PROJECT SUMMARY Alcohol consumption is a major risk factor for both head-and-neck and esophageal squamous cell carcinomas (SCCs), which are both lethal and globally prevalent. The lethality of these cancers may stem from cancer cells exhibiting high CD44 expression (CD44H), which can survive genotoxic stress and drive tumor initiation, invasion, metastasis, therapy resistance and poor prognosis. Acetaldehyde, a major EtOH metabolite, is a potent inducer of DNA interstrand crosslinks (ICLs). ICLs can also arise from cancer-specific processes such as hyperactive DNA replication and cisplatin-based chemotherapy. CD44H cells can tolerate both EtOH and cisplatin exposure. Our preliminary data suggest that ICLs accumulate in oral and esophageal preneoplasia (PreN) and SCC lesions, both of which are recapitulated in patient- and murine-derived organoids (PDO and MDO, respectively), and that these cells display activation of the Fanconi Anemia (FA) pathway, which is responsible for ICL repair. CRISPR-mediated genetic depletion of FA pathway member FANCT, a druggable ubiquitin E2 ligase of the FA pathway, impairs formation of SCC, but not normal MDO. Moreover, pharmacological FANCT inhibitor kills SCC cells, but not normal cells in PDO. In organoids with dysfunctional aldehyde dehydrogenase (Aldh)-2, a mitochondrial enzyme linked to activation of FA pathway, exposure to EtOH gives rise to enrichment of CD44H cells, which in turn survive EtOH-induced mitochondrial damage and oxidative stress via autophagy, a cytoprotective mechanism responsible for removing damaged mitochondria and reducing reactive oxygen species (ROS). Inhibition of both the FA pathway and autophagy may offer a novel therapeutic approach for the selective sensitization of CD44H cells to ICL-mediated cell death by cisplatin. The overall objective of this renewal application is to further elucidate the mechanism of how CD44H cells survive ICLs accumulation, which promotes cell death if left unrepaired. The central hypothesis is that CD44H cells survive via FA pathway-mediated ICLs repair and autophagy, which may present a potential cancer cell-specific vulnerability. We will test our hypothesis by pursuing the following interrelated Specific Aims: (1) To determine how alcohol and ALDH2 dysfunction promote genotoxicity in PreN and SCC lesions; (2) To elucidate how the FA pathway facilitates alcohol-mediated SCC progression; (3) To delineate how CD44H cells survive alcohol- driven ICL burden in PreN and SCC lesions. The present proposal will make use of genetically engineered mice, MDO, PDO and xenograft tumors. We have built a comprehensive platform for development and validation of novel translational applications to target the FA pathway and autophagy in PreN and SCCs. Our innovative approaches will reveal novel insights about the role of alcohol metabolism in malignant transformation of head- and-neck and esophageal epithelial cells with a therapeutic implication. These findings have the...