GAS6 inhibition induces platinum sensitivity through increased replication stress in ovarian cancer

Objectives: More than 80% of women with high grade serous ovarian carcinoma (HGSOC) ultimately develop platinum resistance. There are no FDA approved agents to improve sensitivity to carboplatin. On candidate target is growth arrest-specific 6 (GAS6) which has been associated with prognosis in HGSOC. We hypothesized that GAS6 inhibition with AVB-500 (AVB) sensitizes platinum-resistant cells to platinum chemotherapy by stalling replication forks and increasing replication stress. We also hypothesized that GAS6 expression would predict response to neoadjuvant chemotherapy. Methods: AVB was supplied by Aravive Biologics. Both homologous recombination (HR)-proficient (OVCAR3-TPMES, CAOV3, COV362) and HR-deficient (OVCAR8) cells were used for all experiments. In vitro clonogenic assays were done on chemoresistant ovarian tumor cells treated with carbo±AVB. The effect of carbo + AVB on intraperitoneal tumor burden was evaluated in mouse models. For DNA fiber assays, cells were labeled with the thymidine analog IdU for 20 minutes followed by CldU for 60 minutes and treatment with carbo, cisplatin (cis), or AVB. Immunofluorescent (IF) assays targeting γH2AX for DNA damage, RAD51, BRCA1, and BRCA2 for HR and 53BP1 for non-homologous end joining (NHEJ) were performed. Human HGSOC samples were obtained pre- and post-neoadjuvant chemotherapy. GAS6 expression was measured by tissue immunohistochemistry (IHC) and serum ELISA. Results: Carbo + AVB decreased survival of platinum-resistant cells as measured by clonogenic colonies than carbo alone (p<0.05). Platinum-resistant mouse models treated with chemotherapy + AVB had significantly less tumor burden than those treated with chemotherapy alone (50mg vs 357mg, P<0.01). Combenefit analyses confirmed AVB and carboplatin were synergistic. Treatment with carbo or cis plus AVB led to replication fork perturbation and increased replication fork stress than carbo/cis or AVB alone. Specifically, there was significant shortening of the CldU label and decrease of CldU/IdU ratios in cells treated with carbo/cis plus AVB. Additionally, tumor cells treated with carbo + AVB compared to carbo alone demonstrated an increase in γH2AX and 53BP1 foci (P<0.01) and a decrease in RAD51, BRCA1, and BRCA2 foci (P<0.05). Patients with high pretreatment tumor GAS6 IHC expression (>80%) (n=7) or serum GAS6 concentrations (>25ng/mL) (n=13) were more likely to have a poor response to neoadjuvant chemotherapy than those with low GAS6 (P=0.002). Additionally, high GAS6 concentration was associated with decreased overall survival (24.4 months versus median not reached, P=0.009). Conclusions: Inhibition of the GAS6 pathway with AVB improves sensitivity of platinum-resistant cells to platinum chemotherapy by increasing replication stress and DNA damage and decreasing HR. GAS6 is a potential biomarker predictive of poor response to platinum-based neoadjuvant chemotherapy and might identify patients who would benefit from treatment with AVB. More than 80% of women with high grade serous ovarian carcinoma (HGSOC) ultimately develop platinum resistance. There are no FDA approved agents to improve sensitivity to carboplatin. On candidate target is growth arrest-specific 6 (GAS6) which has been associated with prognosis in HGSOC. We hypothesized that GAS6 inhibition with AVB-500 (AVB) sensitizes platinum-resistant cells to platinum chemotherapy by stalling replication forks and increasing replication stress. We also hypothesized that GAS6 expression would predict response to neoadjuvant chemotherapy. AVB was supplied by Aravive Biologics. Both homologous recombination (HR)-proficient (OVCAR3-TPMES, CAOV3, COV362) and HR-deficient (OVCAR8) cells were used for all experiments. In vitro clonogenic assays were done on chemoresistant ovarian tumor cells treated with carbo±AVB. The effect of carbo + AVB on intraperitoneal tumor burden was evaluated in mouse models. For DNA fiber assays, cells were labeled with the thymidine analog IdU for 20 minutes followed by CldU for 60 minutes and treatment with carbo, cisplatin (cis), or AVB. Immunofluorescent (IF) assays targeting γH2AX for DNA damage, RAD51, BRCA1, and BRCA2 for HR and 53BP1 for non-homologous end joining (NHEJ) were performed. Human HGSOC samples were obtained pre- and post-neoadjuvant chemotherapy. GAS6 expression was measured by tissue immunohistochemistry (IHC) and serum ELISA. Carbo + AVB decreased survival of platinum-resistant cells as measured by clonogenic colonies than carbo alone (p<0.05). Platinum-resistant mouse models treated with chemotherapy + AVB had significantly less tumor burden than those treated with chemotherapy alone (50mg vs 357mg, P<0.01). Combenefit analyses confirmed AVB and carboplatin were synergistic. Treatment with carbo or cis plus AVB led to replication fork perturbation and increased replication fork stress than carbo/cis or AVB alone. Specifically, there was significant shortening of the CldU label and decrease of CldU/IdU ratios in cells treated with carbo/cis plus AVB. Additionally, tumor cells treated with carbo + AVB compared to carbo alone demonstrated an increase in γH2AX and 53BP1 foci (P<0.01) and a decrease in RAD51, BRCA1, and BRCA2 foci (P<0.05). Patients with high pretreatment tumor GAS6 IHC expression (>80%) (n=7) or serum GAS6 concentrations (>25ng/mL) (n=13) were more likely to have a poor response to neoadjuvant chemotherapy than those with low GAS6 (P=0.002). Additionally, high GAS6 concentration was associated with decreased overall survival (24.4 months versus median not reached, P=0.009). Inhibition of the GAS6 pathway with AVB improves sensitivity of platinum-resistant cells to platinum chemotherapy by increasing replication stress and DNA damage and decreasing HR. GAS6 is a potential biomarker predictive of poor response to platinum-based neoadjuvant chemotherapy and might identify patients who would benefit from treatment with AVB.