G-S checkpoint loss contributes to carcinogenesis and increases reliance upon the G-M checkpoint for adaptation to stress and DNA repair, making G-M checkpoint inhibition a target for novel therapeutic development. AZD1775, an inhibitor against the critical G-M checkpoint protein WEE1, is currently in clinical trials across a number of tumor types. AZD1775 and DNA-damaging agents have displayed favorable activity in several preclinical tumor models, often in the molecular context of loss. Whether AZD1775 efficacy is modulated by other molecular contexts remains poorly understood. The tumor suppressor serine/threonine kinase 11 () is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC) and is commonly comutated with oncogenic mutations. We investigated the preclinical effects of AZD1775 in the context of/ in NSCLC. Using NSCLC cell lines, we found that AZD1775 alone and in combination with DNA-damaging agents (e.g., cisplatin and radiation) decreased tumor cell viability in -deficient NSCLC cells. deficiency enhanced DNA damage and apoptosis in response to AZD1775 exposure compared with wild-type cells. In a genetically engineered mouse model of mutant with concomitant loss of , combined AZD1775 and cisplatin extended overall survival compared with cisplatin alone. Our data suggest that lack of phosphorylation of LKB1 by ATM was involved in AZD1775-mediated cytotoxicity. Collectively, these findings provide a clinical application for AZD1775 with DNA-damaging agents in/ NSCLC. .

Within eukaryotic cells, the G1/S and G2/M cell cycle checkpoints activate in response to intracellular or extracellular stress to prevent defects in DNA synthesis and mitosis. Deregulation of the cell cycle is a fundamental characteristic of cancer, with loss of the G1/S checkpoint playing an important role in carcinogenesis. Phenotypically, loss of the G1/S checkpoint within tumor cells results in increased reliance upon the G2/M checkpoint for adaptation to stress and DNA damage repair. This dependence has led to interest towards inhibition of the G2/M checkpoint for therapeutic treatment. Efforts have produced several clinically relevant compounds that target the regulatory proteins of the G2/M checkpoint. One of these compounds the WEE1 kinase inhibitor, AZD1775 displays favorable activity in a variety of pre-clinical tumor models in combination with DNA damaging agents and in particular, AZD1775 shows enhanced activity in tumor cells harboring inactivating mutations to p53. However, whether AZD1775 or other G2 checkpoint inhibitors have improved activity in the context of other mutations remains poorly understood. The tumor suppressor, LKB1 (STK11) is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC) and is commonly co-mutated with oncogenic mutations to KRAS (mtKRAS). Functional studies have revealed that mtKRAS/LKB1-deficient (LKB1-) NSCLC cells display increased activation of the G2/M checkpoint and this characteristic of mtKRAS/LKB1- NSCLC has been proposed as a point for therapeutic intervention. Based upon the reported response of tumor cells to AZD1775, we investigated the pre-clinical effects of AZD1775 in mtKRAS/LKB1- NSCLC. Employing NSCLC cell lines, we find that AZD1775 has increased activity in mtKRAS/LKB1- cells in vitro. mtKRAS/LKB1- NSCLC cells display increased protein levels of markers of DNA damage (phosphorylated γ-H2AX) and apoptosis (cleaved PARP) and reduced cell viability with AZD1775 treatment, compared to mtKRAS NSCLC with functional LKB1. The effects of AZD1775 in mtKRAS/LKB1- NSCLC cells were enhanced in the presence of DNA damaging agents (radiation, cisplatin). Using a genetically-engineered mouse model of mtKRAS/LKB1- NSCLC, combined treatment of AZD1775 and cisplatin were found to increase overall survival, compared to cisplatin mono-therapy in vivo. Collectively, these findings suggest that application of AZD1775 with DNA damaging agents may have therapeutic efficacy in mtKRAS/LKB1- NSCLC, a genomic subgroup of lung cancer with no effective therapeutic regimens. Citation Format: Amanda L. Richer, Jacqueline M. Cala, Kelley O’Brien, Vashti M. Carson, Timothy G. Whitsett, Landon J. Inge. An inhibitor to the WEE1 kinase has pre-clinical activity in mutant KRAS/LKB1-deficient non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2817.