Abstract B21: Suppression of adaptive resistance mechanisms to trametinib by inhibition of BET bromodomains in TNBC

Abstract

Abstract The RAF-MEK-ERK signaling pathway regulates the proliferation of Triple Negative Breast Cancer (TNBC). Although MEK inhibition by trametinib in TNBC cell lines and mouse models inhibits growth, the response is not durable and the tumor develops resistance. The lack of durability of targeted kinase inhibitors is a common problem in many tumor types due to induction of alternative bypass signaling pathways that lead to resistance. We have profiled the response of TNBC to MEK inhibition using trametinib in order to identify adaptive signaling pathways which drive resistance. We used a chemical proteomics method [multiplexed inhibitor beads coupled to mass spectrometry (MIB/MS)], to quantify global changes in kinase activity coupled with transcriptional changes in response to trametinib using RNAseq. Our studies revealed that adaptive resistance to trametinib occurs through increased expression and activation of multiple receptor tyrosine kinases, leading to reactivation of ERK. These adaptive kinases are heterogeneous across TNBC including PDGFRβ, VEGFR2, DDR1, AXL, KIT and FGFR2. There is no single targeted kinase inhibitor able to block all the adaptive kinases induced and activated, making the design of combination targeted kinase inhibitor therapies impractical. We therefore hypothesized an alternative therapeutic approach was required to inhibit adaptive reprogramming. Our goal was to block the initial transcriptional response to trametinib which upregulates adaptive kinase expression. Several studies in the lab determined that we could block adaptive kinase expression by inhibiting BET family bromodomain proteins that interact with acetylated histones and facilitate the formation of transcriptional regulatory complexes. Our work demonstrated that BET family bromodomain inhibitors (JQ1 and iBET151), synergize with trametinib to prevent TNBC growth in short-term (4 days) and long-term (4 weeks) growth assays. Using MIB/MS we found that combination trametinib/BET bromodomain inhibitor treatment blocks upregulation of adaptive kinase expression and activation. RNAseq demonstrated that BET bromodomain inhibitors synergize with trametinib to block the induction of kinases expressed as part of the adaptive bypass response. ChIP-seq of the BET bromodomain protein BRD4 and H3K27ac, a marker of active enhancers, showed BRD4 is recruited to super-enhancer regions proximal to adaptive response kinases as early as 4hr following trametinib treatment. Furthermore, the recruitment of BRD4 to enhancer regions is maintained in cells made resistant to trametinib by constant culture in low-dose drug. In sum, these results suggest that BRD4 recruitment to enhancer regions, following trametinib treatment, leads to the formation of transcriptional complexes which maintain adaptive gene expression. Ongoing studies seek to understand the mechanism by which BRD4 is recruited to super-enhancers following trametinib treatment in TNBC. Mouse xenograft studies also show promising evidence of enhanced tumor growth suppression in vivo using combination trametinib/iBET151 treatment. The results presented provide a novel treatment strategy to block adaptive resistance and have the potential to lead to durable clinical responses of TNBC. Citation Format: Samantha M. Miller, Jon S. Zawistowski, Timothy J. Stuhlmiller, Noah Sciaky, Charlene M. Santos, David B. Darr, Gary L. Johnson. Suppression of adaptive resistance mechanisms to trametinib by inhibition of BET bromodomains in TNBC. [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 B21.