Summary HPV-negative head and neck squamous cell carcinomas (HNSCC) typically lose G1/S cell cycle checkpoints, with most tumors having mutations in TP53, and many also mutating other tumor suppressors such as CDKN2A. Such tumors become dependent on checkpoints associated with G2/M to repair DNA damage arising from replication stress and other genomic insults. This dependency suggests a tumor-selective vulnerability to synthetic lethal strategies controlling progress through G2/M. In extensive preliminary data, we show that AZD1775/adavosertib, an inhibitor of the G2/M checkpoint kinase WEE1, potently sensitizes TP53mut HNSCC cell lines to inhibition of Aurora A kinase (AURKA). WEE1 induces an inhibitory Y15-phosphorylation of CDK1, blocking M-phase entry and thus blunting the cytotoxic effects AURKA inhibition. WEE1 inhibition abrogates this arrest, accelerating mitotic entry for cells bearing highly disruptive spindle abnormalities and other defects arising from AURKA inhibition, resulting in mitotic catastrophe and apoptosis. We found combined AURKA/WEE1 inhibition is potent in HNSCC xenografts, while well-tolerated in normal tissue and cells. We have extended this concept, identifying additional promising drug combinations between WEE1 and other G2/M regulatory kinases (PLK1 and CHK1). Notably, the limited number of cells surviving synthetic lethal treatment are characterized by aneuploidy and other defects suggesting they may have increased tumor mutation burden (TMB), express neoantigens, and upregulated inflammatory signaling associated with sensitivity to immune checkpoint inhibition. This project will take this observation directly to the clinic. We will conduct a pre-operative window phase I and expansion clinical trial of the late generation, high potency selective AURKA inhibitor LY3295668 with adavosertib combination, establishing pharmacodynamic proof of concept, identifying biomarkers for patient selection in future studies, and placing synergistic combinations in context of genomic alteration and treatment resistance. In Aim 1, we will evaluate mechanisms of combination lethality, and use single cell sequencing and Luminex profiling to query TMB, predict neoantigens, and measure inflammatory signaling. Aim 2 will query the effect of classes of common HNSCC TP53 and CDKN2A mutations, and cisplatin resistance, on response to a WEE1-AURKA inhibitor combination, using defined cell line models and patient derived xenografts (PDXs). In Aim 3, we will perform a pre-operative window trial to establish recommended phase 2 doses, determine activity and establish pharmacodynamic proof of concept, and to evaluate putative predictive biomarkers for response to combination WEE1/AURKA inhibition in HNSCC.