Synergistic efficacy of gedatolisib and darolutamide in prostate cancer to overcome resistance to androgen-targeted therapy.

383 Background: The PI3K/AKT/mTOR (PAM) pathway is frequently dysregulated in prostate cancer (PC), often in association with the loss of PTEN. Since PAM pathway activation is a key adaptive resistance mechanism to androgen-targeted therapy, concomitant inhibition of both the PAM pathway and the androgen receptor (AR) pathway is a promising treatment strategy for castration resistant PC (CRPC). However, many current inhibitors that only target single PAM pathway components have limited effectiveness because other components of the pathway can circumvent their activity. Our prior research showed that gedatolisib, a multi-target PAM inhibitor that targets all Class I PI3K isoforms and mTORC1/2, exerts greater growth inhibitory effects than single-target inhibitors in PC cell lines, irrespective of their PTEN or AR status. In the present study, we tested gedatolisib in combination with darolutamide in PC cell models. Methods: The cellular and molecular effects of the gedatolisib/darolutamide combination were tested in both PTEN-positive and PTEN-deficient PC cell lines. AR-positive PC lines adapted to long-term darolutamide treatment were also developed to model progression after AR-targeted therapy. To assess the effects of gedatolisib, darolutamide, or their combination, multiple assays were employed, including GR metrics analysis, flow cytometry analysis of PAM pathway activity, cell cycle and death, analysis of glucose and lipid metabolism, and qPCR analysis of AR, AR-target genes, and E2F-target genes. Results: The combination of gedatolisib and darolutamide demonstrated stronger anti-proliferative and cytotoxic effects across most AR-positive PC cell lines compared to either drug alone, regardless of the cells’ PTEN status. Mechanistically, the drug combination blocked cell cycle progression and DNA proliferation in association with decreased E2F-target gene transcription, induced apoptosis, and reduced glucose and lipid metabolism. Importantly, the combination was effective in cell lines adapted to darolutamide, suggesting a potential benefit for prostate tumors that have progressed following androgen-targeted therapies. Conclusions: The combination of gedatolisib and darolutamide shows a combinatorial benefit in multiple AR-positive PC cell models, regardless of PTEN status or sensitivity to AR inhibitors. These results provide a strong mechanistic rationale for clinical studies evaluating gedatolisib in combination with AR inhibitors in CRPC.