The BCL-2-specific inhibitor, ABT-199 (venetoclax) has exhibited remarkable clinical activity in nearly all cases of chronic lymphocytic leukemia. In contrast, responses are usually much less in diffuse large B-cell lymphoma (DLBCL), despite high level expression of BCL-2 in over 40% of cases, indicating that co-expression of related anti-apoptotic BCL-2 family proteins may limit the activity of ABT-199. We have investigated the roles of BCL-2 proteins in DLBCL cells using a panel of specific BCL-2 homology 3 (BH3)-mimetics and identified subgroups of these cells that exhibited marked and specific dependency on either BCL-2, BCL-X L or MCL-1 for survival. Dependency was associated with selective sequestration of the pro-apoptotic proteins BIM, BAX and BAK by the specific anti-apoptotic BCL-2 protein which was important for cellular survival. Sensitivity to BH3-mimetics was independent of genetic alterations involving the BCL-2 family and only partially correlated with protein expression levels. Treatment with ABT-199 displaced BAX and BIM from BCL-2, subsequently leading to BAK activation and apoptosis. In contrast, apoptosis induced by inhibiting BCL-X L with A1331852 was associated with a displacement of both BAX and BAK from BCL-X L and occurred independently of BIM. Finally, the MCL-1 inhibitor S63845 induced mainly BAX-dependent apoptosis mediated by a displacement of BAK, BIM and NOXA from MCL-1. In conclusion, our study indicates that in DLBCL, the heterogeneous response to BH3-mimetics is mediated by selective interactions between BAX, BAK and anti-apoptotic BCL-2 proteins.

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Interferons (IFNs) are key players in the tumor immune response and act by inducing the expression of IFN-stimulated genes (ISGs). Here, we identify the mixed-lineage kinase domain-like pseudokinase (MLKL) as an ISG in various cancer cell lines. Both type I and type II IFNs increase the expression of MLKL indicating that MLKL up-regulation is a general feature of IFN signaling. IFNγ up-regulates mRNA as well as protein levels of MLKL demonstrating that IFNγ transcriptionally regulates MLKL. This notion is further supported by Actinomycin D chase experiments showing that IFNγ-stimulated up-regulation of MLKL is prevented in the presence of the transcriptional inhibitor Actinomycin D. Also, knockdown of the transcription factor IFN-regulatory factor 1 (IRF1) and signal transducer and activator of transcription (STAT) 1 as well as knockout of IRF1 significantly attenuate IFNγ-mediated induction of MLKL mRNA levels. Up-regulation of MLKL by IFNγ provides a valuable tool to sensitize cells towards necroptotic cell death and to overcome apoptosis resistance of cancer cells.