A novel CerS2 activator, DH20931, synergizes with doxorubicin to overcome therapeutic resistance in breast cancer

Background: Doxorubicin (DOXO) efficacy in triple-negative breast cancer (TNBC) is severely limited by acquired resistance and dose-limiting cardiotoxicity. We identified ceramide synthase 2 (CerS2), producer of tumor-suppressive very long-chain ceramides (VLCCs), as a prognostic marker and therapeutic target in breast cancer (BC). We previously developed DH20931, a CerS2 activator that inhibits growth across BC subtypes by inducing lipotoxic endoplasmic reticulum (ER) stress. We hypothesized that DH20931 could sensitize BC cells to DOXO and reverse resistance. Methods: The synergistic efficacy of DH20931 and DOXO was evaluated in parental and DOXO-resistant BC cell lines, e.g., MDA-MB-231, 4T1. Synergy was quantified using the Chou-Talalay method (Combination Index, CI). Mechanisms were assessed via confocal microscopy (DOXO uptake), immunoblotting (ER stress, apoptosis markers) and in vivo efficacy. Results: The DH20931 combination significantly reduced the required IC 50 of DOXO by 3-8-fold. DH20931 demonstrated strong synergy with DOXO (CI <1) across all tested BC cell lines, including DOXO-resistant models. The synergy was eliminated in CerS2-knockout cells indicating CerS2 target engagement. Mechanistically, the combination therapy resulted in hyper-activation of the UPR, maximized ATF4/CHOP/PUMA expression, and a marked increase in cleaved caspase-3 compared to either agent alone. In vivo , the DH20931+DOXO combination significantly inhibited tumor growth compared to DH20931 and DOXO monotherapy. Conclusion: DH20931 effectively sensitizes BC cells to Doxorubicin and reverses DOXO-resistance, likely by enhancing CerS2-mediated lipotoxic ER-stress. This synergistic approach offers a promising clinical strategy to improve DOXO efficacy, reduce its cardiotoxicity by allowing dose reduction, and improve outcomes for patients with TNBC and other BC subtypes.