Abstract PARP1/2 inhibitors (PARPi) are the first approved targeted DNA damage response (DDR) inhibitors and have been shown to have clinical benefit, particularly in tumors harboring mutations in BRCA1/BRCA2 and other genes of homologous recombination repair (HRR). However, resistance to PARPi monotherapy will impact on both the breadth and depth of response. We showed that HRR-mutant TNBC and ovarian cancer patient-derived tumor xenograft (PDX) models frequently exhibit innate or acquired PARP inhibitor resistance and that this resistance is linked to a proficient or reactivated HRR, indicated by the ability to form RAD51 foci. A key component of PARPi mechanism of action results from trapping PARP onto DNA, which has the potential to generate replication stress. Moreover, recent data demonstrate that PARP1, along with components of the HRR, are associated with the re-start and protection of stalled replication forks. Here, we assessed whether combinations of the PARPi olaparib together with inhibitors of the replication stress response (RSR) could reverse PARPi resistance in our PDX cohort and we analyzed the effects on RSR by immunofluorescence and immunoblotting. Our data demonstrate that 7/27 models exhibit WEE1i (AZD1775) single agent activity measured as tumor regressions (≤ -30% change in tumor volume), all of which were PARPi resistant. Of the PARPi and WEE1i resistant models, 5 responded to combination treatment and demonstrated a significant increase in the RSR markers pRPA32 and pan-γH2AX compared to either single agent treatment alone. For monotherapy ATRi treatment, there were three models showing tumor regression with AZD6738, all harbored ATM mutations, two also responding to WEE1i monotherapy and one responded to olaparib monotherapy. There were an additional two models that responded to the combination of PARPi plus ATRi, and one of these also responded to the WEE1i/PARPi combination. Together, our analysis demonstrates that by targeting the replication stress response we could cause tumor regression in 13/20 PARPi resistant PDX models and in 6 of these cases the response required PARP inhibition, with DDR signalling indicating that the PARPi was impacting on the RSR. Further insights will be presented into which genetic backgrounds and acquired PARPi resistant mechanisms are reversed by targeting either WEE1 or ATR, thus highlighting the potential for how PARPi resistance can be reversed by targeting alternative DDR dependencies. Citation Format: Mark J. O'Connor, Cristina Cruz, Marta Castroviejo-Bermejo, Urszula M. Polanska, Gemma N. Jones, Anderson Wang, Zhongwu Lai, Josep Forment, Krishna Bulusu, Alba Llop-Guevara, Brian Dougherty, Cristina Saura, Rachel Brough, Chris J. Lord, Alejandra Bruna, Carlos Caldas, Stephen Fawell, J Carl Barrett, Susan E. Critchlow, Judith Balmaña, Elaine Cadogan, Violeta Serra. Reversing PARP inhibitor resistance by targeting the replication stress response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 932.