PROJECT SUMMARY/ABSTRACT Homologous recombination (HR) repair-deficient cancers are dependent on polymerase theta (POLq)-mediated DNA end joining, so that POLq is a candidate target for these cancers. This reliance extends to the PARP inhibitor-resistant state, where POLq limits excessive DNA end resection and RAD51 loading, preventing the accumulation of toxic intermediates. POLq deficiency has also been linked to micronucleation, a process associated with activation of innate immunity. Through high-throughput small molecule screens, we have recently identified the antibiotic novobiocin (NVB) as a specific POLq inhibitor that selectively kills HR-deficient cells in vitro and in vivo, including those with acquired PARP inhibitor resistance, suggesting that NVB may be useful alone or in combination with PARP inhibition in treating cancers with HR alterations. NVB also induces micronucleation and consequent cGAS/STING pathway activation in HR-deficient cells. In the first Specific Aim, multiple cell line and patient-derived xenograft models of BRCA-mutant breast, ovarian and pancreatic cancer, including those that are PARP inhibitor-sensitive and those with acquired PARP inhibitor resistance, will be studied for response and pharmacodynamics of NVB combined with the PARP inhibitor talazoparib to compare combinatorial antitumor and biological efficacy to that achieved by monotherapy. Pharmacodynamic effects will be assessed using g-H2AX, pRPA and RAD51 foci as biomarkers of DNA damage, end resection and RAD51 loading, respectively, to pilot their utility for clinical trial. In the second Specific Aim, we will conduct Phase 1 clinical trials of NVB monotherapy and NVB combined with talazoparib in patients with advanced solid tumors harboring HR alterations. The monotherapy study will utilize a BOIN design to determine the recommended phase 2 dose of chronic monotherapy and the combination trial will utilize a waterfall design to define the MTD contour over a two-dimensional dose matrix. Both studies will evaluate pharmacokinetic parameters and incorporate paired biopsies for pharmacodynamic endpoints to establish proof-of-mechanism. The minimum biological effective dose of NVB will be determined in the monotherapy trial, which will guide dosing in the combination study. In the third Specific Aim, NVB used alone and combined with talazoparib will be studied for induction of cGAS/STING activation in vitro, as well as in an immunocompetent genetically engineered mouse model of BRCA-deficient breast cancer. The effects of NVB and NVB/talazoparib on the immune microenvironment will be comprehensively characterized and antitumor activity will be assessed in the absence and presence of immune checkpoint blockade. Taken together, the preclinical and clinical studies in this project are designed to advance NVB-mediated POLq inhibition in the armamentarium for HR-deficient cancers.