Single Centre Analysis of JAK2 V617F and Exon 12 Negative Idiopathic Erythrocytosis

Abstract Introduction The molecular categorisation of myeloproliferative neoplasms (MPNs) has changed the landscape of diagnosis and treatment. Polycythaemia vera (PV) is characterised by red blood cell proliferation and JAK2 V617F or Exon 12 mutations in up to 98% of patients 1. However, some patients without such mutations have an arduous diagnostic course with varying management and prognostic outcomes 2. We present our experience in managing this challenging cohort and aim to illuminate a potential diagnostic pathway for patients. Method We searched electronic records of patients attending haematology clinics over the last 20 years at University College Hospital with a prior diagnosis of PV / erythrocytosis (presenting with raised Haemoglobin (Hb) &/ Haematocrit (Hct)) with no evidence of JAK2 exon 12 or 14 mutations on bone marrow or peripheral blood molecular analysis (multiplex PCR sensitivity 0.1%). We reviewed their diagnostic workup, which included full blood count & where available, bone marrow myeloid (Illumina TruSight Myeloid) & erythroid next generation sequencing (NGS) panels. Results 37 patients with JAK2 V617F & Exon 12 mutation negative erythrocytosis were identified. Patients were categorised in to 3 groups 2; idiopathic erythrocytosis (IE), secondary polycythaemia (SP) & high affinity haemoglobinopathies (HAH); patient characteristics are summarised in Table 1. The median age of IE & HAH was younger, their presenting Hb/Hct levels was higher compared to SP, with a male predominance. Constitutional symptoms were only reported in the IE cohort. Erythropoietin (EPO) was elevated in HAH & IE patients but within normal range in SP. Thrombotic events occurred in all cohorts, most frequently in IE. Splenomegaly was reported in 4/21 IE, 1/13 SP, but was not a feature in HAH patients. When performed, IE red cell mass (RCM) studies were raised but within normal range in SP patients. Table 2 details IE cohort erythroid mutations. Myeloid NGS only identified MPL and BCOR mutations of pathogenic significance and multiple single nucleotide polymorphisms of no known significance. No abnormalities were demonstrated in 15% of SP patients that underwent bone marrow myeloid mutational analysis. Venesections (VS) were instigated in 95% of the IE cohort, antiplatelets (AP) in 52%, anticoagulation (AC) in 14% and cytoreductive therapy (CT) in 19% due to intolerance/failure of VS. VS programme was instigated in 46% of SP patients, AP in 7% and AC in 47%. Discussion The median age of our IE cohort was 48 yrs with a 19% incidence of thrombosis. Where performed, bone marrow histology demonstrated hypercellularity but was not consistent with MPN diagnostic criteria. Myeloid NGS panel mutations such as BCOR may represent clonal haematopoieis of indeterminate potential. Heterozygous VHL C598T & C376A mutations, in keeping with Chuvash polycythaemia, were demonstrated. Mutations in EGLN1 & BPGM,were detected in our patients, however there was an absence of correlating haematological parameters or family history to support a diagnosis of congenital erythrocytosis (2). Variants of unknown significance were also detected in SH2B3, BMP6 & EGLN3 gene duplication. SP patients were older (median age 68 years) and where performed had normal RCM and no myeloid mutations identified. The initial approach adopted at our centre for diagnosing and managing JAK2 V617F & Exon 12 negative erythocytosis begins with clinical evaluation for secondary causes. This is followed by assessment of EPO level, RCM study, extended molecular mutational analysis involving screening for high affinity haemoglobins, congenital erythrocytosis and bone marrow histology. Where patients are symptomatic or considered at high risk for thrombosis, we venesect to a personalised target and patients are offered AP and/or AC. CT is instigated where VS is not tolerated or ineffective, however this approach is not corroborated in the literature. Our experience highlights the clinical heterogeneity of JAK2 negative erythrocytosis and the need to develop a robust and systematic diagnostic and treatment algorithm with further clarification of the role of molecular profiling. 1. William W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017 Feb; 129(6):667-679 2. McMullin MF. Idiopathic erythrocytosis: a disappearing entity. Hematology Am Soc Educ Program. 2009; 2009(1):629-635 Figure 1 Figure 1. Disclosures Sekhar: Novartis: Consultancy, Research Funding.

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Congenital JAK2 V617F Polycythaemia Vera Cured with Sibling Allogeneic Bone Marrow Transplantation

Blood ◽

10.1182/blood.v112.11.5227.5227 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5227-5227

Author(s):

Kevin R Kelly ◽

Corrina McMahon ◽

Stephen Langabeer ◽

Hassan Eliwan ◽

Aengus O’Marcaigh ◽

...

Keyword(s):

Bone Marrow ◽

Oral Mucosa ◽

Blood Count ◽

Allogeneic Bone Marrow Transplantation ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Full Blood Count ◽

Polycythaemia Vera ◽

Allogeneic Bone ◽

V617f Mutation

Abstract Polycythaemia vera (PV) is extremely rare in young children. The Janus Kinase 2 V617F mutation is present in 95 % of adult PV patients. Testing is now widely available and has simplified the diagnostic workup. A previously healthy 7 month old girl was admitted to her local hospital with tonsillitis. Full blood count showed polycythaemia (Hb 19 g/dl) along with an elevated platelet (946 ×109/L) and white cell count (19.7 ×109/L). Oxygen saturations, arterial blood gases, chest x-ray, abdominal ultrasound and P50 were all normal. Serum erythropoietin was low. No mutations were identified in exons 7 and 8 of the EPO receptor gene, the Von Hipple Lindau tumour suppressor gene and the Prolyl hydroxylase domain 2 gene. Bone marrow aspirate and biopsy were showed hypercellularity, megakaryocyte hyperplasia and clustering along with erythroid hyperplasia. Cytogenetic analysis was normal. Spontaneous erythroid colonies were demonstrated. The JAK2 V617F mutation was found by polymerase chain reaction in her blood and in her Guthrie card taken at 2 days of age. Both parents and two of her siblings had normal full blood counts. Two further siblings have not had full blood counts. There is no known family history of myeloproliferative disorders. The JAK2 V617F mutation was not detected in the peripheral blood or the oral mucosa of either parent or in the oral mucosa of the patient. She was treated with regular venesection and aspirin 45mg OD was started when her platelet count rose above 1,500 × 109/L. Her clinical course and laboratory parameters remained stable with this treatment and no thromboembolic complications occurred. Due to the long term risks of malignant transformation and thromboembolism a sibling allogeneic bone marrow transplantation was performed with her 6 year old brother as a fully HLA matched, JAK2 V617F negative donor with a normal full blood count. A total nucleated count of 13 ×108 per kg recipient body weight, bone marrow was infused. The conditioning regimen was busulphan (4mg/kg/day × 5 days) and cyclophosphamide (50mg/kg/day × 4 days). Neutrophils rose to 2.6 ×109/L by day 24. No venous occlusive disease or graft versus host disease occurred. Complete donor chimerism and undetectable JAK2 V617F mutation have been observed from day + 14 to present. She remains in remission 15 months post transplant. She now has a normal full blood count and has not suffered any adverse sequelae. To the best of our knowledge this is the first report of prenatal JAK2 V617F PV. It further highlights the genotype-phenotype diversity that is seen amongst this group of JAK2 V617F positive myeloproliferative neoplasms. The frequency of the mutation in pediatric PV has been variably reported in the literature but this report proves that it can occur at all ages. The absence of the mutation in either parent or the oral mucosa of the child shows that this was likely an acquired somatic event that occurred in utero. The JAK2 V617F mutation is thought to be acquired in both familial and sporadic MPD. In a study of 22 families with PV the mutation was present in variable amounts in affected members and absent in unaffected members. Analysis of another single large family with PV showed the presence of JAK2 V617F in affected family members but not in an obligate carrier. This suggests that other genetic abnormalities, possibly inherited, precede the acquisition of the JAK2 V617F mutation. Inheritance of as yet unknown germline mutations may have predisposed towards the in utero acquisition of the somatic JAK2 V617F mutation in our particular case. Young children with PV face a considerable lifetime risk of arterial and venous thrombosis and of malignant transformation. Hemopoetic stem cell transplantation normalises the full blood count and eradicates the pre malignant clone thereby reducing these risks and should be considered early if a suitable donor is available.

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Effects of the Sympathicomimetic Agonist Mirabegron on Disease Course, Mutant Allele Burden, Marrow Fibrosis, and Nestin Positive Stem Cell Niche in Patients with JAK2-Mutated Myeloproliferative Neoplasms. a Prospective Multicenter Phase II Trial SAKK 33/14

Blood ◽

10.1182/blood.v128.22.3108.3108 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3108-3108 ◽

Cited By ~ 3

Author(s):

Beatrice Drexler ◽

Jakob Passweg ◽

Martin Bigler ◽

Alexandre PA Theocharides ◽

Nathan Cantoni ◽

...

Keyword(s):

Bone Marrow ◽

Phase Ii ◽

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Nerve Fibers ◽

Allele Burden ◽

Sympathetic Nerve Fibers ◽

Bone Marrow Histology ◽

Cell Niche

Abstract Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders characterized by aberrant proliferation of erythroid, megakaryocytic and myeloid lineages. They are associated with decreased survival, thromboembolic complications, hemorrhage and leukemic transformation. MPN can be subdivided into polycythemiavera(PV), essentialthrombocythemia(ET) and primary myelofibrosis (PMF). The JAK2-V617F mutation is present in 70-80% of all MPN patients. MPN is initiated and maintained by mutated hematopoietic stem and progenitor cells (HSPC). Bone marrow mesenchymal stem cells expressing the intermediate filament proteinnestin(nestin+ MSCs) that are innervated by sympathetic nerve fibers constitute an important component of the stem cell niche and regulate normal HSCs. Thesenestin+ MSCs are strongly reduced in bone marrow of JAK2-V617F positive MPN patients and in mice expressing JAK2-V617F due to damage of the sympathetic nerve fibers triggered by cytokines from the mutant cells. In a JAK2-V617F mouse model of MPN, treatment with a beta-3sympathicomimeticagonist corrected the damage inflicted by the MPN clones on their niches and ameliorated the MPN phenotype. To test the potentially beneficial effect on disease-control by modulating bone marrow niche cells in patients with MPN, we performed a phase II trial with the beta-3sympathicomimeticagonistmirabegron. Patients and Methods: The trial consisted ofmirabegrontreatment with 25 mg daily during the first week, followed by 50 mg daily for at least 24 weeks. Patients with acytohistologicallyconfirmed diagnosis of MPN and a JAK2-V617F allele burden >20% in granulocytes at study entry were eligible, if not treated with JAK2 inhibitors or interferon. Reduction of the JAK2-V617F mutant allele burden ³50% in granulocytes was defined as the primary end point. Secondary end points included changes in blood counts or MPN related symptoms. As a side study, bone marrow biopsies were quantified fornestin+ MSCs, fibrosis and CD34+ HSPCs. N=39 patients have been accrued in 10 institutions in Switzerland. Eight (21%) had ET, 22 (56%) PV, and 9 (23%) PMF. N=27 (69%) were male, the median age was 62 (Q1-Q3 53-72) years. Median mutated allele burden at study onset was 52% (Q1-Q3 33-73%). All patients had prior treatment, N=28 (72%) patients hadcytoreductivetreatment, the remaining patients hadantiaggregation, anticoagulation or phlebotomy. Results: No patient reached the primary endpoint of 50% reduction in allele burden, one patient achieved a 25% reduction by 24 weeks of treatment. Adverse events were mostly grade I or II on the CTCAE scale. Three patients had grade III events: two were considered to be at least possibly related to study medication. In the side study, 24 patients agreed to bone marrow biopsy prior to and at the end ofmirabegrontreatment and for 20 patients both measurements are available. In these patients an increase in thenestin+ MSCs cells from a median of 1.09 (Q1-Q3 0.38-3.27)/mm2 to 3.95 (Q1-Q3 1.98-8.79)/mm2 (p<0.0001, Wilcoxon signed-rank test) and a slight decrease of myelofibrosis from a median grade of 1.00 (Q1-Q3 0.50-3.00) to 0.75 (Q1-Q3 0.50-2.00) (p=0.02), were observed. The mean change in thenestin+ cells from baseline to week 24 was 3.52 (95% confidence interval 1.65-5.39)/mm2. Morphometric changes in thenestin+ MSCs were significant for PV (n=13, p=0.007) and PMF (n=5, p=0.04). Bone marrow CD34+ cells slightly increased from a median 2.50 (Q1-Q3 2.00-3.25) to 3.00 (Q1-Q3 2.00-3.75) (p=0.06). Conclusion: In this prospective phase II clinical trial treatment with the beta-3-sympathicomimetic agonistmirabegronfor 24 weeks failed to achieve the primary endpoint to reduce the JAK2-V617F mutant allele burden >50% in patients with MPN. However, an increase in thenestin+ MSCs in bone marrow and a slight decrease of myelofibrosis were found, which will be further investigated. Figure 1 Bone marrow histology of a patient before (week 0) and at the end ofmirabegron treatment (week 24). Upper panel,reticulin fibers are stained black by silver impregnation (Gomori). Lower panel, immunohistochemistry staining with antibodies against humannestin protein (brown staining). Note decrease inreticulin fibrosis and increase innestin+ cells after 24 weeks of treatment. Magnification: 200x. Figure 1. Bone marrow histology of a patient before (week 0) and at the end ofmirabegron treatment (week 24). Upper panel,reticulin fibers are stained black by silver impregnation (Gomori). Lower panel, immunohistochemistry staining with antibodies against humannestin protein (brown staining). Note decrease inreticulin fibrosis and increase innestin+ cells after 24 weeks of treatment. Magnification: 200x. Disclosures Theocharides: Novartis: Consultancy, Honoraria. Rüfer:Novartis: Consultancy, Speakers Bureau. Benz:Celgene: Consultancy. Tzankov:Novartis: Speakers Bureau; Abbott: Speakers Bureau. Skoda:Novartis: Consultancy, Speakers Bureau; Baxalta: Speakers Bureau; Shire: Consultancy, Speakers Bureau.

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Abstract 12726: Crucial Role of Jak2 V617f-positive Myeloproliferative Neoplasm in the Development of Aortic Aneurysm

Circulation ◽

10.1161/circ.142.suppl_3.12726 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Tetsuro Yokokawa ◽

Tomofumi Misaka ◽

Yusuke KIMISHIMA ◽

Kento Wada ◽

Keiji Minakawa ◽

...

Keyword(s):

Bone Marrow ◽

Angiotensin Ii ◽

Aortic Aneurysm ◽

Mrna Expression ◽

Abdominal Aorta ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Jak2 V617f ◽

Vascular Complication ◽

The Impact

Objective: To investigate the impact of hematopoietic JAK2V617F, which causes myeloproliferative neoplasms (MPNs), on the development of aortic aneurysm (AA). Approach and Results: We applied a bone marrow transplantation (BMT) strategy using the donor cells from Jak2 V617F transgenic (JAK2 V617F ) mice into the lethally irradiated apolipoprotein E-deficient mice. To induce the AA formation and progression, the recipient mice (BMT mice) were subjected to continuous angiotensin II infusion. Abdominal aortic diameter in JAK2 V617F -BMT mice was significantly enlarged compared to the control wild-type (WT)-BMT mice in response to angiotensin II. The incidence of abdominal AA was significantly higher in JAK2 V617F -BMT mice than in WT-BMT mice. Hematopoietic JAK2V617F accelerated aortic elastic lamina degradation as well as activation of matrix metalloproteinase (MMP)-2 and MMP-9 in the abdominal aorta. The numbers of CD68 + macrophages and Ly6B.2 + neutrophils and cytokine expressions such as Ccl6 and Tgfb1 were significantly increased in the abdominal aorta of JAK2 V617F -BMT mice accompanied by STAT3 activation. Bone marrow-derived macrophages carrying JAK2V617F showed elevations of both Mmp2 and Mmp9 mRNA expression. Finally, we found that 23% of MPN patients with JAK2 V617F mutation showed the presence of AA and increases in TGFB3 and IL-8 mRNA expression of the peripheral leukocytes. Conclusions: Hematopoietic JAK2V617F was involved in the development of AA through increases in the infiltration of inflammatory cells and MMP activation. Our findings provide a novel feature of vascular complication of AA in MPNs due to constitutive activation of the hematopoietic JAK-STAT pathway.

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Identification of oncostatin M as a JAK2 V617F‐dependent amplifier of cytokine production and bone marrow remodeling in myeloproliferative neoplasms

The FASEB Journal ◽

10.1096/fj.11-193078 ◽

2011 ◽

Vol 26 (2) ◽

pp. 894-906 ◽

Cited By ~ 30

Author(s):

Gregor Hoermann ◽

Sabine Cerny‐Reiterer ◽

Harald Herrmann ◽

Katharina Blatt ◽

Martin Bilban ◽

...

Keyword(s):

Bone Marrow ◽

Cytokine Production ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Oncostatin M

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Cultivation of Megakaryocytes On a Collagen Matrix: A More Sensitive and Reproducible Test for the Diagnosis of Patients with Essential Thrombocythaemia.

Blood ◽

10.1182/blood.v114.22.4970.4970 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4970-4970

Author(s):

Adrian Emanuel Schmidt ◽

Patricia Darlington ◽

Lucie Kopfstein ◽

Elisabeth Ischi ◽

Elisabeth Oppliger Leibundgut ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Negative Predictive Value ◽

Predictive Value ◽

Healthy Subjects ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Essential Thrombocythaemia ◽

Jak2 Mutation ◽

Spontaneous Proliferation

Abstract Abstract 4970 Background Essential thrombocythaemia (ET) is one of the chronic myeloproliferative neoplasms (MPN), along with polycythaemia vera (PV), primary myelofibrosis (PMF) and chronic myeloid leukaemia (CML). Their common feature is excessive proliferation of a certain stem or progenitor cell in the bone marrow; in the case of ET, the megakaryocytic lineage is affected. Clinical manifestations include thrombotic events and haemorrhage. Diagnosis of ET according to new WHO-criteria requires a sustained high platelet count, bone marrow biopsy showing proliferation of the megakaryocytic lineage with large and mature morphology, demonstration of JAK2 V617F (although only present in about 50% of patients with ET) or another clonal marker and explicit exclusion of other myeloid and myeloproliferative neoplasms as well as signs of reactive thrombocytosis. Additionally, spontaneous proliferation of megakaryocytes obtained from peripheral blood can be detected in in vitro culture assays. Presently, we use agar as a matrix for megakaryocyte cultivation, although this assay has never been validated in connection with ET. The identification of megakaryocytic colonies grown on agar can sometimes be quite difficult. Our aims were therefore to technically evaluate the use of a collagen based matrix and to investigate its suitability to identify patients with ET. Patients and Methods We have examined 63 patients (26 with ET, 21 with PV, 8 with myelofibrosis [MF; including PMF and post-ET/PV-MF], 6 with secondary or idiopathic erythrocytosis and 2 with secondary thrombocytosis; mean age=59.8, male=33, female=30, mean platelet count 457 G/l) and 5 healthy subjects. Following informed consent, both clinical and laboratory data was collected. Medication intake, phlebotomies, smoking habits and regular haemogram results were noted in order to recognise possible confounding factors influencing laboratory results. Results of megakaryocyte cultivation on both agar and collagen matrixes were recorded, considering both spontaneous growth and growth stimulated by megakaryocyte derived growth factor (MDGF). Results Based on our collagen culture results we were able to define 2 or more spontaneously grown megakaryocyte colonies as the most optimal cut-off for the identification of patients with MPN (sensitivity 71%, specificity 100% with positive and negative predictive values of 100% and 45%, respectively). Compared to the agar culture results (where a specificity and a positive predictive value of 100% were demonstrated at a cut-off value of ≥ 10 CFU-Mega) we found a higher accuracy and better reproducibility. In addition, we observed an improved negative predictive value (45% with collagen versus 25% with agar cultures) reducing false negative results. Healthy subjects and patients with secondary thrombocytosis showed no significant spontaneous megakaryocyte proliferation. In patients with MF, we observed strong spontaneous and MDGF-stimulated growth of megakaryocytic colonies. At a cut-off value of ≥ 50 CFU-Mega (after stimulation with MDGF), the collagen assay showed a sensitivity of 100% and a specifity of 70% for this special form of MPN, resulting in a negative predictive value of 100%. We found no confounding clinical or laboratory parameters such as medication intake (particularly cytoreductive treatment with hydroxyurea) or phlebotomies influencing our culture results, and no significant effect of the Jak2-V617F mutation on the growth behaviour of megakaryocytic colonies. Conclusion The results of this ongoing study imply that the collagen based assay is more sensitive, specific, time efficient and user friendly regarding the detection of spontaneous proliferation of megakaryocytes than the currently used agar based culture assay. In addition, the collagen based assay also has the great advantage that it allows isolation of single megakaryocytic colonies for further analyses, for example PCR-based identification of a JAK2 mutation. Furthermore, the collagen based assay facilitates the diagnosis of patients with MPN, especially in cases where conventional diagnostic criteria are lacking, such as in ET without a JAK2 mutation. Ultimately, the new assay may well be able to detect transformation from PV/ET to MF. Disclosures No relevant conflicts of interest to declare.

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Direct Activation of STAT5 by TEL-Lyn Fusion Protein Promotes Induction of Myeloproliferative Neoplasms with Myelofibrosis

Blood ◽

10.1182/blood.v116.21.4114.4114 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4114-4114

Author(s):

Yusuke Takeda ◽

Chiaki Nakaseko ◽

Hiroaki Tanaka ◽

Masahiro Takeuchi ◽

Makiko Yui ◽

...

Keyword(s):

Bone Marrow ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Signaling Molecules ◽

Janus Kinase ◽

Idiopathic Myelofibrosis ◽

Lyn Kinase

Abstract Abstract 4114 Background Myeloproliferative neoplasms (MPN), a group of hematopoietic stem cell (HSC) disorders, are often accompanied by myelofibrosis. The V617F somatic mutation in the Janus kinase 2 (JAK2) gene has recently been found in the majority of patients with polycythemia vera (PV) and more than half of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). The expression of JAK2 V617F causes a PV-like disease with myelofibrosis in a murine bone marrow (BM) transplant model. In addition, a gain-of-function c-MPL W515 mutation was described in nearly 10% of patients with JAK2 V617F-negative IMF. However, the mechanism responsible for MPD and the formation of myelofibrosis in patients without JAK2 or c-MPL mutations is still unclear. We previously identified the fusion of the TEL gene to the Lyn gene (TEL-Lyn) in idiopathic myelofibrosis with ins(12;8)(p13;q11q21). The introduction of TEL-Lyn into HSCs resulted in fatal MPN with massive myelofibrosis in mice, implicating the rearranged Lyn kinase in the pathogenesis of MPN with myelofibrosis. However, the signaling molecules directly downstream from and activated by TEL-Lyn remain unknown. Design and Methods We examined the signaling pathways activated by TEL-Lyn by Western blotting, immunoprecipitation, and in vitro kinase assay using a TEL-Lyn kinase-dead mutant as a control. We further characterized the functional properties of Stat5-deficient HSCs transduced with TEL-Lyn by colony-forming assay and bone marrow transplantation to evaluate the role of STAT5 in TEL-Lyn-induced MPN. Results TEL-Lyn was demonstrated to be constitutively active as a kinase through autophosphorylation. In TEL-Lyn-expressing cells, STAT5, STAT3, and Akt were constitutively activated. Among these signaling molecules, STAT5 was activated most prominently and this occurred without the activation of Jak2, the major kinase for STAT5. TEL-Lyn was co-immunoprecipitated with STAT5, and STAT5 was phosphorylated when incubated with TEL-Lyn, but not with TEL-Lyn kinase-dead mutant. These results indicate that TEL-Lyn interacts with STAT5 and directly activates STAT5 both in vitro and in vivo. Of note, the capacity of TEL-Lyn to support the formation of hematopoietic colonies under cytokine-free conditions in vitro and to induce MPN with myelofibrosis in vivo was profoundly attenuated in a Stat5-null background. Conclusions In this study, we clearly showed that TEL-Lyn directly activates STAT5 and the capacity of TEL-Lyn to induce MPN with myelofibrosis was profoundly attenuated in the absence of STAT5. Our findings of TEL-Lyn in this study support the role of the Src family kinases in the regulation of STAT pathways and implicate active Lyn in the alternative pathway for STAT activation in pathological cytokine signaling. Our mouse model of MPD with myelofibrosis would be beneficial for the analysis of therapeutic approaches for myelofibrosis. Disclosures: No relevant conflicts of interest to declare.

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Combination Treatment with JAK2 and PI3K Inhibitors in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v120.21.180.180 ◽

2012 ◽

Vol 120 (21) ◽

pp. 180-180

Author(s):

Meng Ling Choong ◽

Christian Pecquet ◽

Shi Jing Tai ◽

Jacklyn WY Yong ◽

Vishal Pendharkar ◽

...

Keyword(s):

Bone Marrow ◽

Combination Treatment ◽

Kinase Inhibitors ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Pi3k Inhibitor ◽

Spleen Weight ◽

Pi3k Inhibitors ◽

Jak2 Inhibitor ◽

Jak2 Inhibitors

Abstract Abstract 180 Background and Aims. The main pathogenic molecular events associated with myeloproliferative neoplasms (Polycythemia Vera, Essential Thrombocytosis, and Primary Myelofibrosis) are mutations in Janus kinase 2 (JAK2) or in the thrombopoietin receptor that arise in the hematopoietic stem/progenitor cells. Both type of mutations lead to constitutive activation of the JAK2 signaling pathways. The approved JAK2 inhibitor (Ruxolitinib) is not expected to be selective for the mutant JAK2/receptor signaling or to completely suppress the multiple signaling pathways activated by the aberrant JAK2 signaling. We postulate that myeloproliferative neoplasms can be treated more effectively if we target the constitutive JAK2 signaling by a JAK2 inhibitor together with another kinase inhibitor targeting a specific pathway that is co-activated by the aberrant JAK2 signaling. This should increase targeting specificity, reduce JAK2 inhibitor dosages, and minimize potential side effects of these drugs. To this end, we constructed cell line models of myeloproliferative neoplasms and tested the models using a JAK2 inhibitor in combination with a panel of kinase inhibitors to identify combination pairs that give the best synergism. The synergistic pair was further confirmed in mouse models of myeloproliferative neoplasms. Methods. Mouse Ba/F3 cells were engineered to express either JAK2 WT, or JAK2 V617F, or TpoR W515L, or TpoR JAK2 WT, or TpoR JAK2 V617F, or Bcr-Abl. The effect of two JAK2 inhibitors (Ruxolitinib and TG101348) in combination with a panel of 15 various kinase inhibitors (one JNK, one B-Raf, one ROCK-1, one TIE-2, one PI3K, two CDK, two MAPK, three p38, and three mTOR inhibitors). An 8×8 constant ratio Latin square design were used for testing inhibition of cell proliferation/survival in these cell line models. Calculations were carried out using the Chou-Talalay method to determine which drug-pair demonstrated synergism in inhibiting cell growth. Further eight PI3K inhibitors were acquired and tested when we found strong synergism between the JAK2 inhibitors and the PI3K inhibitor ZSTK474 in the first panel. The engineered Ba/F3 cells were also inoculated into female BALB/c nude mice to generate the JAK2 mutant mouse model. These mice were treated intravenously with Ruxolitinib and the PI3K inhibitor GDC0941. Blood profile and physical parameters of the mice were measured for 14 days post treatment. Bone marrow cells from mice reconstituted with bone marrow from JAK2 V617F knock-in mice were plated for colony formation in the presence or absence of Ruxolitinib and the PI3K inhibitor GDC0941. Primary Epo-independent colonies from CD34+ cells of one PV patient were assessed in two independent experiments in the presence or absence of combination drugs. Results. Out of 15 kinase inhibitors tested, three PI3K inhibitors (ZSTK474, GDC0941 and BEZ235), synergized with JAK2 inhibitors (Ruxolitinib and TG101348) in inhibiting cell growth. The combination index was less than 0.5 in all 8×8 dose combination ratios. The JAK2-PI3K inhibitors combination was specific for JAK2 signaling as growth of Ba/F3 cells expressing Bcr-Abl (at equivalent STAT5 activation levels) was unaffected by this combination treatment. Balb/c mice inoculated with Ba/F3 cells expressing TpoR JAK2 V617F were found to have increased spleen weight due to proliferation of autonomous cells. Our combination treatment using Ruxolitinib and GDC0941 could drastically reduce spleen weight compared to treatment with either compound alone. Endogenous erythroid colony forming unit (CFU-E) and burst forming unit (BFU-E) formation from JAK2 V617F knock-in bone marrow cells was reduced significantly by the combined use of Ruxolitinib and GDC0941 compared to individual drugs. Similarly, Epo-independent BFU-E colony formation from peripheral CD34+ cells of one JAK2 V617F-positive PV patient was reduced significantly by the drug combination. Conclusions. Our findings of strong synergy between the JAK2 inhibitors and PI3K inhibitors suggested that we may be able to administer these drugs at lower concentrations than when the drugs are used individually. It provides a framework for combination trials using compounds in these two classes in patients with myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

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JAK2 Signaling Specifies Phenotypic Pleiotropy In Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v122.21.1608.1608 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1608-1608

Author(s):

Lily Huang ◽

Huiyu Yao ◽

Yue Ma

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Phenotypic Diversity ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Wild Type ◽

Gain Of Function ◽

Hematopoietic Stem ◽

Phenotypic Differences ◽

V617f Mutation

Abstract Myeloproliferative neoplasms (MPNs) are a phenotypically diverse group of pre-leukemic diseases characterized by overproduction of one or more of the myeloid cell lineages. Gain-of -function mutations in the Janus tyrosine kinase 2 (JAK2) are major determinants in MPNs, These include the V617F mutation and mutations in exon 12. Interestingly, MPN phenotype in patients with exon 12 mutations is distinct from that of patients with the V617F mutation. Mechanisms underlying the phenotypic differences are not well understood. We performed an unbiased screen for residues essential for JAK2 auto-inhibition, and identified a panel of novel gain-of-function mutations. Interestingly, three of them with similar kinase activities in vitro elicited distinctive hematopoietic abnormalities in mice. Specifically, JAK2(K539I) results primarily in erythrocytosis, JAK2(N622I) predominantly granulocytosis, and JAK2(V617F) in both. These phenotypes are consistent with clinical data showing that patients with the V617F mutation exhibit erythrocytosis and granulocytosis, whereas those with mutations in exon 12 (where K539 resides) exhibit erythrocytosis only. To determine the mechanisms underlying the phenotypic differences by different JAK2 mutants, we characterized hematopoietic progenitors and precursor subsets in these mice for their proliferation, apoptosis and differentiation. Quantification of the hematopoietic stem and progenitor population showed an increased percentage of granulocyte-monocyte progenitors (GMP) and skewing of differentiation towards the granulocytic lineage in JAK2(V617F) and JAK2(N622I) mice compared to JAK2(K539I) or wild-type JAK2 mice. Because no difference was observed in the proliferation or apoptosis of bone marrow progenitors from JAK2 mutant mice, differentiation of the common myeloid progenitors (CMP) was likely skewed towards GMP by JAK2(V617F) and JAK2(N622I). Consistent with this hypothesis, similar results were observed in colony forming assays from sorted CMP populations. In the spleen, a decrease in GMP apoptosis and an increase in apoptosis of the megakaryocyte-erythrocyte progenitors (MEP) also contributed to the skewing towards the granulocytic lineage in JAK2(N622I) mice. Similar to MPN patients, mice expressing JAK2 mutants exhibited splenomegaly. We found that JAK2 mutants caused redistribution of hematopoietic stem and progenitors from the bone marrow to spleen. As a result, more differentiated precursors were expanded in the spleens of JAK2 mutants mice compared to mice expressing wild-type JAK2. Consistent with their phenotypes, the percentage of Annexin V＋7AAD－erythroblasts in JAK2(K539I) and JAK2(V617F) mice was significantly less than in JAK2(N622I) or wild-type JAK2 mice. On the other hand, both proliferation and apoptosis contribute to the differential degrees of granulocytosis among mice expressing different JAK2 mutants. In line with the different effects elicited by different JAK2 mutants in progenitor and precursor cells, signal transduction pathways were differentially activated downstream of different JAK2 mutants. In summary, our results showed that JAK2 mutants differentially skew differentiation in early stem and progenitor compartments, and also regulate apoptosis and proliferation of distinct precursor subsets to cause erythrocytosis or granulocytosis in mice. These results provide the mechanistic basis for the phenotypic diversity observed in MPNs with different JAK2 mutants. Disclosures: No relevant conflicts of interest to declare.

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The Red Cell Mass (RCM) Value In Polycythaemia Vera Disease In The JAK2 V617F Era

Blood ◽

10.1182/blood.v122.21.5249.5249 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5249-5249 ◽

Cited By ~ 1

Author(s):

Hassan A. Al-Jafar ◽

Leena M Aytoglu ◽

Issa Loutfi ◽

Iman Al-Shemmari ◽

Salem H Alshemmari

Keyword(s):

Polycythemia Vera ◽

Cell Mass ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Jak2 V617f Mutation ◽

Polycythaemia Vera ◽

Red Cell ◽

Red Cell Mass ◽

V617f Mutation ◽

Idiopathic Erythrocytosis

Abstract Introduction In Polycythaemia Vera (PV), the RBC lineage is involved with increased haemoglobin, RBC count and haematocrit. WHO diagnostic criteria for PV are JAK2 V617F mutation and elevated red cell mass (RCM) > 25% of mean normal value. In addition, tests of marrow hypercellularity, blood erythropoietin and colony formation, are minor criteria. However, the diagnostic role of RCM test is still controversial and requires clarification. In this work, PV patients who had both an RCM study and JAK2 V617F mutation test, and routine laboratory tests, are evaluated to check if RCM was essential in the diagnostic work up for PV. Methods Over 2 years, 75 patients with abnormal haematocrit (men ≥ 0.50, women ≥ 0.45) had RCM and JAK2 V617F mutation tests (except JAK2 exon 12 mutation). All subjects consented to the study approved by the ethics committee. RCM was done by Cr-51 RBC radiolabeling method (no prior venesection at least 1 month). Statistical analysis involved descriptive statistics and chi-square test. Results There were 71 males and 4 females, mean age 46 y (range 17-75 y). Increased RCM was found in 41/75 (55%). Positive JAK2 V617F was found in 13/75 patients (17%), who also had RCM above the mean normal predicted value, however, when the WHO RCM criteria were applied, only 7/13 (54%) could be considered as having “truly” increased RCM. In the patient group with negative JAK2 V617F test, 12/28 (43%) had RCM results as per WHO criteria. There was no statistical association between presence of JAK2 V617F and the RCM values. Conclusion In patients with negative JAK2 V617F but with high clinical suspicion for PV and all other causes of secondary and idiopathic erythrocytosis excluded, an increase in RCM would support the diagnosis of PV (about 10 % PV cases). In patients with JAK2 positive mutation and high haematocrit but RCM below the WHO cut-off level, an increased RCM would still count to confirm the diagnosis as the current standard level seems too stringent. References James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C et al. A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera. Nature 2005; 434(7037): 1144-8. Kralovics R, Passamonti F, Buser AS, Soon-Siong T, Tiedt R, Passweg JR, et al. A Gain-of-Function Mutation of JAK2 in Myeloproliferative Disorders. Merck Manual of Diagnosis and Therapy. 16th Edition, 1992 McMullin MF, Bareford D, Campbell P, Green AR, Claire Harrison C, Hunt B, Oscier D, et al. Guidelines for the diagnosis, investigation and management of polycythaemia/erythrocytosis. British Journal of Haematology 2005; 130(2): 174-95. Scott LM, Tong W, Levine RL, et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med. 2007;356:459-468. Pardanani A, Lasho TL, Finke C, et al. Prevalence and clinicopathologic correlates of JAK2 exon 12 mutations in JAK2V617F-negative polycythemia vera. Leukemia. 2007;21:1960-1963. Pancrazzi A, Guglielmelli P, Ponziani V, et al. A sensitive detection method for MPLW515L or MPLW515K mutation in chronic myeloproliferative disorders with locked nucleic acid-modified probes and real-time polymerase chain reaction. J Mol Diagn. 2008;10:435-441. Disclosures: No relevant conflicts of interest to declare.

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