A Novel Activating JAK2 Mutation, JAK2R564Q, Causes Familial Essential Thrombocytosis (fET) Via Mechanisms Distinct From JAK2V617F

Abstract

123 Activating mutations in JAK2 are responsible for the majority of myeloproliferative diseases (MPDs) by stimulating aberrant signaling and hyperproliferation of one or more cell lineages. Although JAK2V617F is the most common activating mutation, a number of other point mutations also appear to have clinical relevance. Here we describe JAK2R564Q, a novel mutation in the pseudokinase domain that causes fET and determine its biological function in vitro, compared to JAK2V617F. A 6 year old male was referred for evaluation of thrombocytosis (initial platelet count 961k/mcL). The patient was otherwise asymptomatic with no past medical history, no recent illnesses and on no medications. The family history, review of systems and physical examination were unremarkable and workup for secondary thrombocytosis was negative. While the patient did not have the JAK2V617F, MPLW515L/K or S505N mutations, we discovered a novel JAK2 mutation, R564Q. His mother and sister also presented with elevated platelet counts (500–600k/mcL) and were also found to have the JAK2R564Q mutation, whereas the father presented with a normal platelet count and displayed two wild type (WT) JAK2 alleles. The arginine residue at 564 is highly evolutionarily conserved in the autoinhibitory domain of JAK2. To determine the biological significance of JAK2R564Q and compare it to JAK2V617F, we stably expressed WT, R564Q, V617F and R564Q+V617F JAK2 in Ba/F3 cells stably expressing the thrombopoietin receptor c-Mpl (BaF-Mpl). These cells express comparable levels of both JAK2 and c-Mpl. TPO-dependent proliferation assays demonstrated striking differences between the different cell types. JAK2R564Q exhibits significant increases in cell survival in the absence of TPO and at low concentrations compared to WT, while cells expressing JAK2V617F and R564Q+V617F are growth factor independent. Interestingly, the double mutant (R564Q+V617F) exhibits higher maximal cell proliferation than V617F alone, suggesting that R564Q is functioning through alternative mechanisms to that of V617F. Next, we analyzed annexin V expression following growth factor withdrawal to determine the effects of mutated JAK2 on apoptosis. Concurrent with our proliferation assays, JAK2R564Q inhibited apoptosis compared to WT, while JAK2V617F and R564Q+V617F exhibited even less apoptosis. These data suggest that JAK2R564Q is important for cell survival in the absence of cytokines, but it does not elicit the proliferation-promoting effects of JAK2V617F. To elucidate the mechanisms through which JAK2R564Q and JAK2V617F mediate their actions we determined their effects on intracellular signaling. Cells were starved prior to stimulation with TPO. Interestingly, we found that cells expressing JAK2R564Q have considerably higher levels of phospho-JAK2 (Y1007/8) and phospho-STAT5, signals which are normally associated with proliferation, than WT, V617F alone and the double mutant. We also observed differences in the phosphorylation of several other JAK2 tyrosine residues that are important for regulating its activity. Intriguingly, hyperphosphorylation of the negative regulator JAK2Y570, was by far the most robust in JAK2R564Q mutants, which could potentially contribute to the reduced factor-independent proliferation observed, compared to JAK2V617F. Interestingly, we also found differential phosphorylation of JAK2 at Y831, which positively regulates JAK2 signaling via interactions with SH2-Bβ. JAK2Y831 was also hyperphosphorylated in R564Q mutants compared to V617F mutants, especially in the absence of cytokines. Levels of phospho-ERK1/2 and phospho-Akt were comparable in all JAK2 mutants and significantly reduced compared to WT cells, characteristic of cells that fail to undergo starvation induced cell cycle arrest. Taken together, these data demonstrate that the JAK2R564Q mutation causes fET most likely by inhibiting apoptosis in hematopoietic stem cells and megakaryocytic progenitors. Importantly, even though this mutation is localized in the same pseudokinase domain as V617F, its effect on cell survival and signaling in response to TPO is significantly different. This work provides an insight into the functionality of alternative, clinically-relevant JAK2 mutations and how they have separate and additive effects on cell growth and survival. Disclosures: No relevant conflicts of interest to declare.

A novel germline JAK2 mutation in familial myeloproliferative neoplasms

“…Two cases of hereditary thrombocytosis associated with novel JAK2 germline mutations (R564Q and V617I) have been recently reported . According to these previous reports and to our study, it seems that JAK2 germline mutations might account for some cases of familial ET that are indeed hereditary thrombocytosis.…”

Inherited predisposition to myeloproliferative neoplasms

“…Interestingly, MPL K39N is a functional polymorphism restricted to the African American population, whereby the presence of one risk allele was reported to correlate with elevated platelets in the peripheral blood and presence of two copies was linked to severe thrombocytosis [Moliterno et al 2004]. Finally, there are isolated examples where inherited JAK2 mutations segregate with features of disease; JAK2 mutations V617I, R564Q, and H608N are associated with a phenotype characterized by thrombocytosis and high penetrance [Etheridge et al 2011; Mead et al 2012; Rumi et al 2012]. It appears that these mutations are more weakly activating that V617F, which may explain their ability to be passed through the germline.…”

Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines for the detection of JAK2 V617F and other relevant mutations

“…Inherited forms of thrombocythaemia may be caused by mutations within the 5′ untranslated region of THPO (also termed TPO ) or within the MPL locus itself, including K39N (MPL‐Baltimore), P106L and S505N mutations (Skoda, ). Recently, two families with inherited thrombocytosis and activating mutations within JAK2 (V617I and R564Q) have been reported (Etheridge et al , ; Mead et al , ). By contrast, the V617F mutation itself has not been reported to be inherited in familial cases although family members may acquire the mutation independently (Cario et al , ).…”