JAK2V617F Activity Research Articles

JAK2V617F Activates IL-10R Signalling Potentially through Transactivation of JAK1

Classical myeloproliferative neoplasms (MPNs) arise from somatic mutations in haematopoietic stem cells (HSCs) that lead to unregulated proliferation of mature myeloid cells. The most common mutation associated with MPNs is JAK2 V617F, this causes constitutive JAK-STAT signalling leading to excessive production of mature myeloid cells. In humans, for MPN to arise, a JAK2 V617F HSC must have a selective advantage over WT HSC. However, in mouse MPN models Jak2 V617F HSCs do not display an overt selective advantage. This suggests that unique selective pressures may be present among those predisposed to acquire MPN that allow for the emergence of Jak2 V617F clones. We have identified defects in the IL-10R signalling pathway among MPN patients, with evidence that this defect is an intrinsic not acquired abnormality. In mice, we found that the blockade of IL-10R extends the cycling of WT but not Jak2 V617FHSC in response to inflammatory stimuli and hastens proliferation-induced WT HSC exhaustion. We hypothesized that IL-10R blockade may create an environment that affords Jak2 V617F HSC a selective advantage, indeed we found that IL-10R blocking antibody allowed Jak2 V617F cells to outcompete WT cells in mouse transplant experiments. From these mouse data, we hypothesized that Jak2 V617Fenhances IL-10R signalling. To test this hypothesis, we created Ba/F3 cell lines co-expressing IL-10Rα and Jak2 V617F. We found that Jak2 V617F enhances growth at limiting concentrations of IL-10. We also found that cytokine-independent IL-10Rα Jak2 V617F clones (but never IL-10Rα Jak2 WTor IL-10Rα cells) emerge at day 8-10 in culture after withdrawal of cytokines. We performed a limiting dilution assay to pinpoint the proportion of IL-10R Jak2 V617F cells capable of surviving IL-3 withdrawal. One in 126 IL-10Rα Jak2 V617F cells has transforming ability, whereas 1 in 1.4 MPL Jak2 V617F has transforming ability. Together, these data support our hypothesis that Jak2 V617F enhances IL-10R signalling and can activate IL-10Rα, however with weaker transformation potential compared to Jak2V617F's activation of MPL signalling. JAK2 canonically does not participate in the IL-10R pathway. It has been previously shown that JAK1 can transactivate JAK2 when JAK2 V617F cells are under the stress of persistent JAK2 inhibitor exposure, and this is the purported mechanism of JAK inhibitor resistance among MPN patients. We hypothesized that JAK2 V617F can transactivate JAK1, leading to the activation of IL-10R. The same transfected BaF3 cells aforementioned were serum starved for 4 hours and stimulated with mIL-10 for 15 minutes to induce signalling. By way of Western Blot analysis, cytokine-independent Jak2 V617F IL-10Rα Ba/F3 cells showed constitutive activation of phosphorylated JAK1 and JAK2 as well as STAT3 and STAT5, consistent with transactivation. Studies to demonstrate direct interactions between JAK2 V617F and JAK1 are currently ongoing.

Preclinical Evaluation of INCB160058 - a Novel and Potentially Disease-Modifying Therapy for JAK2V617F Mutant Myeloproliferative Neoplasms

The Janus kinase 2 ( JAK2) mutation, JAK2V617F, is the most common oncogenic driver in myeloproliferative neoplasms (MPNs), with nearly all cases of polycythemia vera (PV) and over half of primary myelofibrosis (MF) and essential thrombocythemia (ET) patients positive for the somatic mutation. Approved therapies for MPNs such as ruxolitinib, which act by directly inhibiting activity of the kinase domain (JH1) of JAK2, have demonstrated impressive clinical efficacy and safety in patients with MPNs; however, they do not address JAK2V617F allelic burden or achieve molecular remission of disease. A targeted JAK2V617F selective agent sparing wild-type (WT) JAK2 activity has potential to eliminate mutant cells, induce molecular remission, and theoretically lead to functional cure of MPNs. We report herein the preclinical development of pseudokinase (JH2)-targeting INCB160058, a first-in-class, orally bioavailable small molecule with the ability to selectively target JAK2V617F + cell populations derived from patients with JAK-mutant MPNs. Using structure- and function-guided molecular design, INCB160058 was designed to bind with picomolar affinity to the JH2 domain of JAK2V617F at the canonical ATP-binding site, with high specificity (>2500-fold) relative to binding at the active kinase domain (JH1) targeted by currently approved JAK inhibitors. Live cell single-molecule fluorescence microscopy showed that INCB160058 binding to JAK2V617F blocked ligand-independent thrombopoietin receptor dimerization induced by the mutation, and consequently led to loss of JH1 domain kinase activity. X-ray crystallography analysis indicates that the observed inhibition is likely driven by conformational disruption of the ⍺C helix motif at Phe 594 and Phe 595 in conjunction with a shift of the upstream region from Leu 583 to Asn 589 upon INCB160058 binding to the JH2 domain of JAK2V617F. We utilized both CD34 + human multipotent hematopoietic stem cells derived from patients with JAK2-mutant MF, engineered JAK2-mutant human hematopoietic cancer cell lines (eg, SET2 and UKE-1), and murine BA/F3 cell lines to explore the selective effects of INCB160058 on JAK2V617F compared with WT JAK2. INCB160058 treatment selectively reduced pathogenic phospho-STAT5 levels, decreased abnormal megakaryopoiesis, and suppressed colony formation only in JAK2V617F + CD34 + cells but not in CD34 + cells from healthy volunteers. Importantly, continuous exposure of mutant and WT JAK2 cells to INCB160058 in co-cultures at concentrations below IC 50 resulted in progressive elimination of JAK2V617F + cells without affecting WT cells. At the end of the testing period, the JAK2V617F-harboring population was no longer detectable in the co-culture assay. In NSG mice subcutaneously inoculated with JAK2V617F-expressing SET2 cells, INCB160058 was tolerated and exhibited significant antitumor activity. In addition, following INCB160058 treatment, a significant reduction in the engraftment of total human cells, particularly human erythroid progenitors (hCD45 − mCD45 − Ter119 − hCD71 + hCD235a +), was observed in NSGS mice xenotransplanted with JAK2V617F + CD34 + cells. Moreover, INCB160058 treatment also led to the normalization of various pathogenic cytokines, such as interleukin (IL)-6 and IL-8. Importantly, these observations were absent in NSGS mice engrafted with CD34 + cells from healthy volunteers following INCB160058 treatment, further demonstrating the selectivity of INCB160058 for JAK2V617F. In summary, our results indicate a novel mechanism of action of INCB160058, a high-affinity pseudokinase (JH2) binding inhibitor of JAK2V617F that blocks cytokine-independent activity of JAK2V617F while preserving cytokine-dependent signaling. Extended treatment with INCB160058 at low therapeutic doses results in the specific elimination of mutant JAK2V617F-harboring cells in mouse models and human cancer cells with minimal impact on WT counterparts. Clinical testing of INCB160058 may allow patients with MPNs to achieve molecular remission by eliminating cells with the main genetic aberration and afford an opportunity to overcome the disease.

Modulation of the Human Thrombopoietin Receptor Conformation Uncouples JAK2 V617F-Driven from Cytokine-Induced Activation

Myeloproliferative Neoplasms are clonal diseases where mutations in hematopoietic stem cells (HSCs) result in cytokine-independent activation of the Thrombopoietin Receptor (TpoR) and aberrant proliferation and differentiation of HSCs and myeloid progenitors1. The most prevalent mutation, JAK2 V617F, is the basis of >95% of Polycythemia Vera (PV) and 60% of Essential Thrombocythemia (ET) and Myelofibrosis cases2. Current treatment strategies have focused on direct inhibition of JAK2 with little or no success and with poor specificity for the V617F mutant. Unlike JAK2, the TpoR is present at the cell surface and is more easily targetable. Using a system of controlled dimerization, we previously reported that different dimeric orientations of murine TpoR result in distinct signaling pathways and variable activation intensity3. Recently, others have demonstrated that extracellular ligands (diabodies) are able to modulate human TpoR conformation to influence downstream signaling4. Here, we investigated the possibility to modulate human TpoR conformation to specifically target JAK2 V617F driven activation while preserving natural cytokine-induced signaling. Using our system of controlled dimerization of TpoR transmembrane (TM) domain, we characterized the active and inactive conformation of human TpoR in presence of JAK2 WT or JAK2 V617F in hematopoietic cells. While murine TpoR is active in almost all (6 out of 7) dimeric conformations, only 3 conformations allowed activation of human TpoR. The discrepancy between murine and human TpoR is due to sequence differences in the TM domain that modulates dimerization. Remarkably, we discovered that different dimeric conformations mediate activation of JAK2 WT and JAK2 V617F in human but not in murine TpoR. Using a cysteine cross-linking assay on live cells, we validated that TpoR dimers exhibit a different conformation in presence of JAK2 V617F than this induced by the thrombopoietin (Tpo). Given the different dimeric conformations of TpoR induced by Tpo and JAK2 V617F, we hypothesize that modulation of TpoR conformation could allow specific inhibition of JAK2 V617F-driven activation while preserving Tpo-induced activation. We probed this by introducing a glycine zipper GxxxG motif to favor dimerization in a conformation that renders JAK2 V617F inactive but allows Tpo induced activation. Conversely, we mutated residues of the TM domain to specifically disrupt the conformation required for JAK2 V617F-mediated activation. Both strategies resulted in complete inactivation of JAK2-V617F driven activation of TpoR but preserved its full activation by Tpo and cell surface localization. Together, these results provide a first demonstration that modulation of TpoR dimeric conformation is a suitable strategy for specific inhibition of the JAK2 V617F-driven activation and opens the road for novel therapeutic avenues. 1. Vainchenker W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017;129(6):667-679. 2. Constantinescu SN, Vainchenker W, Levy G, Papadopoulos N. Functional Consequences of Mutations in Myeloproliferative Neoplasms. Hemasphere. 2021;5(6):e578. 3. Staerk J, Defour JP, Pecquet C, et al. Orientation-specific signalling by thrombopoietin receptor dimers. Embo j. 2011;30(21):4398-4413. 4. Cui L, Moraga I, Lerbs T, et al. Tuning MPL signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia progenitors. Proceedings of the National Academy of Sciences. 2021;118(2):e2017849118. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

JAK: Not Just Another Kinase.

The JAK/STAT axis is implicated in cancer, inflammation, and immunity. Numerous cytokines/growth factors affect JAK/STAT signaling. JAKs (JAK1, JAK2, JAK3, and TYK2) noncovalently associate with cytokine receptors, mediate receptor tyrosine phosphorylation, and recruit ≥1 STAT proteins (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6). Tyrosine-phosphorylated STATs dimerize and are then transported into the nucleus to function as transcription factors. Signaling is attenuated by specific suppressor of cytokine signaling proteins, creating a negative feedback loop. Both germline mutations and polymorphisms of JAK family members correlate with specific diseases: Systemic lupus erythematosus (TYK2 polymorphisms); severe combined immunodeficiency (JAK3 mutations); pediatric acute lymphoblastic leukemia (TYK2 mutations); and hereditary thrombocytosis (JAK2 mutations). Somatic gain-of-function JAK mutations mainly occur in hematologic malignancies, with the activating JAK2 V617F being a myeloproliferative disorder hallmark; it is also seen in clonal hematopoiesis of indeterminate potential. Several T-cell malignancies, as well as B-cell acute lymphoblastic leukemia, and acute megakaryoblastic leukemia also harbor JAK family somatic alterations. On the other hand, JAK2 copy-number loss is associated with immune checkpoint inhibitor resistance. JAK inhibitors (jakinibs) have been deployed in many conditions with JAK activation; they are approved in myeloproliferative disorders, rheumatoid and psoriatic arthritis, atopic dermatitis, ulcerative colitis, graft-versus-host disease, alopecia areata, ankylosing spondylitis, and in patients hospitalized for COVID-19. Clinical trials are investigating jakinibs in multiple other autoimmune/inflammatory conditions. Furthermore, dermatologic and neurologic improvements have been observed in children with Aicardi-Goutieres syndrome (a genetic interferonopathy) treated with JAK inhibitors.

JAK2 Variant Signaling: Genetic, Hematologic and Immune Implication in Chronic Myeloproliferative Neoplasms.

The JAK2V617F variant constitutes a genetic alteration of higher frequency in BCR/ABL1 negative chronic myeloproliferative neoplasms, which is caused by a substitution of a G ˃ T at position 1849 and results in the substitution of valine with phenylalanine at codon 617 of the polypeptide chain. Clinical, morphological and molecular genetic features define the diagnosis criteria of polycythemia vera, essential thrombocythemia and primary myelofibrosis. Currently, JAK2V617F is associated with clonal hematopoiesis, genomic instability, dysregulations in hemostasis and immune response. JAK2V617F clones induce an inflammatory immune response and lead to a process of immunothrombosis. Recent research has shown great interest in trying to understand the mechanisms associated with JAK2V617F signaling and activation of cellular and molecular responses that progressively contribute to the development of inflammatory and vascular conditions in association with chronic myeloproliferative neoplasms. Thus, the aim of this review is to describe the main genetic, hematological and immunological findings that are linked to JAK2 variant signaling in chronic myeloproliferative neoplasms.

Abstract 3117: CD80 can be the marker of chronic myeloid leukemia stem cells and regulated by BCR-ABL signaling

Abstract Chronic myeloid leukemia (CML) is a hematological malignancy caused by an abnormal tyrosine kinase, BCR-ABL1. Leukemic stem cells (LSCs) in this disease proliferate indefinitely. Also, the LSCs normally differentiate into mature neutrophils. Although tyrosine kinase inhibitors (TKIs) that target the kinase activity of BCR-ABL1 improved prognosis of CML patients, many patients still have residual diseases and need life-long treatment with the TKIs. This is due to a small number of CML LSCs that cannot be completely eradicated by the TKIs. It has been hypothesized that not only chemotherapy alone, but also immune surveillance systems of patients are needed to completely eradicate cancerous cells from their body. We hypothesized that residual CML cells might escape from the immune surveillance. Surfaces molecules associated with immune surveillance are examined on CML cells. At first, we confirmed expression levels of CD80 and PD-L1 by flowcytometry using CML cell lines. Our data showed that CD80 was expressed on surfaces of CML derived cell, K562, not PD-L1. Moreover, an engineered K562 subline is generated with the constitutive active JAK2V617F gene and became dependent on JAK-STAT signaling, but not BCR-ABL. This subline did not show CD80 on the surface. The TKIs inhibited expression of CD80 on the parental K562. Meanwhile, an ERK inhibitor decreased CD80 expression in K562. These results suggested that CD80 was downstream of BCR-ABL-ERK signaling pathway, not JAK-STAT pathway. When K562 cells were differentiated into mature neutrophils by ATRA, levels of CD80 decreased. Cytotoxicity assays using K562 and NK cells from normal individuals showed that the parental K562 cells and K562 cells with decreased levels of CD80 were similarly killed. These data suggested that CD80 on K562 played a minimum role in NK cell immune surveillance. We also found that only a small fraction of CD11b positive monocytes in normal peripheral blood showed expression of CD80. Also, blastic cells of CML blastic phase showed CD80 expression. These results suggested that only blastic cells of CML and a fraction of monocytes expressed CD80. These cells were distinguished by expression of CD11b as a marker. Although it is currently impossible to distinguish normal stem cells from CML LSCs, our study suggested that CD80/CD11b might be helpful to distinguish the CML LSCs in patients' bone marrow. It may be possible to collect a very small number of CML LSCs in CML patients under the TKI treatment using these antibodies and characterize the residual LSCs. Citation Format: Huixin Li, Shunichiro Yasuda, Satoru Aoyama, Chenyang Zhang, Yohei Kawano, Miyuki Azuma, Norihiko Kawamata. CD80 can be the marker of chronic myeloid leukemia stem cells and regulated by BCR-ABL signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3117.

A Novel JAK2 Mutation Confers Cytokine-Independence in the Absence of a Type I Dimeric Cytokine Receptor

JAK2 fusions reported in hematologic malignancies are often associated with eosinophilia. In addition to JAK2 fusions such as PCM1-JAK2, BCR-JAK2 and ETV6-JAK2, the JAK2V617Fpoint mutant has been recurrently observed in patients with hypereosinophilic syndrome (HES), occurring in ~4% of patients with hypereosinophilia of unknown significance (Schwaab, Am J Hematol, 2015).Here, we describe a novel JAK2 mutation in a 69 year-old woman with a history of immune thrombocytopenia referred for steroid-refractory HES. Peripheral blood eosinophilia was present for at least five years, with absolute eosinophil counts ranging up to 17,500/μL. Bone marrow biopsy revealed hypercellular marrow with trilineage hematopoiesis, increased atypical megakaryocytes, and markedly increased eosinophils with abnormal morphology, consistent with myeloproliferative neoplasm. Results from an eosinophilia FISH panel for FIP1L1/CHIC2/PDGFRα, FGFR, PDGFRβ, and CBFβ fusions were negative, as were T-cell clonality studies by next-generation sequencing (NGS). An NGS-based myeloid malignancies panel revealed a novel mutation in JAK2 consisting of a ten base deletion and insertion of a single thymine (JAK2 c.1747_1756delinsT, p.Leu583_Ala586delinsSer;JAK2indel). Treatment with ruxolitinib improved the peripheral blood eosinophilia, but dose intensity was limited by thrombocytopenia (Table 1).For transformation of cells to cytokine-independence, JAK2V617F requires co-expression of a type I dimeric cytokine receptor, such as Epo-R, to enable downstream signaling in the absence of growth factors (Lu et al, Proc Natl Acad Sci, 2005). To characterize the signaling properties of the new JAK2indel mutant, we cloned JAK2WT, JAK2V617F, and JAK2indel into the MSCV-IRES-GFP (MIG) retroviral vector. IL-3-dependent Ba/F3 cells lacking type I cytokine receptors were infected with retrovirus to assess cytokine independence.Parental Ba/F3 cells and those transduced with JAK2WT, JAK2V617F, or JAK2indel proliferated at similar rates when grown in WEHI conditioned medium (CM). However, upon IL-3 (WEHI) withdrawal, only GFP-positive JAK2indel Ba/F3 cells demonstrated growth factor independence (n=3). Furthermore, parental and GFP-sorted JAK2WT- and JAK2V617F-expressingBa/F3 cells display acceleration of growth when cultured in graded dilutions of WEHI CM, while growth of JAK2indel Ba/F3 cells is not further enhanced by WEHI CM. JAK2indel-expressing Ba/F3 cells also demonstrated cytokine-independent growth in colony assays, whereas parental, JAK2WT-, and JAK2V617F-expressing cells displayed little to no colony formation in the absence of cytokines. Immunoblot analyses revealed that after 4 hours of culture ± IL-3, all cells grown in IL-3 demonstrated activation of STAT5 and ERK, but only JAK2indel Ba/F3 cells demonstrated cytokine-independent signaling activation.JAK2indel activates STAT5 and ERK without co-expression of a type I cytokine receptor, implicating a mechanism of JAK2 kinase activation that is distinct from JAK2V617F. Unexpectedly, preliminary structural analysis suggests that the conformational change induced by JAK2indel resembles that of JAK2V617F. Mutation of glutamic acid 596 to arginine (E596R) has been shown to abolish the constitutive activity of JAK2V617F via modulation of the C-terminal residues of the SH2-JH2 linker, thereby restoring pseudokinase domain autoinhibitory function (Leroy et al, Biochem J, 2016). To test whether this regulatory mechanism is preserved in JAK2indel, we introduced E596R into JAK2indel and are currently characterizing JAK2indel/E596R in cellular and biochemical assays. Additional modeling and biochemical work is underway to clarify whether JAK2indel confers a new functionality that supports signaling activation in the absence of a type I cytokine receptor.In summary we report a novel JAK2 mutation in a patient with myeloproliferative-variant HES that results in cytokine-independent growth, is associated with STAT5 and ERK phosphorylation and may be employing an alternative mechanism to achieve constitutive activation of signaling. The fact that the patient's eosinophilia improved with ruxolitinib treatment illustrates the importance of performing NGS in patients with HES to identify potentially targetable genetic lesions. [Display omitted] DisclosuresDeininger:Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Research Funding; Pfizer: Consultancy; BMS: Consultancy, Research Funding; ARIAD: Consultancy.

Mechanism of Escape from LNK Negative Regulation: Constitutively Active JAK2V617F Retains Lysine63-Ubiquitin

IntroductionThe myeloproliferative neoplasms (MPN), polycythemia vera (PV), essential thrombocytosis and primary myelofibrosis are clonal hematopoietic stem cell disorders that arise from gain of function mutations that directly or indirectly activate JAK2, the cognate tyrosine kinase of the growth factor receptors for erythropoietin, thrombopoietin, and granulocyte colony-stimulating factor. Normally, JAK2 activation is modulated by the adapter protein LNK. However, this mechanism and how MPN mutations that activate JAK2 constitutively bypass LNK modulation are unknown. Clinically, loss of function LNK mutations mimic the MPN phenotype. LNK interacts with BRISC (BRCC36 isopeptidase complex) a lysine63-specific four member deubiquitinating complex. MERIT40 (Mediator of RAP80 Interactions and Targeting 40 kDa), the core subunit of BRISC, is necessary for both stability and activity of the BRISC deubiquitinating complex. This LNK-BRISC interaction suggested that deubiquitination might be a potential mechanism by which LNK could negatively regulate JAK2. We, therefore, examined the behavior of LNK, BRISC and MERIT40 in the context of JAK2 V617F.MethodsWe used a JAK2 V617F transgenic mouse PV model and a JAK2 V617F-expressing human cell line, DAMI, to examine JAK2 ubiquitination. The JAK2 V617F transgenic mouse, whether heterozygotic (VF) or homozygotic (VF/VF), authentically recapitulated human PV in a gene dosage-dependent manner: VF mice developed PV by 6 weeks, and splenomegaly and myelofibrosis by 30 weeks, while VF/VF mice demonstrated greater thrombocytosis, neutrophilia and erythrocytosis, with corresponding splenomegaly and fibrosis evident by 21 weeks.ResultsMERIT40 protein expression was found to be decreased in the JAK2 V617F transgenic mouse. Given the critical role of MERIT40 in BRISC protein stability and function, protein expression of both BRCC36 and LNK were assessed and also found to be decreased. Since we previously observed that BRISC activity is targeted to different proteins and cellular locations by interacting with different adaptor proteins (Patterson-Fortin et al. 2010; Zheng et al. 2013), we asked if LNK targeted BRISC deubiquitinating activity to JAK2. Lysates of unfractionated bone marrow from WT and VF mice were immunoprecipitated with a JAK2 antibody. The blots were probed with antibodies targeting lysine63-linked ubiquitin. JAK2 was robustly deubiquitinated in bone marrow samples from WT mice, whereas not only was this debubiquitination lost in in bone marrow samples from VF mice, there was also increased JAK2 phosphorylation. In the human DAMI cell model, MERIT40, LNK and BRISC protein levels were also all reduced, and JAK2 retained dramatic levels of lysine63-linked ubiquitin. Furthermore, we were able to rescue JAK2 deubiquitination in DAMI cells by transfecting MERIT40.We next performed immunohistochemistry on bone marrow samples from WT, VF and VF/VF age-matched mice (Figure). As expected, a JAK2 V617F gene dosage-dependent increase in megakaryocytes was observed with VF/VF mice having the greatest increase. Importantly, in contrast to WT mice, VF-positive megakaryocytes demonstrated cytoplasm localization of lysine63-linked ubiquitin and loss of this signal in the nucleus, most striking in VF/VF mice.ConclusionsWe employed a gene dosage-dependent JAK2 V617F transgenic mouse model of PV and human DAMI cells to demonstrate for the first time that activated JAK2 avoids LNK inhibition because both LNK and MERIT40, a core subunit of the BRISC deubiquitinating complex, are downregulated. This leads to increased JAK2 lysine63-linked ubiquitination and increased JAK2-mediated signaling as evidenced by increased JAK2 phosphorylation. Additionally, there is a marked translocation of the lysine63-linked ubiquitination complex from the nucleus to the cytoplasm in the JAK2 V617F context. These results establish that normally LNK negatively regulates JAK2 activity by targeting a lysine63-deubiquitinating complex to the JAK2 protein. This leads to deubiquitination and suppression of JAK2 activity. In contrast, in the constitutively-activated JAK2 V617F context, down regulation of LNK and MERIT40, and possibly lysine63-linked ubiquitin redistribution, results in loss of negative regulation, perpetuating JAK2 signaling. These data suggest that deubiquitinating agents could be a novel targeted therapy in MPN. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ruxolitinib Reduces Endothelial Pro-Adhesive Interactions: Implications for JAK2V617+ MPN Thrombosis

Introduction The JAK2V617F+ mutation occurs in up to 95% of patients with polycythemia vera (PV) and increases the risk of thrombosis 6-fold. Recent studies demonstrate that JAK2V617F+ endothelial cells express pro-adhesive proteins, suggesting that the endothelium may contribute to increased thrombosis.1-3 The targeted JAK1/JAK2 inhibitor ruxolitinib is an approved second-line therapy for PV patients and is effective in alleviating constitutional symptoms, lowering hematocrit, and reducing cell number. However, there is limited data regarding efficacy of ruxolitinib in reducing thrombosis. Although recent work has demonstrated ruxolitinib reduces neutrophil extracellular trap formation,4 the vascular effects of ruxolitinib are unknown. Therefore, we hypothesize that ruxolitinib reduces endothelial cell pro-adhesive activation leading to decreased rolling and adhesion of JAK2V617F+ samples. Methods To mimic JAK2V617F activation, primary human umbilical vein endothelial cells (HUVEC, passage 1-5) were treated with TNF-α (10 ng/mL) +/- ruxolitinib (400 nM-4 µM, Selleckchem) and characterized 4h later. For confocal microscopy, cells were fixed with paraformaldehyde, permeabilized and stained for either VWF, VCAM-1, or P-selectin along with 2-(4-amidinophenyl)-1H-indole-carboxamide (DAPI, Thermo Fisher). Images were captured on Olympus FluoView FV1000 IX2 Inverted Confocal microscope. Secretion of VWF, VCAM-1 and P-selectin into conditioned media was assessed with ELISA (Molecular Innovations; BioLegend). Citrated normal and JAK2V617F+whole blood was obtained per IRB and labeled with calcein AM (ThermoFisher). To quantify leukocyte and platelet velocity and adhesion, an endothelialized poly-di-methyl-siloxane (PDMS) microchannel was prepared and treated with TNF-α (10 ng/mL) +/- ruxolitinib (400 nM-4 µM). Samples were perfused through the microfluidic at a shear stress of 0.35 dynes/cm2, and 10 s images were captured using a fluorescence microscope (Olympus). Cell velocity and adhesion were quantified using FIJI (NIH). Results Compared to TNF-α treated HUVEC alone, the combination of ruxolitinib + TNF-α reduced expression of VWF, VCAM-1 and P-selectin using both immunofluorescence and ELISA. In endothelialized microfluidic devices treated with TNF-α alone, both normal (n=3) and JAK2V617F+ (n=9) leukocyte and platelet velocity was significantly decreased and cell adhesion increased. This result was independent of hematocrit and platelet levels. In normal controls (n=2), TNF-α+ruxolitinib lead to a trend toward normal leukocyte and platelet velocity with decreased cell adhesion. Lastly, in a small subset of JAK2V617F+ patients (n=4), addition of ruxolitinib increased cell velocity. Conclusions In conclusion, in TNF-α-activated endothelial cells, treatment with ruxolitinib decreases pro-adhesive VWF, VCAM-1, and P-selectin expression. Using normal controls and JAK2V617F+ MPN blood samples, our TNF-α-activated endothelialized microfluidics model demonstrates significant reduction in leukocyte and platelet velocity and increased cell adhesion. In normal and JAK2V617F+ MPN whole blood, treatment of TNF-α-stimulated endothelium with ruxolitinib improves cell velocity. Further evaluation using JAK2V617F+ endothelial cells is planned. Collectively, these results suggest that ruxolitinib may reduce JAK2V617F+ thrombotic risk through reduction of pro-adhesive endothelial activation. Disclosures Vercellotti: CSL Behring: Research Funding.

New Paradigms for the Mechanisms of Thrombopoietin Receptor Activation and Dysregulation By the JAK2V617F Mutation

Janus kinase (JAK2)V617F is the most common mutation found in patients with Philadelphia chromosome negative myeloproliferative neoplasms (Ph- MPNs). The discovery of this mutation over 15 years ago revolutionised MPN diagnosis and inspired the development of JAK inhibitors as new therapeutic interventions. However, despite extensive structural and biophysical studies using JAK2 domains in isolation, the exact molecular mechanisms of JAK2V617F activation remains elusive. We have previously demonstrated that expression of the thrombopoietin (TPO) receptor, MPL, which interacts directly with JAK2, is essential for disease development in a mouse model of a JAK2V617F-positiveMPN (Blood 2014 124:3956-3963). Using total internal reflection fluorescence (TIRF) microscopy, we visualized MPL interaction dynamics in live cells on single molecule level. Effective cell surface MPL fluorescence labelling and dual-color imaging allowed us to determine the level of MPL dimerization under various experimental conditions. Using this assay, we clearly established that MPL is monomeric at physiologically relevant receptor densities. However, TPO stimulation results in significant dimerization of MPL (>50%) and an equilibrium between monomers and dimers. This counters the current dogma that MPL exists at the membrane as a pre-formed dimer. Strikingly, we found that JAK2V617F shifts this monomer-dimer equilibrium leading to significant TPO-independent MPL dimerization providing a novel mechanistic model of oncogenic JAK2 activation. To highlight the role of ligand-independent receptor dimerization in JAK2 activation, we compared three groups of autoactivating mutations in the PK domain covering the FERM-SH2 (FS2)-PK linker region (Group I), residues in the proximity of the αC helix (Group II) and at the autoinhibitory PK-TK interface (Group III). Consistent MPL dimerization was only observed for mutations in groups I and II. Mutations in these groups both localize to a potential homomeric PK/PK interface that has been implicated as a switch of JAK activation. Using MD simulations, we also found that the FERM domain of JAK2 strongly interacts with the inner leaflet of the lipid bilayer of the plasma membrane via a single hydrophobic residue (L224) surrounded by several positively charged residues that allows the region to act as a membrane anchor. This tight coupling to the membrane enforces an appropriate orientation between the JAKs within the receptor dimers required for optimal intermolecular PK/PK interaction that is critical for receptor dimerization. To interfere with membrane anchoring, we introduced a negative charge in this position (L224E). Strikingly, ligand-independent MPL dimerization and activation by JAK2V617F was dramatically reduced upon introducing L224E, supporting the vital importance of L224 for orienting JAK2 at the membrane to allow productive PK-PK interactions. Here, we demonstrate that JAK2V617F mutation acts by altering and strengthening the intermolecular interactions involving the PK/PK dimerization interface. In essence, these mutations drive cytoplasmic stabilization of receptor-JAK dimers, bypassing extracellular stabilization of dimers via cytokine binding. These results provide critical and entirely novel mechanistic insights into signal initiation in MPNs and readdress the roles of receptor-associated proteins. Disclosures Hubbard: Ajax Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Co-Founder.

JAK2-V617F promotes venous thrombosis through β1/β2 integrin activation.

JAK2-V617F-positive chronic myeloproliferative neoplasia (CMN) commonly displays dysfunction of integrins and adhesion molecules expressed on platelets, erythrocytes, and leukocytes. However, the mechanism by which the 2 major leukocyte integrin chains, β1 and β2, may contribute to CMN pathophysiology remained unclear. β1 (α4β1; VLA-4) and β2 (αLβ2; LFA-1) integrins are essential regulators for attachment of leukocytes to endothelial cells. We here showed enhanced adhesion of granulocytes from mice with JAK2-V617F knockin (JAK2+/VF mice) to vascular cell adhesion molecule 1- (VCAM1-) and intercellular adhesion molecule 1-coated (ICAM1-coated) surfaces. Soluble VCAM1 and ICAM1 ligand binding assays revealed increased affinity of β1 and β2 integrins for their respective ligands. For β1 integrins, this correlated with a structural change from the low- to the high-affinity conformation induced by JAK2-V617F. JAK2-V617F triggered constitutive activation of the integrin inside-out signaling molecule Rap1, resulting in translocation toward the cell membrane. Employing a venous thrombosis model, we demonstrated that neutralizing anti-VLA-4 and anti-β2 integrin antibodies suppress pathologic thrombosis as observed in JAK2+/VF mice. In addition, aberrant homing of JAK2+/VF leukocytes to the spleen was inhibited by neutralizing anti-β2 antibodies and by pharmacologic inhibition of Rap1. Thus, our findings identified cross-talk between JAK2-V617F and integrin activation promoting pathologic thrombosis and abnormal trafficking of leukocytes to the spleen.

MicroRNA-101 expression is associated with JAK2V617F activity and regulates JAK2/STAT5 signaling.

FP was supported by Fondazione Umberto Veronesi, and Institute Pasteur - Fondazione Cenci Bolognetti.

Abstract 954: TGF-ß signaling inhibition counteracts myelofibrosis in MPN

Abstract Myelofibrosis (MF) is a common feature of the Philadelphia-Chromosome negative (Ph-) myeloproliferative neoplasms (MPN), a group that includes Primary Myelofibrosis (PMF), Essential Thrombocytosis (ET) and Polycythemia Vera (PV). Current treatment with Jak2 inhibitors reduces splenomegaly, but does not eliminate malignant clones nor improve bone marrow (BM) or spleen fibrosis, underlining the need for alternative therapies. One promising target is TGF-β1, a cytokine crucial for the development of myelofibrosis that is elevated in PMF patient plasma, progenitors and megakaryocytes (MK), and is known to promote fibrosis through stimulation of fibroblasts and their progenitors, mesenchymal stroma cells (MSC). Dysregulation of TGF-β1 signaling has been described in a number of mouse models of myelofibrosis exhibiting aberrant megakaryopoiesis, one of the hallmarks of MPNs. Preliminary data from 93 MPN patients showed overexpression of TGF-ß in PMF samples (p=0.001), with increased activation of the canonical target SMAD2 (p=0.005). We tested the efficacy of the orally available TGF-β1 signaling inhibitor Galunisertib (LY2157299) in counteracting fibrosis in two MF mouse models of common MPN oncogenes Jak2 and Mpn. Mice in the first model express the constitutively active human Jak2V617F mutant under the control of the ubiquitous hematopoietic promoter vav1 and show a PV-like phenotype at 6 weeks old, progressing to fibrosis of bone marrow (BM) and spleen around 25-30 weeks. Thirty week old mice were treated with Galunisertib for 4 weeks and showed a significant decrease in fibrosis compared to control mice. Similar results were observed in 50 week old mice treated with a murine antibody against TGFβRII (IMC-TR1,LY3022859) for 4 weeks.Our second model relies on transplantation of mice with BM cells transduced with MPLW515L, a mutant mpl variant that is active in the absence of TPO. These mice rapidly develop MF, myeloproliferation and splenomegaly. Flow cytometry showed an expansion of immature and mature megakaryocytes with overexpression of TGF-β1 in MPLW515L cells. Treatment from day 12 to day 26 after transplantation reduced both white blood cell counts (a measure of myeloproliferation) and fibrosis as measured by reticulin staining. In vitro, Galunisertib counteracted the pro-fibrotic effects of TGF-ß1 on both murine and human MSC by antagonizing its stimulatory effects on collagen I and III production. Analysis of MSCs from Galunisertib-treated mice also showed reduced collagen I and III production compared to controls. In conclusion, we offer further evidence that aberrant megakaryopoiesis drives TGF-β1 overproduction in MPN by increasing megakaryocyte numbers and overexpressing TGF-β1 in mutant MKs. Galunisertib counteracts TGF-ß1 induced fibrosis in two MPN mouse-models and is a promising candidate for symptomatic treatment of myelofibrosis. Citation Format: Matthias Bartenstein, Lanzhu Yue, Wanke Zhao, Wanting Tina Ho, Cem Murdun, Adam W. Mailloux, Ling Zhang, Anjali Budhakoti, Kith Pradhan, Franck Rapaport, Huaquan Wang, Zonghong Shao, Ulrich Steidl, Ross L. Levine, Zhizhuang Joe Zhao, Amit K. Verma, Pearlie K. Epling-Burnette. TGF-ß signaling inhibition counteracts myelofibrosis in MPN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 954. doi:10.1158/1538-7445.AM2017-954

Abstract 3034: A programmed ribosomal frameshifting defect potentiates the transforming activity of the JAK2-V617F mutation

Abstract The JAK-STAT pathway is one of the major mechanisms through which cellular proliferation, differentiation, migration, survival and apoptosis are regulated in response to external stimuli. Promiscuous activation of this pathway is a major driver in the pathogenesis of myeloproliferative neoplasms, often characterized by clonal expansion of mature myeloid cells. The JAK2-V617F allele is the most common and well characterized mutation linked to this class of leukemias. A change of G to T at nucleotide 1849 of the coding sequence (CDS) results in substitution of valine to phenylalanine at codon 617. JAK2-V617F mutant cells have strongly increased activation of JAK-STAT signaling, which can partially be explained by its ability abolish inhibition of kinase activity and increase the Km of the enzyme. It is however unclear if these effects are sufficient to explain the strong activation of the JAK-STAT pathway induced by the V617F mutation. Here, we demonstrate that this mutation destabilizes a programmed -1 ribosomal frameshift (-1 PRF) signal in the JAK2 mRNA, leading to decreased rates of -1 PRF promoted by this element. There is an inverse relationship between -1 PRF and mRNA stability. Thus, decreased -1 PRF results in increased abundance of the JAK2 mRNA and increased JAK2 protein expression. Prior studies established that elevated expression levels of JAK proteins are sufficient to transform cells in vitro, suggesting that -1 PRF may normally help to limit JAK2 expression. Silent protein coding changes in the -1 PRF signal slippery sequence ablating JAK2 frameshifting also increased JAK2 expression and phenocopied the transforming activity of JAK2-V617F in cell culture. Further, the combination of the V617F and slippery site mutations conferred an additive effect on cellular transformation. The presence at least three additional -1 PRF signals in the JAK2 mRNA suggests that -1 PRF plays a significant role in controlling JAK2 expression. Citation Format: Yousuf A. Khan, Sergey O. Sulima, Joe Kendra, Vivek M. Advani, John E. Jones, Joseph Briggs, Kim de Keersmaecker, Jonathan D. Dinman. A programmed ribosomal frameshifting defect potentiates the transforming activity of the JAK2-V617F mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3034. doi:10.1158/1538-7445.AM2017-3034

Activation of JAK/STAT Signaling in Megakaryocytes Is Necessary and Sufficient for Myeloproliferation In Vivo

Aberrant megakaryopoiesis is a hallmark of the myeloproliferative neoplasms (MPN). It is has been long known that abnormal megakaryocytes secrete elevated levels of cytokines such as TGFβ, resulting in pathologies including bone marrow fibrosis. Two recent studies showed that megakaryocytes regulate the quiescence of HSCs, raising the possibility that megakaryocytes may promote the MPNs by influencing the biology of non-malignant HSCs. However, the mechanism by which megakaryocytes regulate the initiation and progression of MPNs is largely unknown.To study the role of megakaryocytes in the MPNs, we analyzed the phenotype of PF4-Cre/Jak2V617F mice in which Jak2 is expressed in the megakaryocyte lineage from the endogenous locus, in contrast to previous studies, which used transgenic models. Selective activation of Jak2V617F was confirmed by allele-specific qPCR. CD41+ cells were positive for mutant Jak2, whereas sorted stem/progenitor cells and erythroid cells were Jak2 wild-type. Furthermore, flow cytometry showed that Stat5 activation was present in megakaryocytes, but not in erythroid or myeloid cells. Activation of JAK-STAT signaling caused an expansion of megakaryocytes in the bone marrow and spleen and a modest increase in the platelet count. Surprisingly, PF4-Cre/Jak2V617F mice also displayed a robust expansion of TER119(low)/CD71(high) and TER119(high)/CD71(high) red cells in the spleen, increased hematocrit and splenomegaly. Histological examination of the spleen revealed expansion of the erythroid lineage coupled with disrupted splenic architecture and fibrosis. This PV-like phenotype was fully penetrant and comparable to that of Vav-Cre/Jak2V617F mice, which express mutant Jak2 in all hematopoietic lineages. Profiling of hematopoietic progenitors by flow cytometry demonstrated that myeloid progenitor populations were expanded and skewed toward the erythroid-megakaryocyte lineage with a significant increase in Pre Meg-E, Pre CFU-E and MKPs in the PF4Cre/Jak2V617F mice. In addition, LSK cells were increased in both the bone marrow and spleen. Cytokine profiling of the plasma revealed increased levels of several cytokines, including Il-6, which is known to be upregulated in human JAK2 mutant PV megakaryocytes. Significant increases in Cxcl1, Cxcl2, and Ccl11 were also detected. Real-time qPCR analysis confirmed increased expression of these cytokines/chemokines in Jak2V617F-mutant CD41+ cells. Furthermore, IL6 treatment increased EPO-dependent colony formation of wild type LSKs and MEPs, and also enhanced expression of the erythroid cell markers CD71 and Ter119.To further explore the role of megakaryocytes in the MPNs, we used a strategy in which expression of the diphtheria toxin receptor (DTR) sensitizes cells to diphtheria toxin (DT). We transduced c-Kit+ cells from PF4-Cre/iDTR+/- mice with MPLW515L and transplanted the cells to irradiated mice. As expected, both iDTR+/- and PF4-Cre/iDTR+/- mice developed a PMF-like phenotype, including leukocytosis, thrombocytosis, splenomegaly and myelofibrosis (Fig 1). Treatment of these animals with DT caused significant reductions in megakaryocytes in the bone marrow and spleen as well as a decrease in the platelet count of PF4-Cre/iDTR+/- mice. Of note, DT also significantly reduced the white count and spleen weight, while restoring splenic architecture. PF4Cre/iDTR+/- mice also showed significant reduction of c-Kit+ myeloid progenitor cells. Therefore, depletion of megakaryocytes significantly attenuated the disease phenotype of MPLW515L induced MPN in vivo. Together, these two model systems reveal that JAK2 activation in megakaryocytes is sufficient and necessary for MPNs and support the development of megakaryocyte differentiation therapy in the disease. Moreover our data resonate with studies in MPN patients in which a JAK2V617F low allele burden in the setting of full-blown, clinical MPN. [Display omitted] DisclosuresLevine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Crispr-Cas9 Mediated Disruption of Dnmt3a in JakV617F Hematopoietic Stem Cells Accelerates Disease Phenotype and Induces Lethal Myelofibrosis

Myeloid malignancies arise following the sequential acquisition of somatic mutations within hematopoietic stem and progenitor cells (HSPC). JAK2V617F is commonly found in myeloproliferative neoplasms (MPN) such as polycythemia vera, essential thrombocythemia and myelofibrosis. While other mutations (e.g. TET2, DNMT3A) have been found to co-occur in MPN HSPC, it remains unclear how they impact disease biology or progression from early stage disease (i.e. polycythemia or essential thrombocythemia) to advanced stage disease such as myelofibrosis or acute myeloid leukemia. DNMT3A methylates cytosine rich DNA residues (known as CpG islands, and often found in promoters of genes) leading to transcriptional repression. DNMT3A is also recurrently mutated at relatively low frequency in polycythemia vera (5-7%) but mutations are more common in advanced MPN (approximately 15% of MF and 17% of AML, Stegelmann et al. Leukemia 2011; Abdel-Wahab et al. Leukaemia 2011). These mutations are found in the methyltransferase domain and cluster around arginine 882 (e.g. R882H), resulting in loss of DNA binding and reduced catalytic activity. We used CRISPR-Cas9 gene editing technology to disrupt Dnmt3a function in mouse HSPC and assessed for cooperativity together with a conditional, knockin Jak2V617F allele.Jak2V617F/ΔDnmt3a-Cas9 but not Jak2V617F/Cas9 controls demonstrated increased HSPC self-renewal and proliferation properties in vitro as evidenced by serial replating in methylcellulose (>5 weeks) and increased colony forming unit capacity. Flow-cytometry analysis of Jak2V617F/ΔDnmt3a-Cas9 revealed enrichment in LKS+ (Lin-Sca-1highKithigh) cells 5 weeks after CRISPR-Cas9disruption of Dnmt3a, and this was associated with increased expression of stemness markers Kit and Cd34 in Jak2V617F/ΔDnmt3a-Cas9 cells. RNAseq was performed on early (week 1, P1) and late culture HSPC (week 5, P5) from Jak2V617F-Cas9 (P1 only) and Jak2V617F/ΔDnmt3a-Cas9 (P1, P5). This confirmed deletion of Dnmt3a in Jak2V617F/ΔDnmt3a-Cas9 but not in Jak2V617F/Cas9 controls. Transcriptional upregulation of Kit and Cd34 were confirmed, as well as other key stem cell genes such as Erg and Angpt1 in Jak2V617F/ΔDnmt3a-Cas9 P5. We observed denovo expression of imprinted genes Igf2 and H19 in Jak2V617F/ΔDnmt3a P5, suggesting impaired DNA methylation in this group. Jak2V617F/ΔDnmt3a-Cas9 P5 were significantly enriched for transcriptional pathways controlling cell cycle progression, oncogenic signatures, and DNA damage. Conversely, Jak2V617F/Cas9 controls were enriched for myeloid differentiation and normal progenitor cell signatures.To assess the effect of Dnmt3a loss on Jak2V617F driven MPN, we transplanted Jak2V617F/ΔDnmt3a-Cas9 or Jak2V617F/Cas9 LKS+ into irradiated B6 recipients. Recipients of Jak2V617F/Cas9 LKS+ developed early stage MPN reminiscent of polycythemia vera with high hemoglobin, white cell count and platelets and was sustained >32 weeks. In contrast, Jak2V617F/ΔDnmt3-Cas9 recipients exhibited a biphasic disease, reminiscent of human myelofibrosis. At 8 weeks, Jak2V617F/ΔDnmt3-Cas9 showed panmyelosis with thrombocytosis (1.38x106/µl vs. 1.14x106/µl controls, p=0.057). However, by 32 weeks, this mice became severely pancytopenic with progressive bone marrow failure (Hemoglobin 121g/L vs. 210g/L controls, p =0.0011; platelets 0.338x106/µl vs. 1.343x106/µl controls, p <0.0001). Jak2V617F/ΔDnmt3-Cas9 mice exhibited extreme splenomegaly associated with reticulin fibrosis and the accumulation of myeloid cells. Bone marrow histology of Jak2V617F/ΔDnmt3-Cas9 revealed osteosclerosis and disorganized architecture and a dense fibrocellular infiltrate and reticulin fibrosis. Flow cytometry revealed impaired erythropoiesis and blocked differentiation. AML was not seen.These data demonstrate new evidence linking loss of Dnmt3a with acquisition of self-renewal in combination with constitutively active Jak2V617F. Importantly, in vivo loss of Dnmt3a accelerates or induces myelofibrotic transformation of the underlying MPN. This work provides new understanding to the factors that promote advanced disease in MPN. Ultimately, such knowledge has the potential to inform the development of novel targeted therapeutic approaches for the treatment of transformed MPN, a highly chemorefractory disease associated with extremely poor prognosis in patients. DisclosuresLane:Janssen: Other: i have done consulting (once) for janssen..

Metformin inhibits JAK2V617F activity in MPN cells by activating AMPK and PP2A complexes containing the B56α subunit.

Metformin suppresses the growth of a variety of malignant hematologic cells. It is widely accepted that metformin inhibits the growth of malignant cells primarily by suppressing the mTOR pathway or regulating autophagy. In contrast, we found another possible mechanism that inhibits the growth of malignant cells, suppression of the activity of the oncogenic kinase JAK2V617F. We identified at least two distinct mechanisms involved in metformin-induced JAK2V617F inhibition. First, metformin increases reactive oxygen species levels in these cells, leading to the inhibition of SHP-2, a positive regulator of JAK2V617F. These effects of metformin require AMPK. Second, metformin activates protein tyrosine phosphatase PP2A, a negative regulator of JAK2V617F. Furthermore, we determined that among the numerous PP2A subfamily members, the PP2A complex containing the B56α subunit is responsible for the inhibition of JAK2V617F. In contrast, the B56α-containing PP2A complex functions as a positive regulator of JAK2V617F by inhibiting AMPK. Finally, we determined that metformin enhances the antileukemic action of ruxolitinib in HEL and SET-2cells. Our present observations suggest that the combination of metformin with ruxolitinib might be a new therapeutic option for treating JAK2V617F-induced myeloproliferative neoplasms. In addition, activators specific for PP2A complexes containing the B56α subunit may be usefulfor the treatment of JAK2V617F-induced myeloproliferative neoplasms.

The loss of Ezh2 drives the pathogenesis of myelofibrosis and sensitizes tumor-initiating cells to bromodomain inhibition

EZH2 is a component of polycomb repressive complex 2 (PRC2) and functions as an H3K27 methyltransferase. Loss-of-function mutations in EZH2 are associated with poorer outcomes in patients with myeloproliferative neoplasms (MPNs), particularly those with primary myelofibrosis (MF [PMF]). To determine how EZH2 insufficiency is involved in the pathogenesis of PMF, we generated mice compound for an Ezh2 conditional deletion and activating mutation in JAK2 (JAK2V617F) present in patients with PMF. The deletion of Ezh2 in JAK2(V617F) mice markedly promoted the development of MF, indicating a tumor suppressor function for EZH2 in PMF. The loss of Ezh2 in JAK2(V617F) hematopoietic cells caused significant reductions in H3K27 trimethylation (H3K27me3) levels, resulting in an epigenetic switch to H3K27 acetylation (H3K27ac). These epigenetic switches were closely associated with the activation of PRC2 target genes including Hmga2, an oncogene implicated in the pathogenesis of PMF. The treatment of JAK2(V617F)/Ezh2-null mice with a bromodomain inhibitor significantly attenuated H3K27ac levels at the promoter regions of PRC2 targets and down-regulated their expression, leading to the abrogation of MF-initiating cells. Therefore, an EZH2 insufficiency not only cooperated with active JAK2 to induce MF, but also conferred an oncogenic addiction to the H3K27ac modification in MF-initiating cells that was capable of being restored by bromodomain inhibition.

Uncoupling JAK2 V617F activation from cytokine-induced signalling by modulation of JH2 αC helix

The mechanisms by which JAK2 is activated by the prevalent pseudokinase (JH2) V617F mutation in blood cancers remain elusive. Via structure-guided mutagenesis and transcriptional and functional assays, we identify a community of residues from the JH2 helix αC, SH2-JH2 linker and JH1 kinase domain that mediate V617F-induced activation. This circuit is broken by altering the charge of residues along the solvent-exposed face of the JH2 αC, which is predicted to interact with the SH2-JH2 linker and JH1. Mutations that remove negative charges or add positive charges, such as E596A/R, do not alter the JH2 V617F fold, as shown by the crystal structure of JH2 V617F E596A. Instead, they prevent kinase domain activation via modulation of the C-terminal residues of the SH2-JH2 linker. These results suggest strategies for selective V617F JAK2 inhibition, with preservation of wild-type function.

Thrombocytosis and STAT5 activation in chronic myelogenous leukaemia are not associated with JAK2 V617F or calreticulin mutations

AimsMarked thrombocytosis is uncommon in chronic myelogenous leukaemia (CML) but may be associated with mutation of JAK2 V617F, calreticulin (CALR) and/or phospho-STAT5 (p-STAT5) activation in other myeloproliferative neoplasms (MPNs), particularly...