Abstract RB1 is a tumor suppressor gene that is frequently mutated in various tumors, including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. In addition, RB1 is one of the most prevalent tumor suppressor genes driving metastasis. Despite extensive studies of pRb family proteins, a therapeutic approach that specifically targets defects in this tumor suppressor is currently not available, representing a critical knowledge gap and the need to search for alternative strategies for treating cancers with inactivated RB1. Synthetic lethality (SL) presents a viable approach to target RB1-mutated tumors. A pair of genes can be defined as having a SL interaction when perturbation of either gene alone is not lethal but simultaneous perturbation of both genes is lethal. We previously performed a genetic screen for SL partners of Rb in the Drosophila eye and then confirmed the validity of identified targets (splicing machinery, RAN, eIF4A3, and others) in human cancer cell lines and patient tumor samples. Moreover, we demonstrated that these SL interactions are preserved in the presence of additional strong oncogenic alterations such as activation of Ras and loss of Pten (Parkhitko et al., 2021). We hypothesize that combined targeting of two synthetic lethal partners belonging to different pathways would have an additive/synergistic effect on the proliferation of RB1- deficient cells and the growth of xenograft tumors. In this application, we propose to test a small library of drugs targeting the identified SL targets/pathways alone and pairwise in isogenic pairs of RB1-relevant cancer cell lines (Aim 1); and to test whether the combined targeting of two SL partners selectively and more efficiently to single drugs slows down the growth of xenograft tumors (Aim 2). We expect that our proposal will have a high translational impact as we will prioritize the FDA-approved drugs that can be easily translated to a human clinical trial and provide a personalized approach to RB1-mutated cancers. In addition, we will get new insights into the biology of RB1- deficient cells that belong to the top most often mutant tumor suppressor genes in cancer cells.