Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis in NSCLC
Katherine R. Amato, Shan Wang, Andrew K. Hastings, Victoria M. Youngblood, Pranav R. Santapuram, Haiying Chen, Justin M. Cates, Daniel C. Colvin, Fei Ye, Dana M. Brantley-Sieders, Rebecca S. Cook, Li Tan, Nathanael S. Gray, Jin Chen
Katherine R. Amato, Shan Wang, Andrew K. Hastings, Victoria M. Youngblood, Pranav R. Santapuram, Haiying Chen, Justin M. Cates, Daniel C. Colvin, Fei Ye, Dana M. Brantley-Sieders, Rebecca S. Cook, Li Tan, Nathanael S. Gray, Jin Chen
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Research Article Oncology

Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis in NSCLC

Abstract

Genome-wide analyses determined previously that the receptor tyrosine kinase (RTK) EPHA2 is commonly overexpressed in non–small cell lung cancers (NSCLCs). EPHA2 overexpression is associated with poor clinical outcomes; therefore, EPHA2 may represent a promising therapeutic target for patients with NSCLC. In support of this hypothesis, here we have shown that targeted disruption of EphA2 in a murine model of aggressive Kras-mutant NSCLC impairs tumor growth. Knockdown of EPHA2 in human NSCLC cell lines reduced cell growth and viability, confirming the epithelial cell autonomous requirements for EPHA2 in NSCLCs. Targeting EPHA2 in NSCLCs decreased S6K1-mediated phosphorylation of cell death agonist BAD and induced apoptosis. Induction of EPHA2 knockdown within established NSCLC tumors in a subcutaneous murine model reduced tumor volume and induced tumor cell death. Furthermore, an ATP-competitive EPHA2 RTK inhibitor, ALW-II-41-27, reduced the number of viable NSCLC cells in a time-dependent and dose-dependent manner in vitro and induced tumor regression in human NSCLC xenografts in vivo. Collectively, these data demonstrate a role for EPHA2 in the maintenance and progression of NSCLCs and provide evidence that ALW-II-41-27 effectively inhibits EPHA2-mediated tumor growth in preclinical models of NSCLC.

Authors

Katherine R. Amato, Shan Wang, Andrew K. Hastings, Victoria M. Youngblood, Pranav R. Santapuram, Haiying Chen, Justin M. Cates, Daniel C. Colvin, Fei Ye, Dana M. Brantley-Sieders, Rebecca S. Cook, Li Tan, Nathanael S. Gray, Jin Chen

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Figure 6

Structure and properties of ALW-II-41-27, a small-molecule kinase inhibitor of EPHA2.

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Structure and properties of ALW-II-41-27, a small-molecule kinase inhibi...
(A) Chemical structures for ALW-II-41-27 and its structural analog, NG-25. (B) H358 cells were treated with 1 μM NG-25 or ALW-II-41-27 over a time course, and cells were stimulated with EPHRIN-A1 ligand (EFNA1, 100 ng/ml) for the last 15 minutes of treatment. EPHA2 was immunoprecipitated, and tyrosine phosphorylation of EPHA2 was determined by Western blot analysis. (C) Dose-dependent effect of ALW-II-41-27 on EPHA2 phosphorylation. Cells were treated with inhibitors for 72 hours, including a 15-minute stimulation with EFNA1 at the end of the incubation. Tyrosine phosphorylation of EPHA2 was determined as in B. (D and E) H358 or H2009 cells transduced with lentiviruses containing an empty vector or an EPHA2-specific shRNA were treated with ALW-II-41-27, NG-25, or DMSO, and the percentage of viable cells was assessed at 72 hours via the MTT assay. Cells with wild-type levels of EPHA2 exhibited a marked loss of cell viability in the presence of ALW-II-41-27, while EPHA2 knockdown cells displayed minimal decrease in cell viability upon ALW-II-41-27 treatment. Data are presented as average percent of cell viability ± SEM.