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Interferon alpha (IFNα) based therapies can induce hematologic and molecular responses in polycythemia vera (PV); however, patients do not respond equally. Germline genetic factors have previously been implicated in differential drug response. We addressed the effect of common germline polymorphisms on hematologic and molecular response (HR/MR) in PV therapy within the PROUD-PV and CONTINUATION-PV studies including 122 patients with PV receiving ropeginterferon alfa-2b. Genome-wide association studies using longitudinal data on HR and MR over 36 months follow-up did not reveal any associations at genome-wide significance. Further, we performed targeted association analyses at the interferon lambda 4 (IFNL4) locus, well known for its role in hepatitis C viral clearance and recently reported to influence HR during therapy of myeloproliferative neoplasms. While we did not observe any association of IFNL4 polymorphisms with HR in our study cohort, we demonstrated a statistically significant effect of the functionally causative IFNL4 diplotype (haplotype pair including the protein-coding variants rs368234815/rs117648444) on MR (p=3.91x10-4; OR=10.80; 95%CI:[2.39-69.97]) as reflected in differential JAK2V617F mutational burden changes according to IFNL4 diplotype status. Stratification of PV patients based on IFNL4 functionality may allow for optimizing patient management during IFNα treatment.

Somatic mutations of calreticulin (CALR)have been identified as one of the main disease drivers of myeloproliferative neoplasms (MPNs), suggesting that developing drugs targeting mutant CALR is of great significance. Site-directed mutagenesis in the N-glycan binding domain (GBD)abolishes the ability of mutant CALRto oncogenically activate the thrombopoietin receptor (MPL).We thus hypothesized that a small molecule targeting the GBD might inhibit the oncogenicity of the mutant CALR. Using an in-silico molecular docking study, we identified candidate binders to the GBD of CALR. Further experimental validation of the hits identified a group of catechols inducing selective growth inhibitory effect on cells that depend on oncogenic CALRs for survival and proliferation. Apoptosis-inducing effects by the compound were significantly higher in the CALR mutated cells than in CALR wild type cells. Additionally, knockout or C-terminal truncation of CALR abolished the drug hypersensitivity in CALR mutated cells. We experimentally confirmed the direct binding of the selected compound to CALR, the disruption of the mutant CALR-MPL interaction, the inhibition of the JAK2-STAT5 pathway, and reduction of intracellular level of mutant CALR upon the drug treatment. Our data conclude that small molecules targeting the GBD of CALR can selectively kill CALR mutated cells by disrupting the CALR-MPL interaction and inhibiting the oncogenic signaling.

Background:Treatment options for Myeloproliferative Neoplasms (MPN) have mainly been limited to disease managing risk of thromboembolic events and long‐term transformation to acute myelogenous leukemia and myelofibrosis. To date, type I interferons (IFNs) are the only class of drugs with curative potential in MPN as they can actually reduce the allelic burden of mutant clones in patients. The mechanism of action of IFN in MPNs remains elusive. This is mainly because modelling IFN treatment in mouse models has proven to be difficult due to the short half‐life of murine non‐pegylated IFNs. This shortcoming was solved by the murine pegylated IFN‐α (murine ropeginterferon‐a, mRopeg) developed by PharmaEssentia (Taipei, Taiwan) which can be used to model IFN treatment in mouse models of MPN.Aims:Generation of a conditional transgenic mouse model for CALR‐del52 induced MPN and modelling IFN treatment in these mice using murine pegylated IFN‐α (mRopeg).Methods:The CALR‐del52 transgenic mice (with human mutant sequence) were generated on a C57BL/6 background by homologous recombination of embryonic stem cells at Ozgene (Perth, Australia). The CALR‐del52 floxed mice were bred with vavCre transgenics, thereby allowing the expression of CALR‐del52 specifically in the hematopoietic cells. MPN phenotype in the mice was characterized by peripheral blood counts and flow cytometry. IFN treated mice received mRopeg weekly (600 ng) by subcutaneous injection for 4 weeks. Control mice received the same volume of vehicle (in this case, phosphate buffered saline – PBS).Results:Heterozygous CALR‐del52fl/+;vavCre mice displayed elevated platelet counts starting from 6 weeks of age, with normal white blood cell and red blood cell counts. However, homozygous CALR‐del52fl/fl;vavCre mice developed much higher platelet counts associated with increased white blood cell and decreased red blood cell counts. Homozygous CALR‐del52 mice also developed splenomegaly starting from 6 months of age. Also at 6 months, there was significant increase in the megakaryocyte population in the bone marrow, along with significant increase in LSK cells (Lin− Sca+ kit+) and the quiescent (Fraction A) stem cell population. IFN‐α treatment of CALR‐del52 transgenic mice for 4 weeks resulted in the normalization of the platelet counts to wild type levels. Interestingly, the total stem cell population (LSK cells) in the bone marrow increased upon IFN‐α treatment. However, the percentage of quiescent stem cells (Fraction A) reduced, while the percentage of cycling stem cells (Fraction B) and multipotent stem cells (Fraction C) was increased.Summary/Conclusion:Expression of CALR‐del52 in hematopoietic cells leads to the development of MPN phenotype in mice, and the severity positively correlated with the number of mutant alleles. Heterozygous CALR‐del52 mice developed an Essential Thrombocythemia (ET) like phenotype, with high platelet counts but normal white blood and red blood cell counts. However, the homozygous CALR‐del52 mice developed signs of myelofibrosis with age, including reduced red blood cell counts and splenomegaly. IFN‐α treatment resulted in normalization of platelet numbers to wild type levels within 4 weeks of treatment. Importantly, it also resulted in significant reduction of the quiescent hematopoietic stem cells, while cycling and multipotent stem cells were increased. This suggests that IFN‐α induces proliferation of quiescent stem cells into cycling and multipotent stem cells in CALR‐del52 mutant cells.image