Abstract B24: Triple-negative breast cancer adaptive response to MEK inhibition is regulated by the induction of super-enhancers
Authors: Zawistowski Jon S. , Miller Samantha M. , Goulet Daniel R. , Stuhlmiller Timothy J. , Singh Darshan , Sciaky Noah , Velarde Sara H. , Angus Steven P. , Johnson Gary L. 
Published: May 2016

Abstract
A predominant mechanism by which cancer cells circumvent the action of targeted kinase inhibitors is the activation of bypass signaling networks that reactivate cellular proliferation. In triple negative breast cancer (TNBC), a chief component of the adaptive response to MEK inhibition is the transcriptional upregulation and activation of receptor tyrosine kinases (RTKs), which contributes to drug resistance by reactivating ERK signaling as well as by initiating growth signaling through other kinase nodes. The cohort of RTKs activated differs between TNBC molecular subtypes and is heterogeneous even among cell lines of the same subtype; thus designing effective inhibitor combinations is challenging. Targeting the chromatin reader protein BRD4 via BET bromodomain inhibition attenuates the transcriptional adaptive response to MEK inhibition, yielding a more durable growth suppression than kinase inhibitor combinations. Yet, a detailed understanding of molecular mechanisms underlying BET bromodomain inhibitor synergy with MEK inhibitor remain unclear. We show by ChIP-seq the dynamic formation of BRD4-dense super-enhancers in response to 24 h MEK inhibition. In SUM159PT claudin-low breast cancer cells, de novo super-enhancers, whose identity were validated by histone H3 lysine 27 acetylation and MED1 co-occupancy with BRD4, were found proximal to genomic loci of DDR1, KDR, and MYLK kinases previously implicated in the adaptive MEK inhibitor response. In sum we identified 110 putative MEK inhibitor-induced SUM159PT super-enhancers, 40% of which reside within 100 kilobases 5' or 3' of a gene whose transcript is upregulated 2 fold or greater by MEK inhibition as assessed by RNA-seq, suggesting that de novo superenhancer formation is a general mode of transcriptional regulation of the adaptive response. Importantly, the MEK inhibitor-induced super-enhancers are disrupted by the BET bromodomain inhibitor JQ1, providing one potential mechanism for the growth suppression synergism obtained by combining MEK + BET bromodomain inhibition. To identify candidate direct DNA binding factors that may be seeding the observed super-enhancer formation, Multiple EM for Motif Elicitation (MEME) analysis identified an enrichment of consensus DNA binding motifs for the enhancer factors CEBPB/D in de novo super-enhancer regions, and the presence of CEBPB was confirmed to be a component of these regions by ChIP-seq. Additionally, we have achieved MEK inhibitor growth suppression synergy with inhibitors of the lysine acetyl transferases CBP/p300, known super-enhancer components. These data provide a new mechanism underlying the transcriptional control of the adaptive response to MEK inhibitor and suggest that targeting drug-induced super-enhancer elements may be a potential therapeutic avenue for TNBC.
Citation Format: Jon S. Zawistowski, Samantha M. Miller, Daniel R. Goulet, Timothy J. Stuhlmiller, Darshan Singh, Noah Sciaky, Sara H. Velarde, Steven P. Angus, Gary L. Johnson. Triple-negative breast cancer adaptive response to MEK inhibition is regulated by the induction of super-enhancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B24.