Synergistic Effect of Chidamide and Venetoclax on Apoptosis in Acute Myeloid Leukemia Cells and Its Mechanism
Authors: Li Gangping , Li Dongbei , Yuan Fangfang , Cheng Cheng , Chen Lin , Wei Xudong 
Published: July 2021

Abstract
Background: Acute myeloid leukemia (AML) is a hematological malignancy with a low remission rate and a high recurrence rate. The 5-year overall survival rate is only approximately 25%. The overexpression of the antiapoptotic protein Bcl-2 is associated with a lower overall survival rate in AML patients. Venetoclax (ABT199) is a selective inhibitor of Bcl-2 that has a significant effect in AML, but single-drug resistance often occurs due to the high expression of Mcl-1 protein. Chidamide is a histone deacetylase inhibitor that was independently developed in China. Studies have confirmed that chidamide can downregulate the expression levels of Bcl-2 and Mcl-1 and induce apoptosis. We examined whether the combination of venetoclax and chidamide could synergistically inhibit AML.Methods: This study aimed to use AML cell lines and primary cells to study the effects of venetoclax and chidamide combination therapy on AML cell apoptosis, the cell cycle and changes in related signaling pathways in vitro; establish an AML mouse model to observe the efficacy and survival time of combination therapy in vivo; and analyze the drug effects with multi-omics sequencing technology. The changes in gene and protein expression before and after treatment were examined to clarify the molecular mechanism driving the synergistic effect of the two drugs.Results: (1) Both venetoclax and chidamide promoted apoptosis in AML cell lines and primary cells in a time- and concentration-dependent manner. The effect was further enhanced when the two drugs were combined, and they had a synergistic effect (Combination Index < 1). (2) The cell cycle of the AML cell line was arrested in the G1 phase by chidamide monotherapy. The primary cells were arrested in S phase by monotherapy with chidamide or venetoclax. The cell-cycle-blocking effect was further strengthened by the combination of the two drugs. (3) At both the mRNA and protein levels, the expression of Mcl-1 was upregulated by venetoclax and downregulated by chidamide. The expression of Mcl-1 decreased further after combination treatment. (4) Transcriptome sequencing showed that the differentially expressed genes in the combination group compared with the venetoclax monotherapy group were mainly enriched in the PI3K-AKT pathway and JAK2/STAT3 pathway. Moreover, qRT-PCR and Western blotting confirmed the following results. 1) Chidamide downregulated the expression of the AKT gene and P-AKT protein, upregulated the expression of p21, and downregulated the expression of CDK2 and c-myc. 2) The expression of the SOCS3 gene and protein was upregulated, whereas the expression of the JAK2 gene and p-Jak2 protein was downregulated, by chidamide. 3) The expression of the HDAC1 gene and protein was upregulated in the chidamide monotherapy and combination therapy groups. (5) Compared with the monotherapy groups and control group, the combination therapy group exhibited significantly inhibited disease progression and a prolonged survival time among AML mice.Conclusion: First, venetoclax combined with chidamide synergistically promoted apoptosis in AML cell lines and primary cells. Second, transcriptome sequencing, qRT-PCR and Western blotting showed that chidamide synergistically promoted the apoptosis of AML cells by inhibiting the activation of the PI3K/AKT pathway and Jak2/STAT3 pathway. Third, compared with monotherapy, combination therapy with chidamide and venetoclax significantly inhibited tumor progression and prolonged the survival time of mice.、