Through targeting the two different fork stabilizing mechanisms, described above, we hypothesize that combining PARP and ATR inhibition (PARPiCATRi) will lead to increase DNA double-strand breaks and an increase in tumor cell death in cells regardless of HR status. We previously showed that PARPi treatment activates ATR/CHK1 and ATRi added to PARPi treatment decreased ATR/CHK1 signaling, release of G2/M checkpoint, increased DNA breaks, and tumor regression in deficient in vitro and PDX models40. the existence of alternative resistance mechanisms. However, regardless of the mechanisms of resistance, complete and durable therapeutic responses to PARPi-ATRi that significantly increase survival are observed in clinically relevant platinum and acquired PARPi-resistant patient-derived xenografts (PDXs) models. These findings indicate that PARPi-ATRi is a highly promising strategy for OVCAs that acquire resistance to PARPi and platinum. mutant (gene amplification, copy-number gain, or elevated protein expression. These CCNE1HIGH tumors are associated with poor overall survival and platinum resistance1,11,12. Overcoming drug resistance is the ultimate obstacle for curing this disease. Multiple resistance mechanisms to platinum and PARPi have been described. Platinum resistance may emerge due to reduced intracellular drug accumulation, intracellular inactivation of the agent, increased DNA repair, or impaired apoptotic signaling pathways, to name a few13. Mechanisms of PARPi resistance can be HR-dependent or independent. HR-dependent mechanisms include restoration of function either through secondary or reversion mutations14C16 or restoration of HR by other means (loss of 53BP1, RIF1, REV7, PTIP, Artemis, or the Shieldin complex) that are independent of loss or amplification), ATR inhibition not only leads to replication fork collapse, but also loss of the G2-M checkpoint, allowing cells with damaged DNA to progress prematurely into M phase, leading to mitotic catastrophe and cell death37C40. As such, potent and selective ATR inhibitors (ATRi) such as AZD673841 and M662042 are in phase I/II clinical trials (clinicaltrials.gov). Through targeting the two different fork stabilizing mechanisms, described above, we hypothesize that combining PARP and ATR inhibition (PARPiCATRi) will lead to increase DNA double-strand breaks and an increase in PROTAC Mcl1 degrader-1 tumor cell death in cells regardless of HR status. We previously showed that PARPi treatment activates ATR/CHK1 and ATRi added to PARPi treatment decreased ATR/CHK1 signaling, release of G2/M checkpoint, increased DNA breaks, and tumor regression in deficient in vitro and PDX models40. Using our preclinical drug development platform, we established acquired and de novo PARPi and platinum-resistant cell and PDX models encompassing various pathogenic genetic alterations common in the clinic. Here, we report PARPi-resistant cells with varying genetic context and reversion mutation and amplification), all of which exhibit PARPi or platinum resistance. These studies support the use of PARPiCATRi for the treatment of ovarian cancers that progress on PARPi in the clinic. Results Genomic instability and increased ATR/CHK1 with PARPi resistance PARPi and platinum-resistant (platinum/PARPi-resistant) models were developed from copy normal, OVKATE), acquired PARPi-resistant (PEO1-PR and JHOS4-PR), de novo PARPi-resistant (PEO4, Kuramochi, and UWB/BRCA1+/?), and platinum-resistant cells (PEO1-CR; were treated with PARPi 1?M and lysates were collected at 0, 2, 6?h. Cells were selected in PARPi or carboplatin and tested after a 10-day drug washout (except nonsense mutation (c.6952C>T)43 were intrinsically PARPi-resistant despite being amplification ((missense change; ubiquitin-dependent DNA repair regulation), (splice variant; sister chromatid separation), (splice variant; localization of DNA repair proteins), (splice variant; DNA mismatch repair)(LOH; base excision repair), (LOH; epigenetic regulator of DNA damage PROTAC Mcl1 degrader-1 checkpoint), and (LOH; nonhomologous end joining). Alterations in genes related to epigenetic regulation involving transcription factors and regulators (were also detected. We confirmed there was no functional BRCA2 protein expression in all germline (ggenes (Supplementary Table?1). MDR1 protein efflux pump level was also increased in both PARPi/platinum-resistant (amplification; nucleotide excision repair) and (loss of EIF4EBP1 heterozygosity (LOH); nonhomologous end joining). XPC knockdown with siRNA in JHOS4-PR partially restored PARPi sensitivity suggesting functional relevance of this alteration in contributing to PARPi resistance (Supplementary Fig.?2c). Artemis (and copy normal cells such as OVKATE (pCHK1 increased 4-fold in OVCAR3, 6-fold in FUOV1; and 3-fold in COV318 vs OVKATE, copy normal cells; Fig.?1d). Given the increase in phosphor-ATR/CHK1 either with acquired PARPi- resistance or from PROTAC Mcl1 degrader-1 increased CCNE1 expression, these data support targeting the ATR/CHK1 signaling pathway in these models. Combination is synergistic in PARPi and platinum-resistant cells Given that PARPi and carboplatin-resistant cells have increased baseline pCHK1 levels and that PARPis exert toxicity during S phase, PROTAC Mcl1 degrader-1 we examined if combining PARPi with ATRi.