Approximately 20% of patients with myeloproliferative neoplasms (MPNs) harbor mutations in the gene calreticulin (CALR), with 80% of those mutations classified as either type 1 or type 2. While type 2 CALR mutant proteins retain many of the Ca2+ binding sites present in the wild type protein, type 1 CALR mutant proteins lose these residues. The functional consequences of this differential loss of Ca2+ binding sites remain yet unexplored. Here, we show that the loss of Ca2+ binding residues in the type 1 mutant CALR protein directly impairs its Ca2+ binding ability, which in turn leads to depleted endoplasmic reticulum (ER) Ca2+ and subsequent activation of the IRE1a/XBP1 pathway of the unfolded protein response. Genetic or pharmacological inhibition of IRE1a/XBP1 signaling induces cell death only in type 1 mutant but not type 2 mutant or wild type CALR-expressing cells, and abrogates type 1 mutant CALR-driven MPN disease progression in vivo.

Cells use a variety of mechanisms to maintain optimal mitochondrial function including the mitochondrial unfolded protein response (UPRmt). The UPRmt mitigates mitochondrial dysfunction by differentially regulating mitoprotective gene expression through the transcription factor ATFS-1. Since UPRmt activation is commensurate with organismal benefits such as extended lifespan and host protection during infection, we sought to identify pathways that promote its stimulation. Using unbiased forward genetics screening, we isolated novel mutant alleles that could activate the UPRmt. Interestingly, we identified one reduction of function mutant allele (osa3) in the mitochondrial ribosomal gene mrpl-2 that activated the UPRmt in a diet-dependent manner. We find that mrpl-2(osa3) mutants lived longer and survived better during pathogen infection depending on the diet they were fed. A diet containing low levels of vitamin B12 could activate the UPRmt in mrpl-2(osa3) animals. Also, we find that the vitamin B12-dependent enzyme methionine synthase intersects with mrpl-2(osa3) to activate the UPRmt and confer animal lifespan extension at the level of ATFS-1. Thus, we present a novel gene-diet pairing that promotes animal longevity that is mediated by the UPRmt.

Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR). 80% of CALR mutations are classified as either type 1 or type 2, exemplified by a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5), respectively. Despite their shared mutant C-termini and mutual ability to bind and activate MPL, patients with type 1 and type 2 CALR mutations display significant clinical and prognostic differences. Type 1 mutations are primarily associated with an MF phenotype and a higher risk of fibrotic transformation from ET, while type 2 mutations are more common in ET. Molecularly, type 2 CALR mutant proteins retain many of the calcium binding sites present in the wild type protein, while type 1 CALR mutant proteins lose these residues. The functional consequences of this differential loss of calcium binding sites remain yet unexplored. Current targeted therapies for CALR mutated MPN are not curative, and treatment does not differentiate between type 1 versus type 2 mutant CALR-driven disease, despite the different phenotypic and prognostic outcomes in these patients. In order to improve treatment strategies for CALR mutated MPN patients, it is critical to identify specific dependencies unique to each CALR mutation type that can be exploited for therapeutic gain. Here, we show that type 1 CALRdel52 but not type 2 CALRins5 mutations lead to activation of and dependency on the IRE1α-XBP1 pathway of the unfolded protein response (UPR). Mechanistically, we found that the loss of calcium binding residues in the type 1 mutant CALR protein directly impairs its calcium binding ability, which in turn leads to depleted ER calcium and subsequent activation of the IRE1α-XBP1 pathway. Using cell lines and primary MPN patient samples, we identified two novel transcriptional targets of XBP1 specific to type 1 CALRdel52-expressing cells - the anti-apoptotic protein BCL-2 and the calcium efflux channel IP3R. We show that BCL-2 acts downstream of XBP1 to promote survival in the face of depleted ER calcium, while IP3R is up-regulated downstream of XBP1 to promote continued ER calcium efflux in order to sustain IRE1α-XBP1 pathway activation and survival. We found that genetic or pharmacological inhibition of IRE1α-XBP1 signaling induced cell death only in type 1 mutant but not type 2 mutant or wild type CALR-expressing cells. Moreover, we show that in vivo inhibition of IRE1α significantly abrogates type 1 mutant CALR-driven disease in a bone marrow transplantation model, but has no effect on type 2 mutant CALR-driven disease. This work is the first to demonstrate that type 1 and type 2 mutant CALR-expressing cells display differential molecular dependencies that can be exploited for therapeutic gain. Moreover, this study answers an enduring question regarding the functional consequence of the loss of calcium binding sites on the type 1 mutant CALR protein, and demonstrates how type 1 CALR mutant-expressing cells rewire the UPR, downstream calcium signaling, and apoptotic pathways to drive MPN. Figure 1 Figure 1. Disclosures Koschmieder: BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Shire: Honoraria, Other; Karthos: Other: Travel support; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Geron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Abbvie: Other: Travel support; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Alexion: Other: Travel support; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Baxalta: Membership on an entity's Board of Directors or advisory committees, Other; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); CTI: Membership on an entity's Board of Directors or advisory committees, Other; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; AOP Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Image Biosciences: Other: Travel support.

Supplementary Data from Type I but Not Type II Calreticulin Mutations Activate the IRE1α/XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms

<div>Abstract<p>Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (<i>CALR</i>), with 80% of those mutations classified as either type I or type II. While type II CALR-mutant proteins retain many of the Ca²⁺ binding sites present in the wild-type protein, type I CALR-mutant proteins lose these residues. The functional consequences of this differential loss of Ca²⁺ binding sites remain unexplored. Here, we show that the loss of Ca²⁺ binding residues in the type I mutant CALR protein directly impairs its Ca²⁺ binding ability, which in turn leads to depleted endoplasmic reticulum (ER) Ca²⁺ and subsequent activation of the IRE1α/XBP1 pathway of the unfolded protein response. Genetic or pharmacologic inhibition of IRE1α/XBP1 signaling induces cell death in type I mutant but not type II mutant or wild-type <i>CALR</i>-expressing cells, and abrogates type I mutant CALR-driven MPN disease progression <i>in vivo</i>.</p>Significance:<p>Current targeted therapies for <i>CALR</i>-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type–specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I <i>CALR</i>-mutated MPNs.</p><p><i><a href="https://aacrjournals.org/bloodcancerdiscov/article/doi/10.1158/2643-3230.BCD-3-4-ITI" target="_blank">This article is highlighted in the In This Issue feature, p. 265</a></i></p></div>