Summary KRAS mutations activate Raf/MEK/ERK1/2 that can directly phosphorylate Mcl-1 at T163, enhancing Mcl-1’s function. KRAS mutations also activate PI3K/AKT that can inactivate GSK-3 and inhibit GSK-3-mediated pMcl-1 at S159 to reduce Mcl-1 degradation. We hypothesize that KRAS mutation-activated ERK1/2 and PI3K/AKT pathways contribute to stabilization of Mcl-1 via upregulation of pMcl-1 at T163 and downregulation of pMcl-1 at S159 in lung cancer. Our preliminary data show increased pMcl-1 at T163 in tumor tissues from NSCLC patients, which associated with worse survival outcome, suggesting that pMcl-1 at T163 may provide a new therapeutic target and a prognostic biomarker in NSCLC patients. We found that Mcl-1, in addition to its canonical antiapoptotic function, plays a critical role in supporting homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs). Based on this novel function, we discovered an entirely new class of small molecule Mcl-1 inhibitor, MI-223, that interacts with the BH1 pocket of Mcl-1 and inhibits HR activity. MI-223 has potent anti-tumor activity against lung cancer in vitro and in vivo. Olaparib is an FDA-approved PARP-1 inhibitor with anti-cancer efficacy; however, only patients with HR deficiency (e.g. BRCA1/2 mutations) respond to olaparib therapy. Since MI-223 inhibits HR-mediated DNA repair, this provides a rationale for combining MI-223 and olaparib to treat various cancers, including those without BRCA1/2 mutations. Combined treatment with MI-223 and olaparib synergistically suppresses lung cancer growth in vitro and in vivo. Since our data indicate that KRAS mutations can activate Mcl-1, we hypothesize that MI-223 alone or in combination with olaparib may be effective against lung cancers with KRAS mutations. MI-223-induced DSBs upregulate PD-L1 in tumor tissue from mutant KRAS driven lung cancer model, suggesting combination of MI- 223 with anti-PD-L1 may overcome PD-1 inhibitor resistance in KRAS-mutant lung cancer. To characterize and develop this novel Mcl-1 inhibitor MI-223 for the treatment of lung cancer, we propose two specific aims: (1) Determine whether and how KRAS mutations activate Mcl-1 leading to treatment resistance in human lung cancer cells. Studies will determine whether pMcl-1 at T163 is a novel prognostic biomarker and therapeutic target in patients with NSCLC; (2) Determine mechanism of action of novel Mcl-1 inhibitor MI-223 in killing human lung cancer cells. Studies will test the potency of MI-223 alone or in combination with PARP inhibitor olaparib in patient-derived lung cancer xenograft (PDX), radioresistant, and KRAS-mutant lung cancer xenografts. Determine whether MI-223 synergizes with olaparib or anti-PD-L1 to more effectively suppress tumor growth and prolong survival in genetically engineered mutant KRAS-driven lung cancer animal models. By targeting Mcl-1, we expect to develop a new class of anti-cancer agents and combination strategies for lung cancer ...