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MEK Inhibitors Overcome Resistance to BET Inhibition Across a Number of Solid and Hematologic Cancers Anastasia Wyce1, Jeanne J

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MEK Inhibitors Overcome Resistance to BET Inhibition Across a Number of Solid and Hematologic Cancers Anastasia Wyce1, Jeanne J Wyce et al. Oncogenesis (2018) 7:35 DOI 10.1038/s41389-018-0043-9 Oncogenesis ARTICLE Open Access MEK inhibitors overcome resistance to BET inhibition across a number of solid and hematologic cancers Anastasia Wyce1, Jeanne J. Matteo1,ShawnW.Foley1, Daniel J. Felitsky2, Satyajit R. Rajapurkar1, Xi-Ping Zhang1, Melissa C. Musso1,SusanKorenchuk1, Natalie O. Karpinich1, Kathryn M. Keenan1, Melissa Stern1, Lijoy K. Mathew1, Charles F. McHugh1, Michael T. McCabe1, Peter J. Tummino1,3,RyanG.Kruger1, Christopher Carpenter1 and Olena Barbash1 Abstract BET inhibitors exhibit broad activity in cancer models, making predictive biomarkers challenging to define. Here we investigate the biomarkers of activity of the clinical BET inhibitor GSK525762 (I-BET; I-BET762) across cancer cell lines and demonstrate that KRAS mutations are novel resistance biomarkers. This finding led us to combine BET with RAS pathway inhibition using MEK inhibitors to overcome resistance, which resulted in synergistic effects on growth and survival in RAS pathway mutant models as well as a subset of cell lines lacking RAS pathway mutations. GSK525762 treatment up-regulated p-ERK1/2 levels in both RAS pathway wild-type and mutant cell lines, suggesting that MEK/ERK pathway activation may also be a mechanism of adaptive BET inhibitor resistance. Importantly, gene expression studies demonstrated that the BET/MEK combination uniquely sustains down-regulation of genes associated with mitosis, leading to prolonged growth arrest that is not observed with either single agent therapy. These studies 1234567890():,; 1234567890():,; highlight a potential to enhance the clinical benefit of BET and MEK inhibitors and provide a strong rationale for clinical evaluation of BET/MEK combination therapies in cancer. Introduction inhibitors including transcriptional suppression of onco- BET proteins (BRD2, BRD3, BRD4, and BRDT) mod- genes3, there is no consensus and it is likely that ulate expression of genes involved in cell growth and mechanisms vary, thus making the identification of pre- oncogenesis by binding to acetylated chromatin via their dictive biomarkers difficult. bromodomains, which in turn recruit downstream effec- Although BET inhibitors show broad activity in many tors that promote transcription. Selective BET inhibitors, cancer types, within each there are resistant models. such as I-BET151 and the clinical molecule GSK525762 Understanding the basis of BET inhibitor sensitivity and (I-BET762; I-BET)1,2, abrogate binding of BET proteins to resistance is important to inform the clinical development acetylated chromatin thereby inhibiting BET-dependent of BET inhibitors as monotherapies and to identify transcription1,2. BET inhibitors exhibit broad anti- rational combinations. To this end, we analyzed genetic proliferative activity in cancer models3. Although several data from a large set of cell lines treated with GSK525762 mechanisms have been implicated in the efficacy of BET to identify biomarkers of sensitivity and resistance. From these studies, we identified KRAS mutations as a sig- nificant predictor of resistance to BET inhibition. This led Correspondence: Olena Barbash ([email protected]) us to hypothesize that combinations with inhibitors of 1 Cancer Epigenetics DPU, Oncology R&D, GlaxoSmithKline, Collegeville, PA, RAS signaling, such as MEK inhibitors, may further USA 2Target Sciences, GlaxoSmithKline, Upper Merion, PA, USA improve upon BET inhibitor efficacy. Indeed, we observed Full list of author information is available at the end of the article © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Oncogenesis Wyce et al. Oncogenesis (2018) 7:35 Page 2 of 12 broad synergistic effects for BET/MEK combinations inhibitor combinations in cancer cell lines with and across cancer models, which we attribute to profound and without RAS pathway mutations (mutations in RAS, sustained inhibition of MEK/ERK signaling that is speci- BRAF, or NF1), and with varying levels of sensitivity to fically observed with the combination ultimately leading GSK525762. For these studies, we utilized the BET inhi- to growth arrest and cell death. bitor I-BET151, (pharmacological profile highly similar to GSK5257621; Supplemental Figure S3), and the clinical Results MEK inhibitor PD0325901. Combination of I-BET151 RAS mutations are novel biomarkers of resistance to and PD0325901 produced synergistic effects (CI < 0.78; GSK525762 see Methods) most commonly in, but not limited to, RAS To identify genetic predictors of sensitivity or resistance pathway mutant models. Synergy was observed in multi- to BET inhibitors we first examined the anti-proliferative ple tumor types (Fig. 2a, Supplemental Table S3), with the activity of GSK525762 in ~230 cancer cell lines. Hema- combination improving growth inhibition (gIC50, gIC100) tologic cancer cell lines were highly sensitive (low growth and/or cytotoxicity (dEC50) even in cell lines sensitive to IC50 values and net cell death) to GSK525762, whereas single agent BET inhibition (Fig. 2b, c, Supplemental solid tumor models exhibited a wide range in sensitivity Table S1, S3). Across all cell lines evaluated, there was a (gIC50 13 nM to > 29.3 µM; partial, cytostatic, and cyto- significant correlation (p = 0.022; Fisher’s exact test) toxic responses), providing an opportunity to compare the between RAS pathway mutations and synergy (Fig. 2a; genetic profiles of a large number of sensitive and resis- Supplemental Table S3). However, synergy was not tant lines (Fig. 1a, b, Supplemental Table S1). Using restricted to mutant models, suggesting that additional publicly available data for 19,312 genes with protein- mechanisms may drive combination effects. changing mutations, we performed unbiased analyses of Given the reported variability in biological activity of genetic predictors of sensitivity or resistance to MEK inhibitors based on differing mechanisms of inhi- 4 GSK525762 based on gIC50 values. These analyses iden- bition , we evaluated combinations with additional MEK tified 634 genes with protein-changing mutations that inhibitors in cell lines that exhibited synergistic responses correspond to resistance or sensitivity to GSK525762 to I-BET151 and PD0325901. Synergy was observed with (Wilcoxon rank sum test p < 0.05; Supplemental Table S2, a variety of structurally distinct MEK inhibitors, including Supplemental Figure S1). Notably, KRAS mutations GDC-0623, trametinib, and cobimetinib (Supplemental were among the top five most significant (p = 0.0001) Figs. S4A-4B). Importantly, combination of the clinical associations, with a clear enrichment of KRAS mutations BET inhibitor GSK525762 with therapeutic concentra- 5 among the more resistant cell lines (median gIC50 tions of trametinib resulted in decreased cell viability KRAS mutant = 1667 nM vs WT = 550 nM; Fig. 1c). (Supplemental Figure S4C). Analysis of mutations at the amino acid level further To determine if in vitro synergy translates into identified KRAS G12 missense mutations as significantly improved in vivo efficacy, we profiled the activity of the (p = 0.00006) associated with resistance (Supplemental BET/MEK combination in xenografts representing var- Figure S2). A similar association (p = 0.004) between ious tumor types and RAS pathway mutations. Combi- KRAS mutations and resistance was observed in colorectal nation of GSK525762 and trametinib significantly delayed cancer (CRC) cell lines, where KRAS mutations are fre- tumor growth in xenograft models of CRC (RKO; quent (mutations in 13/22 lines; 59%) (Fig. 1d), indicating BRAFmut, NF1mut), triple-negative breast cancer (TNBC; that the association with resistance is not driven by the MDA-MB-231; KRASmut, BRAFmut, NF1mut), and multi- general responsiveness of individual tumor types. Finally, ple myeloma (MM; RPMI-8226; KRASmut) (Fig. 2d; Sup- KRAS mutations were significantly depleted (p = 0.003; plemental Figures S5-S7), and significantly improved Fisher’s exact test) among the cell lines exhibiting net cell tumor growth inhibition in a xenograft model of pan- death in response to GSK525762 (Supplemental Table creatic cancer (PaCa; HPAF-II; KRASmut) (Supplemental S1), suggesting that activated RAS signaling may be a Figure S8) at well-tolerated doses (Supplemental Figure mechanism by which cancer cells are able to survive BET S9). inhibition. Synergy for BET/MEK combinations in RAS pathway wild Broad synergy for combinations of BET and MEK inhibitors type cell lines in RAS pathway mutant cancer cell lines Although RAS pathway mutations predict resistance to The strong association between KRAS mutations and BET inhibitors and sensitivity to BET/MEK combinations, resistance to GSK525762 suggested that agents targeting we also observed synergy
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