Studies of JAK2 mutations in myeloproliferative disorders

Myeloproliferative disorders (MPD) are diseases characterized by clonal hematopoiesis with overprodution of mature cells from erythroid, megakaryocytic and myeloid lineages. Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) constitute classic MPDs. Activating somatic JAK2 mutations are frequently found in patients with myeloproliferative disorders. These mutations lead to constitutive activation of the JAK-STAT signaling pathway, which plays essential roles in hematopoiesis. The JAK2-V617F mutation is involved in the pathogenesis of 95% of PV and about 50% of ET and PMF patients. JAK2 exon 12 mutations surrounding amino acids 539-545 are found in the majority of PV patients who are negative for the JAK2V617F mutation. Most of PV patients with JAK2-V617F have homozygous erythroid colonies as a result of mitotic recombination, which is rare in ET patients and PV patients with JAK2 exon 12 mutations. JAK2 exon 16 mutant alleles affecting a highly conserved arginine residue at position 683 (R683) are found in 18%-28% of patients with Down’s syndrome-associated acute lymphoblastic leukemia (DS-ALL). In addition to JAK2, MPLW515 mutations are identified in about 5% of PMF patients and 1-9% of ET patients through screening other players in hematopoiesis, which could lead to activation of JAKSTAT signaling. In the first part of my thesis, I compared JAK2-V617F positive PV patients with those carrying JAK2 exon 12 mutations in regard to the lineage distribution of these mutations and the presence of the mutations in erythroid progenitors in these PV patients. JAK2V617F and JAK2 exon 12 mutations represent clonal markers useful for tracking the hematopoietic lineages involved in MPD. The results provided clues about the stage (such as hematopoietic stem cells or committed progenitors) at which the transformation of hematopoietic progenitors occurred, which may cause different phenotypes. I developed a novel and sensitive assay to quantitate the amount of JAK2 exon 12 mutations in purified platelets, granulocytes, monocytes, B lymphocytes, T lymphocytes and natural killer cells (NK cells). The lineage distributions of JAK2 exon 12 mutations and JAK2-V617F were similar in platelets, granulocytes, and monocytes, which always carried the mutations, while the involvement of lymphoid cells showed large interindividual variations and T cells were rarely involved. This similarity does not explain why exon 12 mutations and JAK2-V617F result in divergent phenotypes. Analysis of erythroid progenitors indicated clonal heterogeneity in PV patients. One patient displayed erythroid colonies homozygous for the exon 12 mutation, which is very rare in patients with JAK2 exon 12 mutations, with evidence for mitotic recombination on chromosome 9p. In several patients with exon 12 mutations or JAK2-V617F, a substantial proportion of erythroid endogenous colonies (EECs) were JAK2 wild type. One patient carried two independent clones: one with an exon 12 mutation and another clone carrying JAK2-V617F. The finding of clonal heterogeneity is compatible with the hypothesis that additional clonal events are involved in the pathogenesis of PV. From the first part of my work, we noticed that in some patients the frequency of JAK2V617F mutation in peripheral blood is very low, and can only be detected with very sensitive methods such as allele-specific PCR. It has also been observed that in about half of PV patients with JAK2-V617F, the homozygous erythroid colonies only constituted a small proportion of the total number of BFU-Es, and more than half of patients with JAK2 exon 12 mutations had only a small percentage of BFU-Es carrying the mutation. To answer how such a small proportion of mutant cells can lead to a substantial increase in red cell population, we hypothesized that JAK2-V617F homozygous BFU-Es or JAK2 exon 12 mutant BFU-Es proliferate more efficiently and prevail over wild type BFU-Es during terminal erythroid differentiation. In the second part of my thesis work, I performed a pilot experiment by comparing the amount of JAK2 mutation in BFU-Es with that in reticulocytes from the same patient sample to address this question. Preliminary data showed that in some PV patients who had a higher ratio of mutated JAK2 in reticulocytes than in granulocytes, the frequency of mutant allele increased during terminal differentiation from BFU-Es to reticulocytes, indicating a substantial amplification occurred at this stage. However, this phenomenon cannot be solely attributed to the presence of homozygous JAK2-V617F colonies since some patients who did not have homozygous JAK2-V617F colonies also had the mutation amplified. Future directions including analysis of a larger cohort of samples and examination of the clonal origin of reticulocytes using X-chromosome inactivation assays will further elucidate the impact of JAK2 mutations on erythroid terminal differentiation. To define the pathologic role of various JAK2 mutations, and investigate the functional differences between different JAK2 mutations, the third part of my thesis work was to generate transgenic mouse models with inducible expression of JAK2 exon 12 or exon 16 mutations. The most frequent JAK2 exon 12 mutations (N542-E543del and E543D544del) and JAK2 exon 16 R683G mutation were chosen as our candidates. Using a highly efficient recombination engineering technique with bacterial artificial chromosomes (BACs), we generated the JAK2 exon 12 mutant transgene constructs with the exon 12 sequence placed in the inverse orientation and flanked by antiparallel loxP sites. Similarly, the JAK2 transgene construct with R683G was made to have the sequences encoding the kinase domain placed in the inverse orientation and flanked by antiparallel loxP sites. The JAK2 R683G transgene construct is ready for microinjection. The JAK2 exon 12 mutant transgene constructs were microinjected into the pronucleus of zygotes from C57/BL6 mice and transferred to foster mice. Three transgenic founders with JAK2 exon 12 N542-E543del and two transgenic founders with JAK2 exon 12 E543D544del have been obtained. These founders will be crossed with VavCre or MxCre transgenic mice in order to induce expression of mutant human JAK2. Detailed blood counts, pathological abnormality assessment and genotype-phenotype relationship analysis will be performed.