Proteasome inhibitors and Smac mimetics cooperate to induce cell death in diffuse large B‐cell lymphoma by stabilizing NOXA and triggering mitochondrial apoptosis

Abstract

Copy number gains and increased expression levels of cellular Inhibitor of Apoptosis protein (cIAP)1 and cIAP2 have been identified in primary diffuse large B-cell lymphoma (DLBCL) tissues. Second mitochondria-derived activator of caspases (Smac) mimetics were designed to antagonize IAP proteins. However, since their effect as single agents is limited, combination treatment represents a strategy for their clinical development. Therefore, we investigated the Smac mimetic BV6 in combination with proteasome inhibitors and analyzed the molecular mechanisms of action. We discovered that BV6 treatment sensitizes DLBCL cells to proteasome inhibition. We show a synergistic decrease in cell viability and induction of apoptosis by BV6/Carfilzomib (CFZ) treatment, which was confirmed by calculation of combination index (CI) and Bliss score. BV6 and CFZ acted together to trigger activation of BAX and BAK, which facilitated cell death, as knockdown of BAX and BAK significantly reduced BV6/CFZ-mediated cell death. Activation of BAX and BAK was accompanied by loss of mitochondrial membrane potential (MMP) and activation of caspases. Pretreatment with the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Aspfluoromethylketone (zVAD.fmk) rescued BV6/CFZ-induced cell death, confirming caspase dependency. Treatment with CFZ alone or in combination with BV6 caused accumulation of NOXA, which was required for cell death, as gene silencing by siRNA or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated NOXA inactivation inhibited BV6/CFZinduced cell death. Together, these experiments indicate that BV6 and CFZ cooperatively induce apoptotic cell death via the mitochondrial pathway. These findings emphasize the role of Smac mimetics for sensitizing DLBCL cells to proteasome inhibition with important implications for further (pre)clinical studies. M.V. and S.F. shared equally to the senior authorship.

BIRC3 and BIRC5: multi‐faceted inhibitors in cancer

“…SMAC mimetics have been tested as single agents and in combination to proteasome inhibitors, leading to promising results. SMAC mimetics are also under investigation for the therapy of diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL) [4,6].…”

Proteasome Inhibitors and Their Potential Applicability in Osteosarcoma Treatment

“…More recently, expression levels of BAX, cleaved caspases, and cleaved PARP were increased dose-dependently in PI-1840-treated OS cells [ 57 ]. Moreover, the level of cytochrome c in the mitochondria decreased, which confirmed the presence of apoptosis in the OS cells at the mitochondrial level [ 56 ]. These findings indicate that PIs induce apoptosis in OS cells by activating both extrinsic and intrinsic pathways [ 56 ].…”

“…The direct binding of NOXA facilitates the oligomerization of BAX and BAK, causing the release of mitochondrial intermembrane space proteins such as cytochrome c into the cytosol [ 26 , 30 ]. Cytochrome c release enables the formation of the apoptosome followed by cleavage of initiator and effector caspases, executing apoptotic cell death [ 30 , 56 ]. More recently, expression levels of BAX, cleaved caspases, and cleaved PARP were increased dose-dependently in PI-1840-treated OS cells [ 57 ].…”

Smac mimetics and TRAIL cooperate to induce MLKL-dependent necroptosis in Burkitt's lymphoma cell lines

“…Our findings have important implications for the development of necroptosis-inducing therapies based on Smac mimetic combinational treatments. Both Smac mimetics and recombinant TRAIL have been tested in several cancer models [10 , [46] , [47] , [48] , [49] , [50] , [51] , but very little data are available on their therapeutic potential in BL. Besides BV6 combinations, in the present study, several other Smac mimetics that have already entered clinical trials [30 , 31 , 52] were tested in combination with TRAIL.…”