Co-Expression of JAK2-V617F and Calr-del52 In Vivo Enhances Myeloproliferative Phenotype in Mice and Does Not Influence Competitive Fitness of Hematopoietic Stem Cells

Abstract

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.

Acquisition of JAK2 V617F to CALR‐mutated clones accelerates disease progression and might enhance growth capacity

“…The current report is the first to show a case with the co‐occurrence of JAK2 V617F and CALR mutations in the same clone. In mice, HSCs carrying the JAK2 V617F and CALR mutations developed more severe MPN constitutional symptoms such as splenomegaly, leukocytosis and thrombocytosis without bone marrow fibrosis, compared with single‐mutant mice 16 . That study also showed murine JAK2 / CALR double‐mutated HSCs did not have a competitive advantage in transplantation settings 16 .…”

“…In mice, HSCs carrying the JAK2 V617F and CALR mutations developed more severe MPN constitutional symptoms such as splenomegaly, leukocytosis and thrombocytosis without bone marrow fibrosis, compared with single-mutant mice. 16 That study also showed murine JAK2/CALR double-mutated HSCs did not have a competitive advantage in transplantation settings. 16 Interestingly, our case suggested that adding a JAK2 V617F mutation to the CALR-mutated HSC compartment occurred during the progression from ET to MF.…”

“…16 That study also showed murine JAK2/CALR double-mutated HSCs did not have a competitive advantage in transplantation settings. 16 Interestingly, our case suggested that adding a JAK2 V617F mutation to the CALR-mutated HSC compartment occurred during the progression from ET to MF. In addition, the allele frequency of myeloid and erythroid progenitors was increased.…”