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

Int J Cancer. 2020 Sep 1;147(5):1485-1498. doi: 10.1002/ijc.32976. Epub 2020 Apr 8.

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-Asp-fluoromethylketone (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/CFZ-induced 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.

Keywords: NOXA; Smac mimetics; diffuse large B-cell lymphoma; intrinsic apoptosis; proteasome inhibitor.

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Apoptosis / drug effects*
  • Apoptosis Regulatory Proteins / pharmacology*
  • Caspases / metabolism
  • Cell Death / drug effects
  • Cell Line, Tumor
  • Drug Synergism
  • Humans
  • Inhibitor of Apoptosis Proteins / metabolism
  • Lymphoma, Large B-Cell, Diffuse / metabolism
  • Lymphoma, Large B-Cell, Diffuse / pathology*
  • Membrane Potential, Mitochondrial / drug effects
  • Mitochondrial Proteins / pharmacology*
  • Oligopeptides / pharmacology
  • Proteasome Inhibitors / pharmacology*
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*
  • bcl-2 Homologous Antagonist-Killer Protein / genetics
  • bcl-2 Homologous Antagonist-Killer Protein / metabolism
  • bcl-2-Associated X Protein / genetics
  • bcl-2-Associated X Protein / metabolism

Substances

  • Antineoplastic Agents
  • Apoptosis Regulatory Proteins
  • BAK1 protein, human
  • BAX protein, human
  • BV6 peptide
  • DIABLO protein, human
  • Inhibitor of Apoptosis Proteins
  • Mitochondrial Proteins
  • Oligopeptides
  • PMAIP1 protein, human
  • Proteasome Inhibitors
  • Proto-Oncogene Proteins c-bcl-2
  • bcl-2 Homologous Antagonist-Killer Protein
  • bcl-2-Associated X Protein
  • carfilzomib
  • Caspases