Ps1436 Ifn‐α Treatment Normalizes the Platelet Counts and Reduces Quiescent Stem Cells in a Calr‐del52 Transgenic Mouse Model, in Vivo

Abstract

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