Transcriptional readout downstream of canonical Wnt signaling is known to be mediated by β-catenin activation of well-described targets, but potential transcriptional readout in response to noncanonical Wnt signaling remains poorly understood. Here, we define a transcriptional pathway important in noncanonical Wnt signaling. We have found that Wnt11 is a direct target of a canonical β-catenin pathway in developing heart and that Wnt11 mutants show cardiac outflow tract defects. We provide genetic and biochemical evidence that Wnt11 signaling affects Reprints and permissions information is available online at

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.

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1737 Myeloproliferative disorders (MPDs) are a heterogeneous group of bone marrow disorders characterized by increases in one or more blood cell lineages. A single, somatic mutation in JAK2 (V617F) is responsible for many of the features of Philadelphia chromosome-negative MPDs (Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis). Clinically, the most common cause of death in these patients is arterial thrombosis; however some patients display a bleeding diathesis. Although the incidence and potential causes of dysfunctional hemostasis in patients with MPDs has been studied extensively, the critical regulating factors are unclear and therefore it has been difficult to develop an effective therapeutic regimen for these complications. As the formation of stable thrombi requires interactions between endothelial cells, platelets and leukocytes, we have recently generated mice that express human JAK2V617F in each of these cell lineages by crossing a JAK2V617F/Flip-Flop (FF1) mouse with mice expressing lineage-specific Cre recombinases. These crosses have generated the following mice; 1) Tie2-Cre/FF1, where JAK2V617F is expressed in all hematopoietic and endothelial cells, 2) Pf4-Cre/FF1, where JAK2V617F expression is limited to platelets, and 3) LysM-Cre/FF1, where JAK2V617F expression is limited to leukocytes. Expression of human JAK2V617F was confirmed in megakaryocytes, platelets, leukocytes and endothelial cells (Tie2-Cre/FF1), megakaryocytes and platelets (Pf4-Cre/FF1) and leukocytes (LysM-Cre/FF1) by conventional and real-time PCR. Of the 3 mouse strains, only Tie2-Cre/FF1 exhibited a MPD phenotype. Platelet counts were significantly increased compared to Tie2-Cre controls (at 3 months, Tie2-Cre: 779 (±61)/ml;Tie2-Cre/FF1: 2943 (± 217)/ml) without significant increases in any other cells types. Tie2-Cre/FF1 mice also exhibit greatly increased number of CFU-MKs and bone marrow derived megakaryocytes. Therefore, Tie2-Cre/FF1 mouse exhibits an ET-like phenotype. Although circulating platelet counts did not increase in Pf4-Cre/FF1 mice, we did observe an increase in the number of CFU-MKs in colony assays. Next we determined the roles of the lineage-restricted JAK2V617F expression on hemostasis in vitro and in vivo. Aggregometry on washed platelets showed no significant difference between any group and their controls in response to PAR4 (100–400mM), ADP (2–20mM) or collagen (1–10mg/ml). Additionally, we were unable to show a significant difference in GPIIbIIIa activation or surface expression of P-selectin in response to the same agonists. Despite no clear platelet abnormalities in any of the 3 mouse lineages, we identified significant hemostatic abnormalities in vivo in Tie2-Cre/FF1 mice. Tail bleeding time was significantly increased in Tie2-Cre/FF1 mice compared to Tie2-Cre controls (Tie2-Cre average, 2min 47secs; Tie2-Cre/FF1, 6mins 37secs) while Tie2-Cre/FF1 mice also exhibited an increased occurrence of re-bleeding compared to Tie2-Cre controls. Additionally, we performed FeCl3 carotid artery occlusion assays to better determine in vivo thrombosis. We found that at 10% FeCl3, Tie2-Cre control mice exhibited complete artery occlusion in approximate 6 min. In contrast, Tie2-Cre/FF1 mice failed to show any sign of arterial occlusion throughout the duration of the experiment (30 min). Given the significant increase in platelet numbers in Tie2-Cre/FF1 mice, we next determined if acquired von Willibrand Disease (VWD) could account for prolonged bleeding and reduced clotting; plasma vW Factor levels by ELISA were normal. In contrast to Tie2-Cre/FF1 mice, neither the PF4-Cre/FF1 or LysM-Cre/FF1 mice exhibit dysfunctional thrombosis. These data provide compelling evidence that expression of JAK2V617F in cells other than just platelets or just leukocytes is necessary to generate the hemostatic abnormalities seen in patients with MPDs. Recent findings show that some patients express endothelial JAK2V617F and patients with ET exhibit increased numbers of circulating endothelial progenitors. Thus, our data is consistent with the hypothesis that expression of JAK2V617F in endothelial cells, in addition to hematopoietic cells results in the bleeding diathesis seen in patients with MPDs. Disclosures: No relevant conflicts of interest to declare.