Background: GP-2250, a novel analog of taurultam (TRLT), has emerged as a potent anti-neoplastic drug; however, the mechanisms underlying its effects are not well understood. Here, we investigated the mechanism of action and the biological effects of GP-2250 using in vitro and in vivo models. Methods: We carried out a series of in vitro experiments including MTT assay, Annexin V/PI assay, colony formation assay, reverse-phase protein array (RPPA), and HRLC/IC analysis to determine the biological activity of GP-2250 and investigate the mechanism of action. In vivo experiments were carried out to determine the therapeutic efficacy of GP-2250 alone and in combination with standard-of-care drugs (e.g., paclitaxel, cisplatin topotecan, and poly ADP-ribose polymerases (PARP) inhibitors. Results: We investigated the cytotoxic effect of GP-2250 in 10 ovarian cancer cell lines and found that HRD ovarian cancer cells (e.g., Kuramochi, OVCAR4, and OVCAR8) were more vulnerable to GP-2250 than HRP ovarian cancer cells (e.g., A2780 and OVCAR5). In addition, GP-2250 combination with a PARP inhibitor showed the most synergistic effects. There was no difference among the PARP inhibitors (e.g., olaparib, niraparib, and rucaparib) with regard to the combinatorial effect with GP-2250. RPPA analyses revealed that GP-2250 inhibited hypoxia-inducible factor-1α, AKT, and mTOR activation and expression level. Ultra-high resolution mass spectrometry (HRMS) analysis also revealed that hexokinase2 activity and expression was significantly reduced by GP-2250 treatment. Furthermore, GP-2250 also reduced glycolysis and ATP synthesis in cancer cells. In vivo pharmacodynamic experiment using the OVCAR8 mouse model demonstrated that a dose of 500 mg/kg GP-2250 was the most effective in downregulating AKT and mTOR activation and expression In the in vivo therapy experiment using an orthotopic mouse model, a combination of GP-2250 and PARP inhibitors (olaparib, niraparib, or rucaparib) or bevacizumab showed a significant reduction of tumor weights (0.16 ± 0.05 g, 0.13 ± 0.06 g, 0.29 ± 0.05 g, and 0.07 ± 0.03 g, respectively) and nodules (1.56 ± 0.44, 1.89 ± 0.59, 3.11 ± 0.59, and 0.78 ±0.2, respectively) compared to those treated with a vehicle (tumor weight, 0.95 ± 0.1 g and nodules, 8.4 ± 0.65) or the monotherapy groups, GP-2250 (tumor weight, 2.9 ± 0.48 g, and nodules, 2.9 ± 0.48), olaparib (tumor weight, 0.53 ± 0.09 g, and nodules, 3.3 ± 0.64), niraparib (tumor weight, 0.38 ± 0.05 g, and nodules, 3.4 ± 0.44), rucaprib, (tumor weight, 0.52 ± 0.1 g, and nodules, 4.85 ± 0.79), and bevacizumab (tumor weight, 0.43 ± 0.08 g, and nodules, 3.8 ± 0.71), respectively. Conclusions: Taken together, our data indicate that GP-2250 exerts profound effects on tumor metabolism and combination with PARP inhibitors or bevacizumab showed promising anti-tumor efficacy. These findings could have implications for the clinical development of GP-2250. Citation Format: Mark S. Kim, Deanna Glassman, Adrian Lankenau Ahumada, Emine Bayraktar, Nicholas B. Jennings, Robiya Joseph, Sanghoon Lee, Robert L. Coleman, Anil K. Sood. Mechanisms and rational combinations with gp-2250, a novel oxathiazine derivative, in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 528.

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Patients with high-grade serous ovarian cancer (HGSC) who have no visible residual disease (R0) after primary surgery have the best clinical outcomes, followed by patients who undergo neoadjuvant chemotherapy (NACT) and have a response enabling interval cytoreductive surgery. Clinically useful biomarkers for predicting these outcomes are still lacking. Extracellular vesicles (EVs) have been recognized as liquid biopsy-based biomarkers for early cancer detection and disease surveillance in other disease settings. In this study, we performed extensive molecular characterization of serum-derived EVs and correlated the findings with therapeutic outcomes in patients with HGSC. Using EV-DNA whole-genome sequencing and EV-RNA sequencing, we identified distinct somatic EV-DNA alterations in cancer-hallmark genes and in ovarian cancer genes, as well as significantly altered oncogenic pathways between the R0 group and NACT groups. We also found significantly altered EV-RNA transcriptomic variations and enriched pathways between the groups. Taken together, our data suggest that the molecular characteristics of EVs could enable prediction of patients with HGSC who could undergo R0 surgery or respond to chemotherapy.

Purpose Tumor-associated macrophages (TAMs) are known to contribute to adaptive resistance to anti-vascular endothelial growth factor (VEGF) antibody (AVA) therapy in ovarian cancer. BET (bromodomain and extra-terminal domain) inhibitors (BETi) may have unique roles in targeting TAMs. Our objective was to examine the effects of BETi on TAMs, especially in the context of enhancing the efficacy of AVA therapy. Methods We conducted a series of in vitro (MTT assay, apoptosis, flow cytometry, and RNA sequencing) and in vivo (xenograft ovarian cancer model) experiments to determine the biological effects of BETi combined with AVA in ovarian cancer. For statistical analysis, a two-tailed Student’s t test (equal variance) or ANOVA was used for multiple groups’ comparison, and p < 0.05 was considered significant. Results BETi resulted in a dose-dependent decrease in cell viability and induced apoptosis (p < 0.01) in ovarian cancer cells (SKOV3ip1, OVCAR5, and OVCAR8). Treatment with BETi significantly increased apoptosis in THP-1 monocytes and macrophages (PMA-differentiated THP-1; p < 0.01). Furthermore, BETi selectively induced greater apoptosis in M2-like macrophages (PMA and IL-4, IL-13-differentiated THP-1) (31.3%-36.1%) than in M1-like macrophages (PMA and LPS-differentiated THP-1) (12.4%-18.5%) (p < 0.01). Flow cytometry revealed that the percentage of M1-like macrophages (CD68+/CD80+) was significantly increased after treatment with low-dose BETi (ABBV-075 0.1 µM; p < 0.05), whereas the percentage of CD68+/CCR2+ macrophages was significantly decreased (p < 0.001); these findings suggest that BETi may selectively inhibit the survival of CCR2+ macrophages and re-polarize the macrophages into an M1-like phenotype. RNA-seq analysis revealed that BETi selectively targeted macrophage infiltration-related cytokines/chemokines in ovarian cancer (adjusted p < 0.05 and Log2 fold change ≥ 1.5). Finally, using in vivo ovarian cancer models, compared with control or monotherapy, the combination of BETi (ABBV-075) and bevacizumab resulted in greater inhibition of tumor growth and macrophage infiltration (p < 0.05) and longer survival of tumor-bearing mice (p < 0.001). Conclusions Our findings indicate a previously unrecognized role for BETi in selectively targeting CCR2+ TAMs and enhancing the efficacy of AVA therapy in ovarian cancer.