Small Molecule Inhibition of OXA1L Selectively Kills ZFHX3 Mutant NSCLC from Imbalanced Cytidine Pool Expansion Thomas W. Hanigan, Junichiro Takaya, Haoxin Li, Jarret R. Remsberg, Verena Albert, J.C. Ducom, Christopher M. Joslyn, Melissa A Dix, Kim Masuda, Enrique Saez, Kenji Sasaki, Christopher G. Parker, Benjamin F. Cravatt* Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA. *To whom correspondence should be addressed; Email: email@example.com Targeting Oxidative Phosphorylation (Ox Phos) for treatment of cancer is an overlooked approach due to the common perception that tumor cells depend on anaerobic glycolysis for proliferation. Here we disclose a first-in-class reversible inhibitor targeting the mitochondrial insertase OXA1L. Akin to OXA1L mutations, we show its inhibition selectively prevents translation and expression of complex IV (COXIV) of Ox Phos, leading to COXIV deficiency and complete inhibition of oxygen consumption. Despite a general metabolic rescue through upregulated glycolysis, we found OXA1L inhibition to be selectively cytotoxic to a subset of NSCLC, due to both a commitment to de novo pyrimidine biosynthesis from ammonia through CPSI, and from a loss of expression of the pyrimidine catabolic enzyme cytidine deaminase (CDA). This combined defect prevents regulation of bicarbonate buffering capacity, and leads to imbalanced cytidine pool expansion, causing loss of transcriptional fidelity, G2 cell cycle arrest, and cell death. Fundamental to the ability of resistant cells to adapt to both the acidosis and pyrimidine flux is expression of a wildtype ZFHX3, an understudied DNA/RNA DEAD-box helicase mutated in ~13% of lung cancers, which we show downregulates cytidine deaminase expression in opposition to the RecQ helicase family. Ultimately, ZFHX3 mutations leave a T>C mutational signature and render cells more sensitive to OXA1L inhibition, but more resistant to thymidine synthase or dihydroorotate dehydrogenase inhibition, which may serve as a broad biomarker for several FDA approved targeted therapies in conjunction with OXA1L inhibition.