Identification of a synergistic combination of Smac mimetic and Bortezomib to trigger cell death in B-cell non-Hodgkin lymphoma cells.

Recently, copy number gains and increased expression levels of cIAP1 and cIAP2 have been reported in B-cell non-Hodgkin lymphomas (NHL). Therefore, we investigated the therapeutic potential of the Smac mimetic BV6 that antagonizes cIAP1/2 and XIAP. Here, we discover that subtoxic concentrations of BV6 prime B-cell NHL cells to proteasome inhibitor Bortezomib-induced cell death. Synergistic induction of cell death by BV6 and Bortezomib is confirmed by calculation of combination index in different cell lines, emphasizing the broader relevance of this combination. Interestingly, addition of the caspase inhibitor zVAD.fmk provides no or only partial protection from BV6/Bortezomib-stimulated cell death. Consistently, BV6/Bortezomib co-treatment alone or in combination with zVAD.fmk increases phosphorylation of MLKL, a typical marker of necroptosis. Importantly, genetic silencing of key components of necroptosis signaling such as MLKL or RIP3 significantly protects DG-75 cells from BV6/Bortezomib-induced cell death in the presence and even the absence of zVAD.fmk. Similarly, pharmacological inhibitors of MLKL (i.e. NSA), RIP3 (i.e. GSK'872, Dabrafenib) or RIP1 (i.e. Necrostatin-1s) significantly rescue DG-75 cells from BV6/Bortezomib-induced cell death irrespective of the presence of zVAD.fmk. In addition, NSA or Nec-1s act in concert with zVAD.fmk to reduce BV6/Bortezomib-induced cell death in U-2932 cells. Together, these findings demonstrate that BV6 and Bortezomib synergize to induce cell death in B-cell NHL cells. BV6/Bortezomib co-treatment primarily triggers necroptosis or, alternatively, engages both apoptotic and necroptotic cell death. The discovery of this synergistic combination that is effective even when apoptosis is blocked has important implications for the development of new treatment strategies for B-cell NHL.