Summary Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are an exciting and promising new class of anticancer drugs, which have been approved by the FDA for recurrent ovarian cancer with BRCA1 or BRCA2 mutations, and as maintenance therapy after frontline therapy for platinum sensitive ovarian cancer regardless of BRCA mutation. PARPi selectively kill BRCA1/2-deficient cancer cells through synthetic lethality. However, patients receiving PARPi eventually develop cancer progression, and acquired PARP inhibitor resistance remains a clinical hurdle. One of the mechanisms underlying acquired PARPi resistance is the restoration of DNA repair capacity, mainly through the secondary mutations of BRCA1/2. Our preliminary study has demonstrated that PARPi can enhance the Aldehyde dehydrogenase (ALDH) activity in ovarian cancer cells, mainly through inducing expression of ALDH1A1, an isoform of the ALDH family. In addition, we also found that ALDH1A1 is able to augment the microhomology-mediated end joining (MMEJ), one of the pathways for repairing DNA double-strand breaks (DSBs), enhance the expression of DNA polymerase θ (Pol θ), a key player in the MMEJ pathway, as well as reduce the sensitivity of BRCA2 mutated ovarian cancer cells to PARPi. Based on this scientific premise, we generate a hypothesis that PARPi-induced overexpression of ALDH1A1 enhances MMEJ via increasing the expression of Pol θ, and promotes cell survival after PARPi treatment in HR-deficient cancer cells, eventually leading to acquired PARPi resistance. Consequently, inhibition of ALDH1A1 should be able to synergize with PARPi in treating HR-deficient EOC, and reverse resistance to PARPi in HR-deficient EOC. The main objective of this proposal is to determine a novel mechanism that contributes to PARPi resistance in BRCA1/2-mutated EOC cells, and test the efficacy of targeting this mechanism in preventing and reversing PARPi resistance in these cells. Two specific aims are proposed to test this hypothesis and achieve our goal. In specific aim 1, we will determine the mechanism underlying PARPi-induced augmentation of MMEJ in HR-deficient EOC cells and its contribution to PARPi resistance. In specific aim 2, we will determine the therapeutic potential of an ALDH1A1 inhibitor, NCT-505, in preventing and reversing PARPi resistance in BRCA1/2-mutated EOC in vitro and in vivo. It is our expectation that at the conclusion of this project, we will have demonstrated a new mechanism contributing to the development of PARPi resistance in HR-deficient EOC. We will have also shown the therapeutic potential of an ALDH1A1 inhibitor in treating these patients.