Abstract 3020: Targeting both canonical and non-canonical NF-kB pathways by the IAP antagonist birinapant potentiates bortezomib anti-myeloma activity
Authors: Zhou Liang , Chen Shuang , Zhang Yu , Kmieciak Maciej , Leng Yun , Li Lihong , Lin Hui , Turner Joel , Sullivan Daniel , Dai Yun , Grant Steven 
Published: July 2016

Abstract
Inhibitor of apoptosis proteins (IAPs e.g., XIAP, cIAP1, cIAP2) inhibit apoptosis through diverse mechanisms. Recent attention has focused on novel functions of cIAP1/2, including activation of the canonical and non-canonical NF-κB pathways and inhibition of the extrinsic apoptotic pathway through the ripoptosome. These considerations have stimulated the development of IAP antagonists such as the bivalent IAP inhibitor birinapant (TL-32711). Proteasome inhibitors such as bortezomib are highly active in multiple myeloma (MM), and act, at least in part, by inhibiting NF-κB, raising the possibility of cooperative interactions with IAP antagonists. Synergistic induction of apoptosis was observed in human MM cell lines, including drug-naïve U266 cells, their bortezomib-resistant counterparts (PS-R), and multiple other human MM lines. Birinapant (500 nM) sharply down-regulated cIAP1/2 in MM cells, accompanied by reduced expression of TRAF2, RIP1, and p-IKKβ, as well as robust TRAF3 up-regulation, in association with down-regulation of NIK, p-IKKα, and p52, consistent with canonical and non-canonical NF-κB pathway inactivation, respectively. Notably, U266 cells overexpressing TRAF2 or in which TRAF3 was knocked down by shRNA displayed significant resistance to the birinapant/bortezomib regimen, indicating that disruption of both canonical and non-canonical NF-κB signaling contributes functionally to anti-myeloma activity. Moreover, Bcl-2 or Bcl-xL overexpression and dominant-negative FADD markedly blocked apoptosis, suggesting the involvement of both intrinsic and or extrinsic apoptotic pathways. 3D co-culture studies with HS-5 stromal cells revealed no protection of MM cells from the birinapant/bortezomib regimen, indicating circumvention of micro-environmental forms of drug-resistance. Markedly enhanced cell killing by the combination was also observed in primary CD138+ MM cells and primitive CD138-/19+/20+/27+ progenitors, but not normal CD34+ cells. HS-5 cells also failed to protect primary CD138+ MM cells from this regimen. Finally, co-administration of birinapant and bortezomib significantly reduced tumor burden and enhanced animal survival compared to single-agent treatment with minimal toxicity in NSG mice inoculated with U266 cells. Together, these findings indicate that targeting cIAP1/2 by birinapant significantly increases bortezomib anti-MM activity in MM cells, including bortezomib-resistant and primary MM cells, circumvents microenvironment-related resistance, and mechanstically involves concommitant inhibition of the canonical and non-canonical NF-κB pathways as well as activation of the intrinsic and extrinsic apoptotic pathways. Finally, the regimen is well tolerated and active in in vivo animal models. Collectively, these findings support exploring an IAP/proteasome inhibitor strategy in MM.
Citation Format: Liang Zhou, Shuang Chen, Yu Zhang, Maciej Kmieciak, Yun Leng, Lihong Li, Hui Lin, Joel Turner, Daniel Sullivan, Yun Dai, Steven Grant. Targeting both canonical and non-canonical NF-kB pathways by the IAP antagonist birinapant potentiates bortezomib anti-myeloma activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3020.