Compound Heterozygosity of Two Novel JAK2 Mutations in Hereditary Essential Thrombocythemia Implicates Important Monomer-Monomer Interactions in Thrombopoiesis Signaling

Essential thrombocythemia (ET) is a myeloproliferative neoplasm that is characterized by pathological overproduction of platelets with an increased risk of thromboembolic events, progression to myelofibrosis and leukemic transformation. Mutations of the Janus Kinase 2 (JAK2) gene, most commonly JAK2V617F, are known driving factors of ET pathogenesis. However, the molecular processes involved in JAK2 activation and regulation and the specific mechanism(s) by which JAK2 mutations disrupt hematopoiesis remain unclear. Most mutations identified in hereditary ET involve the thrombopoietin or thrombopoietin receptor gene; only 4 germline JAK2 mutations have been reported, all of which are inherited in an autosomal dominant pattern. We report the first case in which 2 novel germline JAK2 mutations, JAK2L815P and JAK2V1123G, were found in the compound heterozygous state in 2 siblings with ET. Family members who carry either one of these variants have normal blood counts. The purpose of this study was to demonstrate causality and to explore the mechanism(s) by which these JAK2 mutations, in compound heterozygous state, result in thrombopoietic dysregulation.

To study signaling in the JAK2-signal transducer and activator of transcription (STAT) pathway, wild-type (WT) and mutated forms of murine JAK2 were transduced into murine pro-B cells (Ba/F3) stably expressing the thrombopoietin receptor (MPL), Ba/F3-MPL cells. Immunoblotting analysis was performed on Ba/F3-MPL cells that were starved of cytokines overnight and then treated with 10 ng/mL thrombopoietin (TPO) or vehicle for 5 minutes. At baseline (under starved conditions), JAK2L815P + JAK2V1123G Ba/F3-MPL cells demonstrated increased STAT3 and STAT5 phosphorylation compared to WT JAK2 Ba/F3-MPL cells. However, JAK2 double mutant Ba/F3-MPL cells had less JAK2, STAT1 and STAT3 phosphorylation in comparison to JAK2V617F Ba/F3-MPL cells at baseline. After TPO stimulation, both JAK2 double mutant and JAK2V617F Ba/F3-MPL cells showed increased phosphorylated JAK2, STAT1 and STAT3 compared to WT JAK2 Ba/F3-MPL cells.

To investigate JAK2-STAT signaling in the patients' cells, immunoblotting analyses of platelets and megakaryocytes grown in vitro from patients' CD34 cells were performed. Our patients' platelets demonstrated increased JAK2, STAT1, STAT3 and STAT5 phosphorylation compared to platelets from a normal control but less JAK2, STAT1 and STAT3 phosphorylation compared to the platelets from a pediatric ET patient with a known JAK2V617F mutation, a similar signaling pattern to that seen in JAK2 double mutant Ba/F3-MPL cells. Similarly, in vitro megakaryocytes from the patients' CD34 cells showed increased STAT5 activation compared to those from normal volunteers in the unstimulated state.

To assess cell proliferative capability, IL-3 dependent Ba/F3 cells were starved of cytokines and viable cells were counted every 24 hours for a total of 72 hours. The JAK2V617F and JAK2 double mutant Ba/F3-MPL cells easily proliferated without cytokines and had a large fold-increase at 24, 48 and 72 hours compared to 0 hours. The JAK2L815P + JAK2V1123G Ba/F3-MPL cells had a significantly higher proliferation rate compared to JAK2V617F and WT JAK2 Ba/F3-MPL cells. As expected, the number of viable parental Ba/F3 cells declined over time in the absence of cytokines.

To evaluate response to ruxolitinib, a JAK1 and JAK2 inhibitor, IL-3 independent Ba/F3-MPL cells were grown under starved conditions in the presence of 0 to 1 mM ruxolitinib. After 48 hours, the number of viable Ba/F3-MPL cells was measured as a percentage of the dimethylsulfoxide (DMSO) control. The 50% inhibitory concentration (IC50) value was higher in JAK2L815P + JAK2V1123G Ba/F3-MPL cells (147+/-20 nM) compared to JAK2V617F Ba/F3-MPL cells (93+/-1 nM).

In the compound heterozygous state, the novel germline JAK2 mutations, JAK2L815P and JAK2V1123G, appear to function cooperatively to increase cell proliferation through activation of the JAK2-STAT pathway. This finding implicates that within the JAK2 dimer in the MPL-JAK2-STAT pathway, the monomer-monomer interactions are important in JAK2 activation and regulation. Additional in vitro and in vivo studies are currently underway to elucidate the exact mechanism(s) of thrombopoietic dysregulation resulting in the siblings' ET and to gain further insight into JAK2 activation and regulation.