ALBERT

Description: Myeloproliferative neoplasms (MPNs) are a group of hematopoietic stem cell disorders driven by mutations that constitutively activate physiologic signal transduction pathways essential for hematopoiesis. The majority of patients with classical MPNs harbor mutations within the Janus activated kinase 2 (JAK2), calreticulin (CALR), or thrombopoietin receptor (MPL) genes. The occurrence of driver mutations among patients is mutually exclusive but rare double positive cases have been reported. Employment of targeted sequencing methods for diagnostics revealed more double positive cases and reviewing published studies we estimate the CALR and JAK2 double positive MPN frequency to be about 0.5% in all MPNs and 2% in essential thrombocythemia. However, the mutual exclusivity of CALR and JAK2 mutations in double positive cases was confirmed at single cell level in few studies where clonogenic assays were performed with subsequent genotyping of colonies. In our MPN biobank of over 800 samples, we identified one case diagnosed with PMF, carrying both in JAK2 and CALR, with allelic burdens of 8% and 41%, respectively. Using a clonogenic assay, we confirmed mutual exclusivity of the mutations at CFU level confirming previous findings. Mutations can be mutually exclusive due to their synthetic lethal interaction. Such synthetic lethal interaction has been recently described in splicing factor mutated MDS, showing that SF3B1 and SRSF2 double mutant hematopoietic cells (HSC) have reduced fitness in vivo providing explanation why such patients are never observed. In this study, we tested the hypothesis that JAK2-V617F and CALR-del52 mutations are synthetic lethal if they occur in the same HSC. We have generated mice that co-expresses both JAK2-V617F and CALR-del52 mutations in hematopoietic lineages and analyzed their phenotype. First, we co-expressed JAK2-V617F and CALR-del52 on the Vav1-Cre backgound in which Cre recombinase activates the floxed transgenes in embryonic HSC. Double positive offspring were born at expected Mendelian frequency compared to single positive littermates, suggesting no signs of synthetic lethality in utero. The phenotype of the JAK2-V617F and CALR-del52 double positive mice was significantly more severe compared to single mutant mice. More specifically, double positive mice showed more pronounced splenomegaly, higher white blood cell, lymphocyte, granulocyte, monocyte, and platelet counts in peripheral blood. In the bone marrow, double positive mice had more prominent megakaryocyte dyspoiesis and altered myeloid to erythroid ratios, without evident myelofibrosis as observed in histological sections. This increase in megakaryocyte numbers was also confirmed by FACS. In addition, double positive mice had more obscured follicular architecture and more signs of enhanced extramedullary hematopoiesis in the spleen, and more pronounced megakaryocytic sequestration in the lungs when compared to the JAK2-V617F histology findings. These mice also had lower overall survival compared to the JAK2-V617F and CALR-del52 mice. Next, we performed competitive bone marrow transplantation (BMT) to examine HSC fitness in primary and secondary transplants. Wild type bone marrow (BM) derived from F1 hybrid CD45.1/CD45.2 mice was mixed with BM form either mice bearing single mutation or double mutations (CD45.2), and ingrafted into CD45.1 recipients. The changes in chimerism were followed in peripheral blood by FACS. Double positive BM engrafted recipients equally well as JAK2-V617F or CALR-del52 cells suggesting no functional defect at HSC level. Same results were seen also in secondary BMT. In summary, double positive mice have an enhanced MPN phenotype with lower overall survival compared to single positive JAK2-V617F and CALR-del52 animals. Our results suggest that the mutual exclusivity of MPN driver mutations JAK2-V617F and CALR-del52 is not due to synthetic lethality or loss of HSC fitness. It is possible that once the second mutation is acquired, JAK2-V617F and CALR-del52 double positive cells do not gain additional competitive advantage over single positive HSCs, and therefore, do not grow out into a significant population. Another reason why we do not observe JAK2-V617F and CALR-del52 double positive colonies in patients is the very low likelihood of such HSC arising. Our data shows that such MPN patients may be found and very likely will have more severe MPN. Disclosures No relevant conflicts of interest to declare.

Description: The curative potential of Type I interferons for patients suffering from Myeloproliferative Neoplasms (MPNs) has been reported and these are the only class of drugs that can lead to reduction of the mutant allelic burden in patients. However, modelling IFN treatment in mice has been challenging. Here, we report the use of murine pegylated IFNα (murine ropeginterferon-a, mRopeg) developed by PharmaEssentia (Taipei, Taiwan) to model IFN treatment in transgenic MPN mouse models. We started treating JAK2V617Ff/+;vavCre and control vavCre mice (n=6-8) with PBS or mRopeg (600 ng/mouse/week), by subcutaneous injections from the time they were 4 weeks old. The mice were bled every 2 weeks from the facial vein and the blood parameters were monitored. We observed significant normalization of platelet and WBC counts in Jak2-V617F fl/+ vavCre mice to wild type levels. No effect on hematocrit and hemoglobin level was observed in the Jak2-V617F fl/+ vavCre mice. VavCre control animals showed no sign of negative effect such as cytopenia during the entire treatment course. We observed a highly significant prolongation of the survival of mRopeg treated JAK2V617Ff/+;vavCre mice over a duration of 80 days of treatment. While all the PBS treated JAK2V617Ff/+;vavCre mice died within 60 days, all the mRopeg treated mice were still alive till the end of the treatment duration. We also generated a novel transgenic mouse model that conditionally expresses hybrid mutant CALR protein (murine exons 1-8 and human CALR del52 exon9) from the endogenous murine Calr locus. We bred them into vavCre background (in both heterozyhous and homozygous states) to induce expression of CALR-del52 in hematopoietic cells. Upon Cre recombinase expression, the endogenous murine exon 9 is replaced by the human del52 exon 9 and the expression of the humanized Calr-del52 oncoprotein is detectable by Western blot analysis using mutant CALR specific antibodies. Calr-del52 animals develop an essential thrombocythemia (ET) like phenotype when expressed in a heterozygous state with elevated number of hematopoietic stem cells and megakaryocytes in the bone marrow. In the homozygous state, the thrombocythemia is more severe with splenomegaly and older animals show anemia with increased WBC. Bone marrow histology shows megakaryocytic hyperplasia with no sign of fibrosis up to age of one year. We treated a cohort of animals with 600 ng mRopeg/PBS once a week for 4 weeks. Peripheral blood counts were determined at baseline and at regular intervals during treatment. At the end of treatment, mice were sacrificed, and splenic and bone marrow cells were immunophenotyped and quantified by FACS. We observed correction of thrombocythemia in the homozygous Calr-del52 mice but no unspecific decrease of platelet count in the vavCre mRopeg treated animals. We observed significant specific reduction of the long-term hematopoietic stem cells (LT-HSCs/fraction A) in homozygous CALR-del52 mice. In conclusion, Type I IFN treatment significantly reduces platelet counts to normal levels in both JAK2 and CALR mutant driven MPN mouse models. The prolongation of survival of JAK2V617F transgenic mice upon Type I IFN treatment is particularly remarkable; as no survival data is reported until now in any clinical trials or other animal models. Further experiments are required to understand the mechanism of action of this phenomenon. Disclosures No relevant conflicts of interest to declare.