PARP inhibitors (PARPi) have been effective in high-grade serous ovarian cancer (HGSOC), although clinical activity is limited against BRCA wild type HGSOC. The nearly universal loss of normal p53 regulation in HGSOCs causes dysfunction in the G1/S checkpoint, making tumor cells reliant on Chk1-mediated G2/M cell cycle arrest for DNA repair. Therefore, Chk1 is a reasonable target for a combination strategy with PARPi in treating BRCA wild type HGSOC. Here we investigated the combination of prexasertib mesylate monohydrate (LY2606368), a Chk1 and Chk2 inhibitor, and a PARP inhibitor, olaparib, in HGSOC cell lines (OVCAR3, OV90, PEO1 and PEO4) using clinically attainable concentrations. Our findings showed combination treatment synergistically decreased cell viability in all cell lines and induced greater DNA damage and apoptosis than the control and/or monotherapies (p<0.05). Treatment with olaparib in BRCA wild type HGSOC cells caused formation of Rad51 foci, whereas the combination treatment with prexasertib inhibited transnuclear localization of Rad51, a key protein in homologous recombination repair. Overall, our data provide evidence that prexasertib and olaparib combination resulted in synergistic cytotoxic effects against BRCA wild type HGSOC cells through reduced Rad51 foci formation and greater induction of apoptosis. This may be a novel therapeutic strategy for HGSOC.

The aim of our study was to evaluate possible synergistic cytotoxic effects of the combination treatment with the BH3-mimetic ABT-263 and the PARP inhibitor BMN 673 in high-grade serous ovarian cancer (HGSOC) cells using clinically achievable concentrations of each drug. In vitro cytotoxic effects of ABT-263 and BMN 673 were assessed by XTT assay in three HGSOC cell lines: OVCAR3, OVCAR8, and OV90 cells. Combination index values and synergy/antagonism volumes were used to determine synergy. The drug effects on DNA damage accumulation, cell cycle progression, apoptosis induction, and expression levels of Bcl-2 family proteins were examined to dissect molecular mechanisms. The combination treatment synergistically decreased cell viability in a concentration- and time-dependent manner in all cell lines; combination index values were <0.9 and synergy/antagonism volumes were >100 after 72 h of treatment. Clinically achievable concentrations of ABT-263 2 µM and BMN 673 25 nM were used to investigate mechanisms. No increase in γ-H2AX foci formation was observed with addition of ABT-263 to BMN 673 treatment. The combination treatment increased the sub-G1 and Annexin V-positive cell populations after 48 h compared with the control and each monotherapy. It also induced greater caspase-3/7 activity and PARP cleavage. ABT-263 alone and in combination with BMN 673 induced expression levels of Bim, a pro-apoptotic protein. In conclusion, the ABT-263 and BMN 673 combination resulted in synergistic cytotoxic effects against HGSOC cells through greater induction of apoptosis. This may be a novel therapeutic strategy for HGSOC.

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic malignancy in industrialized countries and has limited treatment options. Targeting ataxia-telangiectasia and Rad3-related/cell-cycle checkpoint kinase 1 (CHK1)-mediated S-phase and G2–M-phase cell-cycle checkpoints has been a promising therapeutic strategy in HGSOC. To improve the efficacy of CHK1 inhibitor (CHK1i), we conducted a high-throughput drug combination screening in HGSOC cells. PI3K/mTOR pathway inhibitors (PI3K/mTORi) showed supra-additive cytotoxicity with CHK1i. Combined treatment with CHK1i and PI3K/mTORi significantly attenuated cell viability and increased DNA damage, chromosomal breaks, and mitotic catastrophe compared with monotherapy. PI3K/mTORi decelerated fork speed by promoting new origin firing via increased CDC45, thus potentiating CHK1i-induced replication stress. PI3K/mTORi also augmented CHK1i-induced DNA damage by attenuating DNA homologous recombination repair activity and RAD51 foci formation. High expression of replication stress markers was associated with poor prognosis in patients with HGSOC. Our findings indicate that combined PI3K/mTORi and CHK1i induces greater cell death in HGSOC cells and in vivo models by causing lethal replication stress and DNA damage. This insight can be translated therapeutically by further developing combinations of PI3K and cell-cycle pathway inhibitors in HGSOC. Significance: Dual inhibition of CHK1 and PI3K/mTOR pathways yields potent synthetic lethality by causing lethal replication stress and DNA damage in HGSOC, warranting further clinical development.

<p>Clean version of supplementary Data</p>

HGSOC is characterized by universal TP53 mutations and consequent G1 cell cycle dysfunction, thus relies on ATR/CHK1-mediated G2/M arrest for survival. CHK1 is also necessary for chromosome stability by mitigating replication stress (RS) and promoting homologous recombination (HR) DNA repair following DNA damage. However, clinical responses to CHK1 inhibitor (CHK1i) monotherapy have been variable in HGSOC, requiring new treatment strategies. We thus conducted a high throughput drug combination screening of a CHK1i, prexasertib (Prex) with 1,912 drugs in HGSOC cell lines (BRCA wild-type [BRCAwt: OVCAR5 and OVCAR8] and BRCA2 mutant [BRCA2m: PEO1]). In this screen, 24 drugs showed synergistic cytotoxicity with Prex, including 14 (58%) chemotherapies, 6 (25%) PI3K pathway inhibitors and 4 (17%) targeted drugs. We prioritized the PI3K pathway inhibitors given the PI3K pathway is activated in &gt;45% of HGSOC and associated with increased cell survival, DNA repair and chemo-resistance. We confirmed the combination of Prex and a dual PI3K/mTOR inhibitor LY3023414 (LY302) yielded synergistic cytotoxicity (combination index&lt;1) in a panel of HGSOC cell lines (OVCAR3, OVCAR5, OVCAR8, OV90 and PEO4 [all BRCA-proficient] and PEO1) by XTT and colony formation assays. OVCAR8 and PEO1 were used for further mechanistic studies. With clinically attainable concentrations of Prex (5 nM) and LY302 (200 nM), Prex+LY302 increased caspase 3/7 activation compared to Prex (increased 2,407% and 39%, respectively; P&lt;0.01) or LY302 (increased 2,457% and 57%, respectively; P&lt;0.001) in both cell lines. Prex+LY302 also enhanced the percentage of cells with ≥5 γH2AX foci relative to Prex (increased 39% and 22%, respectively; P&lt; 0.001) or LY302 (increased 48% and 32%, respectively; P&lt;0.001) in both cells, indicating greater DNA damage. We found Prex+LY302 induced a significant HR deficiency as manifested by decreased DR-GFP reporter activity compared to Prex (decreased 37% and 28%, respectively; P&lt;0.01) or LY302 (decreased 44% and 63%, respectively; P&lt;0.01), suggesting Prex+LY302 causes DNA damage likely via reducing HR functionality. Cell cycle analysis showed an imposed S phase arrest in cells treated with Prex+LY302 compared to those treated with Prex (increased 30% and 35%, respectively; P&lt;0.05) or LY302 (increased 38% and 51%, respectively; P&lt;0.01), implying Prex+LY302 increases RS or DNA damage during S phase. Supporting this notion, Prex+LY302 augmented RS as evidenced by increased phospho-RPA+/γH2AX+ populations compared with Prex (increased 37% and 38%, respectively; P&lt;0.05) or LY302 (increased 80% and 79%, respectively; P&lt;0.001). Overall, our results suggest that dual inhibition of CHK1 and PI3K pathways results in greater RS, DNA damage and subsequent cell death in HGSOC cells independent of BRCA mutation status. Citation Format: Tzu-Ting Huang, Jayakumar Nair, Ethan Brill, Xiaohu Zhang, Kelli Wilson, Lu Chen, Craig J. Thomas, Jung-Min Lee. Combined inhibition of checkpoint kinase 1 (CHK1) and phosphoinositide 3-kinase (PI3K) pathways induces greater replication stress and DNA damage in high-grade serous ovarian cancer (HGSOC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 621.

<p>Clean version of supplementary Data</p>

<div>Abstract<p>High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic malignancy in industrialized countries and has limited treatment options. Targeting ataxia-telangiectasia and Rad3-related/cell-cycle checkpoint kinase 1 (CHK1)-mediated S-phase and G₂–M-phase cell-cycle checkpoints has been a promising therapeutic strategy in HGSOC. To improve the efficacy of CHK1 inhibitor (CHK1i), we conducted a high-throughput drug combination screening in HGSOC cells. PI3K/mTOR pathway inhibitors (PI3K/mTORi) showed supra-additive cytotoxicity with CHK1i. Combined treatment with CHK1i and PI3K/mTORi significantly attenuated cell viability and increased DNA damage, chromosomal breaks, and mitotic catastrophe compared with monotherapy. PI3K/mTORi decelerated fork speed by promoting new origin firing via increased CDC45, thus potentiating CHK1i-induced replication stress. PI3K/mTORi also augmented CHK1i-induced DNA damage by attenuating DNA homologous recombination repair activity and RAD51 foci formation. High expression of replication stress markers was associated with poor prognosis in patients with HGSOC. Our findings indicate that combined PI3K/mTORi and CHK1i induces greater cell death in HGSOC cells and <i>in vivo</i> models by causing lethal replication stress and DNA damage. This insight can be translated therapeutically by further developing combinations of PI3K and cell-cycle pathway inhibitors in HGSOC.</p>Significance:<p>Dual inhibition of CHK1 and PI3K/mTOR pathways yields potent synthetic lethality by causing lethal replication stress and DNA damage in HGSOC, warranting further clinical development.</p></div>

<div>Abstract<p>High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic malignancy in industrialized countries and has limited treatment options. Targeting ataxia-telangiectasia and Rad3-related/cell-cycle checkpoint kinase 1 (CHK1)-mediated S-phase and G₂–M-phase cell-cycle checkpoints has been a promising therapeutic strategy in HGSOC. To improve the efficacy of CHK1 inhibitor (CHK1i), we conducted a high-throughput drug combination screening in HGSOC cells. PI3K/mTOR pathway inhibitors (PI3K/mTORi) showed supra-additive cytotoxicity with CHK1i. Combined treatment with CHK1i and PI3K/mTORi significantly attenuated cell viability and increased DNA damage, chromosomal breaks, and mitotic catastrophe compared with monotherapy. PI3K/mTORi decelerated fork speed by promoting new origin firing via increased CDC45, thus potentiating CHK1i-induced replication stress. PI3K/mTORi also augmented CHK1i-induced DNA damage by attenuating DNA homologous recombination repair activity and RAD51 foci formation. High expression of replication stress markers was associated with poor prognosis in patients with HGSOC. Our findings indicate that combined PI3K/mTORi and CHK1i induces greater cell death in HGSOC cells and <i>in vivo</i> models by causing lethal replication stress and DNA damage. This insight can be translated therapeutically by further developing combinations of PI3K and cell-cycle pathway inhibitors in HGSOC.</p>Significance:<p>Dual inhibition of CHK1 and PI3K/mTOR pathways yields potent synthetic lethality by causing lethal replication stress and DNA damage in HGSOC, warranting further clinical development.</p></div>