ALBERT

Description: 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.

Description: Regulation of growth factor and cytokine signaling is essential for maintaining physiologic numbers of circulating hematopoietic cells. Thrombopoietin (Tpo), acting through its receptor c-Mpl, is required for hematopoietic stem cell maintenance and megakaryopoiesis. Therefore, the negative regulation of Tpo signaling is critical in many aspects of hematopoiesis. In this study, we determine the mechanisms of c-Mpl degradation in the negative regulation of Tpo signaling. We found that, after Tpo stimulation, c-Mpl is degraded by both the lysosomal and proteasomal pathways and c-Mpl is rapidly ubiquitinated. Using site-directed mutagenesis, we were able to determine that c-Mpl is ubiquitinated on both of its intracellular lysine (K) residues (K553 and K573). By mutating these residues to arginine, ubiquitination and degradation were significantly reduced and caused hyperproliferation in cell lines expressing these mutated receptors. Using short interfering RNA and dominant negative overexpression, we also found that c-Cbl, which is activated by Tpo, acts as an E3 ubiquitin ligase in the ubiquitination of c-Mpl. Our findings identify a previously unknown negative regulatory pathway for Tpo signaling that may significantly impact our understanding of the mechanisms affecting the growth and differentiation of hematopoietic stem cells and megakaryocytes.

Description: Key Points MPL is essential for the development of JAK2V617F-positive myeloproliferative neoplasms in vivo. Ablation or reduction of Mpl significantly reduces the pool of neoplastic hematopoietic stem cells.

Description: Mice harboring c-Myb hypomorphic mutations display enhanced thrombopoiesis because of increased numbers of megakaryocytic progenitors (CFU-MK) and mature megakaryocytes (MK). Thrombopoietin (Tpo), the primary regulator of megakaryopoiesis, induces these same effects, which lead us to hypothesize that Tpo might act, at least in part, through modulation of c-Myb expression. We found using quantitative (Q)-PCR that c-Myb mRNA levels were 13-fold reduced during Tpo-induced MK maturation. Micro RNAs (miRs) are ∼22 nucleotide species that down-regulate gene expression by binding to the 3′ untranslated region (UTR) of specific mRNAs, enhancing mRNA degradation, or by reducing mRNA translation efficiency. We noted that the 3′UTR of c-Myb contains a number of miR target sites, including four that bind miR150; using a specific Q-PCR assay we also found that Tpo increased mir-150 expression to 160% of baseline at 24 hr and 250% at 48 hr in UT7/TPO cells (n=2 experiments). To test if miR150 affects c-Myb expression, we introduced the 3′UTR of c-Myb into a luciferase reporter gene (pCMV-luc-3′UTRcMyb), in which CMV promoter-driven luciferase activity would reflect the stability of the 3′UTR of c-Myb, and allow us to test the effects of miR150 on c-Myb expression in transduced cells; Q-PCR and western blotting were used to simultaneously assess endogenous c-Myb mRNA and protein levels in the cells treated with miR-150 and anti-miR-150, and their respective controls (Ambion, ABI). Co-transfection of UT7/TPO cells with pCMV-luc-3′UTRcMyb and miR-150 significantly down-regulated luciferase activity to 40% of baseline 24 hr following transfection (p = 0.035; n=2 experiments) compared to a miR negative control. Luciferase activity in cells transfected with a control luc plasmid lacking the 3′UTR of c-Myb was not modulated by introduction of miR-150. Q-PCR analysis revealed that endogenous c-Myb mRNA was significantly down-regulated to 60% of baseline upon transfection of miR-150 compared to the negative control (p = 0.043), while the essential megakaryocytic transcription factor, AML1/RUNX1, remained unaltered. Western blotting of these cell lysates revealed that c-Myb protein expression was down-regulated to 30% of baseline (n=3 experiments) following transduction with miR150 but not with the miR negative control. Converse experiments utilizing anti-miRs, which inhibit expression of endogenous miRs, revealed that anti-miR150 significantly upregulated luciferase activity to 180% of baseline compared to an anti-miR-negative control (p=0.003; n=2 experiments). These findings establish that miR-150 down-modulates c-Myb mRNA, and to a greater extent protein levels, suggesting effects on both mRNA stability and protein translation efficiency. And since Tpo affects miR-150 expression, our results also suggest that in addition to direct effects on the survival and growth of MK progenitor cells, mediated by the JAK/STAT, PI3K/Akt and MAPK pathways, Tpo down-modulates c-Myb expression during megakaryopoiesis through the induction of miR150. We are currently ascertaining the in vivo role of miR-150 in Tpo-induced megakaryopoiesis, but these studies already establish that hematopoietic growth factors such as Tpo can influence transcription factor expression through modulation of microRNA species.

Description: Thrombopoietin (Tpo), acting through its receptor c-Mpl, is critical for the support of hematopoietic stem and progenitor cell survival and proliferation, in addition to being the principal regulator of megakaryopoiesis. Consequently, Tpo signaling must be tightly regulated to prevent uncontrolled proliferation of a number of hematopoietic lineages. Several mechanisms to negatively regulate hematopoietic signaling have previously been identified, including suppressors of cytokine signaling, protein phosphatases and negative regulatory signaling pathways. However, one of the most effective mechanisms to permanently disable activated signaling proteins is by targeted degradation, via either lysosomes or proteasomes. In this study we investigated the mechanisms that regulate Tpo-mediated c-Mpl degradation in both c-Mpl-expressing cell lines and primary mouse cells. Using a factor-dependent hematopoietic cell line, BaF3, that were engineered to express human c-Mpl, we found that Tpo-stimulation caused both lysosomal and proteasomal degradation of c-Mpl. These findings were subsequently confirmed using murine megakaryocytes. We also found that Tpo-stimulation caused the rapid ubiquitination of c-Mpl, which is essential for targeting proteins to the proteasome. As ubiquitination occurs only on lysine residues of target proteins, we next determined which of the two intracellular domain lysine residues of c-Mpl are ubiquitinated by generating BaF3-Mpl cell lines bearing lysine to arginine (K to R) mutations at K553 and K573. Tpo-mediated ubiquitination was unaltered in both single mutations (K553R or K573R), but ubiquitination was completely ablated in cell lines bearing c-Mpl with both mutations (K553+573R), indicating that both sites are ubiquitinated following Tpo-stimulation. Additionally, c-Mpl K553+573R displays greatly reduced Tpo-stimulated degradation and significantly greater proliferation in response to Tpo, strongly suggesting a critical role for c-Mpl ubiquitination in the negative regulation of Tpo-mediated proliferation. We also found that following Tpo-stimulation, c-MplK553+573R remained phosphorylated significantly longer than wild type (WT), indicating that ubiquitination and degradation, in addition to protein phosphatase activity, is essential for extinguishing the activity of phosphorylated c-Mpl. We observed no significant difference in internalization of c MplK553+573R compared to WT following Tpo stimulation, showing that monoubiquitination, which can mediate endocytosis in a number of receptor types, does not regulate c-Mpl internalization. Ubiquitin E3 ligases are required for covalently attaching ubiquitin to lysine residues of target proteins. C-Cbl is one such E3 ligase that has previously been shown to be activated by Tpo. Using siRNA, we significantly attenuated c-Cbl protein expression in BaF3-Mpl cells, which resulted in a marked reduction of c-Mpl degradation (c-Cbl siRNA = 18% degradation; control siRNA = 51% degradation) and ubiquitination after Tpo-stimulation for 60 minutes, compared to cells transfected with non-targeting siRNA. Taken together, these data demonstrate a significant role for Tpo-stimulated c-Mpl proteasomal degradation in the negative regulation of Tpo signaling and proliferation. We have shown that both c-Mpl intracellular lysine residues are ubiquitinated and are vital for the proteasomal degradation of the activated receptor. Further, we have also identified c-Cbl as a potential E3 ligase responsible for the ubiquitination of c-Mpl. Our findings provide novel insights into the negative regulation of Tpo signaling, which greatly enhance our understanding of the biology of c-Mpl signaling and could potentially highlight new targets for the treatment of myeloproliferative diseases.

Description: Thrombopoietin (TPO), acting through its receptor Mpl, promotes survival and proliferation of hematopoietic progenitor cells and also drives megakaryocyte differentiation. The pro-proliferation and survival signals activated by TPO must therefore be tightly regulated to prevent uncontrolled cell growth. Several mechanisms to down-regulate hematopoietic growth factor signaling have been identified, including increased expression of suppressors of cytokine signaling (SOCS) proteins, activation of protein phosphatases, and endocytosis and degradation of activated growth factor receptors. In this work we determined the mechanisms that control TPO-stimulated Mpl internalization and defined the processes leading to Mpl degradation using IL-3-dependent BaF3 cells engineered to express wild type (WT) and mutant forms of human Mpl. Stimulation of BaF-Mpl cells with TPO lead to rapid endocytosis of Mpl which was blocked by pre-treatment with the clathrin-mediated endocytosis inhibitor monodansylcadaverine, indicating that Mpl internalization is clathrin-mediated. Additionally, we found that inhibition of Janus kinase 2 (Jak2) and Src-family kinases greatly reduced Mpl internalization. Sites of clathrin assembly on the plasma membrane contain the adaptor protein complex AP2, which associates with transmembrane proteins enabling targeted endocytosis. Association of AP2 and transmembrane proteins relies on an interaction between AP2μ2 subunit and internalization motifs YXXΦ (where X=any amino acid and Φ=bulky hydrophobic) expressed by target proteins. Mpl contains two YXXΦ motifs, at cytoplasmic Y8 (Y8RRL) and Y78 (Y78RRL). BaF-Mpl cells transfected with siRNA targeted to AP2 displayed greatly reduced TPO-mediated internalization of Mpl, demonstrating that AP2-Mpl association is critical for Mpl endocytosis. Next, we introduced Y to F point mutations at Mpl cytoplasmic Y8 and Y78 individually, and in combination (Y8+78F). Internalization was greatly reduced in Mpl Y78F and Mpl Y8+78F after TPO stimulation, whereas Mpl Y8F internalization was only slightly attenuated. Furthermore, we found that Mpl Y78F and Y8+78F exhibited increased proliferation and increased strength and duration of activated Jak2 and AKT in response to TPO, compared to Mpl WT and Y8F. In addition to mediating endocytosis, cytoplasmic YXXΦ motifs located between 6 and 9 residues from the transmembrane domain have been shown to mediate lysosomal targeting of proteins following endocytosis. Consequently, we studied the role of Mpl Y8RRL motif in Mpl lysosome targeting. BaF-Mpl WT and Y8F cells were pretreated with cyclohexamide and stimulated with TPO for up to 2 hours. Mpl Y8F displayed greatly reduced Mpl degradation in response to TPO compared to WT. Furthermore, Mpl Y8F recycled back to the plasma membrane following TPO starvation quicker than WT Mpl, suggesting that the Mpl Y8F remains in endosomes, rather than being targeted to lysosomes. Our data shows that Mpl cytoplasmic YRRL motifs are responsible for both TPO-mediated internalization via interactions with AP2 and lysosomal targeting following endocytosis. These findings significantly advance our understanding of normal Mpl function. Further study of internalization motifs, which are present in receptors for other hematopoietic growth factors including; erythropoietin, granulocyte colony stimulating factor, leukemia inhibitory factor and interleukin, may highlight the importance of these sequences in mediating hematopoiesis and potentially aid identification of novel targets in hematological disease.

Description: Thrombopoietin (TPO) is the primary regulator of megakaryopoiesis and therefore also the most important determinant of the number of platelets in circulation. The regulation of TPO blood concentration is complex, with at least a significant component mediated by removal and degradation of the hormone by the mature circulating platelet mass. In this way, if the production of TPO were fixed, when the number of platelets rises, an increased amount of TPO is removed from the plasma, resulting in an inverse relationship between platelet counts and TPO concentration. However, several studies hint at the existence of additional mechanisms in which TPO production is altered in response to physiological and pathologic conditions. We, and others, have previously reported that TPO mRNA is increased in the bone marrow of mice or humans after either immune- or radiation-mediated thrombocytopenia, although hepatic levels remain unchanged by this manipulation. To further explore the mechanism(s) of this effect, we utilized in vitro marrow stromal cell models to study the effects of blood proteins on TPO production. As an initial hypothesis we determined if platelet-derived proteins in serum might suppress TPO production from both a marrow stromal cell line, OP9, or primary murine marrow stromal cells derived from long-term hematopoietic cultures. As assessed by quantitative RT-PCR we found that TPO mRNA levels increase a mean of 2.8-fold±0.7 (n = 3) twelve hours following the removal of serum from the culture. Similar results were obtained from primary murine marrow stromal cells; TPO mRNA levels rose a mean of 2.9-fold±0.9 (n = 4) within sixteen hours of serum deprivation. In contrast, TPO transcript levels were unaffected by the same manipulation of serum in the hepatocyte cell line HEPA1c1c7. As the removal of serum might have induced a stress response in the cells, we tested whether other cell stressors might mimic this response; we found that neither UV irradiation nor treatment with toxic metals such as nickel, cobalt, or cadmium produced any rise in TPO mRNA in marrow stromal cells. Furthermore, to test whether a cell cycle arrest triggered by serum deprivation might mediate these effects, we treated stromal cells with cell cycle inhibitors, but failed to find any affect on TPO transcript levels in stromal cells. Further biochemical fractionation of serum suggested that one or more distinct proteins is responsible for this effect, demonstrated by the ability of both ammonium sulfate precipitation and ion exchange chromatography to partition the suppressive effects of serum. However, the active agent in serum is not TPO itself, as the addition of 150 ng/ml of the pure hormone did not suppress stromal cell TPO transcript levels. Knowledge of this novel regulatory mechanism should be useful in treating platelet disorders and perhaps also during stem cell transplantation, a setting in which TPO is known to play a vital role.

Description: Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. In the previous study, we reported that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. The absence of PI3K activity results in a block of transition from G1 to S phase in these cells (Geddis AE et al. JBC2001276:34473–34479). However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. TPO induces phosphorylation of Akt and FOXO3a in both UT-7/TPO, a megakaryocytic cell line, and primary murine MKs in a PI3K dependent fashion. Cell cycle progression of UT-7/TPO cells is blocked in G1 phase by inhibition of PI3K. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in UT-7/TPO cells and primary MKs in a PI3K dependent fashion. UT-7/TPO stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to induce p27Kip1 expression after TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In an attempt to assess whether FOXO3a has an effect of MK proliferation in vivo, we compared the number of MKs in Foxo3a-deficient mice and in wild type controls. Although peripheral blood cell counts of erythrocytes, neutrophils, monocytes and platelets were normal in the Foxo3a-deficient mice, total nucleated marrow cell count of Foxo3a-deficient mice were 60% increased compared with wild type controls. In addition, the increase of MKs was more profound than that of total nucleated marrow cells; CD41+ MKs from Foxo3a-deficient mice increased 2.1-fold, and mature MKs with 8N and greater ploidy increased 2.5-fold, compared with wild type controls. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings strongly suggest that the effect of TPO on MK proliferation is mediated by PI3K/Akt-induced FOXO3a inactivation and subsequent p27Kip1 down-regulation in vitro and in vivo.

Description: Megakaryocyte (MK) differentiation is marked by the development of progressive polyploidy, facilitating platelet production by the creation of a large cytoplasmic volume. MKs become polyploid through repeated cycles of endomitosis (EnM), in which mitosis is initiated but subsequently aborted in late anaphase with failure to complete karyokinesis and cytokinesis. However, the mechanisms underlying EnM remain poorly understood. Recent hypotheses explored in the literature have focused on the possible absence or mislocalization of the chromosomal passenger protein Aurora-B kinase, as it has a pivotal role in many aspects of cytokinesis. Along with the other passenger proteins, Aurora-B kinase transits from the centromeres of metaphase chromosomes to the bundled microtubules of the spindle midzone and overlying cortex between separating chromosomes in anaphase. The midzone and its associated proteins, are thought to be critical for determining the position of the cleavage furrow. One of these proteins, the kinesin MKLP-2, is required for the translocation of Aurora-B kinase to the midzone, where it co-localizes with the GTPase MgcRacGAP and stimulates its activity towards RhoA, potentially regulating actin dynamics at the cleavage furrow. We have previously demonstrated that several chromosomal passenger proteins including Aurora-B kinase are normally expressed and localized to centromeres in EnM MKs. In this work, we use deconvolution microscopy in primary murine and human MKs to extend those findings and demonstrate that EnM MKs form midzone structures that are characteristic of late anaphase; in addition, Aurora-B kinase is clearly present on the spindle midzone, as are MKLP-2 and MgcRacGAP. Although we found images suggestive of initial cleavage furrow formation with cortical localization of Aurora-B kinase in late phase cells, we were unable to demonstrate enhanced localization of actin or anillin to the furrow in EnM cells, despite their normal localization in diploid control cells. Therefore, many of the components of the central spindle are intact during MK EnM, but the formation of the cleavage furrow appears to be incomplete. These data add to our understanding of the possible mechanisms underlying EnM and offer an alternative hypothesis to that of failed expression or localization of the chromosomal passenger proteins. Ongoing studies will focus on the assembly and function of the cleavage furrow in this enigmatic process.