interact only in the presence of the translocation by chromatin conformation followed by sequencing (4C-seq). Exon-level expression via RNAseq in primary tumors revealed that the final exon of PAX3, not involved in the translocation, was unexpressed, indicating that only the allele influenced by the FOXO1 SE is activated in patients. Finally, CRISPR/Cas9 technologies were employed to functionally interrogate the relative contributions of the enhancer elements and CTCF looping sites at TAD boundaries. Together these data suggest that these newly juxtaposed enhancer elements initiate and continually drive PAX3-FOXO1 expression, implicating that enhancer miswiring is at the heart of the oncogenic process in FP-RMS. Thus, late myogenic factors (MYOG/MYOD) are contributing to drive an early factor (PAX3), changing a “progressive” enhancer logic into an “infinite loop” enhancer logic. Citation Format: Berkley E. Gryder, Marco Wachtel, Hsien-Chao Chou, Young Song, Joana Marques, Beat Schaefer, Javed Khan. Miswired super enhancer logic driving childhood sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4992. doi:10.1158/1538-7445.AM2017-4992