MEK Inhibitor Response Research Articles

Abstract P183: CRISPR screens identify sensitizers to trametinib in KRAS mutant cancer cell lines

Abstract KRAS is the most frequently mutated oncogene in human tumors and drives tumorigenesis across multiple lineages. Specifically, KRAS is mutated in about 30% of lung cancer and over 90% of pancreatic cancer. Despite recent progress in developing mutant selective KRASG12C inhibitors, patient treatment options for RAS activated cancers remain very limited. Targeting the MAPK pathway by means of MEK and ERK inhibitors has been explored as an alternative strategy for KRAS mutant cancer. However, the clinical benefit is modest due to drug resistance caused by reactivation of the MAPK pathway and potentially other pathways and/or bypass mechanisms. Here we used CRISPR-based screens to identify potential combination therapy targets to enhance trametinib response in KRAS mutant cancers. We screened in five KRAS mutant cell lines of lung and pancreatic lineages and identified both known as well as novel modulators of MEK inhibitor response. Consistent with previous reports, gene knockouts that impair the reactivation of ERK downstream of MEK inhibition scored as top hits in our drop-out screens. Knocking out MAPK1, RAF1, BRAF and PTPN11 sensitized all five cell lines to trametinib treatment supporting these as candidate drug combination targets for MAPK pathway inhibition. Our screens also identified multiple genes within the heparan sulfate pathway (EXT1, EXT2, EXTL3, XYLT2, ALG6, B3GAT3, B4GALT7 and HS2ST1) and the MAPK7 pathway (MAPK7 and MAP2K5) that sensitize multiple but not all cell lines to trametinib. As part of our target discovery platform, we further validated these screening results using various phenotypic assays. Altogether, our results suggest that resistance to MEK inhibitors is driven by reactivation of the MAPK pathway as previously demonstrated and that impairing such reactivation restores the sensitivity of KRAS mutant cancer cells to trametinib. The genetic mechanisms driving the MAPK pathway rebound are likely different in different cancers and understanding such mechanisms will be key for achieving clinical success. Citation Format: Silvia Fenoglio, Aileen M. Cristo, James Tepper, Teng Teng, Samuel R. Meier, Ashley Choi, Hongxiang Zhang, Shan-chuan Zhao, Shangtao Liu, Leanne G. Ahronian, Daniel Aird, Nikitha M. Das, Yi Yu, Robert Tjin Tham Sjin, Jannik N. Andersen, Alan Huang, Fang Li, Xuewen Pan. CRISPR screens identify sensitizers to trametinib in KRAS mutant cancer cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P183.

Impact of between-tissue differences on pan-cancer predictions of drug sensitivity.

Increased availability of drug response and genomics data for many tumor cell lines has accelerated the development of pan-cancer prediction models of drug response. However, it is unclear how much between-tissue differences in drug response and molecular characteristics may contribute to pan-cancer predictions. Also unknown is whether the performance of pan-cancer models could vary by cancer type. Here, we built a series of pan-cancer models using two datasets containing 346 and 504 cell lines, each with MEK inhibitor (MEKi) response and mRNA expression, point mutation, and copy number variation data, and found that, while the tissue-level drug responses are accurately predicted (between-tissue ρ = 0.88-0.98), only 5 of 10 cancer types showed successful within-tissue prediction performance (within-tissue ρ = 0.11-0.64). Between-tissue differences make substantial contributions to the performance of pan-cancer MEKi response predictions, as exclusion of between-tissue signals leads to a decrease in Spearman's ρ from a range of 0.43-0.62 to 0.30-0.51. In practice, joint analysis of multiple cancer types usually has a larger sample size, hence greater power, than for one cancer type; and we observe that higher accuracy of pan-cancer prediction of MEKi response is almost entirely due to the sample size advantage. Success of pan-cancer prediction reveals how drug response in different cancers may invoke shared regulatory mechanisms despite tissue-specific routes of oncogenesis, yet predictions in different cancer types require flexible incorporation of between-cancer and within-cancer signals. As most datasets in genome sciences contain multiple levels of heterogeneity, careful parsing of group characteristics and within-group, individual variation is essential when making robust inference.

YAP-Mediated Repression of HRK Regulates Tumor Growth, Therapy Response, and Survival Under Tumor Environmental Stress in Neuroblastoma.

Following chemotherapy and relapse, high-risk neuroblastoma tumors harbor more genomic alterations than at diagnosis, including increased transcriptional activity of the Yes-associated protein (YAP), a key downstream component of the Hippo signaling network. Although YAP has been implicated in many cancer types, its functional role in the aggressive pediatric cancer neuroblastoma is not well-characterized. In this study, we performed genetic manipulation of YAP in human-derived neuroblastoma cell lines to investigate YAP function in key aspects of the malignant phenotype, including mesenchymal properties, tumor growth, chemotherapy response, and MEK inhibitor response. Standard cytotoxic therapy induced YAP expression and transcriptional activity in patient-derived xenografts treated in vivo, which may contribute to neuroblastoma recurrence. Moreover, YAP promoted a mesenchymal phenotype in high-risk neuroblastoma that modulated tumor growth and therapy resistance in vivo. Finally, the BH3-only protein, Harakiri (HRK), was identified as a novel target inhibited by YAP, which, when suppressed, prevented apoptosis in response to nutrient deprivation in vitro and promoted tumor aggression, chemotherapy resistance, and MEK inhibitor resistance in vivo. Collectively, these findings suggest that YAP inhibition may improve chemotherapy response in patients with neuroblastoma via its regulation of HRK, thus providing a critical strategic complement to MEK inhibitor therapy. SIGNIFICANCE: This study identifies HRK as a novel tumor suppressor in neuroblastoma and suggests dual MEK and YAP inhibition as a potential therapeutic strategy in RAS-hyperactivated neuroblastomas.

Abstract B36: Therapy-induced YAP hyperactivation is a mechanism driving the evolution of residual disease and resistance to targeted cancer therapy

Abstract Initial profound responses of precision medicines against cancer are limited in duration in part due to residual disease states that are therapy refractory and serve as a prelude to acquired drug resistance and tumor relapse. A prominent clinical example is in the collection of BRAF mutant cancers, including lung cancer and melanoma, which often respond initially, but incompletely and temporarily, to targeted RAF and MEK inhibition. The Hippo pathway transcriptional coactivator, Yes-associated protein (YAP), is a chief mediator of RAF and MEK inhibitor response in BRAF mutant cancers. However, the underlying mechanism for YAP-driven resistance is not fully understood. Here, we modeled the kinetics of patient-derived and isogenic BRAF mutant (V600E) cell lines in response to BRAF inhibitor and MEK inhibitor treatment. Our analysis indicated that in response to drug treatment the cells evolve through a “persister,” residual disease state that is distinct from therapy-naive cells or cells with acquired resistance. We observed that drug treatment induced early nuclear translocalization of YAP and this response peaked at the “persister” cell state before returning towards baseline upon acquired resistance. Additionally, our functional analysis of the proteomic and transcriptional landscape of the “persister” cells demonstrated drug-dependent reversible and concomitant activation of AKT, YAP target gene expression, and an epithelial mesenchymal transition (EMT). These programs underlay acquired vulnerabilities, as pharmacologic perturbation of AKT and genetic ablation of YAP was lethal specifically in the “persister” cells. Remarkably, AKT was required and sufficient for drug-induced nuclear localization of YAP, indicating that therapy-mediated hyperactivation of AKT is required for YAP induction and YAP-mediated residual disease. Current studies are focused on characterizing the molecular events causing AKT-mediated hyperactivation of YAP in response to MAPK pathway inhibition. Overall, this work aims to deconvolute a novel molecular mechanism of YAP activation in cancer cells and establish the functional implications of YAP signaling on “persister” cell biology and the evolution of targeted cancer therapy resistance. Citation Format: Aubhishek Zaman, Trever Bivona. Therapy-induced YAP hyperactivation is a mechanism driving the evolution of residual disease and resistance to targeted cancer therapy [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B36.

A role for the unfolded protein response stress sensor ERN1 in regulating the response to MEK inhibitors in KRAS mutant colon cancers

BackgroundMutations in KRAS are frequent in human cancer, yet effective targeted therapeutics for these cancers are still lacking. Attempts to drug the MEK kinases downstream of KRAS have had limited success in clinical trials. Understanding the specific genomic vulnerabilities of KRAS-driven cancers may uncover novel patient-tailored treatment options.MethodsWe first searched for synthetic lethal (SL) genetic interactions with mutant RAS in yeast with the ultimate aim to identify novel cancer-specific targets for therapy. Our method used selective ploidy ablation, which enables replication of cancer-specific gene expression changes in the yeast gene disruption library. Second, we used a genome-wide CRISPR/Cas9-based genetic screen in KRAS mutant human colon cancer cells to understand the mechanistic connection between the synthetic lethal interaction discovered in yeast and downstream RAS signaling in human cells.ResultsWe identify loss of the endoplasmic reticulum (ER) stress sensor IRE1 as synthetic lethal with activated RAS mutants in yeast. In KRAS mutant colorectal cancer cell lines, genetic ablation of the human ortholog of IRE1, ERN1, does not affect growth but sensitizes to MEK inhibition. However, an ERN1 kinase inhibitor failed to show synergy with MEK inhibition, suggesting that a non-kinase function of ERN1 confers MEK inhibitor resistance. To investigate how ERN1 modulates MEK inhibitor responses, we performed genetic screens in ERN1 knockout KRAS mutant colon cancer cells to identify genes whose inactivation confers resistance to MEK inhibition. This genetic screen identified multiple negative regulators of JUN N-terminal kinase (JNK) /JUN signaling. Consistently, compounds targeting JNK/MAPK8 or TAK1/MAP3K7, which relay signals from ERN1 to JUN, display synergy with MEK inhibition.ConclusionsWe identify the ERN1-JNK-JUN pathway as a novel regulator of MEK inhibitor response in KRAS mutant colon cancer. The notion that multiple signaling pathways can activate JUN may explain why KRAS mutant tumor cells are traditionally seen as highly refractory to MEK inhibitor therapy. Our findings emphasize the need for the development of new therapeutics targeting JUN activating kinases, TAK1 and JNK, to sensitize KRAS mutant cancer cells to MEK inhibitors.

Proteomic phenotyping of metastatic melanoma reveals putative signatures of MEK inhibitor response and prognosis

BackgroundGenotyping of melanomas is used to identify patients for treatment with BRAF and MEK inhibitors, but clinical responses are highly variable. This study investigated the utility of protein expression phenotyping to provide an integrated assessment of gene expression programs in BRAF/NRAS melanoma which would be useful for prognosis and may predict response to MEK inhibition.MethodsMass spectrometry profiling of early passage cell lines established from Stage III cutaneous melanomas was conducted. Basal protein expression was correlated with in vitro response to the MEK inhibitor, selumetinib. Protein expression in a cohort of 32 drug naïve BRAF/NRAS metastatic melanoma specimens was examined. The prognostic utility of a subset of these proteins and mRNA transcripts from a separate cohort was determined.ResultsUnsupervised analysis of basal cell line protein abundances delineated response to selumetinib, but BRAF/NRAS genotype did not. Resistance was associated with functions including cell motility, cell adhesion and cytoskeletal organization. Several of these response biomarkers were observed in lymph node biospecimens and correlated with melanoma-specific survival. Loss of ICAM-1 protein and mRNA expression was a strong prognosticator of diminished survival in BRAF/NRAS mutant melanoma.ConclusionsThese results demonstrate the utility of proteomic phenotyping to identify both putative biomarkers of response to MEK inhibition and prognostication associated with metastatic melanoma.

Predicting MEK Inhibitor Response in Lung Cancer: A Proper Signature Is Required.

The ERK signaling pathway is one of the most commonly deregulated pathways in cancer. Assays that accurately measure ERK signaling output in clinical specimens would be extremely helpful not only in determining the pharmacodynamic effects of drug treatment but also in selecting those patients most likely to respond to therapy. Clin Cancer Res; 23(6); 1365-7. ©2016 AACRSee related article by Brant et al., p. 1471.

MEK inhibitors block growth of lung tumours with mutations in ataxia-telangiectasia mutated.

Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.

Abstract B24: Triple-negative breast cancer adaptive response to MEK inhibition is regulated by the induction of super-enhancers

Abstract A predominant mechanism by which cancer cells circumvent the action of targeted kinase inhibitors is the activation of bypass signaling networks that reactivate cellular proliferation. In triple negative breast cancer (TNBC), a chief component of the adaptive response to MEK inhibition is the transcriptional upregulation and activation of receptor tyrosine kinases (RTKs), which contributes to drug resistance by reactivating ERK signaling as well as by initiating growth signaling through other kinase nodes. The cohort of RTKs activated differs between TNBC molecular subtypes and is heterogeneous even among cell lines of the same subtype; thus designing effective inhibitor combinations is challenging. Targeting the chromatin reader protein BRD4 via BET bromodomain inhibition attenuates the transcriptional adaptive response to MEK inhibition, yielding a more durable growth suppression than kinase inhibitor combinations. Yet, a detailed understanding of molecular mechanisms underlying BET bromodomain inhibitor synergy with MEK inhibitor remain unclear. We show by ChIP-seq the dynamic formation of BRD4-dense super-enhancers in response to 24 h MEK inhibition. In SUM159PT claudin-low breast cancer cells, de novo super-enhancers, whose identity were validated by histone H3 lysine 27 acetylation and MED1 co-occupancy with BRD4, were found proximal to genomic loci of DDR1, KDR, and MYLK kinases previously implicated in the adaptive MEK inhibitor response. In sum we identified 110 putative MEK inhibitor-induced SUM159PT super-enhancers, 40% of which reside within 100 kilobases 5' or 3' of a gene whose transcript is upregulated 2 fold or greater by MEK inhibition as assessed by RNA-seq, suggesting that de novo superenhancer formation is a general mode of transcriptional regulation of the adaptive response. Importantly, the MEK inhibitor-induced super-enhancers are disrupted by the BET bromodomain inhibitor JQ1, providing one potential mechanism for the growth suppression synergism obtained by combining MEK + BET bromodomain inhibition. To identify candidate direct DNA binding factors that may be seeding the observed super-enhancer formation, Multiple EM for Motif Elicitation (MEME) analysis identified an enrichment of consensus DNA binding motifs for the enhancer factors CEBPB/D in de novo super-enhancer regions, and the presence of CEBPB was confirmed to be a component of these regions by ChIP-seq. Additionally, we have achieved MEK inhibitor growth suppression synergy with inhibitors of the lysine acetyl transferases CBP/p300, known super-enhancer components. These data provide a new mechanism underlying the transcriptional control of the adaptive response to MEK inhibitor and suggest that targeting drug-induced super-enhancer elements may be a potential therapeutic avenue for TNBC. Citation Format: Jon S. Zawistowski, Samantha M. Miller, Daniel R. Goulet, Timothy J. Stuhlmiller, Darshan Singh, Noah Sciaky, Sara H. Velarde, Steven P. Angus, Gary L. Johnson. Triple-negative breast cancer adaptive response to MEK inhibition is regulated by the induction of super-enhancers. [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 B24.

Glutamate receptors: emerging players in melanomagenesis

Glutamate receptors: emerging players in melanomagenesis

Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Selumetinib (AZD6244, ARRY-142886) is a selective, non-ATP-competitive inhibitor of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR-based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.

KRAS/BRAF mutation status and ERK1/2 activation as biomarkers for MEK1/2 inhibitor therapy in colorectal cancer

Phase II clinical trials of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors are ongoing and ERK1/2 activation is frequently used as a biomarker. In light of the mutational activation of BRAF and KRAS in colorectal cancer, inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase are anticipated to be promising. Previous studies in pancreatic cancer have found little correlation between BRAF/KRAS mutation status and ERK1/2 activation, suggesting that identifying biomarkers of MEK inhibitor response may be more challenging than previously thought. The purpose of this study was to evaluate the effectiveness of MEK inhibitor therapy for colorectal cancer and BRAF/KRAS mutation status and ERK1/2 activation as biomarkers for MEK inhibitor therapy. First, we found that MEK inhibitor treatment impaired the anchorage-independent growth of nearly all KRAS/BRAF mutant, but not wild-type, colorectal cancer cells. There was a correlation between BRAF, but not KRAS, mutation status and ERK1/2 activation. Second, neither elevated ERK1/2 activation nor reduction of ERK1/2 activity correlated with MEK inhibition of anchorage-independent growth. Finally, we validated our cell line observations and found that ERK1/2 activation correlated with BRAF, but not KRAS, mutation status in 190 patient colorectal cancer tissues. Surprisingly, we also found that ERK activation was elevated in normal colonic epithelium, suggesting that normal cell toxicity may be a complication for colorectal cancer treatment. Our results suggest that although MEK inhibitors show promise in colorectal cancer, KRAS/BRAF mutation status, but not ERK activation as previously thought, may be useful biomarkers for MEK inhibitor sensitivity.