Project Summary Myeloproliferative neoplasms (MPNs) include primary myelofibrosis (PMF; characterized by the over- proliferation of megakaryocytes and granulocytes with abnormal collagen deposition in the bone marrow stroma), essential thrombocythemia (ET; increased megakaryocyte and platelet production), and polycythemia vera (PV; increased red cell production, hemoglobin, and hematocrit). Although all MPN driver mutations lead to constitutive activation of JAK/STAT signaling, targeted JAK inhibitors are not curative and fail to alter disease progression. Therefore, there is a great unmet need to identify novel curative therapies for MPNs. Mutations in calreticulin (CALR) represent the second most common genetic abnormality in MPN. The CALR gene encodes a calcium (Ca2+)-binding chaperone protein that primarily resides in the endoplasmic reticulum (ER). All CALR mutations share an identical neomorphic C-terminal peptide, which permits binding to the thrombopoietin receptor MPL and the subsequent activation of pathogenic JAK/STAT signaling. The majority of CALR mutations are classified as either type 1 or type 2 based on the extent of homology to the wild type protein, where type 1 proteins exhibit complete loss of C-terminal Ca2+ binding sites that are retained in type 2. Despite their shared mutant C-terminus and ability to bind and activate MPL, type 1 and 2 CALR mutations engender significant phenotypic and prognostic differences. Type 1 mutations are more common in PMF, and are associated with increased risk of myelofibrotic transformation from ET. Conversely, type 2 mutations are primarily associated with ET, exhibit low incidence of myelofibrotic transformation, and are rarely found in PMF. The mechanisms underlying these divergent clinical phenotypes remain unknown. We discovered that the IRE1a/XBP1 pathway of the unfolded protein response (UPR) is differentially activated in type 1 versus type 2 mutant CALR cells, and that type 1 mutant CALR cells are dependent on this pathway for survival and to drive ET. We found that IRE1a/XBP1 is activated only by type 1 and not type 2 mutant CALR due to a loss of calcium binding function specific to the type 1 protein. More recently, we found that the ATF6 pathway of the UPR is differentially up- regulated in type 2 compared to type 1 mutant CALR cells, and that type 2 mutant proteins exhibit loss of molecular chaperone function. These data support the central hypothesis that type 1 and type 2 CALR mutations activate and depend on different arms of the UPR based on their respective losses of function, and that these pathways promote distinct disease phenotypes. Thus, targeting different arms of the UPR based on mutation type may represent a novel, individualized treatment strategy for type 1 versus type 2 CALR+ MPN patients. To test this hypothesis, we will dissect the role of the UPR in type 1 mutant CALR-driven fibrosis (Specific Aim 1) and type 2 mutant CALR-driven ET (Specific Aim 2), and determ...