Page 184 - 2020_08-Haematologica-web

V.M. Smith et al.
RIVA cells also expressed high levels of the BH3-only pro- tein BMF we asked whether BMF could be functionally important, but silencing of BMF did not affect ABT-199- induced apoptosis (Online Supplementary Figure S7).
BAX and BAK were required to mediate ABT-199-induced apoptosis
Next, we explored the role of BAX in BH3-mimetic- induced cell death. Silencing of BAX using siRNA indicat- ed that BAX was essential for the cell death induced by BH3-mimetics, as cell death was significantly reduced in RIVA, U2932, RCK8, SUDHL10 and U2946 cells (Figure 6A-D). In contrast, knockdown of BAK only reduced apoptosis upon treatment with ABT-199 but not upon treatment with A1331852 or S63845, highlighting a prominent role for BAK only in ABT-199-induced apopto- sis (Online Supplementary Figure S8).
We also investigated how BAK was involved in ABT- 199-induced apoptosis. As no direct inhibition of BAK by BCL-2 was observed, we hypothesized that BAX inhibi- tion by BCL-2 is the initial target of ABT-199, and that once BAX is released, BAK is also activated and acceler- ates cell death. To test this hypothesis, the activation of BAK was assessed upon silencing of BAX and treatment with ABT-199. In both RIVA and U2932 cells, silencing of BAX resulted in significantly less active BAK induced by ABT-199, suggesting that BAX contributed to activation of BAK (Figure 6E). To investigate whether BAX could directly activate BAK, the interaction between BAK and BAX was investigated. Treatment with ABT-199 induced complex formation between BAX and BAK in both RIVA and U2932 cells (Figure 6F).
BAX rather than BAK is functionally required for A1331852- or S63845-induced apoptosis
To exclude that the absence of an influence of BAK silencing on A1331852- or S63845-induced apoptosis may be caused by insufficient knockdown, we performed genetic deletion of BAK using CRISPR/Cas9. Deletion of BAK in SUDHL8 cells had only a minor effect on A1331852-induced cell death as compared to cells trans- duced with NHT control gRNA (Online Supplementary Figure S9A, B). To investigate whether BAX could be acti- vated in the absence of BAK, BAX activation was quanti- fied upon treatment with A1331852 using a conforma- tion-specific antibody and flow cytometry. Although the deletion of BAK had a minor influence on the activation of BAX, BAX could clearly still be activated even though BAK was deleted (Online Supplementary Figure S9C).
To interrogate the role of BAK in S63845-induced apop- tosis, BAK was deleted in U2946 cells. In contrast to the data obtained by siRNA-mediated knockdown, genetic deletion of BAK had a significant influence on S63845- induced apoptosis in all BAK-deleted clones investigated (Figure 7A). However, S63845-induced apoptosis was not completely inhibited, suggesting that BAX may play a prominent role also upon S63845 treatment. To confirm that S63845-mediated apoptosis involved BAX, knock- down of BAX was performed in BAK-deleted cells (Figure 7B). Knockdown of BAX by siRNA had a stronger influ- ence than BAK deletion on S63845-induced apoptosis. Combined deletion of BAK and depletion of BAX resulted in complete inhibition of S63845-induced apoptosis (Figure 7C). To investigate how BAX may be activated upon inhibition of MCL-1, we first asked whether BAK
was essential in activating BAX. Analysis of BAX activa- tion in BAK-deleted cells indicated that BAK may be involved in activating BAX, as BAX activation was signif- icantly reduced in BAK-deleted cells. However, some active BAX was still present in BAK-deleted cells, indicat- ing that other factors may be involved in activating BAX. To explore a role of the BH3-only proteins BIM and NOXA, siRNA-mediated knockdown of BIM and NOXA was performed in BAK-deleted cells. In line with the minor reduction of S63845-induced apoptosis by BIM knockdown (Figure 5C), BIM knockdown also reduced S63845-induced apoptosis in NHT- or BAK-deleted U2946 cells (Figure 7E, F). In addition to BIM, NOXA may also be involved in S63845-induced cell death, as knock- down of NOXA partially reduced S63845-induced apop- tosis (Figure 7G,H). These data indicate that NOXA may participate in activating BAX upon S63845 treatment. To explore how NOXA may activate BAK we next investi- gated the binding of NOXA to MCL-1 and observed a prominent displacement of NOXA from MCL-1 by S63845 (Figure 7I). Taken together, these data indicate that BH3-only proteins displaced from MCL-1 by S63845 may contribute to an activation of BAX which primarily mediates S63845-induced apoptosis.
Discussion
By investigating the response to selective BH3-mimet- ics we have identified subgroups of DLBCL cells that depend on either BCL-2, BCL-XL or MCL-1 for survival. Our side-by-side comparison of selective BH3-mimetics targeting the main anti-apoptotic proteins suggests that BCL-2, BCL-XL and MCL-1 are all important therapeutic targets in DLBCL. However, we have not investigated the role of other BCL-2 family proteins, such as BCL2A1 or BCLw, due to the lack of specific inhibitors.
In line with previous studies, our data indicate a corre- lation of ABT-199 sensitivity with high BCL-2 protein expression.7,25 However, in our study sensitivity to ABT- 199 was independent of genetic alterations of BCL-2 and not all cells expressing high BCL-2 levels were sensitive to ABT-199, highlighting the need to better understand the mechanisms of resistance in cells with high expression of BCL-2, such as HBL1 and OCI-LY3. Although RIVA and U2932 also expressed high levels of BCL-XL and MCL-1, BAX and BIM were exclusively sequestered by BCL-2, indicating that in these cells BCL-2 is the preferred bind- ing partner for the pro-apoptotic proteins. The molecular basis for this preferential binding is not known. Increased binding to BCL-2 instead of the related protein BCL-XL cannot be explained by different binding affinities, as BIM BH3-peptides bind more strongly to BCL-XL than to BCL-2,28,32 but may be explained by the amount of acces- sible protein at the mitochondria or by enhanced protein stability.33 Our data indicate that ABT-199 released pro- apoptotic BAX and BIM and that the released BAX induced activation of BAK, as knockdown of BAX signif- icantly reduced BAK activation (Figure 6E). The involve- ment of BIM in ABT-199-induced apoptosis appears to be cell-type-dependent, as BIM knockdown reduced apopto- sis in RIVA but not in U2932 cells (Figure 5).
In contrast, in the BCL-XL-dependent cell lines RCK8 and SUDHL8, BAX and BAK were exclusively bound to BCL-XL. These cell lines expressed high levels of BCL-XL
2160
haematologica | 2020; 105(8)