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Abstract
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. Current targeted therapies for CALR mutated MPNs are not curative, and fail to 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 MPNs patients, it is critical to identify specific dependencies unique to each CALR mutation type that can be exploited for therapeutic gain. 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. Here, we show 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 endoplasmic reticulum (ER) calcium and subsequent activation of the IRE1a/XBP1 pathway of the unfolded protein response (UPR). 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 MPNs disease progression in vivo. In a continued line of research, we aim to define the role of the RNA 2', 3'- cyclic phosphate and 5'-OH ligase (RTCB) in CALR mutant MPNs. RTCB is the primary ligase responsible for the re-joining of XBP1 mRNA exons upon IRE1a splicing. Given our previous findings showing that the IRE1a-XBP1 pathway is critical in type 1 mutant CALR-driven MPNs, we aim at dissecting its XBP1-dependent vs independent functions and whether RTCB targeting represents a novel target in this disease. Separately, given the need to dissect the molecular players and mechanisms involved in myelofibrosis, this work aims to develop a cell-based system that mimics the pathological features of myelofibrosis in vitro. Overall, this work is the first to demonstrate that type 1 and type 2 mutant CALR-expressing cells display differential molecular dependencies that can be targeted 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 MPNs.