Targeting IRE1α-XBP1 Pathway Is a Novel Therapeutic Strategy In Multiple Myeloma
Authors: Mimura Naoya , Hideshima Teru , Gorgun Gullu , Cirstea Diana , Santo Loredana , Hu Yiguo , Fabre Claire , Blumenthal Martina , Tam Victor , Kertesz Nathalie L. , Zeng Qingping , Patterson John B. , Munshi Nikhil C. , Richardson Paul , Anderson Kenneth C. 
Published: October 2019

Abstract
Abstract 4074
Aberrant protein folding results in the accumulation of misfolded/unfolded proteins in the endoplasmic reticulum (ER), which in turn triggers ER stress followed by unfolded protein response (UPR), an adaptive response against ER stress. Since multiple myeloma (MM) cells have high protein synthesis, they are sensitive to ER stress and require strict ER quality control for cell survival. Upon UPR, IRE1α is activated by auto-phosphorylation resulting in activation of its endoribonuclease domain to splice XBP1 mRNA from XBP1 unspliced form (XBP1u: inactive) to XBP1 spliced form (XBP1s: active). Since XBP1 is a transcription factor regulating genes which are responsible for protein folding and ER associated degradation (ERAD), IRE1α-XBP1 pathway acts as a pro-survival signaling pathway under the UPR condition. In this study, we examined whether IRE1α-XBP1 pathway is a potential novel therapeutic option in MM. We first examined IRE1α expression and confirmed its expression in all MM cell lines. In contrast, XBP1s was not detected by RT-PCR in most cell lines except in for RPMI8226 cells. To assess biologic significance of IRE1α in MM cell, we knock-downed its expression using shRNA and found that downregulation of IRE1α inhibited MM cell growth, indicating that IRE1α has a crucial role in MM cell survival. We next examined the impact of inhibition of XBP1 splicing by a small molecule IRE1α endoribonuclease inhibitor MKC-3946 (Mannkind, Valencia CA) in MM cells in vitro. As expected, MKC-3946 significantly inhibited tunicamycin-induced XBP1s without affecting phosphorylation of IRE1α. MKC-3946 induced only modest cytotoxicity in MM cell lines without toxicity in normal mononuclear cells from healthy donors; however, it significantly enhanced cytotoxicity in combination with bortezomib or 17-AAG. Both bortezomib and 17-AAG induced ER stress evidenced by induction of XBP1s; conversely, MKC-3946 blocked XBP1s triggered by these agents. Furthermore, apoptosis induced by these agents was enhanced with MKC-3946 associated with increased CHOP, which is a known pro-apoptotic protein induced in uncompensated ER stress condition. Importantly, MKC-3946 enhanced the cytotoxicity of bortezomib or 17-AAG in INA6 cells, even in the presence of increased IL-6 or bone marrow stromal cells. Finally, MKC-3946 was active inhibiting XBP1 splicing in a model of ER stress and significantly inhibited growth of RPMI8226 plasmacytoma in a xenograft murine model when used in combination with a low dose of bortezomib. Taken together, our results demonstrate that inhibition of XBP1 splicing by blockade of IRE1α is a promising therapeutic option in MM.

Disclosures:
Blumenthal: Mannkind Corporation: Employment, Equity Ownership. Tam:Mannkind Corporation: Employment, Equity Ownership. Kertesz:Mannkind Corporation: Employment, Equity Ownership. Zeng:Mannkind Corporation: Employment, Equity Ownership. Patterson:Mannkind Corporation: Employment, Equity Ownership. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.