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Certain cancers, including gastrointestinal stromal tumor (GIST) and subsets of melanoma, are caused by somatic KIT mutations that result in KIT receptor tyrosine kinase constitutive activity, which drives proliferation. The treatment of KIT-mutant GIST has been revolutionized with the advent of KIT-directed cancer therapies. KIT tyrosine kinase inhibitors (TKI) are superior to conventional chemotherapy in their ability to control advanced KIT-mutant disease. However, these therapies have a limited duration of activity due to drug-resistant secondary KIT mutations that arise (or that are selected for) during KIT TKI treatment. To overcome the problem of KIT TKI resistance, we sought to identify novel therapeutic targets in KIT-mutant GIST and melanoma cells using a human tyrosine kinome siRNA screen. From this screen, we identified lemur tyrosine kinase 3 (LMTK3) and herein describe its role as a novel KIT regulator in KIT-mutant GIST and melanoma cells. We find that LMTK3 regulated the translation rate of KIT, such that loss of LMTK3 reduced total KIT, and thus KIT downstream signaling in cancer cells. Silencing of LMTK3 decreased cell viability and increased cell death in KIT-dependent, but not KIT-independent GIST and melanoma cell lines. Notably, LMTK3 silencing reduced viability of all KIT-mutant cell lines tested, even those with drug-resistant KIT secondary mutations. Furthermore, targeting of LMTK3 with siRNA delayed KIT-dependent GIST growth in a xenograft model. Our data suggest the potential of LMTK3 as a target for treatment of patients with KIT-mutant cancer, particularly after failure of KIT TKIs.

Tyrosine kinase inhibitors (TKI) have improved CML response rates, and some are effective against resistance-promoting point mutations in BCR-ABL1. However, in the absence of point mutations, resistance still occurs. Here, we identify a novel pathway mediating resistance which connects p47phox, the organizer subunit of NADPH oxidase-2 (NOX2), with early growth response-1 (Egr-1) and the Src family kinase Fyn. We found up-regulation of p47phox, Egr-1, and Fyn mRNA and protein using paired isogenic CML cell lines and mined data. Isolation of CD34+ cells and tissue microarray staining from blast crisis CML patients confirmed in vivo over-expression of components of this pathway. Knockdown studies revealed that p47phox modulated reactive oxygen species and Egr-1 expression, which, in turn, controlled Fyn expression. Interestingly, Fyn knockdown sensitized TKI-resistant cells to dasatinib, a dual BCR-ABL1/Src inhibitor. Egr-1 knockdown had similar effects, indicating the utility of targeting Fyn expression over activation. Pointedly, p47phox knockdown also restored TKI-sensitivity, indicating that targeting the NOX2 complex can overcome resistance. The NOX2/Egr-1/Fyn pathway was also conserved within TKI-resistant EGFRΔIII-expressing glioblastoma and patient-derived glioblastoma stem cells. Thus, our findings suggest that targeting the NOX2/Egr-1/Fyn pathway may have clinical implications within multiple cancer types; particularly where efficacy of TKI is compromised.

// Ting-Fang Lee 1 , Yu-Chi Tseng 2 , Wei-Chin Chang 1,3 , Yi-Chen Chen 2 , Yu-Rung Kao 4 , Teh-Ying Chou 1,5 , Chao-Chi Ho 6 and Cheng-Wen Wu 1,2,4 1 Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan 2 Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan 3 Department of Pathology, MacKay Memorial Hospital, Taipei, Taiwan 4 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 5 Department of Pathology, Taipei Veterans General Hospital, Taipei, Taiwan 6 Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan Correspondence to: Cheng-Wen Wu, email: // Keywords : YAP1, EGFR mutation, lung adenocarcinoma, TKI-resistance Received : December 21, 2016 Accepted : June 20, 2017 Published : July 27, 2017 Abstract Epidermal growth factor receptor (EGFR) mutations are found in lung adenocarcinomas leading to tumor cells proliferation and survival. EGFR tyrosine kinase inhibitors (TKIs) that block EGFR activity are effective therapeutics for EGFR-mutant lung adenocarcinoma patients, but TKI-resistance inevitably occurs. The YES-associated protein (YAP1) transcription coactivator has been implicated as an oncogene and is amplified in human cancers and provides tumor cells strong proliferation and survival cues. This study investigated the roles of YAP1 in lung adenocarcinoma by exploring its regulation and functions mediated by EGFR signaling. In this study, we detected a correlation between YAP1 level and EGFR mutation status in lung adenocarcinoma tissues. Using lung adenocarcinoma cell lines, enhanced YAP1 expression and activity mediated by EGFR signaling was detected through enhanced protein stability. A SRC family protein, YES, was involved in EGFR-regulated YAP1 expression and this pathway was crucial for proliferation in EGFR-dependent cells. Small molecules that reduced YAP1 levels by mechanisms bypassing EGFR signaling were effective in reducing viability in EGFR-dependent cells including those with EGFR T790M, the major cause of TKI-resistance. These observations unveiled the significance of YAP1 in EGFR mutant lung adenocarcinomas and identified YAP1 as a promising therapeutic target for EGFR-dependent lung adenocarcinoma patients, including those with EGFR T790M-caused TKI resistance.