Chidamide and venetoclax demonstrate synergistic anti-myeloma effect in HMCLs in vitro
We first investigated the potential inhibition of cell growth by chidamide and venetoclax in MM cells. HMCLs (U266, ARP-1, RPMI-8226 and MM1.S) were respectively treated with increasing concentrations of chidamide and venetoclax for 24 h and 48 h. When used separately, chidamide and venetoclax both decreased the myeloma cell viability in a dose- and time-dependent manner (Fig. 1A). Notably, U266 was the most sensitive to venetoclax, which might be due to U266 harboring t (11,14), thus featuring the highest BCL2 mRNA expression and highest BCL2/MCL1 mRNA ratio compared with the other three MM cells (Supplement Fig. 1A, B). Then, we selected two concentrations of chidamide (0.5µM and 2µM) combined with a series of concentrations of venentoclax treating HMCLs for 48 h to examine the potential synergistic anti-myeloma effect. As shown in Fig. 1B, co-exposure to chidamide and venetoclax resulted in sharply reduced cell viability in all selective cell lines, and the synergy of the two drugs was observed for various venetoclax doses in all cell lines with CI < 1 (Fig. 1C). Moreover, to investigate if the synergistic anti-myeloma effect was sustained over time, cell growth curves were examined every 24 h for consecutive 4 days. As shown in Fig. 1D, the combined treatment showed a more obvious suppressive effect on the growth of HMCLs than chidamide or venetoclax administered individually.
We next examined the synergistic effect of co-treatment on apoptosis induction in HMCLs. As shown in Fig. 2A, treatment with combination therapy markedly increased apoptosis compared to any monotherapies in HMCLs. Moreover, cleaved caspase-3 and cleaved PARP, two apoptosis-related proteins, were significantly increased by combined treatment (Fig. 2B).
To further confirm these findings in primary MM samples, we measured apoptosis in CD138 + plasma cells and treated them with chidamide, venetoclax, their combination, or vehicle for 48 h. Similar to our findings in HMCLs, co-treatment induced a higher level of apoptosis of CD138 + plasma cells from MM patients than chidamide or venetoclax administered alone.
Taken together, these data revealed that chidamide and venetoclax could synergistically exert cytotoxic effects in HMCLs and primary MM samples.
Co-treatment with chidamide and venetoclax induces cell cycle arrest at the G0/G1 phase in HMCLs via activating P21 and P27.
In order to further characterize the role of chidamide and venetoclax-mediated cytotoxicity, we evaluated the cell cycle status. In ARP-1 and MM1.S cell lines, the percentage of cells in the S phase was significantly decreased and the ratio of G0/G1 phase was dramatically increased after exposure to chidamide alone for 48 h, while venetoclax alone did not affect the distribution of cell cycle phases (Fig. 3A, B). Interestingly, co-treatment resulted in a more remarkable cell cycle arrest at the G0/G1 phase compared with chidamide alone in HMCLs, which may be associated with the synergy of inducing MM apoptosis. To further explore the molecular mechanisms of cell cycle arrest induced by chidamide or co-treatment, we performed western blotting to assess the expression of cell cycle related proteins. The results showed that the expression of cyclin-dependent kinase inhibitors (CDKIs) P21 and P27, which can block the formation of dimers from cyclins and cyclin dependent kinases (CDKs), were increased in the mono-chidamide group and the co-treatment group. In addition, the cell cyclins, cyclin D1 and cyclin E1 as well as CDKs, CDK4 and CDK6 were remarkably decreased in the co-treatment group (Fig. 3C). These findings may suggest that chidamide combined with venetoclax induces cell cycle arrest at the G0/G1 phase in HCMLs by increasing the expression of CDKIs (P21 and P27) and decreasing the expression of cyclins (cyclin D1 and cyclin E1) and CDKs (CDK4 and CDK6).
Co-treatment with chidamide and ventoclax disrupts DNA damage response and results in DNA damage in MM cells
Several studies have shown that chidamide can cause DNA damage in tumor cells23–25, which can lead to genomic instability and induce endogenous cell apoptosis. We thus examined whether DNA damage would contribute to enhanced cytotoxic effects of the combination of chidamide and venetoclax. First, we used the Comet assay to detect DNA damage. As shown in Fig. 4A, co-treatment with chidamide and venetoclax resulted in higher levels of DNA damage than treatment with vehicle and chidamide or venetoclax alone in HMCLs, manifested by the highest percentages of tail DNA and tail moment. Next, we confirmed these results by western blotting (Fig. 4B). As expected, γH2A.X, an established marker for DNA double-strand breaks (DSB)26, was sharply increased by co-treatment with chidamide and venetoclax in HMCLs. Finally, we explored the mechanism of increasing DNA damage by combination therapy. Generally, DNA damage response (DDR) can identify and repair DNA damage through various pathways and enzymes, and is critical to maintaining genomic stability and cell survival.27 Notably, combined treatment with these two agents almost completely inhibited the phosphorylation (activation) of DNA damage checkpoints ATM and ATR and thus inhibited the phosphorylation of CHK1 and CHK2, the downstream DNA damage checkpoints of ATM and ATR. Combined treatment also markedly downregulated the expression of DNA repair proteins, Rad51and KU80 (Fig. 4B). Altogether, combined treatment induced abundant DNA damage in HMCLs by the disruption of DNA damage checkpoints (e.g., ATM and ATR and their downstream kinases CHK1 and CHK2), as well as by downregulating DNA repair proteins (e.g., Rad51and KU80), which might contribute to the synergistic interaction between chidamide and venetoclax in HMCLs.
Co-exposure To Chidamide And Venetoclax Induces The Apoptosis Of Hmcls In Connection With Bim Upregulation
The anti-apoptotic proteins BCL-XL and MCL1 play a critical role in venetoclax resistance, and BH3-only protein BIM is important for venetoclax to exert its cytotoxic effects.28–30 Moreover, some studies indicated that HDACi can disrupt the expression of anti-apoptotic and pro-apoptotic proteins in the BCL2 family, including decreasing the expression of MCL1 and BCL-XL and increasing the expression of BIM.18–20 We thus examined the expression change of these BCL2 family proteins after exposure to chidamide and venetoclax. As shown in Fig. 5A, the expression of BCL-XL was reduced and BIM was increased in HMCLs after exposure to chidamide in the presence or absence of venetoclax, however, the expression of MCL1 was unchanged by chidamide or venetoclax or their combination. To further confirm which protein was associated with the synergistic interaction between chidamide and venetoclax in HMCLs, we used lentivirus vectors to knock down the BCL2L1 gene (coding BCL-XL protein) or knock down the BCL2L11 gene (coding BIM protein) respectively in MM1.S and ARP-1 cells. The results showed that the downregulation of BCL-XL expression might not account for the synergistic anti-myeloma effect of the two drugs, since combined treatment with chidamide and venetoclax still induced a markedly higher rate of apoptosis than chidamide or venetoclax monotherapy in BCL-XL knockdown MM1.S cells and the knockdown of BCL-XL did not sensitize MM1.S cells to chidamide or venetoclax monotherapy or combination therapy (Fig. 5D(upper)). In contrast, as shown in Fig. 5D (lower), ARP-1 cells transfected with shBIM lentiviral vector exhibited lower apoptosis rate than ARP-1 cells transfected with the lentivirus vector when they were treated with the combination regime, while the knockdown of BIM did not protect MM cells from apoptosis induced by venetoclax and chidamide administered individually. In conclusion, these results suggested that the upregulated expression of BIM by chidamide might account for or at least contribute to the synergistic anti-myeloma effect of combined treatment with chidamide and venetoclax.
Chidamide combined with venetoclax shows synergistic antitumor efficacy in vivo
A xenograft mouse model was employed to further validate whether combined treatment with chidamide and venetoclax could synergistically inhibit MM growth in vivo. After 7 days of injecting ARP-1 cells subcutaneously in six-week NOD-SCID mice, the animals were divided into four groups, including vehicle control, chidamide, venetoclax, and the combination of the latter two. Interestingly, as shown in Fig. 6, combined treatment resulted in a more obviously decrease of tumor burden, manifested by reduced tumor volume and tumor weight, compared to vehicle control or chidamide/venetoclax administrated separately (Fig. 6A, B and C). Moreover, notable toxicity was not observed in mice subjected to combination treatment, as there were no significant weight decreases of mice during the treatment (Fig. 6D).
We next used immunohistochemistry to validate the expression of cleaved caspase-3, CDK6, γH2A.X, BCL-XL and BIM in tumor masses. As expected, combination treatment with chidamide and venetoclax increased the expression of cleaved caspase-3, γH2A.X and BIM, as well as decreased the expression of CDK6 and BCL-XL (Fig. 6F). Collectively, these findings demonstrated that chidamide combined with venetoclax could synergistically inhibit MM growth in vivo.