PAX3–FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain Vulnerability

Abstract

Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric transcription factor PAX3-FOXO1. The mechanisms by which PAX3-FOXO1 dysregulates chromatin are unknown. We fi nd PAX3-FOXO1 reprograms the cis -regulatory landscape by inducing de novo super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a signifi cant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specifi c vulnerability that can be exploited for precision therapy.SIGNIFICANCE: PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatinlevel functions are critical to understanding its oncogenic activity. We fi nd that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. Cancer Discov; 7(8);

Hotspots of aberrant enhancer activity in fibrolamellar carcinoma reveal molecular mechanisms of oncogenesis and intrinsic drug resistance

“…As super enhancers have been shown to regulate cell identity and oncogenes Lovén et al, 2013), our findings suggest that these FLC-specific enhancer hotspots may regulate genes important in the formation, progression, and/or maintenance of this cancer. Super enhancers are regulated by the bromodomain protein BRD4 (Lovén et al, 2013), and BRD4 inhibitors such as JQ1 (Filippakopoulos et al, 2010) have been shown to disrupt super enhancer function (Gryder et al, 2017;Lovén et al, 2013;Mack et al, 2018;Peeters et al, 2015). Pharmacological disruption of the FLC-specific enhancer hotspots we have described here may represent an alternative therapeutic approach for FLC.…”

Relationship of DNA methylation to mutational changes and transcriptional organization in fusion‐positive and fusion‐negative rhabdomyosarcoma

“…It is proposed that the PAX‐FOXO1 proteins mediate their transcriptional impact by recruiting other transcription factors and chromatin‐binding proteins, such as BRD4, to form super‐enhancers. Moreover, transcription may be driven by three‐dimensional looping that brings PAX‐FOXO1‐associated super‐enhancers to promoters and enables physical interaction of super‐enhancer‐bound proteins with promoters . As the lack of DNA methylation is fundamental to open chromatin states, we hypothesize that promoter DNA hypomethylation may coordinate with PAX‐FOXO1 to drive target gene expression.…”

“…Moreover, transcription may be driven by three-dimensional looping that brings PAX-FOXO1-associated super-enhancers to promoters and enables physical interaction of super-enhancer-bound proteins with promoters. 40 As the lack of DNA methylation is fundamental to open chromatin states, we hypothesize that promoter DNA hypomethylation may coordinate with PAX-FOXO1 to drive target gene expression. In accord with this concept, we observed a significant depletion of DNA methylation in the promoter region in FP compared to FN RMS tumors.…”

Targeting the PI3K/AKT pathway overcomes enzalutamide resistance by inhibiting induction of the glucocorticoid receptor

“…Epigenetic processes such as histone modifications at cis-regulatory elements can affect gene transcription independent of their orientation or distance via enhancers 25 . H3K27ac has been established as an important mark of enhancers which distinguishes between active and inactive regions (active referring to a positive influence on the expression of proximal genes) 26 , hence, regions with deposits of H3K27ac are often associated with enhanced gene activity 26,27 . We performed Chromatin Immunoprecipitation (ChIP) experiments for H3K27ac in the presence of DMSO, ipatasertib, enzalutamide or the combination of both ipatasertib and enzalutamide, with RNA-seq performed in parallel.…”