Chidamide and venetoclax synergistically exert cytotoxicity on multiple myeloma by upregulating BIM expression

Abstract

Background: Multiple myeloma (MM) is the second most common hematologic malignancy with almost all patients eventually having relapse or refractory MM (RRMM), thus novel drugs or combination therapies are needed for improved prognosis. Chidamide and venetoclax, which target histone deacetylase and BCL2, respectively, are two promising agents for the treatment of RRMM.

Results: Herein, we found that chidamide and venetoclax synergistically exert an anti-myeloma effect in vitro in human myeloma cell lines (HMCLs) with a combination index (CI) < 1. Moreover, the synergistic anti-myeloma effect of these two drugs was demonstrated in primary MM cells and MM xenograft mice. Mechanistically, co-exposure to chidamide and venetoclax led to cell cycle arrest at G0/G1 and a sharp increase in DNA double-strand breaks. In addition, the combination of chidamide and venetoclax resulted in BCL-X_L downregulation and BIM upregulation, and the latter protein was proved to play a critical role in sensitizing HMCLs to co-treatment.

Conclusion: In conclusion, these results proved the high therapeutic potential of venetoclax and chidamide combination in curing MM, representing a potent and alternative salvage therapy for the treatment of RRMM.

Keywords: BIM; Cell cycle arrest; Chidamide; DNA damage; HDAC inhibitor; Multiple myeloma; Venetoclax.

Fig. 1

Chidamide and venetoclax demonstrate synergistic anti-myeloma efficacy in HMCLs in vitro. A HMCLs were treated with the indicated concentrations of chidamide and venetoclax for 24 h and 48 h, then CCK-8 assay was performed to test cell viability. B HMCLs were treated with the indicated concentrations of venetoclax ± 0.5 or 2 μM chidamide for 48 h, and the CCK-8 kit was used to test the cell viability. C From the dose–response curves, the CI of the two drugs were calculated using CompuSyn software, with CI < 1 indicating a synergistic interaction. D Cell growth was monitored every 24 h for a consecutive 4 days. The values indicate mean ± SD for at least three independent experiments performed in triplicate

Fig. 2

Chidamide and venetoclax demonstrate synergistic anti-myeloma efficacy in HMCLs in vitro. A HMCLs were incubated with chidamide (2ɥM), venetoclax (concentration was indicated in picture), their combination or vehicle for 48 h and subjected to flow cytometry to analyze cell apoptosis. B HMCLs were incubated with chidamide (1ɥM for U266; 2ɥM for ARP-1, MM1.S and RPMI-8226), venetoclax (2ɥM for U266; 4ɥM for ARP-1, MM1.S and RPMI-8226), their combination or vehicle for 48 h, and the expression of the following apoptosis-related proteins was determined by western blot analysis: PARP1, caspase-3. C Protein levels of cleaved PARP1 and cleaved caspase 3 were normalized to those of GAPDH and presented as fold changes relative to vehicle controls. D Primary MM samples (n = 3) were exposed to chidamide (2ɥM) and/or venetoclax (4ɥM) for 48 h and analyzed by flow cytometry. Data are presented as the mean ± SD of at least three independent experiments (ns P > 0.05; ∗ P < 0.05; ∗  ∗ P < 0.01; ∗  ∗  ∗ P < 0.001; ∗  ∗  ∗  ∗ P < 0.0001)

Fig. 3

Co-treatment with chidamide and venetoclax induces cell cycle arrest at the G0/G1 phase in HMCLs via activating P21 and P27. A HMCLs were incubated with chidamide (1ɥM for U266; 2ɥM for ARP-1 and MM1.S) and/or venetoclax (2ɥM for U266; 4ɥM for ARP-1 and MM1.S) for 48 h, and the cell cycle was analyzed by flow cytometry. B HMCLs were exposed to chidamide (1ɥM for U266; 2ɥM for ARP-1 and MM1.S) and/or venetoclax (2ɥM for U266; 4ɥM for ARP-1 and MM1.S) for 48 h. Western blotting was employed to detect the expression of the following cell cycle-related proteins: P21, P27, cyclin D1, cyclin E1, CDK4 and CDK6. C Protein levels of P21, P27, cyclin D1, cyclin E1, CDK4 and CDK6 were normalized to those of GAPDH and presented as fold changes relative to vehicle controls. Data are presented as the mean ± SD of at least three independent experiments. (ns P > 0.05; ∗ P < 0.05; ∗  ∗ P < 0.01; ***P < 0.001; ∗  ∗  ∗  ∗ P < 0.0001)

Fig. 4

Co-treatment with chidamide and ventoclax disrupts DNA damage response and results in DNA damage in MM cells. A HMCLs were incubated with chidamide (1ɥM for U266; 2ɥM for ARP-1 and MM1.S) and/or venetoclax (2ɥM for U266; 4ɥM for ARP-1 and MM1.S) for 48 h, and DNA damage was detected by Comet assay. B HMCLs were exposed to chidamide (1ɥM for U266; 2ɥM for ARP-1 and MM1.S) and/or venetoclax (2ɥM for U266; 4ɥM for ARP-1 and MM1.S) for 48 h. Western blotting was used to analyze the expressions of γH2A.X, p-ATM, p-ATR, p-CHK1, p-CHK2, Rad51 and KU80. C Protein levels of γH2A.X, p-ATM, p-ATR, p-CHK1, p-CHK2, Rad51 and KU80 were normalized to those of GAPDH and presented as fold changes relative to vehicle controls. Data are presented as the mean ± SD of at least three independent experiments (ns P > 0.05; ∗ P < 0.05; ∗  ∗ P < 0.01; ∗  ∗  ∗ P < 0.001; ∗  ∗  ∗  ∗ P < 0.0001)

Fig. 5

Co-exposure to chidamide and venetoclax induces apoptosis of HMCLs in connection with BIM upregulation. A HMCLs were exposed to chidamide (1ɥM for U266; 2ɥM for ARP-1 and MM1.S) and/or venetoclax (2ɥM for U266; 4ɥM for ARP-1 and MM1.S) for 48 h. The expressions of the following BCL2 family proteins were determined by western blot analysis: BCL2, MCL1, BCL-X_L and BIM. B Protein levels of BCL2, MCL1, BCL-X_L and BIM were normalized to those of GAPDH and presented as fold changes relative to vehicle controls. C BCL-X_L was knocked down by lentivirus in MM1.S cells. D BCL-X_L was knocked down by lentivirus in U266 cells. E BIM was knocked down by lentivirus in ARP-1 cells. F BIM was knocked down by lentivirus in U266 cells. G MM1.S cells (upper) and U266 cells (down) with BCL-X_L knockdown were treated with chidamide (2 μM) ± venetoclax (4 μM) for 48 h and the percentage of apoptosis was determined by flow cytometry H ARP-1 cells (upper) and U266 cells (down) with BIM knockdown were treated with chidamide (2 μM) ± venetoclax (4 μM for U266, 8 μM for ARP-1) for 48 h and the percentage of apoptosis was determined by flow cytometry (ns P > 0.05; ∗ P < 0.05; ∗  ∗ P < 0.01; ∗  ∗  ∗ P < 0.001; ∗  ∗  ∗  ∗ P < 0.0001)

Fig. 6

Chidamide in combination with venetoclax shows synergistical antitumor efficacy in vivo. A Mice were killed, and their tumor masses were fetched and captured by camera. B Tumor weight was measured after detachment. C Tumor volumes were measured every three days after tumor formation. D Mouse body weight was measured every three days after tumor formation. E Immunohistochemistry was performed to investigate the expression of cleaved caspase-3, CDK6, γH2A.X, BCL-X_L and BIM in tumor masses. The values indicate mean ± SD for 6 mice/each group (ns P > 0.05; *P < 0.05; ***P < 0.001; ****P < 0.0001)