Voltage-Dependent Anion Channel 2 Controls Mitochondrial Priming through BAK Stabilization in Multiple Myeloma

Evasion of apoptosis is an essential feature of cancer cells. The BCL2 family has emerged as a promising therapeutic target in cancer due to its ability to tightly regulate apoptosis. The BCL2 family encompasses anti and pro-apoptotic proteins that regulate themselves through protein-protein interactions, the balance between the two groups determines cell fate. Triggering of the apoptotic pathway involves the activation and oligomerization of the BCL2 pro-apoptotic effectors BAX and BAK, which ultimately causes a breach in the outer mitochondrial membrane. Complex mechanisms are involved in the regulation of these effector proteins.

Beyond their sequestration by their anti-apoptotic BCL2 counterparts, non-BCL2 family proteins are emerging as partners of BAX and BAK and are considered as novel regulators of apoptosis. One of them is VDAC2, which belongs to the family of voltage dependent anion channels along with VDAC1 and VDAC3. VDAC2 is abundantly present in the mitochondrial outer membrane and has been implicated in apoptosis regulation. Nevertheless, it is not clear how this mechanistically occurs with contradictory evidence for the role in regulating BAX and BAK effectors. Notably, VDAC2 was reported necessary for efficient BAX dependent apoptosis, while conversely inhibits BAK apoptotic function.

Multiple Myeloma (MM) is a B-cell neoplasia that overexpresses the anti-apoptotic BCL2 members. BAX and BAK effectors are also readily detected in MM patients’ cells. Using 17 Human Myeloma Cell Lines (HMCLs) and 794 primary sample from the Multiple Myeloma Research Foundation (MMRF)-CoMMpass cohort, we found that VDAC1, VDAC2 and VDAC3 were heterogeneously expressed in MM cells. Interestingly, we found that low VDAC2 expression was associated with poor overall survival (OS) in this cohort.

To understand why lower levels of VDAC2 were associated with poor OS, we generated VDAC2 KO myeloma cells. Notably, VDAC2 KO cells exhibited a drastic decrease of BAK protein expression while BAX remained unchanged. We demonstrated that VDAC2 regulated BAK protein expression by preventing its degradation through the proteasome and lysosome pathways. Accordingly, VDAC2 protein expression directly correlated with BAK but not with BAX protein levels and VDAC2 and BAK physically interacted at the mitochondria. Functionally, VDAC2 KO cells completely lost their mitochondrial priming and specific dependences on MCL1 and BCL2, which was due to the shortage of mitochondrial BAK. Conversely, the transient silencing of VDAC2, while maintaining BAK protein levels, increased the global, MCL1 and BCL2 mitochondrial priming, induced BAK activation and in turn enhanced cell death induced by BH3 mimetics targeting MCL1 (S63845) or targeting BCL2 (venetoclax) in MM cells.

Then, we found that efsevin, a novel pharmacological modulator of VDAC2, had a similar impact of VDAC2 knock-down on BH3 mimetics apoptotic response. We demonstrated that Efsevin increased the global, MCL1 and BCL2 mitochondrial priming. The combination of efsevin with S63845 or with Venetoclax demonstrated a synergistic effect, without modifying the protein expression of BAK. Interestingly BAK-KO cells but not BAX-KO cells were refractory to the sensitization effect of efsevin.

We further confirm the effect of Efsevin on cell death sensitization to the MCL1 mimetic S63845 in myeloma cells from 27 consecutive patients from the MYRACLE cohort (Nantes, France). The combination of S63845 and Efsevin was effective in 96% of samples, that is additive (-10%<observed minus expected death<10%) in 16 samples (59%) and synergistic (observed minus expected death>10%) in 10 samples (37%).

Together these results provide evidence of the crucial role of VDAC2 in the regulation of mitochondrial apoptosis in myeloma cells through BAK control. Further investigations are conducted to elucidate if the VDAC2/BAK axis could have a potential therapeutic application in MM and other hematologic cancers, notably with the use of efsevin.