Cephalochromin-Induced Mitochondrial Damage Overcomes Venetoclax-Resistance in Acute Myeloid Leukemia Models

Abstract

The combination of the BCL2 inhibitor venetoclax (VEN) with hypomethylating agents (e.g. azacytidine, AZA) significantly increased complete remission (CR) rates and prolonged overall survival of adults with Acute Myeloid Leukemia (AML) who were ineligible to intensive chemotherapy. However, VEN+AZA is not a curative treatment and resistance to VEN and/or relapses are common. Therefore, the search for new agents able to improve the depth and duration of CR or to overcome VEN resistance are needed. Cephalochromin (CPC), a natural compound easily obtained from Comospora vilior, emerges as a potential candidate. Here, we initially accessed the effects of CPC and VEN monotherapies regarding the cell survival of a large panel of AML cell lines (n=12), covering the main different molecular profiles observed in AML patients. Each cell model exhibited a degree of sensitivity to each drug, with no correlation between the sensitivity for both compounds across the cell models. IC 50 values for CPC ranged from 0.45 to >20 µM, while VEN IC 50 values ranged from <0.001 to 17.5 µM upon 72 h of treatment. To evaluate the cytotoxic effects of CPC in healthy cells, we treated human peripheral blood mononuclear cells from 4 healthy donors, which revealed IC50 values ranging from 3.7 to 8.8 µM at 72 h, suggesting a potential feasible therapeutic window. To evaluate the potential combination effects of CPC + VEN in AML, we performed combination experiments in AML models, chosen based on their VEN sensitivity (Kasumi-1 and U937 cells as partially resistant and OCI-AML3 displaying higher VEN resistance). Combination effects in all AML models revealed an significant improvement on the performance of VEN when combined to CPC, even in low doses of both compounds. We next evaluated the molecular mechanisms associated with the effects of CPC on cell proliferation and survival. Clonogenic assays revealed that CPC treatment was associated with decreased stemness capacity, in a concentration-dependent manner. Cell cycle analysis on CPC-treated cells revealed a progressive increase in sub-G1 cells population, suggesting apoptosis induction in a dose-dependent manner, which was further corroborated by increased Annexin-V levels on treated cells. The effects of CPC regarding the apoptosis induction were more evident in Kasumi-1 cells when compared with U937 and OCI-AML3 cells. In OCI-AML3 cells (which display the highest MCL1 expression across the different AML models), combination of low concentrations of VEN (1 µM) and CPC (1.8 µM) showed an apoptosis induction in 95% of the cells. Similarly, the combination CPC + VEN resulted in higher levels of apoptosis induction in U937 (monocytic leukemia; VEN 2.5 + CPC 2.5 µM) and Kasumi-1 ( TP53 R248Q mutant; VEN 0.6 + CPC 2.5 µM) cells. Metabolic analysis of CPC treated cells revealed extensive mitochondrial damage associated with reduction in the mitochondria mass. Proteomic analysis showed increase PARP1 cleavage (apoptosis) and γH2AX (DNA damage) levels upon CPC treatment in all evaluated models, whereas MCL1 was reduced in Kasumi-1 cells. Moreover, SQSTM1/p62 levels were reduced in U937 and Kasumi-1 cells, suggesting induction of autophagy as a mechanism of response to CPC in these cells. Finally, these findings were confirmed in a cohort of ex vivo treated AML samples (n=10) treated with CPC and VEN alone or in combination, where the combination of VEN 0.1 + CPC 2.5 µM displayed strong synergistic effect when compared to the monotherapies. Treatment with CPC reduced the mitochondrial membrane potential, suggesting the disturbance on the mitochondrial metabolism as one of the key phenomena associated with the efficiency of the CPC+VEN combination therapy. In summary, by using multiple cellular models, we demonstrated that CPC treatment was able to decrease cell viability and survival of AML cells, while it spares healthy counterparts. We uncovered the potential combination effects of CPC with VEN in leukemic models of resistance to VEN. Mechanistically, CPC treatment induced impaired mitochondrial metabolism, DNA damage and decreased the expression of proteins related with VEN-resistance in AML, possibly explaining the synergic effects observed between VEN and CPC.