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• W4248517419 abstract "•During endoplasmic reticulum (ER) stress, ABL kinases localize to the ER membrane•At the ER, ABL scaffolds IRE1α to hyperactivate the unfolded protein response (UPR)•Imatinib blunts the UPR and apoptosis by maintaining ABL in a 14-3-3 cytosolic pool•Direct targeting of IRE1α, using mono-selective KIRA8, reverses autoimmune diabetes In cells experiencing unrelieved endoplasmic reticulum (ER) stress, the ER transmembrane kinase/endoribonuclease (RNase)—IRE1α—endonucleolytically degrades ER-localized mRNAs to promote apoptosis. Here we find that the ABL family of tyrosine kinases rheostatically enhances IRE1α’s enzymatic activities, thereby potentiating ER stress-induced apoptosis. During ER stress, cytosolic ABL kinases localize to the ER membrane, where they bind, scaffold, and hyperactivate IRE1α’s RNase. Imatinib—an anti-cancer tyrosine kinase inhibitor—antagonizes the ABL-IRE1α interaction, blunts IRE1α RNase hyperactivity, reduces pancreatic β cell apoptosis, and reverses type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. A mono-selective kinase inhibitor that allosterically attenuates IRE1α’s RNase—KIRA8—also efficaciously reverses established diabetes in NOD mice by sparing β cells and preserving their physiological function. Our data support a model wherein ER-stressed β cells contribute to their own demise during T1D pathogenesis and implicate the ABL-IRE1α axis as a drug target for the treatment of an autoimmune disease. In cells experiencing unrelieved endoplasmic reticulum (ER) stress, the ER transmembrane kinase/endoribonuclease (RNase)—IRE1α—endonucleolytically degrades ER-localized mRNAs to promote apoptosis. Here we find that the ABL family of tyrosine kinases rheostatically enhances IRE1α’s enzymatic activities, thereby potentiating ER stress-induced apoptosis. During ER stress, cytosolic ABL kinases localize to the ER membrane, where they bind, scaffold, and hyperactivate IRE1α’s RNase. Imatinib—an anti-cancer tyrosine kinase inhibitor—antagonizes the ABL-IRE1α interaction, blunts IRE1α RNase hyperactivity, reduces pancreatic β cell apoptosis, and reverses type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. A mono-selective kinase inhibitor that allosterically attenuates IRE1α’s RNase—KIRA8—also efficaciously reverses established diabetes in NOD mice by sparing β cells and preserving their physiological function. Our data support a model wherein ER-stressed β cells contribute to their own demise during T1D pathogenesis and implicate the ABL-IRE1α axis as a drug target for the treatment of an autoimmune disease. Diverse perturbations compromise folding and structural maturation of secretory proteins in the endoplasmic reticulum (ER). If uncorrected, such “ER stress” promotes cell degeneration and apoptosis. ER stress activates unfolded protein response (UPR) signaling pathways that determine cell fate. Remediable ER stress activates adaptive (“A”)-UPR outputs that favor cell survival. But under irremediably high, chronic ER stress, these adaptive measures wane, as alternate terminal (“T”)-UPR outputs trigger apoptosis. High/chronic ER stress promotes numerous diseases of premature cell loss (Oakes and Papa, 2015Oakes S.A. Papa F.R. The role of endoplasmic reticulum stress in human pathology.Annu. Rev. Pathol. 2015; 10: 173-194Crossref PubMed Scopus (671) Google Scholar). For example, pancreatic islet β cells, responsible for synthesizing and secreting sufficient quantities of insulin to maintain blood glucose homeostasis, commonly experience high ER stress and secretory exhaustion (Scheuner and Kaufman, 2008Scheuner D. Kaufman R.J. The unfolded protein response: a pathway that links insulin demand with beta-cell failure and diabetes.Endocr. Rev. 2008; 29: 317-333Crossref PubMed Scopus (426) Google Scholar). Peripheral insulin resistance further elevates β cell insulin secretory demand during development of type 2 diabetes (T2D) (Back and Kaufman, 2012Back S.H. Kaufman R.J. Endoplasmic reticulum stress and type 2 diabetes.Annu. Rev. Biochem. 2012; 81: 767-793Crossref PubMed Scopus (419) Google Scholar). Insulin gene mutations cause encoded proinsulin to become structurally arrested in the β cell ER, and various UPR gene deletions debilitate insulin production by β cells. Dysregulated UPR signaling promotes β cell autonomous apoptosis in these diverse diabetic syndromes (Ozcan et al., 2004Ozcan U. Cao Q. Yilmaz E. Lee A.H. Iwakoshi N.N. Ozdelen E. Tuncman G. Görgün C. Glimcher L.H. Hotamisligil G.S. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.Science. 2004; 306: 457-461Crossref PubMed Scopus (2972) Google Scholar, Papa, 2012Papa F.R. Endoplasmic reticulum stress, pancreatic β-cell degeneration, and diabetes.Cold Spring Harb. Perspect. Med. 2012; 2: a007666Crossref PubMed Scopus (136) Google Scholar). Type 1 diabetes (T1D) is triggered by immune dysregulation and autoreactive T cell responses against β cells. However, the autoreactivity does not inevitably result in direct β cell destruction (fratricide), but will also induce β cells to autonomously undergo apoptosis (suicide) during disease progression (Atkinson et al., 2011Atkinson M.A. Bluestone J.A. Eisenbarth G.S. Hebrok M. Herold K.C. Accili D. Pietropaolo M. Arvan P.R. Von Herrath M. Markel D.S. Rhodes C.J. How does type 1 diabetes develop?: the notion of homicide or β-cell suicide revisited.Diabetes. 2011; 60: 1370-1379Crossref PubMed Scopus (162) Google Scholar, Bottazzo, 1986Bottazzo G.F. Lawrence lecture. Death of a beta cell: homicide or suicide?.Diabet. Med. 1986; 3: 119-130Crossref PubMed Scopus (70) Google Scholar). Both human and mouse studies have suggested that, at the time of T1D diagnosis, as much as 30%–40% of β cells remain and are functionally unresponsive but can recover following removal of stress, suggesting that a window of opportunity may exist for therapies that prevent further β cell deterioration and restore β cell function (Alanentalo et al., 2010Alanentalo T. Hörnblad A. Mayans S. Karin Nilsson A. Sharpe J. Larefalk A. Ahlgren U. Holmberg D. Quantification and three-dimensional imaging of the insulitis-induced destruction of beta-cells in murine type 1 diabetes.Diabetes. 2010; 59: 1756-1764Crossref PubMed Scopus (65) Google Scholar, Krogvold et al., 2015Krogvold L. Skog O. Sundström G. Edwin B. Buanes T. Hanssen K.F. Ludvigsson J. Grabherr M. Korsgren O. Dahl-Jørgensen K. Function of Isolated Pancreatic Islets From Patients at Onset of Type 1 Diabetes: Insulin Secretion Can Be Restored After Some Days in a Nondiabetogenic Environment In Vitro: Results From the DiViD Study.Diabetes. 2015; 64: 2506-2512Crossref PubMed Scopus (65) Google Scholar). The non-obese diabetic “NOD” mouse develops β cell failure subsequent to innate immune and T cell islet infiltration (as in humans with T1D) (Anderson and Bluestone, 2005Anderson M.S. Bluestone J.A. The NOD mouse: a model of immune dysregulation.Annu. Rev. Immunol. 2005; 23: 447-485Crossref PubMed Scopus (840) Google Scholar). We found that treating NOD mice with the anti-cancer drug imatinib both prevents and reverses diabetes, inducing prolonged remission (Louvet et al., 2008Louvet C. Szot G.L. Lang J. Lee M.R. Martinier N. Bollag G. Zhu S. Weiss A. Bluestone J.A. Tyrosine kinase inhibitors reverse type 1 diabetes in nonobese diabetic mice.Proc. Natl. Acad. Sci. USA. 2008; 105: 18895-18900Crossref PubMed Scopus (183) Google Scholar). This remarkable efficacy in the NOD has prompted a phase II clinical trial to repurpose imatinib for new-onset T1D. However, a full understanding of the underlying mechanism of imatinib’s efficacy has remained unclear. Enigmatically, imatinib shows minimal effects on T cell effector function and trafficking in the NOD. Insulitis scores, CD4+/CD8+ ratios in spleen and pancreatic lymph nodes, and regulatory T cell function remained unchanged, supporting the notion that imatinib’s anti-diabetic effect is not simply due to immune modulation (Louvet et al., 2008Louvet C. Szot G.L. Lang J. Lee M.R. Martinier N. Bollag G. Zhu S. Weiss A. Bluestone J.A. Tyrosine kinase inhibitors reverse type 1 diabetes in nonobese diabetic mice.Proc. Natl. Acad. Sci. USA. 2008; 105: 18895-18900Crossref PubMed Scopus (183) Google Scholar). Recently, investigators reported high ER stress signaling in autoimmune-targeted β cells of the NOD (Engin et al., 2013Engin F. Yermalovich A. Nguyen T. Hummasti S. Fu W. Eizirik D.L. Mathis D. Hotamisligil G.S. Restoration of the unfolded protein response in pancreatic β cells protects mice against type 1 diabetes.Sci. Transl. Med. 2013; 5: 211ra156Crossref PubMed Scopus (203) Google Scholar, Tersey et al., 2012Tersey S.A. Nishiki Y. Templin A.T. Cabrera S.M. Stull N.D. Colvin S.C. Evans-Molina C. Rickus J.L. Maier B. Mirmira R.G. Islet β-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model.Diabetes. 2012; 61: 818-827Crossref PubMed Scopus (251) Google Scholar), which prompted us to inquire whether imatinib may instead protect β cells in the NOD by modulating the UPR. The ER transmembrane kinase/endoribonuclease (RNase), IRE1α, determines cell fate based on ER stress severity. Under ER stress, IRE1α monomers in the ER membrane undergo trans-autophosphorylation and RNase activation, thereby initiating frameshift splicing of the mRNA encoding XBP1 transcription factor to trigger adaptive UPR transcriptional programs (Yoshida et al., 2001Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor.Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar). If ER stress remains unrelieved, IRE1α organizes into high-order, oligomeric complexes as its autophosphorylation and RNase activation state rise further, thereby causing endonucleolytic degradation of many ER-localized mRNAs and apoptosis (Han et al., 2009aHan D. Lerner A.G. Vande Walle L. Upton J.P. Xu W. Hagen A. Backes B.J. Oakes S.A. Papa F.R. IRE1alpha kinase activation modes control alternate endoribonuclease outputs to determine divergent cell fates.Cell. 2009; 138: 562-575Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar). Despite this mechanistic understanding, the exact components of the IRE1α complex remain largely unresolved. Here we find that imatinib’s anti-diabetogenic effects in the NOD derive from ameliorating pro-apoptotic terminal UPR signaling in β cells through an unexpected link between IRE1α and the non-receptor ABL tyrosine kinases, which play diverse, intracellular signaling functions but have not previously been characterized as UPR components. We find that this ABL-IRE1α axis functions upstream in the UPR to potentiate apoptosis during ER stress, and that imatinib reduces apoptosis by attenuating a stimulatory interaction of ABL with IRE1α. These findings predicted that direct inhibition of IRE1α should prove anti-diabetogenic in the NOD. Enabled with newly optimized compounds called “KIRA”s (kinase-inhibitory RNase attenuators) that inhibit IRE1α kinase/RNase activity, we found that a mono-selective KIRA induces near-complete reversal of established diabetes in the NOD model. High ER stress signaling has been reported in autoimmune-targeted NOD islets (Engin et al., 2013Engin F. Yermalovich A. Nguyen T. Hummasti S. Fu W. Eizirik D.L. Mathis D. Hotamisligil G.S. Restoration of the unfolded protein response in pancreatic β cells protects mice against type 1 diabetes.Sci. Transl. Med. 2013; 5: 211ra156Crossref PubMed Scopus (203) Google Scholar, Tersey et al., 2012Tersey S.A. Nishiki Y. Templin A.T. Cabrera S.M. Stull N.D. Colvin S.C. Evans-Molina C. Rickus J.L. Maier B. Mirmira R.G. Islet β-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model.Diabetes. 2012; 61: 818-827Crossref PubMed Scopus (251) Google Scholar). To characterize signature A- and T-UPR events in NOD islets, we examined IRE1α activation state prior to diabetes onset. IRE1α became progressively more induced and activation-loop autophosphorylated in NOD islets at 10 and 12 weeks of age, coinciding with increasing pre-diabetic insulitis (Figure 1A) (Anderson and Bluestone, 2005Anderson M.S. Bluestone J.A. The NOD mouse: a model of immune dysregulation.Annu. Rev. Immunol. 2005; 23: 447-485Crossref PubMed Scopus (840) Google Scholar). Islets from 12-week-old NOD mice show elevated thioredoxin-interacting protein (TXNIP), a key T-UPR mediator induced by IRE1α hyperactivation, that activates the NLRP3 inflammasome to promote islet inflammation and β cell death (Figure 1A) (Lerner et al., 2012Lerner A.G. Upton J.P. Praveen P.V. Ghosh R. Nakagawa Y. Igbaria A. Shen S. Nguyen V. Backes B.J. Heiman M. et al.IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress.Cell Metab. 2012; 16: 250-264Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar). TXNIP mRNA progressively rose, and proinsulin-encoding Ins1 and Ins2 mRNAs, which we identified as IRE1α RNase substrates (Han et al., 2009aHan D. Lerner A.G. Vande Walle L. Upton J.P. Xu W. Hagen A. Backes B.J. Oakes S.A. Papa F.R. IRE1alpha kinase activation modes control alternate endoribonuclease outputs to determine divergent cell fates.Cell. 2009; 138: 562-575Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar), progressively decayed in pre-diabetic NOD islets compared to age-matched immune-deficient NSG controls, which develop neither immune infiltrates nor β cell dysfunction (Figure 1B). During the pre-diabetic window of rising T-UPR outputs, A-UPR mediators, including spliced XBP1 mRNA, and mRNAs encoding the ER chaperone BiP and cytoprotective MANF (Lindahl et al., 2014Lindahl M. Danilova T. Palm E. Lindholm P. Võikar V. Hakonen E. Ustinov J. Andressoo J.O. Harvey B.K. Otonkoski T. et al.MANF is indispensable for the proliferation and survival of pancreatic β cells.Cell Rep. 2014; 7: 366-375Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), waned (Figure 1C). By the time of overt disease, insulin mRNAs declined further, while TXNIP mRNA continued to rise (Figure 1D). Thus, NOD islets morph from an A- to a T-UPR signature after development of insulitis and before progression to frank diabetes (Figure 1E). As imatinib can prevent and reverse diabetes in the NOD (Louvet et al., 2008Louvet C. Szot G.L. Lang J. Lee M.R. Martinier N. Bollag G. Zhu S. Weiss A. Bluestone J.A. Tyrosine kinase inhibitors reverse type 1 diabetes in nonobese diabetic mice.Proc. Natl. Acad. Sci. USA. 2008; 105: 18895-18900Crossref PubMed Scopus (183) Google Scholar), we hypothesized that its efficacy may derive in part from attenuating the T-UPR in islets. Daily oral dosing of pre-diabetic NOD mice with imatinib for 4 weeks significantly decreased islet TXNIP mRNA, preserved first-phase serum insulin, and doubled insulin-positive area in pancreata, without changing body weight (Figures 1F–1I, S1A). Newly diabetic NODs started on daily imatinib showed 80% reversal within 3 weeks (Figure 1J). Random and fasting blood glucose levels were significantly lower in the imatinib group within 2 and 4 weeks, respectively, while their islets showed attenuated TXNIP and preserved insulin mRNAs (Figures 1K–1N). While imatinib blunts the T-UPR in NOD islets, whether it confers a direct, immune-independent, cytoprotective effect on β cells was unclear. To address this, we asked whether immune-deficient NSG mice islets are protected by imatinib from toxic doses of tunicamycin (Tm), a protein glycosylation inhibitor that causes apoptosis (Ghosh et al., 2014Ghosh R. Wang L. Wang E.S. Perera B.G. Igbaria A. Morita S. Prado K. Thamsen M. Caswell D. Macias H. et al.Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.Cell. 2014; 158: 534-548Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). In Tm-exposed NSG islets, imatinib blocks elevations in spliced XBP1 and TXNIP mRNAs and declines in insulin mRNAs, with similar results in human islets (Figures 2A, 2B, and S1B). In NSG islets, Tm-promoted decreases in insulin content and glucose-stimulated insulin secretion (GSIS) are prevented by imatinib (Figures 2C and 2D). In the absence of ER stress agents, imatinib did not change TXNIP and Ins1/2 mRNA and insulin secretion (Figures S1C–S1E). Also, imatinib reduces Tm-promoted TXNIP induction in C57BL/6 islets (Figure 2E), showing that its effects are not strain specific. Finally, imatinib decreases Tm-promoted apoptosis in NSG islets (Figures 2F and 2G). We next used a β cell-derived insulinoma line, INS-1, which can be genetically manipulated, to study imatinib’s mechanistic effects. In INS-1 cells, Tm-promoted IRE1α autophosphorylation, XBP1 mRNA splicing, and TXNIP mRNA/protein induction are all inhibited by imatinib (Figures 2H–2K). Nilotinib, an equally selective but more potent inhibitor than imatinib, reduced Tm-promoted XBP1 splicing (Figures S1F and S1G), TXNIP mRNA induction (Figure S1H), and apoptosis at lower concentrations than imatinib (Figures 2M and S1I). Imatinib’s salutary effects extend broadly to other ER stress regimes, including SERCA pump inhibition by thapsigargin (Tg) and anterograde trafficking blockage by brefeldin A (Figures S1J and S1K). Glucotoxicity-promoted insulin mRNA decay (Lipson et al., 2006Lipson K.L. Fonseca S.G. Ishigaki S. Nguyen L.X. Foss E. Bortell R. Rossini A.A. Urano F. Regulation of insulin biosynthesis in pancreatic beta cells by an endoplasmic reticulum-resident protein kinase IRE1.Cell Metab. 2006; 4: 245-254Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar), TXNIP induction, and proinsulin depletion are all also prevented by imatinib (Figures 2L and S1L). As in islets, imatinib significantly reduces ER stress-induced INS-1 cell apoptosis (Figure 2M). Suggesting that it inhibits the upstream-most UPR signaling step of IRE1α oligomerization, imatinib blocks ER membrane focal aggregation of a superfolder (sf) GFP-IRE1α reporter during DTT-induced ER stress (Figure 2N) (Ghosh et al., 2014Ghosh R. Wang L. Wang E.S. Perera B.G. Igbaria A. Morita S. Prado K. Thamsen M. Caswell D. Macias H. et al.Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.Cell. 2014; 158: 534-548Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). But while imatinib inhibits IRE1α in cells, it does not directly inhibit either the kinase or RNase activities of a recombinant IRE1α∗ mini-protein (Figures S1M–S1P). Therefore, imatinib’s inhibitory effects in cells appear to occur through a different target than IRE1α. As imatinib was optimized for BCR-Abl (Capdeville et al., 2002Capdeville R. Buchdunger E. Zimmermann J. Matter A. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug.Nat. Rev. Drug Discov. 2002; 1: 493-502Crossref PubMed Scopus (1236) Google Scholar), we next monitored the activation state of its non-oncogenic counterpart, c-Abl, in NOD islets. Like IRE1α, c-Abl becomes progressively more induced and activation-loop phosphorylated in pre-diabetic NOD islets (Figures 1A and 3A ). Also, in INS-1 cells, high glucose triggered acute c-Abl mRNA induction and c-Abl activation, which was abrogated by imatinib (Figures 3B, 3C, S2A, and S2B). c-Abl staining was evident in β cells, but not in infiltrating immune cells based on lack of co-localization with CD45 and DAPI (Figures S2C–S2T), with higher protein and mRNA levels in NOD compared to NSG islets (Figures S3A and S3B). Other imatinib targets, PDGFRα, PDGFRβ, and c-KIT, were not detectable in β cells (Figure S3C). In islets, c-Abl co-localizes with synaptophysin in neuroendocrine cells and with glucagon in α cells, but is undetectable in cells expressing pancreatic polypeptide or somatostatin (Figures S2C–S2T). Together, these data suggested that imatinib’s efficacy in the NOD may derive through targeting β cell-expressed c-Abl. The ABL tyrosine kinase family comprises c-Abl (ABL1) and Arg (ABL2), which display redundant and unique functions (Wang, 2014Wang J.Y. The capable ABL: what is its biological function?.Mol. Cell. Biol. 2014; 34: 1188-1197Crossref PubMed Scopus (111) Google Scholar). To study the UPR roles of c-Abl and Arg, we subjected Abl/Arg (−/−) double-knockout (DKO) mouse embryonic fibroblasts (MEFs) to ER stress. XBP1 mRNA splicing, dose-dependently induced by Tm, is significantly blunted in Abl/Arg DKO MEFs (Figures 3D and S4A); c-Abl and Arg appear to play redundant roles because c-Abl (−/−) single-knockout MEFs show no XBP1 splicing defect (Figure S4B). Under ER stress, Abl/Arg DKO MEFs show crippled TXNIP mRNA induction and apoptosis (Figures 3E, 3F, and S4C). Given the necessity of c-Abl/Arg in promoting the T-UPR, we next tested their sufficiency in isogenic INS-1 and T-REx293 lines that stably overexpress c-Abl or Arg under doxycycline (Dox). In T-REx293 cells, induction of c-Abl, capable of phosphorylation of endogenous Crkl, causes spontaneous autophosphorylation of endogenous IRE1α—without ER stress—and rapidly triggers XBP1 mRNA splicing and nuclear accumulation of XBP1 transcription factor (Figures S4D–S4H). Similarly, c-Abl induction in INS-1 cells leads to nuclear XBP1 enrichment and TXNIP induction, while imatinib inhibits IRE1α autophosphorylation, TXNIP elevation, and apoptosis (Figures 3G–3L). Overexpression of Arg is also sufficient to induce TXNIP (Figure S4I). In contrast, overexpression of other imatinib targets, PDGFRα and c-kit, did not induce T-UPR events (Figures S4J and S4K). c-Abl and Arg did not affect PERK, another UPR sensor kinase, based on unchanged phosphorylation of its substrate eiF2α in Abl/Arg DKO MEFs under ER stress, or in INS-1 cells overexpressing c-Abl (Figures S4L and S4M). The IRE1α RNase inhibitor, STF-083010 (Papandreou et al., 2011Papandreou I. Denko N.C. Olson M. Van Melckebeke H. Lust S. Tam A. Solow-Cordero D.E. Bouley D.M. Offner F. Niwa M. Koong A.C. Identification of an Ire1alpha endonuclease specific inhibitor with cytotoxic activity against human multiple myeloma.Blood. 2011; 117: 1311-1314Crossref PubMed Scopus (373) Google Scholar), blocks c-Abl-induced TXNIP mRNA and protein elevation (Figures 3M and 3N), further supporting that c-Abl-driven T-UPR signaling proceeds through IRE1α RNase activation. Tyrosine phosphorylation has not previously been reported to play a role in the UPR, despite the necessity and sufficiency of ABL kinases for stimulating IRE1α. Therefore, the necessity of c-Abl’s tyrosine kinase activity was tested in an INS-1 line conditionally expressing a kinase-dead mutant, K290R. Surprisingly, K290R c-Abl triggers nuclear accumulation of XBP1s and TXNIP induction to similar levels as WT c-Abl (Figures 4A–4D). Remarkably, imatinib reduces K290R c-Abl-induced TXNIP protein elevation (Figure 4E), like WT c-Abl (Figure 3K). These results suggest that c-Abl phosphotransfer-independently stimulates IRE1α and that imatinib counters this effect apart from kinase catalytic inhibition. We therefore reasoned that under ER stress c-Abl may co-localize with IRE1α to scaffold and stimulate IRE1α’s activity, with imatinib countering this interaction. To test this, we performed live-cell imaging with sfGFP-IRE1α and a c-Abl-mCherry fusion. In INS-1 cells, c-Abl-mCherry is diffusely localized, but with significant ER enrichment, consistent with previous reports (Figures 4F and 4G) (Qi and Mochly-Rosen, 2008Qi X. Mochly-Rosen D. The PKCdelta -Abl complex communicates ER stress to the mitochondria - an essential step in subsequent apoptosis.J. Cell Sci. 2008; 121: 804-813Crossref PubMed Scopus (84) Google Scholar). Under ER stress, c-Abl-mCherry co-localizes with sfGFP-IRE1α in ER punctate foci, while imatinib co-treatment prevents c-Abl-mCherry focal re-localization, as with sfGFP-IRE1α (Figures 2N and 4G). We next tested whether c-Abl’s association with cytosolic 14-3-3 proteins that modulate localization of c-Abl and other kinases is disrupted under ER stress, as during oxidative stress (Nihira et al., 2008Nihira K. Taira N. Miki Y. Yoshida K. TTK/Mps1 controls nuclear targeting of c-Abl by 14-3-3-coupled phosphorylation in response to oxidative stress.Oncogene. 2008; 27: 7285-7295Crossref PubMed Scopus (36) Google Scholar, Yoshida et al., 2005Yoshida K. Yamaguchi T. Natsume T. Kufe D. Miki Y. JNK phosphorylation of 14-3-3 proteins regulates nuclear targeting of c-Abl in the apoptotic response to DNA damage.Nat. Cell Biol. 2005; 7: 278-285Crossref PubMed Scopus (204) Google Scholar). Indeed, ER stress causes rapid dissociation of the c-Abl/14-3-3 complex, which imatinib prevents (Figures 4H, 4I, and S5A). These data further support a model of c-Abl re-localizing from the cytosol to an IRE1α complex at the ER membrane under stress. Also, c-Abl co-immunoprecipitates (coIPs) either endogenous or transgenic IRE1α, which imatinib abrogates (Figures 4J, S5B, and S5C). The N-terminal myristoyl group of c-Abl is necessary for directing c-Abl to IRE1α because a non-myristoylated splice variant—Abl1a—cannot rescue XBP1 splicing or TXNIP mRNA elevation when reconstituted in Abl/Arg DKO MEFs, unlike N-terminally myristoylated Abl1b (Figures S5D–S5G). We then asked if the N-terminal SH2, SH3, and kinase domains of c-Abl are sufficient for IRE1α stimulation by testing an N-terminal truncation, c-Abl(3D). XBP1 splicing is abrogated in cells expressing c-Abl(3D) (Figures 4K–4N, S5F, and S5H), showing that c-Abl’s C-terminal domains are also required for IRE1α activation. Also, unlike full-length c-Abl, c-Abl(3D) cannot coIP recombinant IRE1α∗ (Figures 4O–4Q). Finally, to determine if c-Abl and IRE1α directly interact, we conducted biochemical experiments using recombinant c-Abl (Figure S5I) and IRE1α∗. Purified, full-length c-Abl, immobilized on beads with an ATP-competitive inhibitor, coIPs IRE1α∗ (Figure S5J). Finally, purified c-Abl or c-Abl K290R can—with equal potency—directly stimulate IRE1α∗’s RNase catalytic activity in vitro, whereas c-Abl(3D) cannot (Figures 4R–4T and S5K–S5M). Thus, under ER stress, c-Abl scaffolds and stimulates IRE1α at the ER membrane independent of its phosphotransfer activity, but requires both its N-myristoyl group and domains C-terminal to its kinase. Thus, we predicted that forcibly directing c-Abl into IRE1α foci should hyperactivate IRE1α without ER stress. To test this, we used GNF-2 (Figure 5A), an inhibitor that interacts with the myristate-binding pocket in c-Abl’s catalytic domain, thereby displacing c-Abl’s N-terminal myristoyl group and enhancing ER localization (Choi et al., 2009Choi Y. Seeliger M.A. Panjarian S.B. Kim H. Deng X. Sim T. Couch B. Koleske A.J. Smithgall T.E. Gray N.S. N-myristoylated c-Abl tyrosine kinase localizes to the endoplasmic reticulum upon binding to an allosteric inhibitor.J. Biol. Chem. 2009; 284: 29005-29014Crossref PubMed Scopus (45) Google Scholar). Consistent with previous studies, we further find that GNF-2 promotes spontaneous formation of ER foci containing both c-Abl-mCherry and sfGFP-IRE1α, correlating with diminished c-Abl/14-3-3 interaction, without ER stress (Figures 5B and 5C). In INS-1 cells, GNF-2 reduces Crkl phosphorylation while increasing IRE1α autophosphorylation, XBP1 splicing, TXNIP mRNA/protein induction, Ins1 mRNA decay, and apoptosis (Figures 5D–5K); GNF-2 does not increase XBP1 splicing in Abl/Arg DKO MEFs or PERK-mediated eiF2α phosphorylation (Figures S6A and S6B). In summary, although GNF-2 allosterically inhibits c-Abl’s phosphotransfer activity, its ability to promote c-Abl co-localization with IRE1α suffices to hyperactivate IRE1α’s RNase, which can be inhibited with STF-083010 (Figure S6C). By investigating the mechanism of imatinib’s efficacy in the NOD, we identified an ABL-IRE1α signaling axis, leading to a final prediction: if IRE1α hyperactivity promotes β cell death in the NOD, then direct IRE1α inhibition should be anti-diabetogenic. To test this, we treated pre-diabetic NOD females with KIRA6, an ATP-competitive ligand that allosterically inhibits IRE1α’s RNase by breaking oligomers. Using an intraperitoneal (i.p.) dosing regime that reduces diabetes in the Akita mouse—which expresses an oxidative-folding defective proinsulin mutant, Ins2(C96Y), that triggers autonomous β cell apoptosis (Ghosh et al., 2014Ghosh R. Wang L. Wang E.S. Perera B.G. Igbaria A. Morita S. Prado K. Thamsen M. Caswell D. Macias H. et al.Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.Cell. 2014; 158: 534-548Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, Lerner et al., 2012Lerner A.G. Upton J.P. Praveen P.V. Ghosh R. Nakagawa Y. Igbaria A. Shen S. Nguyen V. Backes B.J. Heiman M. et al.IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress.Cell Metab. 2012; 16: 250-264Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar)—we noted significant reduction of TXNIP and recovery of Ins1/Ins2, BiP, and MANF mRNAs within 1 week of treatment in 10-week-old NODs (Figures S6D–S6H). After a 6-week KIRA6 treatment of 8-week-old pre-diabetic NODs, preserved first-phase insulin response—and significantly greater pancreatic insulin protein staining—was evident in the KIRA6 group compared to vehicles (Figures S6I–S6K). Diabetes reversal was attempted by dosing KIRA6 upon disease onset. Random blood glucoses stabilized in the KIRA6 cohort, whereas hyperglycemia continued to rise in controls, without" @default.
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• W4248517419 title "Targeting ABL-IRE1α Signaling Spares ER-Stressed Pancreatic β Cells to Reverse Autoimmune Diabetes" @default.
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