The Endocannabinoid N-Arachidonoyl Dopamine Induces Leukemia Cell Pyroptosis to Overcome the Venetoclax Resistance

Bcl-2 inhibitor has largely changed the treatment options in elderly acute myeloid leukemia patients, while drug insensitivity and resistance still hinder the overall survival. In our previous study, a natural product, N-arachidonoyl dopamine (NADA), was discovered to inhibit NRAS membrane translocation and induced “oncosis-like” cell death. In this study, we found that NADA induced pyroptosis of the leukemia cells by promoting Caspase -1 (CASP1) mediated Gasdermin D (GSDMD) cleavage. Cell dynamic monitoring and western blot analysis revealed that NADA induced typical pyroptosis cell blebbing and the cleavage of GSDMD and CASP1 in leukemia cells. Additionally, the activity of NADA on GSDMD cleavage and pyroptosis induction could be selectively rescued by the CASP1 inhibitor. Moreover, in GSDMD and CASP1 knock-down/knock-out leukemia cells and GSDMD^-/- mice, the cell viability inhibition effect of NADA was impaired significantly. To explored the molecular target of NADA in pyroptosis induction, we performed a click chemistry assay with N3 tagged NADA (N3-NADA). In the analysis of cell death pathway proteins binding to N3-NADA, GSDMD was identified in three independent assays, suggesting a direct or indirect binding of NADA to GSDMD. Subsequently, molecular docking analysis showed a direct binding between NADA and GSDMD, with NADA embedding into the pocket between the N and C terminal of GSDMD with lower binding free energy. Molecular dynamics simulation further revealed the GSDMD N domain in the NADA-GSDMD-CASP1 complex is more rigid than that in the GSDMD-CASP1 complex, resulting in a shorter distance between the GSDMD P1 site residue D275 and the key residues of catalytic site in CASP1, leading to higher cleavage efficiency. Thus, NADA could directly bind to GSDMD and promote its CASP1 dependent cleavage, leading to cell pyroptosis. As the overexpression of anti-apoptotic protein other than BCL-2 is one of the main causes of venetoclax (ABT-199) resistance, therapy surpasses the apoptosis pathway could be effective. Therefore, we used NADA in combination with ABT-199 in both in vitro and in vivo studies to test its anti-tumor effects. The results showed that NADA significantly increased the sensitivity of ABT-199 resistant (VENR) cells to ABT-199. In summary, these findings provide a new strategy to overcome drug resistance and help to develop novel therapies for leukemia.