Targeting Polθ Helicase with RP-3467: Preclinical Validation and Clinical Response in a g BRCA1 ovarian cancer patient with an acquired PARPi resistance mutation in TP53BP1

Homologous recombination (HR) deficiency underlies a range of cancers, and PARP inhibitors (PARPi) targeting this vulnerability have demonstrated clinical efficacy in BRCA1/2 -mutant tumors. However, resistance mechanisms and tolerability issues limit their long-term clinical effectiveness. Here, we characterize RP-3467, a novel, selective small-molecule inhibitor targeting the ATPase domain of DNA polymerase theta (Polθ), the key mediator of the error-prone microhomology-mediated end joining (MMEJ) pathway. We demonstrate that RP-3467 potently suppresses Polθ enzymatic activity, abrogates MMEJ, and disrupts mitotic DNA repair in HR-deficient (HRD) models, inducing synthetic lethality. In preclinical studies, we show that RP-3467 synergizes strongly with PARPi (olaparib and rucaparib), achieving sustained tumor regression across multiple xenograft and patient-derived xenograft (PDX) models without added systemic toxicity. Mechanistically, we find that RP-3467 increases mitotic DNA damage in vivo, as evidenced by elevated micronuclei and CIP2A foci. RP-3467 was evaluated in a phase 1 clinical trial ( NCT06560632 ) and has shown preliminary signs of clinical activity. A heavily pretreated germline BRCA1 -mutant ovarian cancer patient achieved a confirmed partial response to RP-3467 plus olaparib (RECIST v1.1), with retrospective profiling revealing a TP53BP1 loss-of-function mutation previously linked to both PARPi resistance and POLQ dependency. Although broader clinical validation is needed, these early findings suggest that Polθ inhibition has the potential to address PARPi resistance and offer therapeutic benefit in HRD cancers.