Abstract B25: BET bromodomain inhibition targets adaptive responses to lapatinib in HER2-positive breast cancer
Authors: Stuhlmiller Timothy J. , Miller Samantha M. , Zawistowski Jon S. , Duncan James S. , Angus Steven P. , Granger Deborah A. , Reuther Rachel A. , Collins Kyla A.L. , Shawn Gomez M. , Kuan Pei-Fen , Chen Xin , Sciaky Noah , Johnson Gary L. 
Published: May 2016

Abstract
Small molecule kinase inhibitors that target oncogene-driven cancers often elicit dramatic initial clinical responses, but adaptive responses from the kinome and transcriptome limit their efficacy and generate resistance. Such adaptive bypass responses are driven by the disruption of regulatory feedback and feedforward loops that govern many signaling networks. To understand these responses in the context of HER2-addicted cancer, we used a chemical proteomics method to assay whole-kinome activation dynamics in cells treated with the small molecule HER2/EGFR tyrosine kinase inhibitor lapatinib. We observed a rapid reactivation of oncogenic signaling and a global re-wiring of kinase signaling networks including activation of receptor tyrosine kinases (RTKs) HER3, DDR1, FGFRs, IGF1R, INSR, and multiple Ephrin receptors, as well as intracellular nodes PKC, SRC family kinases and FAK. Targeting compensatory kinases with a series of kinase inhibitors provided variable and incremental enhancement of growth inhibition but could not prevent resistant colony formation. Cell line heterogeneity and functional redundancy among compensatory kinases added further difficulty to effectively predict the best combination therapy.
This presents a dilemma where combinations of two or even three kinase inhibitors would be insufficient to suppress all the bypass tracks that were rapidly induced by HER2 inhibition. RNAseq revealed a 2-fold dysregulation of approximately 20% of expressed genes within 2 days of lapatinib treatment, with many responsive kinases being transcriptionally upregulated. We argued that in order for lapatinib to have a durable effect, the adaptive response itself must be inhibited. We found that by targeting the BET bromodomain family of chromatin readers, we could suppress the transcriptional upregulation of RTKs that reactivate MAPK/AKT and drive the bypass survival tracks through SRC family kinase and FAK signaling. Treatment of HER2-positive cells with three different small molecule BET bromodomain inhibitors (JQ1, I-BET151, and I-BE762) had little effect on HER2 and downstream signaling pathways in the absence of lapatinib and accordingly allowed resistant colony formation in long-term growth assays. Combining BET bromodomain inhibitors with lapatinib caused a loss of signature RTK expression, prevented MAPK/AKT reactivation, and suppressed resistant colonies from forming. BET bromodomain inhibitors also downregulated responsive RTKs in cells made resistant to lapatinib and thus re-sensitized the cells to lapatinib.
Transcriptional profiling defined the effects of BET bromodomain inhibition to be less pronounced than lapatinib, with only 8% of expressed genes being downregulated 2-fold or greater. However, more than 25% of lapatinib-induced genes were downregulated 2-fold, indicating BET bromodomains preferentially modulated the induced gene expression. Current literature describes a crucial role for the BET bromodomain protein BRD4 in the regulation and stability of large enhancer regions (super-enhancers) responsible for oncogene regulation and defining cell fate. ChIPseq analysis of enhancer dynamics in response to lapatinib and JQ1 identified a reorganization of the chromatin landscape within 24 hours of drug treatment, with multiple super-enhancers formed and decommissioned. Importantly, the combination of lapatinib and JQ1 caused a loss of the majority of super-enhancers observed in untreated cells. Together, these results demonstrate epigenetic targeting of adaptive response mechanisms can prevent resistance to small molecule kinase inhibitors and provide a more durable effect for oncogene targeting in the treatment of cancer.
Citation Format: Timothy J. Stuhlmiller, Samantha M. Miller, Jon S. Zawistowski, James S. Duncan, Steven P. Angus, Deborah A. Granger, Rachel A. Reuther, Kyla A.L. Collins, Gomez M. Shawn, Pei-Fen Kuan, Xin Chen, Noah Sciaky, Gary L. Johnson. BET bromodomain inhibition targets adaptive responses to lapatinib in HER2-positive breast cancer. [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 B25.