ABSTRACT This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 24-029. Entitled ‘PROTAC-based direct and oncolytic killing of EBV+ DLBCLs through synthetic lethality’, this is an administrative supplement to our U01 award entitled ‘Synthetic lethal targeting of EBV-positive diffuse large B cell lymphomas in persons living with HIV’. Diffuse large B-cell lymphoma (DLBCL), the commonest type of non-Hodgkin lymphoma (NHL), is highly aggressive and associated with significantly worse outcomes when infected with Epstein-Barr virus (EBV) particularly in immunosuppressed individuals such as those with HIV. Thus, understanding how EBV contributes to cancer is essential to discovering new therapeutic approaches. In the U01 funded project, we are investigating the hypothesis that EBV+ DLBCL are vulnerable to synthetic lethal therapeutic targeting. This is based on our observation that EBV rewires cellular DNA repair mechanisms such that cancer cells lose homologous recombination repair, becoming dependent on microhomology-mediated end-joining (MMEJ) and therefore susceptible to synthetic lethal agents that target MMEJ and other forms of DNA repair. Through our U01 project, we have confirmed EBV’s influence on DNA repair mechanisms in DLBCLs and their susceptibility to synthetic lethal agents such as PARP inhibitors and inhibitors of polymerase theta (POLθ), the MMEJ polymerase – and continue to investigate mechanisms and novel synthetic lethal vulnerabilities of EBV+ DLBCL. This application takes advantage of a new collaboration and a novel observation: Dr. Guangrong Zheng, at the University of Florida College of Pharmacy, is a medicinal chemist with expertise in PROTAC (proteolysis targeting chimera) chemistry. PROTACs are designed to degrade proteins of interest. Our novel observation uses a PROTAC developed by Dr. Zheng to selectively degrade HDACs 3 & 8 – using this, we are able to reactivate EBV into the lytic/replicative phase which should enable killing of EBV+ cancer cells by oncolysis. With PROTACs in clinical trials for cancer and oncolytic therapies also in clinical trials for EBV+ lymphomas, we build on our existing hypothesis together with Dr. Zheng to expand our synthetic lethal approach for EBV+ DLBCL. Our new alliance with Dr. Zheng will allow us to 1) investigate the combined (i.e. synthetic lethal) effects of inhibiting POLθ to directly kill and the HDAC3 & 8 PROTAC to indirectly kill tumor cells by forcing EBV out of the latent state and 2) construct a first of its kind POLθ-PROTAC using existing POLθ inhibitors as warheads along with Dr. Zheng’s expertise and library of PROTAC linkers and ligands. We anticipate this multi-pronged approach to pave the way for more effective management of EBV+ DLBCLs and a POLθ-PROTAC to benefit many with cancers known to be susceptible to synthetic lethal approaches.