PROJECT SUMMARY Diffuse large B-cell lymphoma (DLBCL), the commonest type of non-Hodgkin lymphoma (NHL), is highly aggressive and despite antiretrovirals continues to be a leading cause of cancer-related death in persons living with HIV. Notably, up to 90% of HIV-DLBCL are positive for the cancer-causing Epstein-Barr virus (EBV). Thus, understanding how EBV contributes to cancer is essential to discovering new therapeutic approaches. Cancer cells require DNA repair but how EBV engages and reshapes cellular DNA repair is an underexplored area. Our studies on EBV-cancer cells and EBV-transformed human B cells (lymphoblastoid cell lines), the latter an important model of EBV-driven lymphomas in immunosuppressed hosts, converge on STAT3. An oncoprotein, STAT3 is frequently activated in cancer. Several studies have also shown that EBV+ HIV-DLBCL frequently exhibit activating mutations in the Janus kinase (JAK)-STAT3 pathway. We have found that EBV activates STAT3 to circumvent the S phase checkpoint barrier, thereby ensuring cell proliferation but in the process, loses homologous recombination (HR) that repairs DNA double strand breaks (DSB). As a result, EBV- transformed and cancer cells become dependent on other forms of DNA repair, in particular, the error-prone microhomology-mediated end-joining (MMEJ) type of repair. This creates a therapeutic vulnerability to synthetic lethal agents that would otherwise be non-toxic to cells with intact HR. PARP [poly (ADP-ribose) polymerase] inhibitors are among such synthetic lethal agents that target MMEJ. Indeed, we find that EBV-transformed and cancer cells are highly susceptible to MMEJ inhibitors that target PARP and the MMEJ-specific DNA polymerase, POLθ. Supporting this dependence on MMEJ, EBV-transformed cells exhibit genome-wide scars of MMEJ repair, and, EBV+ HIV-DLBCL display higher abundance of STAT3 and POLQ transcripts compared to EBV- tumors; POLQ encodes POLθ. Further, by multiomic analyses of several hundred cancer cell lines, we have identified a STAT3-related gene expression signature that points to a mechanistic link between STAT3 and reliance on MMEJ repair while predicting susceptibility to synthetic lethal therapies. We now propose to investigate how EBV uses the JAK-STAT3 pathway to reshape DNA repair and render EBV+ HIV-DLBCL vulnerable to synthetic lethal therapeutic targeting. Using cell lines, xenografts, and patient-derived EBV+ & EBV- HIV-DLBCL from the NCI AIDS and Cancer Specimen Resource (ACSR), we investigate the link between JAK-STAT3 pathway and DSB repair in EBV+ HIV-DLBCL (Aim 1) and synthetic-lethally exploit JAK- STAT3-dependent DNA repair deficiency to kill EBV+ HIV-DLBCL (Aim 2). These studies specifically address PAR-21-348 by identifying mechanisms and generating new paradigms to reveal how EBV contributes to NHL. In the long-term, these mechanistic insights will uncover novel vulnerabilities and enable the prediction of responses to synthetic lethal therapies to imp...