Objectives/Goals: The Fanconi anemia (FA) pathway is responsible for faithful DNA damage repair. FA mutations are common in multiple types of cancer, including HPV-negative head and neck cancers. We sought to identify synthetic lethal genes within this pathway to target treatment of FA-mutant tumors through siRNA- and chemical-compound-based screens. Methods/Study Population: First, we completed siRNA-based and chemical compound-based screening assays to identify gene targets that reduce patient derived Fanconi pathway mutant cell (FA-D2) viability compared to Fanconi pathway proficient cells (FA-D2 + FANCD2). Five aurora kinase (AURK) inhibitors from the compound screen were chosen for further evaluation. Cell lines were treated with AURK inhibitors or siRNA-based AURK knockdown to assess viability, proliferation, DNA repair, and cell cycle progression differences. Patient mutational, mRNA expression, and outcome data were accessed through The Cancer Genome Atlas (TCGA) portal and the Caris CODEai portal. We stratified patients by tumor AURKA and AURKB mRNA levels and assessed differences in patient survival, tumor grade, and DNA repair proficiency. Results/Anticipated Results: In both screens, AURKA came up as a target to selectively reduce the growth of FA-D2 cells compared to FA-D2 + FANCD2 cells. All five AURK inhibitors identified showed selective growth inhibition (~50–75%) in FA-D2 cells at low nanomolar doses. We narrowed our selection to hesperadin, an AURKB-specific inhibitor, which showed the highest selectivity. siRNA knockdown of AURKA and AURKB decreased cell viability by 50% and 20%, respectively. Patients with FA-mutated tumors from the TCGA pan-cancer dataset had high AURKA (twofold) and AURKB (threefold) mRNA expression. AURKA and AURKB tumor mRNA expression was significantly associated with poor patient survival. Homologous recombination deficiency scores were increased ~5-fold (p Discussion/Significance of Impact: We hypothesize that in FA-deficient backgrounds, loss of AURKA or AURKB leads to heightened genomic instability due to cell cycle dysregulation and accumulated DNA damage. Our findings warrant investigation of the therapeutic potential for AURK inhibitors, specifically hesperadin, in FA-mutant head and neck cancers.