ERK Mutations and Amplification Confer Resistance to ERK-Inhibitor Therapy

Published OnlineFirst May 14, 2018; DOI: 10.1158/1078-0432.CCR-17-3674 Cancer Therapy: Preclinical Clinical Cancer Research ERK Mutations and Amplification Confer Resistance to ERK-Inhibitor Therapy Bijay S. Jaiswal1, Steffen Durinck1, Eric W. Stawiski1, Jianping Yin2, Weiru Wang2, Eva Lin3, John Moffat4, Scott E. Martin3, Zora Modrusan1, and Somasekar Seshagiri1 Abstract Purpose: MAPK pathway inhibitors targeting BRAF and MEK types to generate resistant lines. We have used in vitro modeling, have shown clinical efficacy in patients with RAF- and/or structural biology, and genomic analysis to understand the RAS-mutated tumors. However, acquired resistance to these development of resistance to ERK inhibitors and the mechanisms agents has been an impediment to improved long-term survival leading to it. in the clinic. In such cases, targeting ERK downstream of Results: We have identified mutations in ERK1/2, amplifica- BRAF/MEK has been proposed as a potential strategy for tion and overexpression of ERK2, and overexpression of overcoming acquired resistance. Preclinical studies suggest that EGFR/ERBB2 as mechanisms of acquired resistance. Structural ERK inhibitors are effective at inhibiting BRAF/RAS-mutated analysis of ERK showed that specific compounds that induced on- tumor growth and overcome BRAF or/and MEK inhibitor resis- target ERK mutations were impaired in their ability to bind tance. However, as observed with other MAPK pathway inhib- mutant ERK. We show that in addition to MEK inhibitors, ERBB itors, treatment with ERK inhibitors is likely to cause resistance in receptor and PI3K/mTOR pathway inhibitors are effective in the clinic. Here, we aimed to model the mechanism of resistance overcoming ERK-inhibitor resistance. to ERK inhibitors. Conclusions: These findings suggest that combination therapy Experimental Design: We tested five structurally different with MEK or ERBB receptor or PI3K/mTOR and ERK inhibitors ATP-competitive ERK inhibitors representing three different scaf- may be an effective strategy for managing the emergence of folds on BRAF/RAS-mutant cancer cell lines of different tissue resistance in the clinic. Clin Cancer Res; 1–12. Ó2018 AACR. Introduction human cancers (7, 8). However, efforts to directly target RAS have not been successful so far (9–11). Several small-molecule inhi- The RAS/RAF/extracellular signal–regulated kinase (ERK) path- bitors that target key effector kinases of MAPK signaling cascade way is extensively studied owing to its involvement in the regu- downstream of RAS have been successfully developed (9, 12). Key lation of cell proliferation, differentiation, and survival (1). The FDA-approved MAPK pathway inhibitors include vemurafenib RAS–MAPK signaling cascade involves an upstream receptor and dabrafenib, which target BRAF, and trametinib, AZD6244 tyrosine kinase (RTK) that upon activation sequentially activates (selumetinib), and GDC-0973 (cobimetinib), which target MEK RAS GTPase, which in turn activates the RAF kinases (MAP3K; (13–16). In the clinic, these inhibitors have led to improved ref. 2). The RAF kinases phosphorylate and activate MEK progression-free survival and overall survival of melanoma and (MAP2K), which then phosphorylates ERK (MAPK) leading to colorectal cancer patients, either as single agents or as combina- its activation (1, 3, 4). Activated ERK then phosphorylates many tion therapy (13–16). However, despite their effectiveness and downstream targets, thereby controlling cellular proliferation, therapeutic successes, a majority of patients relapse within a year differentiation, and survival (1, 5, 6). due to acquired resistance to these agents (17). Analysis of drug- Gain-of-function mutations in RAS and BRAF leading to con- resistant tumors from patients showed reactivation of MEK/ERK stitutive activation of the MAPK pathway occur in about a third of signaling and sustained ERK activation involving multiple mechanisms (18–22). Acquired resistance to BRAF inhibitors has been shown to occur through acquisition of NRAS or KRAS 1Molecular Biology Department, Genentech Inc., South San Francisco, California. mutations (18, 23, 24), amplification of BRAF V600E (24), 2Department of Structural Biology, Genentech Inc., South San Francisco, Cali- alternative splicing of BRAF (20), mutations that arise in MEK1 fornia. 3Discovery Oncology Department, Genentech Inc., South San Francisco, 4 or MEK2 (25), and loss of CDKN2A (23). Resistance to MEK California. Department of Biochemical and Cellular Pharmacology, Genentech inhibitors is known to occur due to MEK mutations (26, 27) or Inc., South San Francisco, California. BRAF amplification (28). Note: Supplementary data for this article are available at Clinical Cancer Preclinical studies suggest that ERK inhibition may be effec- Research Online (http://clincancerres.aacrjournals.org/). tive in targeting RAS-mutated tumors (29–31). Also, ERK Corresponding Author: Bijay S. Jaiswal, Genentech Inc., South San Francisco, inhibition has been shown to be effective in overcoming CA 94080. Phone: 1-650-4671898; E-mail: [email protected]; and Somasekar acquired resistance to BRAF/MEK inhibitors (29, 30). Several Seshagiri, [email protected] ERK inhibitors including GDC-0994, MK-8353, LTT462, and doi: 10.1158/1078-0432.CCR-17-3674 BVD-523areinvariousstagesofclinicaldevelopment(32–35). Ó2018 American Association for Cancer Research. ERK inhibitors will expand the choice of targeted therapy for www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst May 14, 2018; DOI: 10.1158/1078-0432.CCR-17-3674 Jaiswal et al. concentration of indicated inhibitors. Cell growth was assessed Translational Relevance after 4 days using Cell Titer-Glo Luminescent cell viability assay kit Acquired resistance to targeted cancer therapy remains a (Promega). All cell viability data shown were mean Æ SEM of at major challenge in the clinic. ERK inhibitors are under inves- least 3 to 6 replicates of a representative experiment that was tigation for treatment of RAF/RAS-mutated tumors or those repeated at least 2 times with similar results. IC50 values were resistant to BRAF/MEK inhibitors. Understanding the evolu- determined by fitting nonlinear regression curves using GraphPad tion of resistance to current ERK inhibitors will help guide the Prism 5.00 Software (GraphPad). development of better inhibitors and also aid in identifying strategies for combination therapy that can overcome clinical Western blot analysis resistance development. Western blotting was performed as described earlier (41). Briefly, 24 hours after treatment with the indicated drugs, cells were washed with cold PBS and lysed in the RIPA lysis buffer containing protease inhibitor (Roche) and PhosStop phosphatase MAPK pathway–deregulated cancers and also for treating inhibitor (Roche). Lysates were centrifuged at 10,000 Â g for tumors resistant to BRAF/MEK inhibitors. However, as 20 minutes at 4C. Proteins were resolved by SDS-PAGE and observed with other small-molecule inhibitors, tumors treated transferred to a nitrocellulose membrane using iBlot (Thermo with ERK inhibitors will likely develop resistance. Consistent Fisher Scientific), immunoblotted with indicated antibodies, with this, recent studies using mutagenesis and in vitro experi- HRP-conjugated secondary antibodies (Thermo Fisher Scientific), ments showed development of on-target resistance to ERK and detected with super signal chemiluminescence (Thermo inhibitors (36, 37). Fisher Scientific) as described earlier (41). Using ERK-inhibitor–sensitive cancer cell lines, we have follow- ed the development of resistance upon treatment with multiple Extraction of DNA/RNA ERK inhibitors. In this study, we applied whole-exome sequencing Genomic DNA and total RNA were simultaneously extracted (WES), transcriptome sequencing (RNA-seq), and whole-genome from cell pellets using the All Prep DNA/RNA mini kit (Qiagen). sequencing (WGS) to understand mechanisms of acquired resistance to ERK inhibition. We found on-target and off-target mechan- WES and variant calling isms of resistance and identified strategies for overcoming or We performed WES of parental and resistant cells to identify managing ERK resistance using the resistant cell lines. acquired resistance mutations. Exome capture was performed using the SureSelect Human All Exome kit (50 Mb; Agilent Technologies), and resulting libraries were sequenced on HiSeq Materials and Methods 2500 (Illumina) to generate 2 Â 75-bp-long paired-end data. Cell lines and antibodies A targeted mean coverage of 111Â with 80% bases covered at A375, HCT116, MIA PaCa-2, and Panc1 cell lines were pur- 20Â was achieved for the exome libraries. Sequencing reads chased from the ATCC. SKMEL30 and IPC298 were obtained from were mapped to the human genome (GRCh38) using BWA German Collection of Microorganisms and Cell Cultures software set to default parameters. Local realignment, duplicate (DSMZ). MelBR1 cell line was generated as described previously marking, and raw variant calling were performed as described (38, 39). Antibodies used in this study are as follows: p-ERK1/2 previously (42). Somatic variants were called by both Strelka (43) (Thr202/Tyr204), pS6-ribosomal protein (Ser235/6), ERK1/2, and MuTect (44), and mutants reported by both programs were RSK, and S6- ribosomal protein (Cell Signaling Technology); included for further evaluation. For on-target mutations, we pRSK (Ser359/363; Abcam); FLAG-M2 and

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