Table S1. sgRNA and PAM sequences; Table S2. The IC50 values for AZD2014 and AZD1775 in NSCLC cells; Table S3. Mutational status of human cell line models; Table S4. CalcuSyn combination indices; Figure S1. Dose response curves; Figure S2. Combined mTOR and WEE1 inhibition induces synergistic apoptotic caspase activity; Figure S3. Combination treatment suppresses human KRAS-driven tumor xenografted growth; Figure S4. Mice with active KRAS-driven lung cancer show significant tumor response upon dual AZD1775 and AZD2014 therapy; Figure S5. Rad51 foci-forming ability of NSCLC cells in response to gemcitabine and etoposide treatment; Figure S6. Ectopic expression of LKB1 is sufficient to overcome the loss of the LKB1-mediated sensitization to dual AZD2014 and AZD1775 therapy; Figure S7. Loss of LKB1 in KRAS-mutant cell lines confers partial resistance to dual WEE1 and mTOR therapy; Figure S8. Ectopic expression of LKB1 is sufficient to overcome the loss of the LKB1-mediated sensitization to dual AZD2014 and AZD1775 therapy.
ARTICLE ABSTRACTPurpose: KRAS-activating mutations are the most common oncogenic driver in non–small cell lung cancer (NSCLC), but efforts to directly target mutant KRAS have proved a formidable challenge. Therefore, multitargeted therapy may offer a plausible strategy to effectively treat KRAS-driven NSCLCs. Here, we evaluate the efficacy and mechanistic rationale for combining mTOR and WEE1 inhibition as a potential therapy for lung cancers harboring KRAS mutations.Experimental Design: We investigated the synergistic effect of combining mTOR and WEE1 inhibitors on cell viability, apoptosis, and DNA damage repair response using a panel of human KRAS-mutant and wild type NSCLC cell lines and patient-derived xenograft cell lines. Murine autochthonous and human transplant models were used to test the therapeutic efficacy and pharmacodynamic effects of dual treatment.Results: We demonstrate that combined inhibition of mTOR and WEE1 induced potent synergistic cytotoxic effects selectively in KRAS-mutant NSCLC cell lines, delayed human tumor xenograft growth and caused tumor regression in a murine lung adenocarcinoma model. Mechanistically, we show that inhibition of mTOR potentiates WEE1 inhibition by abrogating compensatory activation of DNA repair, exacerbating DNA damage in KRAS-mutant NSCLC, and that this effect is due in part to reduction in cyclin D1.Conclusions: These findings demonstrate that compromised DNA repair underlies the observed potent synergy of WEE1 and mTOR inhibition and support clinical evaluation of this dual therapy for patients with KRAS-mutant lung cancers. Clin Cancer Res; 23(22); 6993–7005. ©2017 AACR.