PROJECT SUMMARY This project will investigate the specific mechanisms of DNA lesions that are generated in SCLC during treatment and lead to response or resistance to therapy. This is Project 3 (“Repair Mechanisms of Replication- Fork Lesions”) which is part of a Program Project titled, “Investigating and Targeting Replication Stress in Small Cell Lung Cancer”. Despite the major clinical importance of replication stress related lesions and their repair processes to therapy response, we know surprisingly little about their molecular mechanism and how defects in these processes confer specific vulnerabilities and resistance in tumors. This project aims to fill several fundamental gaps in our knowledge of replication-fork lesions and their mechanisms of repair, and explore novel hypotheses by employing an array of innovative cellular, and single- molecule techniques and assays to define the key steps and molecular mechanisms of DNA lesion formation and repair in SCLC. First, we will define the specific subtypes of replication-fork lesions in naïve SCLC with known genetic backgrounds and establish their signaling and repair processes, and their contribution to cellular state and overall response. Next, we will determine how these processes are affected upon emergence of therapeutic resistance, and the effects of new targeted therapies in resistant SCLC subtypes. In Aim 1 “Define specific fork-lesions and their repair processes in naïve SCLC” we will establish the specific sub-types of DNA lesions in SCLC, and their DDR/R processes, and the formation of specific lesions at unique genomic and chromosomal sites, transcription-replication conflicts (TRCs), G4 DNA structures etc, and their effect on cell state and therapeutic response. In Aim 2, “Establish how formation and repair of fork-lesions is altered in resistant SCLC subtypes”, we will measure the specific modes of resistance in terms of particular lesion formation, suppression and repair, and their contribution to cellular sensitization to immunotherapy. The research work will be highly coordinated within the Program Project with the other three Projects and the three Cores. Our combined diverse approaches include molecular biology, cell biology and engineered cell lines, tumor models, sequencing and scRNA-seq, proteomics and advanced microscopy approaches. Cell lines, targets, molecular player, treatments, and experiments will be designed with Projects 1, 2, and 4, and will be constantly monitored with feedback via Core A. Cellular models will be constructed and validated by Core B, and imaging and analysis will be supported by Core C.