PROJECT SUMMARY Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, with a median survival of 24 months and a five-year overall survival of 7%. Cisplatin-based chemotherapy is the first-line treatment for SCLC, functioning by creating DNA crosslinks leading to cancer cell death. While initially effective, many patients ultimately develop cisplatin resistance and experience tumor recurrence. The DNA damage response (DDR) is a signaling network that recognizes challenges to genome integrity and coordinates diverse DNA repair and cell cycle checkpoint pathways. Targeting DDR proteins critical to the cellular response to cisplatin in SCLC may overcome acquired cisplatin resistance, but the cellular response to cisplatin remains unclear because many proteins that respond to cisplatin-induced DNA damage have yet to be identified. To address this issue, a synthetic lethal screen using a custom siRNA library of 1008 nuclear enzymes was performed in H128 cisplatin- resistant SCLC cells to identify genes critical for mediating cisplatin resistance. Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2), a H3 K27 methyltransferase, was identified as one of the strongest synthetic lethal hits in the screen, where knockdown of EZH2 strongly sensitized H128 cells to cisplatin. EZH2 is overexpressed in SCLC as well as other cancers and has been implicated in cancer progression. Moreover, EZH2 inhibitors have shown efficacy in several cancers, including SCLC;; however, the the molecular underpinnings of the efficacy of EZH2 depletion or inhibition in SCLC and the mechanistic role of EZH2 in mediating cisplatin resistance in SCLC are unclear. In this regard, I validated that EZH2 depletion in cisplatin- resistant H128 and H146 SCLC cell lines results in cisplatin hypersensitivity, and furthermore found that EZH2 localizes to DNA damage sites induced by laser microirradiation, suggesting that EZH2 may function directly in mediating DNA damage resistance in SCLC. In addition, using mass spectrometry analysis of purified EZH2 from cells, I identified a novel interaction between EZH2 and DNA Damage-Binding Protein 1 (DDB1), an E3 ubiquitin ligase component that promotes nucleotide excision repair (NER) of cisplatin-induced DNA intrastrand crosslinks, which I validated by co-immunoprecipitation. As such, I hypothesize that EZH2 plays a critical role in mediating cisplatin resistance in SCLC by promoting DNA damage response (DDR) activities and furthermore that EZH2 inhibition will sensitize resistant SCLC cells and tumors with dysregulated DDR pathways to cisplatin treatment. Aim 1 will determine the mechanism by which EZH2 mediates cisplatin resistance in SCLC. Aim 2 will determine if EZH2 inhibition sensitizes resistant SCLC cells and tumors with dysregulated DDR protein expression t...