Research Article: A CRISPR screen identifies MAPK7 as a target for combination with MEK inhibition in KRAS mutant NSCLC

Date Published: June 18, 2018

Publisher: Public Library of Science

Author(s): Nicholas Dompe, Christiaan Klijn, Sara A. Watson, Katherine Leng, Jenna Port, Trinna Cuellar, Colin Watanabe, Benjamin Haley, Richard Neve, Marie Evangelista, David Stokoe, Aamir Ahmad.

http://doi.org/10.1371/journal.pone.0199264

Abstract

Mutant KRAS represents one of the most frequently observed oncogenes in NSCLC, yet no therapies are approved for tumors that express activated KRAS variants. While there is strong rationale for the use of MEK inhibitors to treat tumors with activated RAS/MAPK signaling, these have proven ineffective clinically. We therefore implemented a CRISPR screening approach to identify novel agents to sensitize KRAS mutant NSCLC cells to MEK inhibitor treatment. This approach identified multiple components of the canonical RAS/MAPK pathway consistent with previous studies. In addition, we identified MAPK7 as a novel, strong hit and validated this finding using multiple orthogonal approaches including knockdown and pharmacological inhibition. We show that MAPK7 inhibition attenuates the re-activation of MAPK signaling occurring following long-term MEK inhibition, thereby illustrating that MAPK7 mediates pathway reactivation in the face of MEK inhibition. Finally, genetic knockdown of MAPK7 combined with the MEK inhibitor cobimetinib in a mutant KRAS NSCLC xenograft model to mediate improved tumor growth inhibition. These data highlight that MAPK7 represents a promising target for combination treatment with MEK inhibition in KRAS mutant NSCLC.

Partial Text

The RAS/MAPK signaling pathway plays a critical role in embryogenesis, tissue growth and repair, and normal tissue homeostasis downstream of growth factor activation. Active RAS signals through the RAF kinases that in turn activate a signaling cascade through the MEK and ERK kinases, resulting in the phosphorylation of numerous effector proteins to promote appropriate cellular programs. Consistent with this role in normal tissue homeostasis, activation of the RAS/MAPK pathway plays a prominent role during oncogenic transformation, tumor growth and maintenance [1]. Widespread mutation and/or amplification of various genes (KRAS, HRAS, NRAS, BRAF) comprising the components of MAPK pathway or its upstream activators (RTKs, such as EGFR, MET) are correlated with constitutive activation of the pathway in a significant proportion of human cancers, including melanomas, non-small cell lung (NSCL), colorectal, and pancreatic cancers [2]. Accordingly, there has been significant effort directed towards developing inhibitors against various components of this pathway [3]. Some of these efforts have shown substantial benefit, such as the development of EGFR inhibitors for the treatment of EGFR mutant NSCLC. The on-target nature of this effect is demonstrated by the fact that second site mutations in EGFR are frequently found upon progression that reduce drug effectiveness [4, 5]. Similarly, BRAF inhibitors result in initial dramatic responses in BRAF mutant melanoma and NSCLC, however these tumors also progress on therapy through additional acquired genetic alterations in BRAF and other factors resulting in pathway reactivation [6]. Addition of MEK inhibitors to BRAF inhibitors has extended the response of BRAF mutant tumors to therapy, illustrating the utility of targeting downstream kinase activation in this context [7]. Despite these successes and the multiple tumor-associated mutations resulting in activation of MEK, clinical benefit to MEK inhibitors has been relatively modest, particularly in the context of tumors harboring mutant RAS [8]. To identify additional druggable proteins and pathways that mediate pathway reactivation following MEK inhibition we performed a CRISPR screen in KRAS mutant NSCLC cell lines. Following extensive validation of hits from this screen we identified MAPK7, also known as ERK5, as a factor that mediates pathway reactivation following MEK inhibition, thereby identifying MAPK7 as a promising target for combination with MEK inhibitors.

CRISPR screens to identify targets for drug sensitization represent a powerful technique to evaluate appropriate combination strategies to treat indications currently underserved by existing therapeutic approaches, such as KRAS mutant NSCLC. Tumors harboring KRAS mutations are resistant to EGFR-directed therapies [25], and in some trials (but not all), showed greater resistance to standard chemotherapy treatment [26]. As MEK is activated in tumors expressing KRAS mutations, MEK inhibitors are a logical treatment option to explore in these patients. Unfortunately, MEK inhibitors have not shown any activity in this setting, either alone or in combination with EGFR inhibitors or chemotherapy [27–29]. As phosphoinositide 3-kinase (PI3K) is also activated by RAS, combinations of MEK and PI3K inhibitors had been proposed as a strategy to block RAS signaling with promise shown for the concept in preclinical models [30]. Unfortunately, these combinations have also been associated with significant toxicity in clinical trials, thereby limiting their benefit [31].

 

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http://doi.org/10.1371/journal.pone.0199264

 

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