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The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor–resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogen-activated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients. Mol Cancer Ther; 11(5); 1143–54. ©2012 AACR.

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Supplementary Table S1: X-ray crystallography data processing and structure refinement statistics Supplementary Table S2: BRAF and RAS mutational status of cell lines used in the study Supplementary Table: S3: Cellular IC50 (nM) of parental and ERKi-resistant lines to ERK inhibitors Supplementary Table S4: Mutations found in ERKi- resistant lines. Supplementary Table-S5- Copy number (log2 ratio) in ERKi-R lines compare to parental cell lines. Supplementary Table S6: Gene expression (RPKM) in parental and ERKi-R cells

Purpose: MAPK pathway inhibitors targeting BRAF and MEK have shown clinical efficacy in patients with RAF- and/or RAS-mutated tumors. However, acquired resistance to these agents has been an impediment to improved long-term survival in the clinic. In such cases, targeting ERK downstream of BRAF/MEK has been proposed as a potential strategy for overcoming acquired resistance. Preclinical studies suggest that ERK inhibitors are effective at inhibiting BRAF/RAS-mutated tumor growth and overcome BRAF or/and MEK inhibitor resistance. However, as observed with other MAPK pathway inhibitors, treatment with ERK inhibitors is likely to cause resistance in the clinic. Here, we aimed to model the mechanism of resistance to ERK inhibitors.Experimental Design: We tested five structurally different ATP-competitive ERK inhibitors representing three different scaffolds on BRAF/RAS-mutant cancer cell lines of different tissue types to generate resistant lines. We have used in vitro modeling, structural biology, and genomic analysis to understand the development of resistance to ERK inhibitors and the mechanisms leading to it.Results: We have identified mutations in ERK1/2, amplification and overexpression of ERK2, and overexpression of EGFR/ERBB2 as mechanisms of acquired resistance. Structural analysis of ERK showed that specific compounds that induced on-target ERK mutations were impaired in their ability to bind mutant ERK. We show that in addition to MEK inhibitors, ERBB receptor and PI3K/mTOR pathway inhibitors are effective in overcoming ERK-inhibitor resistance.Conclusions: These findings suggest that combination therapy with MEK or ERBB receptor or PI3K/mTOR and ERK inhibitors may be an effective strategy for managing the emergence of resistance in the clinic. Clin Cancer Res; 24(16); 4044–55. ©2018 AACR.

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Fig. S1. ERK inhibitors and their effect on MAPK mutant cell lines. A. Structure of ERK inhibitors used in this study. B. Dose response curve of ERK inhibitors on the viability of A375, IPC298, SKMEL30, HCT116, MIA PaCa2 and Panc1 cells. Fig. S2. Western blot analysis of parental, VI-3-R and G994-R HCT116 (A) or MIA PaCa2 (B) cells treated with indicated ERK inhibitors. Fig. S3. Western blot analysis of HCT116 cells (A) and SKMEL30 (B) stably expressing indicated ERK1 or ERK2 mutants. Fig. S4. Copy number and expression analysis of MITF in SKMEL30-ERKi-R cells. A. MITF copy gain in SKMEL30-V11e-R, -VI-3-R and -G994-R. B. MITF copy number vs. expression in SKMEL30-V11e-R, VI-3-R and G994-R cells. Fig. S5. Dose response curve of ERK2 overexpressing IPC298 cells treated with MEK inhibitors GDC-0973 or AZD6244 (A) and ERK2 amplified IPC298-G994-R (B) or MIA PaCa2-S984-R (C) cells treated with indicated ERK-inhibitors from alternate scaffold class. Fig. S6. Dose response curve of parental or ERKi-resistant HCT116 (A) and MIA PaCa2 (B) cells treated with indicated inhibitors. The data shown are same as in Fig. 6B for HCT116 (A) and Fig. 6C for MIA PaCa2 (B) grouped by drug treatment.

The mammalian SWI/SNF complex catalyzes the remodeling of chromatin through the helicase activity of two mutually-exclusive, paralogous subunits, BRG1 and BRM. BRG1 is frequently mutated in cancer and its inactivation results in a cellular dependence on BRM. Despite the attractiveness of BRM as a synthetic lethal therapeutic target, the selective inhibition of BRM represents a considerable challenge due to the high degree of homology between BRM and BRG1. Furthermore, published data indicate that achieving such selectivity is likely essential to afford an acceptable therapeutic index. We sought to mimic the synthetic lethality observed in BRG1-mutant cancers by identifying proteolysis-targeting chimera (PROTAC®) molecules capable of selectively degrading BRM via trimeric complex formation with the von Hippel-Lindau (VHL) E3 ligase. In this disclosure, we report our initial discovery of potent and selective chimeric BRM-degrader molecules which exhibit BRM DC50 values 25. Importantly, selective BRM degradation can be achieved in the absence of selective PROTAC® binding (BRG1/BRM Kd ratios Citation Format: Michael Berlin, Jennifer Cantley, Jing Wang, Mark Bookbinder, Gregory Cadelina, Emily Chan, Huifen Chen, Xin Chen, Kim Davenport, Tharu Fernando, Debbie Gordon, Brian Hamman, Roy Haskell, Alexey Ishchenko, Donald S. Kirkpatrick, Jonathan Maher, Mark Merchant, John Moffat, Alicia Morgan, An Nguyen, Jennifer Pizzano, Connor Quinn, Christopher M. Rose, Emma Rousseau, Vijay Sethuraman, Leanna Staben, Catherine Wilson, Xiaofen Ye, Fabio Broccatelli, Robert Yauch, Peter S. Dragovich. Selective, chemically-induced degradation of BRM (SMARCA2) enables in vivo efficacy in BRG1 (SMARCA4)-deficient xenograft tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5687.

The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor–resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogen-activated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients. Mol Cancer Ther; 11(5); 1143–54. ©2012 AACR.

MAPK pathway inhibitors targeting BRAF and MEK have shown clinical efficacy in patients with RAF and/or RAS mutated tumors. However, acquired resistance to these agents has been an impediment to improved long-term survival in the clinic. In such cases, targeting ERK downstream of BRAF/MEK has been proposed as a potential strategy for overcoming acquired resistance. Preclinical studies suggest that ERK inhibitors are effective at inhibiting BRAF/RAS mutated tumor growth and overcome BRAF or/and MEK inhibitor resistance. However, as with other MAPK pathway inhibitors, treatment with ERK inhibitors is likely to cause resistance in the clinic. In this study, we have used in vitro modeling, structural biology and genomic analysis to understand development of resistance to ERK inhibitors and the mechanisms leading to it. We have identified mutations in ERK1/2, amplification and overexpression of ERK2, and overexpression of EGFR/ERBB2 as mechanisms of acquired resistance. Structural analysis of ERK showed that specific compounds that induced on-target ERK mutations were impaired in their ability to bind mutant ERK. We show that in addition to MEK inhibitor, ERBB-receptor and PI3K/mTOR pathway inhibitors are effective in overcoming ERK-inhibitor resistance. These findings suggest that combination therapy with MEK or ERBB-receptor or PI3K/mTOR and ERK inhibitors may be an effective strategy for managing the emergence of resistance in the clinic. Citation Format: Bijay S. Jaiswal, Steffen Durinck, Eric W. Stawiski, Jianping Yin, Weiru Wang, Eva Lin, John Moffat, Scott E. Martin, Zora Modrusan, Somasekar Seshagiri. ERK mutations and amplification confer resistance to ERK-inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-093.

KRAS mutations occur in approximately 25% of NSCLC (1). Tumors with these mutations are predicted to be sensitive to MEK inhibition due to activation of MAPK signaling. However, MEK inhibitors in multiple clinical trials, either as a monotherapy or in combination with chemotherapies, have not shown superior efficacy in the KRAS mutant subgroup when compared to the KRAS wild-type subgroup, indicating a limitation of utilizing KRAS mutation status as a predictive biomarker of efficacy to MEK inhibition (2, 3). Furthermore, stratification based on KRAS mutation status may inadvertently miss wild-type KRAS tumors that could be addicted to MAPK signaling regardless of KRAS mutation status, thus denying patients potential benefit from MEK inhibitors. Here we describe a novel predictive model that more accurately forecasts the sensitivity of the KRAS wild-type NSCLC subpopulation to MEK inhibitors such as cobimetinib and trametinib. Cell viability data from cobimetinib or trametinib-treated cells, with concomitant gene expression data (RNAseq), from 46 colon, 106 lung, and 37 pancreatic cell lines were used to create an elastic net regression model trained on gene expression features (alpha = 0.5 and optimal lambda chosen by 5-fold cross validation) (4). From the model, we established two distinct predictive gene lists: (1) a longer low cross-validation list, and (2) a shorter low error list. Initial analysis of the model demonstrated that predicted mean viabilities of the cell lines used to create the model correlated well with their actual mean viabilities (R: 0.65-0.7 for trametinib and cobimetinib respectively). Predicted mean viabilities of 40 previously unscreened NSCLC cell lines were then generated by the model based on their expression features (RNAseq). The 40 cell lines were categorized either as sensitive or resistant by the median of predicted mean viabilities derived from the model. Subsequently, the actual experimental GI50 values of cobimetinib were obtained for each of these cell lines. We found that KRAS mutational status predicted that only 8 of the 40 cell lines screened would be sensitive to MEK inhibition. In contrast, our model predicted that 15 of the 40 cell lines screened would be sensitive. Experimentally, we demonstrated that 24 of the cell lines were sensitive to MEK inhibition with a measured GI50 References (1) Blumenschein GR. et al., (2015) Annal Oncol. 26(5):894-901. (2) Gandara DL et al., (2013) J Clin Oncol 31, (suppl; abstr 8028). (3) Laethem JLV et al, (2014) J Clin Oncol 32:5s, (suppl; abstr 4025). (4) Barretina J. et al. (2012) Nature 483, 603-607. Citation Format: Marie Wagle, Christiaan Klijn, Bonnie Liu, Shilpi Mahajan, Peter Haverty, John Moffat, Mark Merchant, Bob Yauch, Garret Hampton, Lukas Amler, Mark Lackner, Shih-Min A. Huang. A novel predictive biomarker model for MEK sensitivity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 417.

The extracellular-signal-regulated kinases (ERK1 and ERK2) represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that commonly is activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling, such as receptor tyrosine kinase (RTK) activation. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Here, we present the discovery and characterization of GDC-0994, an orally bioavailable, small molecule inhibitor of ERK kinase activity. GDC-0994 is highly selective for ERK1 and ERK2, with biochemical potency of 1.1 nM and 0.3 nM, respectively. Daily, oral dosing of GDC-0994 results in significant single-agent activity in multiple in vivo cancer models, including KRAS-mutant and BRAF-mutant human xenograft tumors in mice. PD biomarker inhibition of phospho-p90RSK in these tumors correlates with potency in vitro and in vivo. In contrast to other published ERK inhibitors, GDC-0994 neither increases nor decreases phospho-ERK, suggesting that different ERK inhibitors have alternative mechanisms of action with respect to feedback signaling. Furthermore, we demonstrate a novel approach for targeting the oncogenic signaling through the RAS pathway by combining ERK and MEK inhibitors. GDC-0994 is currently in Phase I clinical development. Citation Format: Kirk Robarge, Jacob Schwarz, Jim Blake, Michael Burkard, Jocelyn Chan, Huifen Chen, Kang-Jye Chou, Dolores Diaz, John Gaudino, Stephen Gould, Jonas Grina, Xin Linghu, Lichuan Liu, Matthew Martinson, David A. Moreno, Christine Orr, Patricia Pacheco, Ann Qin, Kevin Rasor, Li Ren, Sheerin Shahidi-Latham, Jeffrey Stults, Francis Sullivan, Weiru Wang, Peter Yin, Aihe Zhou, Marcia Belvin, Mark Merchant, John G. Moffat. Discovery of GDC-0994, a potent and selective ERK1/2 inhibitor in early clinical development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr DDT02-03. doi:10.1158/1538-7445.AM2014-DDT02-03

Targeting cancer metabolism is currently being evaluated as a potential therapeutic target. Identification of tumor subtypes with specific metabolic requirements will enable the success of such therapies. Using cell line models of pancreatic ductal adenocarcinoma (PDAC), we conducted broad metabolite profiling and identified 3 metabolic subtypes through non-negative matrix factorization (NMF) consensus clustering. Among the most distinguishing features between the subtypes were levels of metabolites important for lipid synthesis, amino acid, and energy-related pathways, suggestive of distinct metabolic dependencies. Using RNAseq we show that these subtypes are defined at the transcriptional level and are associated with known subtypes of PDAC. In metabolic flux experiments, we also show that glucose and glutamine are differentially utilized by each subtype, and as a consequence, exhibit distinct sensitivity to inhibition of various metabolic pathways. Finally, through screening of a large panel of tumor cell lines with various metabolic inhibitors, we cross-validate our finding in PDAC and provide strong evidence that these same subtypes exist and can be identified in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity to metabolic inhibitors in cancer cells. Citation Format: Anneleen Daemen, David Peterson, Nisebita Sahu, Ron McCord, Kaska Kowanetz, Anna Hitz, Xiangnan Du, Bonnie Liu, Min Gao, John Moffat, Rebecca Hong, Deepak Sampath, Mark Merchant, Thomas O'Brien, Bob Yauch, Jeff Settleman, Jing Qing, Georgia Hatzivassiliou, Marie A. Evangelista. Identification of distinct metabolic subtypes within pancreatic ductal adenocarcinoma through broad metabolite profiling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1430. doi:10.1158/1538-7445.AM2014-1430

Lactate Dehydrogenase A (LDHA) is an attractive candidate for targeting glycolysis-addicted tumors. However, due to the inherent plasticity of metabolic networks in cells, there is concern that the benefit of targeting LDHA may be transient and that resistance will quickly emerge. To identify predictive features of LHDA inhibitor sensitivity and to understand how cells adapt to long-term LHDA inhibition, we screened a large panel (∼500) of tumor cell lines with GNE-140, a newly developed LHDA inhibitor. We found that approximately 15% of lines were inherently sensitive to the LDHA inhibitor, with sensitivity correlating with increased expression of glycolysis genes and inversely correlating with expression of oxidative phosphorylation genes. Despite the metabolic plasticity of cells, the timing of acquired resistance to LDHA inhibitors was comparable with other targeted agents. Under long-term LDHAi treatment, glycolytic cells acquired resistance by increased oxidative phosporylation (OX-PHOS) in a mechanism dependent on the AMPK stress response pathway; targeting either AMPK, downstream kinases, or OX-PHOS using tool compounds synergized with and prevented acquired resistance to GNE-140. Taken together, our data suggests that targeting anaerobic glycolysis may benefit a subset of patients across indications and that combinations with agents that block AMPK signaling or the mitochondria will be effective at delaying tumor relapse. Citation Format: Aaron Boudreau, David Peterson, John Moffat, Bonnie Liu, Mandy Kwong, Min Gao, Hans Purkey, Thomas O'Brien, Georgia Hatzivassiliou, Anneleen Daemen, Marie Evangelista. Resistance to LDHA inhibitors requires signaling through the AMPK/mTOR/S6K pathway leading to increased oxidative phosphorylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1423. doi:10.1158/1538-7445.AM2014-1423

The extracellular-signal-regulated kinases (ERK-1 and ERK-2) represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated due to oncogenic mutations in RAF or RAS or due to upstream oncogenic signaling, such as through receptor tyrosine kinase (RTK) signaling. We have discovered and developed potent and selective small molecule inhibitors of both ERK-1 and 2. Optimization of kinase selectivity was a critical goal in order for ATP-competitive ERK inhibitors to maintain parity with the safety profile of highly selective allosteric MEK inhibitors. As a complement to enzymatic kinase panel profiling, we employed an imaged-based cell cycle assay, based on the expected on-target phenotype of cytostatic G1 arrest in B-Raf-mutant cell lines. We were thus able to simultaneously measure on-target and off-target activities. Inhibition of Cdk2, one of the primary off-targets, was associated with a distinct G2 cell cycle arrest, which enabled us to quantify the functional selectivity margin between ERK and Cdk2 in a single assay, and select ERK inhibitors with acceptable selectivity margins for further development. Note: This abstract was not presented at the meeting. Citation Format: Grace Ka-Yan Chan, Tracy Kleinheinz, Matthew Martinson, Hok-Sum Cheung, John G. Moffat. Phenotypic profiling and selectivity optimization of ERK inhibitors using a high-throughput imaged-based cell cycle assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 740. doi:10.1158/1538-7445.AM2014-740

Background: Acquired resistance to antitumor therapies underlies one of the challenges in effectively treating cancer. Understanding the mechanisms of acquired drug resistance may lead to elucidation of redundant oncogenic pathways and the development of more effective therapies. The RAS/RAF/MEK pathway is active in over 30% of human tumors, often due to mutation in BRAF or RAS family members. Several MEK inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in various stages of clinical development. These inhibitors are allosteric and act by binding to a hydrophobic pocket adjacent to the ATP binding pocket to inhibit MEK kinase activity. Preclinical studies using randomly mutagenized MEK cDNAs have identified mutations in MEK that upon transfection in MEK sensitive lines can render them resistant to MEK inhibitors. The strongest effects were observed with mutations affecting the allosteric binding pocket, thus blocking inhibitor binding. Results: To model acquired resistance to MEK inhibitors 3 independent KRAS mutant tumor cell lines (breast MDA-MB-231, colon HCT116 and LoVo) were grown in the presence of increasing concentrations of the selective MEK inhibitor PD0325901 and pools of MEK resistant cells were characterized versus parental cells. MEK resistant cells were still dependent on the MAPK pathway and sensitive to treatment with inhibitors against the downstream kinase ERK. In all three lines, MEK resistance was associated with acquisition of mutations in the MEK allosteric binding pocket, resulting in lack of signaling inhibition and suggesting that such resistance can arise relatively rapidly and in distinct tumor types. To compare mechanisms of acquired resistance to ERK inhibitors, we also generated HCT116 cells resistant to a selective ERK inhibitor. ERK resistant lines remained sensitive to MEK inhibitors and dependent on the MAPK pathway. Conclusions: Acquired resistance to MEK inhibitors in KRAS mutant cells arises through mutations in MEK that block inhibitor binding so that the inhibitor is no longer effective. In this case, tumors remain dependent on the MAPK pathway and can be targeted by inhibitors that act downstream of mutated target. These studies provide the rationale for future screening of patients with acquired MEK inhibitor resistance for mutations in MEK1 and MEK2. Patients whose resistance is driven through MEK mutations may benefit from treatment with an ERK inhibitor. Combination of MEK and ERK inhibitors may potentially prevent or delay the onset of resistance in MAPK-addicted tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4734. doi:10.1158/1538-7445.AM2011-4734