Blockade of XBP1 Splicing by Inhibition of IRE1α Is a Promising Therapeutic Option in Multiple Myeloma

Abstract

133 Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response (UPR). Therefore blockade of UPR could provide a novel therapeutic option in MM. Upon UPR, inositol-requiring enzyme 1α (IRE1α) is activated by auto-phosphorylation, resulting in activation of its endoribonuclease domain to cleave XBP1 mRNA from XBP1 unspliced form (XBP1u: inactive) to generate the XBP1 spliced form (XBP1s: active). XBP1s protein in turn regulates genes responsible for protein folding and degradation, playing a pro-survival signaling role in the UPR. In this study, we specifically examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM. We first examined the biologic significance of IRE1α by knockdown using lentiviral shRNA and observed significant growth inhibition in IRE1α knockdown cells. We next examined the impact of inhibition of XBP1 splicing using a novel small molecule IRE1α endoribonuclease domain inhibitor MKC-3946 (MannKind, Valencia CA). MKC-3946 blocked not only the basal level, but also inducible (by tunicamycin) XBP1s, evidenced by RT-PCR analysis in RPMI8226 cells, without affecting phosphorylation of IRE1α. Importantly, MKC-3946 also inhibited XBP1s in primary tumor cells from MM patients. We also confirmed functional inhibition of XBP1s, with target genes including SEC61A1, p58IPK, and ERdj4 downregulated by MKC-3946 treatment. Importantly, MKC-3946 triggered growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Furthermore, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG in RPMI8226 and INA6 cells, as well as primary tumor cells from MM patients. Both bortezomib and 17-AAG induced ER stress with XBP1s, which was markedly blocked by MKC-3946. Moreover, apoptosis induced by bortezomib or 17-AAG was enhanced by MKC-3946, associated with increased CHOP mRNA and protein, a proapoptotic factor triggered by ER stress. We next demonstrated that XBP1s was induced by bortezomib in INA6 cells co-cultured with bone marrow (BM) stromal cells, which was inhibited by MKC-3946, associated with enhanced cytotoxicity induced by the combination. Finally, MKC-3946 inhibited XBP1s in a model of in vivo ER stress induced by tunicamycin. To evaluate the anti-MM effect of MKC-3946, we used the subcutaneous RPMI8226 xenograft model in mice. MKC-3946 significantly reduced MM tumor growth in the treatment versus control group, associated with prolonged overall survival. We also confirmed that MKC-3946 treatment significantly inhibited XBP1s in excised tumors, assessed by RT-PCR. In order to examine the activity of MKC-3946 on MM cell growth in the context of the human BM microenvironment in vivo, we used the SCID-hu model, in which INA6 cells are directly injected into a human bone chip implanted subcutaneously in SCID-mice. MKC-3946 treatment significantly inhibited tumor growth compared with vehicle control. Moreover, XBP1s in excised tumor cells was inhibited, evidenced by RT-PCR. In conclusion, these data demonstrate that blockade of XBP1s by MKC-3946 triggers MM cell growth inhibition in vivo and prolongs host survival. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential novel therapeutic option in MM. Disclosures: Tam: MannKind Corporation: Employment, Equity Ownership. Zeng:MannKind Corporation: Employment, Equity Ownership. Patterson:MannKind Corporation: Employment, Equity Ownership. Richardson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; MannKind: Membership on an entity's Board of Directors or advisory committees.

Endoplasmic reticulum stress signalling – from basic mechanisms to clinical applications

“…IRE1 RNase-binding site IRE1 RNase inhibitors include salicylaldehydes [305] 4l8C [306], MKC-946 [307], STF-83010 [308], toyocamycin [309] and hydroxyl-aryl-aldehydes [86]. The reported cocrystal structures of murine IRE1a with salicyaldehyde-based inhibitor show that Lys 907 is involved in Schiff base arrangement (PDB code: 4PL3…”

Emerging roles of ER stress in the etiology and pathogenesis of Alzheimer's disease

“…The second most studied UPR pathway to develop new pharmacological molecules is the IRE1-XBP1s axis, especially for the treatment of metabolic diseases and cancer. Inhibition of the IRE1 RNAse activity with MKC-3946 or STF-083010 selectively reduced the growth of myeloma tumors in xenograft models, associated with low toxicity [42,143]. Other compounds that efficiently inhibiting IRE1 still lack a complete pharmacological profile and it is not known if they can cross the blood-brain barrier (reviewed in [41]).…”

Single‐cell RNA sequencing reveals chemokine self‐feeding of myeloma cells promotes extramedullary metastasis

“…Interleukin-6 (IL-6) signaling molecules, including IL-6 signal transducer (IL6st), receptor (IL6R), and transcriptional factor STAT3, necessary for myeloma survival and progression [23][24][25], were upregulated in BMMCs and contribute to PC1. Transcriptional factors specific for myeloma cells, such as IRF4, Blimp-1 (PRDM1), and XBP1, are also transcribed in BMMCs [26][27][28][29]. Upregulation of nourishing growth factors for myeloma, such as HGF, IGF1, and VEGF [30][31][32][33], was also confirmed.…”