Abstract 469: Regulated IRE1-dependent mRNA decay occurs under extreme proteotoxic stress in myeloma, but not under conditions that would affect therapeutic targeting of IRE1α
Authors: Bright Michael D. , Wardell Christopher P. , Itzhak Daniel N. , Bagratuni Tina , Morgan Gareth J. , Davies Faith E. 
Published: May 2015

Abstract
Many secretory cancers rely on cellular mechanisms such as the unfolded protein response (UPR) to survive under conditions that lead to proteotoxic stress. Multiple myeloma (MM) is a plasma cell malignancy that is particularly reliant on this pathway as it controls cell survival under proteotoxic stress. One branch of the UPR is IRE1α, a receptor in the endoplasmic reticulum (ER) membrane that senses misfolded protein and stimulates cell adaptation and survival under proteotoxic stress. The ribonuclease domain of IRE1α cleaves XBP1 mRNA, which is ligated to code for the active transcription factor XBP1s. IRE1α also cleaves other mRNAs resulting in their degradation, termed regulated IRE1α-dependent mRNA decay (RIDD). While IRE1α is a potential therapeutic target in MM and other secretory cancers, little is known about the role of RIDD in malignancy. We set out to determine whether RIDD occurs in MM, and how it would contribute to the mechanism of IRE1-targeting therapeutics. We cross-referenced published expression data to identify consistent RIDD targets and found that 11 transcripts were regulated by RIDD in more than one experimental system. To determine if these were RIDD targets in MM, cell lines were treated with DTT to induce strong proteotoxic stress in the presence or absence of an IRE1α RNAse inhibitor. Transcripts were measured using qPCR. Only BLOC1S1 was regulated by RIDD in this system. Interestingly, BLOC1S1 was only degraded after complete splicing of XBP1 mRNA, suggesting that RIDD occurs when a cell has exhausted its natural substrate and not recovered from ER-stress. Importantly, using bioinformatic analysis we identified a conserved stem loop structure in BLOC1S1 that may be cleaved by IRE1α, adding further weight to its potential as a RIDD target. The effect of mutation of this stem loop to an IRE1-resistant form was analysed. In order to determine the clinical significance of RIDD, we tested whether RIDD occurred as a consequence of drug treatment. A standard treatment for MM, that is believed to induce ER-stress, is the proteasome inhibitor Bortezomib. To test whether this treatment might induce RIDD, MM cell lines were treated with a timecourse of Bortezomib and BLOC1S1 mRNA was measured. Bortezomib did not induce BLOC1S1 degradation, and hence RIDD. Finally, in order to assess whether RIDD occurs in MM patients, we used data from primary patient cells from over 250 patients and correlated expression of conserved RIDD target genes with the ratio of spliced to unspliced XBP1 mRNA (XBP1s/u), as a measure of IRE1α activity. No correlation was found, suggesting that RIDD is not present in clinical samples. Taken together, these data suggest that while RIDD occurs under extreme stress in MM cells in vitro, it is not clinically significant in this disease. Therefore, RIDD should not affect the action of IRE1α-targeting therapeutics in MM or other secretory cancers.
Citation Format: Michael D. Bright, Christopher P. Wardell, Daniel N. Itzhak, Tina Bagratuni, Gareth J. Morgan, Faith E. Davies. Regulated IRE1-dependent mRNA decay occurs under extreme proteotoxic stress in myeloma, but not under conditions that would affect therapeutic targeting of IRE1α. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 469. doi:10.1158/1538-7445.AM2014-469