Lung cancer is the leading cause of cancer-related deaths, demanding improvement in current treatment modalities to reduce the mortality rates. Lung cancer is divided into two major classes with non-small cell lung cancer representing ~84% of lung cancer cases. One strategy widely used to treat non-small cell lung cancer patients includes targeting the epidermal growth factor receptor (EGFR) using EGFR-inhibitors, such as erlotinib, gefitinib, and afatinib. However, most patients develop resistance to EGFR-inhibitors within a year post-treatment. Although some mechanisms that drive resistance to EGFR-inhibitors have been identified, there are many cases in which the mechanisms are unknown. Thus, in this study, we examined the role of microRNAs in driving EGFR-inhibitor resistance. As mediators of critical pro-growth pathways, microRNAs are severely dysregulated in multiple diseases, including non-small cell lung cancer where microRNA dysregulation also contributes to drug resistance. In this work, through screening of 2019 mature microRNAs, multiple microRNAs were identified that drive EGFR-inhibitor resistance in non-small cell lung cancer cell lines, including miR-432–5p.

EGFR inhibitors (EGFRi) are standard-of-care treatments administered to patients with non–small cell lung cancer (NSCLC) that harbor EGFR alterations. However, development of resistance posttreatment remains a major challenge. Multiple mechanisms can promote survival of EGFRi-treated NSCLC cells, including secondary mutations in EGFR and activation of bypass tracks that circumvent the requirement for EGFR signaling. Nevertheless, the mechanisms involved in bypass signaling activation are understudied and require further elucidation. In this study, we identify that loss of an epigenetic factor, lysine methyltransferase 5C (KMT5C), drives resistance of NSCLC to multiple EGFRis, including erlotinib, gefitinib, afatinib, and osimertinib. KMT5C catalyzed trimethylation of histone H4 lysine 20 (H4K20), a modification required for gene repression and maintenance of heterochromatin. Loss of KMT5C led to upregulation of an oncogenic long noncoding RNA, LINC01510, that promoted transcription of the oncogene MET, a component of a major bypass mechanism involved in EGFRi resistance. These findings underscore the loss of KMT5C as a critical event in driving EGFRi resistance by promoting a LINC01510/MET axis, providing mechanistic insights that could help improve NSCLC treatment. Significance: Dysregulation of the epigenetic modifier KMT5C can drive MET-mediated EGFRi resistance, implicating KMT5C loss as a putative biomarker of resistance and H4K20 methylation as a potential target in EGFRi-resistant lung cancer.

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Epidermal growth factor receptor inhibitors (EGFRi) are standard-of-care treatments administered to patients with non-small cell lung cancer (NSCLC) that harbor EGFR alterations.However, development of resistance within a year post-treatment remains a major challenge.Multiple mechanisms can promote survival of EGFRi treated NSCLC cells, including secondary mutations in EGFR and activation of bypass tracks that circumvent the requirement for EGFR signaling. Nevertheless, mechanisms involved in bypass track activation are understudied, and in a subset of cases the mechanisms are unknown. The findings from this study identified an epigenetic factor, SUV420H2 that when lost drives resistance of NSCLC to multiple EGFRi, including erlotinib, gefitinib, afatinib, and osimertinib. SUV420H2 catalyzes trimethylation of histone H4 lysine-20, a modification required for gene repression and maintenance of heterochromatin. Here we show that loss of SUV420H2 leads to upregulation of an oncogenic long non-coding RNA, LINC01510 that promotes transcription of the oncogene MET, a component of a major bypass track involved in EGFRi resistance. Significance:Due to an incomplete understanding of the mechanisms involved in promoting resistance to EGFRi, patients often succumb to their disease. Here we identified a global mediator of EGFRi resistance, SUV420H2 that helps to uncover an additional mechanism involved in resistance driven via a major bypass track involving the protooncogene MET. Supplementary information can be found online at Acknowledgments:The authors gratefully acknowledge the support of multiple core facilities from the Purdue

Lung cancer is the leading cause of cancer-related deaths in the world. Non-small cell lung cancer (NSCLC) accounts for ~85% of the cases. NSCLC patients frequently harbor causal gene mutations. Epidermal Growth Factor Receptor (EGFR), an NSCLC causal gene, is mutated in 10-35% of NSCLC patients. Patients with EGFR activating mutation are treated with a tyrosine-kinase inhibitor, Erlotinib, specifically targeting EGFR. However, most patients develop Erlotinib resistance within a year. Although multiple mechanisms are involved in the development of Erlotinib resistance, the role of microRNAs in mediating Erlotinib resistance is largely unexplored. MicroRNAs (miRNAs) are small non-coding RNAs that regulate normal cellular physiology. In cancers, miRNAs are severely dysregulated, contributing to multiple cancer processes, including drug response. Despite the involvement of miRNAs in cancer, their direct role as drivers of drug resistance remain understudied, therefore, there is a critical need to understand the role of miRNAs in inducing Erlotinib resistance. To this end, we propose an unbiased two-prong approach to identify the miRNAs that drive the development of Erlotinib resistance. Therefore, miRNAs are either being (i) overexpressed, or (ii) silenced in Erlotinib sensitive cells with the hypothesis that perturbed miRNA levels will drive Erlotinib resistance. Erlotinib sensitive NSCLC cell lines, EKVX and H322M were identified from the NCI-60 Developmental Therapeutics Project and their Erlotinib dose response curves were established. To perform the miRNA overexpression screen, EKVX and H322M cells stably expressing renilla and firefly luciferase genes were generated, which will be used to monitor cell number and transfection efficiency, respectively. The luciferase-expressing cells will be transfected with a library of 2,019 individually arrayed human miRNAs and cell growth in the presence of Erlotinib will be monitored. The second prong of the study will identify miRNAs, that when lost, confer Erlotinib resistance. 400-fold coverage of small guide RNA (sgRNA) library of the CRISPR-Cas9 system was transduced in Cas9 overexpressing EKVX cells, to knockout ~21,000 human encoded genes (~1800 miRNA genes). Cells are being cultured in the presence of 75% or 90% growth inhibitory concentrations (GI75 or GI90) of Erlotinib so that only cells with sgRNAs against genes critical for Erlotinib response, survive and grow due to acquired resistance. DNA from these cells will be harvested, the sgRNAs sequenced, and compared to the sgRNAs present in Erlotinib-untreated cells. Successful completion of this project will identify miRNAs that drive Erlotinib resistance and may contribute towards development of miRNA therapeutics to enhance Erlotinib sensitivity of NSCLC tumors. Citation Format: Arpita S. Pal, Alejandra Agredo, Andrea L. Kasinski. Aberrantly expressed microRNAs drive the development of acquired Erlotinib resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3142. doi:10.1158/1538-7445.AM2017-3142