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Supplementary Table 6 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 2 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 3 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Figure 1 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 1 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 5 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Methods, Table Legends 1-6 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 4 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses.

Myeloproliferative diseases (MPDs) represent the commonest cause of splanchnic vein thrombosis (SVT), including Budd-Chiari syndrome (BCS) and portal vein thrombosis (PVT), but their diagnosis is hampered by changes secondary to portal hypertension, while their influence in the outcome of SVT remains unclear. We assessed the diagnostic and prognostic value of JAK2 and MPL515 mutations in 241 SVT patients (104 BCS, 137 PVT). JAK2V617F was found in 45% of BCS and 34% of PVT, while JAK2 exon 12 and MPL515 mutations were not detected. JAK2V617F was found in 96.5% of patients with bone marrow (BM) changes specific for MPD and endogenous erythoid colonies, but also in 58% of those with only one feature and in 7% of those with neither feature. Stratifying MPD diagnosis first on JAK2V617F detection would have avoided BM investigations in 40% of the patients. In BCS, presence of MPD carried significantly poorer baseline prognostic features, required hepatic decompression procedures earlier, but had no impact on 5-year survival. Our results suggest that JAK2V617F testing should replace BM investigations as initial test for MPD in patients with SVT. Underlying MPD is associated with severe forms of BCS, but current therapy appears to offset deleterious effects of MPD on the medium-term outcome.

The MPL (W515L and W515K) mutations have been detected in granulocytes of patients suffering from certain types of primitive myelofibrosis (PMF). It is still unknown whether this molecular event is also present in lymphoid cells and therefore potentially at the hematopoietic stem cell (HSC) level. Toward this goal, we conducted MPL genotyping of mature myeloid and lymphoid cells and of lymphoid/myeloid progenitors isolated from PMF patients carrying the W515 mutations. We detected both MPL mutations in granulocytes, monocytes, and platelets as well as natural killer (NK) cells but not in T cells. B/NK/myeloid and/or NK/myeloid CD34+CD38−-derived clones were found to carry the mutations. Long-term reconstitution of MPL W515 CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice was successful for as long as 12 weeks after transplantation, indicating that MPL W515 mutations were present in HSCs. Moreover, the 2 MPL mutations induced a spontaneous megakaryocytic growth in culture with an overall normal response to thrombopoietin (TPO). In contrast, erythroid progenitors remained EPO dependent. These results demonstrate that in PMF, the MPL W515L or K mutation induces a spontaneous megakaryocyte (MK) differentiation and occurs in a multipotent HSCs.

The release of transforming growth factor-β1 (TGF-β1) in the bone marrow microenvironment is one of the main mechanisms leading to myelofibrosis in murine models and probably in the human idiopathic myelofibrosis (IMF). The regulation of TGF-β1 synthesis is poorly known but seems regulated by nuclear factor κB (NF-κB). We previously described the overexpression of an immunophilin, FK506 binding protein 51 (FKBP51), in IMF megakaryocytes. Gel shift and gene assays show that FKBP51's overexpression in a factor-dependent hematopoietic cell line, induces a sustained NF-κB activation after cytokine deprivation. This activation correlates with a low level of IκBα. A spontaneous activation of NF-κB was also detected in proliferating megakaryocytes and in circulating CD34+ patient cells. In normal cells, NF-κB activation was only detected after cytokine treatment. The expression of an NF-κB superrepressor in FKBP51 overexpressing cells and in derived megakaryocytes from CD34+ of IMF patients revealed that NF-κB activation was not involved in the resistance to apoptosis after cytokine deprivation of these cells but in TGF-β1 secretion. These results highlight the importance of NF-κB's activation in the fibrosis development of this disease. They also suggest that FKBP51's overexpression in IMF cells could play an important role in the pathogenesis of this myeloproliferative disorder.

To the editor: In a recent paper, Perez-Garcia et al described an inherited mutation in the SH2B3 gene associated with the development of acute lymphoblastic leukemia.[1][1] SH2B3 encodes the lymphocyte adaptor protein (LNK) that negatively modulates the signaling of several cytokine receptors

Potentiation of the cytotoxic activity of 5-fluorouracil (FUra) by folinic acid (5-HCO-H4folate) is due to elevation of the methylene tetrahydrofolate (CH2-H4folate) level, which increases the stability of the ternary complex of thymidylate synthase (TS), fluorodeoxyuridine monophosphate, and CH2-H4folate that inactivates the TS. Methionine deprivation results in the production of tetrahydrofolate (H4folate) and, subsequently, CH2-H4folate from methyl tetrahydrofolate, as a consequence of the induction of methionine synthesis. We hypothesized that the efficacy of FUra could be augmented by the combination of high-concentration 5-HCO-H4folate and recombinant methioninase (rMETase), a methionine-cleaving enzyme. Studies in vitro were performed with the cell line CCRF-CEM. Cytotoxic synergism of FUra + rMETase and FUra + 5-HCO-H4folate + rMETase was demonstrated with the combination index throughout a broad concentration range of FUra and rMETase. A subcytotoxic concentration of rMETase reduced the IC50 of FUra by a factor of 3.6, and by a factor of 7.5, in the absence and in the presence of 5-HCO-H4folate, respectively. 5-HCO-H4folate increased the intracellular concentrations of CH2-H4folate and H4folate from their baseline levels. Concentrations of folates were not changed by exposure to rMETase. Levels of free TS in cells treated with FUra + 5-HCO-H4folate and with FUra + rMETase were lower than those in cells exposed to FUra alone. The decrease of TS was still more pronounced in cells treated with FUra + 5-HCO-H4folate + rMETase. The synergism described in this study will be a basis for further exploration of combinations of fluoropyrimidines, folates, and rMETase.

A diagnosis of methylenetetrahydrofolate reductase (MTHFR) deficiency was made in four sibs at different ages. The first three, including a pair of twins, had retarded psychomotor development, poor social contact, and seizures. Biologically, hyperhomocysteinemia and hypomethioninemia were found associated with low folate levels in serum and red cells, especially undetectable methyltetrahydrofolate in red cells. In the fourth child, prenatal diagnosis was not conclusive because of moderate decrease of enzymatic activity in chorionic villi and trophoblast. The girl was also affected, as shown by hyperhomocysteinemia and low folate levels found several days after birth. A 677CT (AlaVal) mutation was found in a homozygous state in the four children and in the father. Additionally, a second homozygous mutation, 1081CT, changing an arginine to cysteine also was identified in all of the children, whereas the distantly consanguineous parents were heterozygous. This amino acid substitution affecting an arginine residue in a sequence located at the end of catalytic domain seems critical for the function of the enzyme. The difficulty of prenatal diagnosis is discussed given the variability found in enzymatic activity and in the clinical phenotypes. Am. J. Med. Genet. 91:363–367, 2000. © 2000 Wiley-Liss, Inc.

Deficiency in methylenetetrahydrofolate reductase (MTHFR), the enzyme involved in the remethylation of homocysteine to methionine using methyltetrahydrofolate as cofactor, induces hyperhomocysteinaemia, homocysteinuria, hypomethioninaemia and low methylfolate levels. Diagnosis usually occurs during infancy because of various neurological abnormalities. We report MTHFR deficiency diagnosed in an adult woman after a pulmonary embolism. Her adult sister, intellectually retarded, suffered from the same disease. Molecular analysis of the MTHFR gene exhibited four different mutations (two missense mutations, one exon skipping and C677T). The impact of these mutations was analysed through the biological abnormalities in the parents and children.

Activating mutations in signaling molecules, such as JAK2-V617F, have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients, suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here, we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells, we demonstrated that Lnk expression is regulated by the TPO-signaling pathway, thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain, but also through an unrevealed JAK2-binding site within its N-terminal region. In addition, the presence of the V617F mutation causes a tighter association with Lnk. Finally, we found that the expression level of the Lnk protein can modulate JAK2-V617F–dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together, our results indicate that changes in Lnk expression and JAK2-V617F–binding regulate JAK2-mediated signals in MPNs.

During normal megakaryocyte development, in response to thrombopoetin, mature cells enter a senescence-like state in which they shed platelets; this state, characterized by cell cycle arrest, is defective in malignant megakaryocytes., Thrombopoietin (TPO) via signaling through its cognate receptor MPL is a key cytokine involved in the regulation of megakaryocyte differentiation leading to platelet production. Mature megakaryocytes are polyploid cells that have arrested DNA replication and cellular proliferation but continue sustained protein synthesis. Here, we show that TPO induces cell-cycle arrest in the megakaryocytic UT7-MPL cell line by the activation of the ERK/MAPK pathway, induction of p21CIP transcription, and senescence markers through EGR1 activation. A similar senescence-like process was also detected in normal primary postmitotic megakaryocytes. In contrast, senescence was not observed in malignant megakaryocytes derived from primary myelofibrosis patients (a form of chronic myeloid hemopathy). Our data indicate that polyploid mature megakaryocytes receive signals from TPO to arrest cell proliferation and enter a senescent-like state. An escape from this physiological process may be associated with certain myeloproliferative neoplasms leading to abnormal megakaryocytic proliferation., Author Summary Megakaryocytes are huge bone marrow cells that shed platelets into the blood stream to promote clotting at sites of injury. Mature megakaryocytes differentiate from precursor cells in response to a hormone called thrombopoetin. Here, we show that as part of this normal differentiation process mature megakaryocytes enter a state called senescence in which cell division stops—a feature normally associated with cell aging and death. By studying megakaryocytes in culture, we were able to determine the biochemical pathway induced by thrombopoetin that leads to gene activation associated with senescence. We conclude that thrombopoietin acts differently at two steps in megakaryocyte differentiation: in the early stages it induces megakaryocyte proliferation, and at a latter stage it arrests the cell division cycle leading to platelet production by these cells. Interestingly, certain malignant megakaryocytes did not undergo senescence in response to thrombopoetin, which might explain the abnormal proliferation of these cancerous cells.

SOCS3 inhibits TPO-stimulated, but not spontaneous, megakaryocytic growth in primary myelofibrosis

International audience; MPL (or thrombopoietin receptor, TPO-R) 515 mutations have recently been described in 5-10% of primitive myelofibrosis (PMF) cases as decisive oncogenic events capable of triggering the disease. Here we report additional mutations located in exon 10 of MPL in PMF patients. We investigated whether these new mutations also lead to cell transformation. MPL exon 10 was systematically sequenced in 100 PMF patients. Seven different mutations were found in eight patients. We introduced each MPL mutant in Ba/F3 cells to determine whether they correspond to gain-of-function mutations. Only MPL W515 mutations induced (1) Ba/F3 proliferation independently of growth factors, (2) tumorigenesis in nude mice, (3) spontaneous activation of JAK/STAT, RAS/MAPK and PI3K transduction pathways and (4) increased S phase of cell cycle. Similar to all other myeloproliferative disorder oncogenic events identified to date, these results demonstrate that only the detected MPL W515 mutations trigger spontaneous MPL activation leading to a G(1)/S transition activation. The other mutations are devoid of significant transforming activity but may synergize with JAK2 V617F or other not yet characterized molecular events.

The JAK2 V617F mutation has recently been described as an essential oncogenic event associated with polycythemia vera (PV), idiopathic myelofibrosis (IMF), and essential thrombocythemia. This mutation has been detected in all myeloid lineages but has not yet been detected in lymphoid cells. This raises the question whether this molecular event occurs in a true lymphomyeloid progenitor cell. In this work, we studied the presence of the mutation in peripheral blood cells and sorted B, T, and natural killer (NK) cells from PV and IMF. We detected the JAK2 V617F mutation in B and NK cells in approximately half the patients with IMF and a minority of those with PV. Moreover, in a few cases patients with IMF had mutated peripheral T cells. The mutation (homozygous or heterozygous) could be subsequently detected in B/NK/myeloid progenitors from PV and IMF, with a much higher frequency in clones derived from IMF. Using the fetal thymus organ culture (FTOC) assay, the mutation was also detected in all T-cell fractions derived from IMF and PV CD34+ cells. These results demonstrate that myeloproliferative disorders take their origin in a true myeloid/lymphoid progenitor cell but that their phenotype is related to a downstream selective proliferative advantage of the myeloid lineages.

Deficiency in methylenetetrahydrofolate reductase (MTHFR), the enzyme involved in the remethylation of homocysteine to methionine using methyltetrahydrofolate as cofactor, induces hyperhomocysteinaemia, homocysteinuria, hypomethioninaemia and low methylfolate levels. Diagnosis usually occurs during infancy because of various neurological abnormalities. We report MTHFR deficiency diagnosed in an adult woman after a pulmonary embolism. Her adult sister, intellectually retarded, suffered from the same disease. Molecular analysis of the MTHFR gene exhibited four different mutations (two missense mutations, one exon skipping and C677T). The impact of these mutations was analysed through the biological abnormalities in the parents and children.

Methionine depletion in the human cell line CCRF-CEM through the action of recombinant methioninase (rMETase), a methionine-cleaving enzyme, was previously demonstrated to produce a strong cytotoxic synergistic effect with fluorouracil (FUra) throughout a broad range of concentrations of FUra and rMETase, including subcytotoxic levels of rMETase. Potentiation was associated with a decrease in free thymidylate synthase from preexisting levels. To further investigate the action of rMETase on CCRF-CEM cells, in the present study we explored the effects of rMETase as a single agent on DNA methylation levels and DNA synthesis, which may be changed as a result of deprivation of methionine. Cells treated with rMETase under subcytotoxic conditions contained significantly lower levels of genomic methylated DNA than did control cells, as demonstrated by incorporation of the methyl radical of [methyl-(3)H]S-adenosylmethionine in DNA and by use of methylation-sensitive arbitrarily primed PCR. DNA hypomethylation produced by rMETase was of similar magnitude as that produced with the DNA methyltransferase inhibitor 5-azacytidine. Cells exposed to rMETase synthesized significantly more DNA than did untreated cells. Incorporation of [6-3H]thymidine and [6-3H]2'-deoxyuridine in these cells was augmented over that in control by mean factors of 1.78 and 2.36, respectively. Increased 3H nucleoside incorporation resulted in greater numbers of nuclear grains as demonstrated by autoradiography. The increase in DNA synthesis induced by rMETase is likely to result from enhancement of DNA repair because it was not accompanied by differences in cell cycle phase distribution or in total DNA content as determined by flow cytometry. We hypothesize that potentiation of FUra cytotoxicity by rMETase may result from increased inhibition of thymidylate synthase, together with DNA hypomethylation and enhanced DNA repair that could be involved in cell responses to drug-induced damage.

Methylenetetrahydrofolate reductase (MTHFR) deficiency is an autosomal recessive disorder resulting in elevated homocysteine levels in plasma and urine. MTHFR catalyses the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, a cofactor for homocysteine remethylation to methionine. MTHFR deficiency may be diagnosed from infancy to adulthood with a broad spectrum of clinical symptoms. A molecular analysis of the MTHFR gene combined with an assessment of MTHFR activity, plasma homocysteine and folate in plasma and red blood cells (RBC), especially methylfolate, was assessed in the members of 11 families from children affected with this disorder. This study was performed to try to define the impact of the mutations found in the MTHFR gene on symptoms and biological abnormalities. A total of 14 mutations were found and 10 of them were identified for the first time. Two were found in two families, two more in two other families and one in three families. The position of the mutation spread all over the gene does not predict the degree of biological abnormalities found in parents or healthy siblings bearing the mutation. Two different mutations located not far apart on the same exon may cause mild or severe abnormalities. The thermolabile variant C677T when expressed in an homozygote state in some parents was associated with lower MTHFR activity, higher homocysteine levels, lower folate levels, mainly methylfolate in RBC than in parents without the mutation; conversely, two or more mutations on the same allele had mild effects when the other allele was normal. Conclusion: given the heterogeneity of mutations, no one seems preponderant to predict neurological and/or vascular symptoms.

Hyperhomocysteinemia, a risk factor for vascular disease, is related to vitamin B12, vitamin B6, and especially folate deficiency, or to genetic factors such as mutations in methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in the remethylation pathway of homocysteine to methionine. Recently, a C677 --> T mutation identified in the MTHFR gene was found to be frequently associated with decreased MTHFR activity and an elevated plasma homocysteine concentration. Since hyperhomocysteinemia seems to be determined by both genetic and environmental factors, we studied the interactions between MTHFR (phenotype and genotype) and folate status, including methyltetrahydrofolate (methylTHF), the product of MTHFR, on the homocysteine concentration in 52 healthy subjects, (28 women and 24 men; mean age, 32.7 years). MTHFR activity seems to be dependent on folate status, as shown by a lower activity in folate-deficient subjects and a return to normal values after supplementation with folic acid, and also by a decreased enzymatic activity on phytohemagglutinin (PHA)-stimulated lymphocytes grown in a folic acid-deficient medium. Conversely, the C677 --> T mutation seems to influence folate metabolism. Subjects who were homozygous for this mutation (+/+) had significantly higher plasma homocysteine and lower plasma folate and total and methylfolate levels in red blood cells (RBCs) than heterozygous (+/-) and normal (-/-) subjects. The ratio of RBC methylfolate to RBC total folate was, respectively, 0.27 in +/+, 0.66 in +/-, and 0.71 in -/-. This mutation seems to have an impact on methylTHF generation. These data illustrate the interactions between nutritional and genetic factors.

Abstract 436 Polycythemia vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF) are myeloproliferative disorders (MPDs) without curative treatment, unless hematopoietic stem cell (HSC) transplantation is performed. However, for several years the use of interferon-alpha (IFNα) has provided an efficient therapeutic alternative for MPD patients. IFNα was shown to induce complete long-term hematologic and molecular remissions in JAK2V617F-positive MPD patients, suggesting a possible curative effect of IFNα. In order to better understand mechanisms of action of this drug, experiments were perfomred on cell lines, patient cells and mice cells harboring a JAK2V617F mutation. We hypothesized that IFNα may target directly MPD cells through binding to its specific receptor, in addition to the potential immunological effect of this molecule and could induce cell cycle entry of the pathological quiescent HSCs. Human cell lines harboring JAK2 mutation or BCR-ABL oncogene were treated with increasing doses of IFNα. A significant anti-proliferative effect at low concentrations (100 IU/ml) on the JAK2V617F-positive HEL cell line was observed. On the contrary, at this low dose IFNα did not influence growth of the BCR-ABL-positive K562 and the non-mutated UT-7 cell lines. This result supported a direct effect of IFNα in JAK2V617F cells. Suppressor of cytokine signaling (SOCS) are potent inhibitors of the JAK-STAT pathway by proceeding to a classic negative regulation loop proteins. Following IFNα stimulation of HEL cells, SOCS1 and SOCS3 mRNAs expression were induced (p=0.00036 and p=0.0012, respectively) and efficient knock-down of either SOCS1 or SOCS3 by shRNAs expression in HEL cells was able to counteract the anti-proliferative effect of IFNα (p=0.028 and p=0.031, respectively). We concluded that SOCS1 and SOCS3 are involved, in IFNα proliferative inhibition activity of HEL cells. To determine whether JAK2V617F confer hypersensitivity to IFNα inhibitory effect, proliferation and genotyping of CD34+ progenitors isolated from the bone marrow of JAK2V617F–positive MPD patients (n=5) and control subjects (n=4) were plated at one cell per well in 96-well plates and counted and genotyped at Day 10-12. A significant decrease of the patients progenitors clonogenicity was observed when exposed to IFNα (10 000 IU/ml, p Lastly, in order to explain the long term molecular responses observed in PV patients treated by IFNα, we investigated the effect of IFNα on the cell cycle in more immature cells. BrdU assay on JAK2V617F Knock-in (KI) mice was performed. Five-months old JAK2V617F KI and wild-type mice received or not 10,000 UI of murine IFNα during 3 days. Sixteen hours before analysis, BrdU was injected i.p. in the animals. Bone marrow Lin-Sca+cKit+cells (LSK) were stained with CD150 and CD48 antibodies before BrdU labelling was analyzed by flow cytometry. Analysis of BrdU postive cells confirmed that i-JAK2V617F induces LSK CD150+CD48- to enter cell cycle ( 8.55+/-3.12% for WT cells versus 19.92+/-3,19% for JAK2V617F KI cells respectively (p = 0.04)) and ii- That IFNα induces CD150+CD48- LSK to enter cell cycle whatever the JAK2 WT (8.55+/-3.12% for non treated animals versus 15.43+/-3.19% for IFNα receiving mice (p=0.02)) or mutated status but this induction was more statistically significant in JAK2V617F mice (19.92+/-1.82% versus 25.23+/-0.57% respectively( p=0.02)). In conclusion, we gave rise to a double effect of IFNα in MPD cells: A direct preferential anti-proliferative effect of IFNα on JAK2V617F–positive MPD progenitor cells, possibly through the induction of SOCS over-expressions and a direct cell cycling effect on JAK2V617F stem cells suggesting that IFNa containing treatments could be of interest for JAK2V617F patients cure. Disclosures: No relevant conflicts of interest to declare.

Abstract 963 Introduction: Flt3, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis. Primary myelofibrosis (PMF) is a Ph-negative (Ph−) myeloproliferative neoplasm (MPN) characterized by a myeloproliferation with increased hematopoietic progenitors (HPs) and a prominent proliferation of “dystrophic” megakaryocytes (MK). The JAK2V617F mutation is present in about 50% of PMF patients. Previous results from our group have revealed a MAPK pathway gene deregulation associated with an Flt3 transcript modulation in PMF patients. Since activation of Flt3 receptor is known to activate MAPK pathway, which plays a role in megakaryopoiesis, we studied the functional impact of MAPK and Flt3 abnormalities on PMF dysmegakaryopoiesis. Patients and Methods: The study included a group of 106 PMF patients. Transcriptome and QRT-PCR studies were performed on MACS selected CD34+ HPs, megakaryocytes (MK)-derived from CD34+ cell cultures and peripheral blood mononuclear cells (PBMNC) from PMF patients and healthy donors. Cell phenotype associated with Flt3 expression as well as phosphorylation levels of Flt3 and MAPK effectors were analyzed by flow cytometry. Functional studies (FL-induced stimulation and migration) were performed on MK-precursors at day 6 of CD34+ culture. Effect of i) MAPK inhibitors: PD98059, targeting ERK1/2; SB202190, SB203580, PD169316, targeting p38 and, SP600129, targeting JNK, ii) Flt3 inhibitors and iii) Flt3 monoclonal antibody was tested on PMF MK cell cultures. MAPK-induced transcripts were quantified by QRT-PCR in MK-precursors during an 18-hour FL-stimulation kinetic. FL mRNA level was evaluated by using QRT-PCR in bone marrow stromal cells and FL protein was quantified in plasma by using ELISA. Results: Comparative transcriptome analysis of CD34+ HPs and MK cells from PMF patients (with or without JAK2 mutation) and healthy donors showed that the MAPK pathway gene deregulation was independent of the presence of the JAK2V617F mutation. This alteration was associated with a modulation of mRNA Flt3 level in both types of cells. PMF patients also had a higher proportion of circulating Flt3+CD34+CD41+ cells as compared to healthy donors. This population demonstrated an increased phosphorylation of Flt3 on tyr591 and of MAPK (p38, p42/p44, JNK). MAPK effector phosphorylation was also increased in PMF CD34+ cells and MK-derived from CD34+ cell cultures, independently of JAK2 mutational status. In contrast to healthy donors, Flt3 membrane expression was maintained at all stages of in vitro megakaryocyte differentiation in PMF patients. The FL level was increased in the plasma of patients and was mainly expressed by bone marrow stromal cells. In contrast to healthy donors, in MK-derived from PMF CD34+ cell cultures, activation of Flt3/FL axis by addition of exogenous FL induced a MAPK hyper-phosphorylation, especially of p38 and p42/p44 as well as an up-regulation of downstream p38 transcripts (ATF-2, NFATC4, p53, AP-1, IL-8). Addition of chemical inhibitors targeting either MAPK or Flt3 and of an antibody directed against Flt3 reduced the phosphorylation of p38 and of its pathway effectors (MKK3/MKK6, MSK1, ATF2, HSP27 and MAPKAPK2) and normalized the PMF altered megakaryopoiesis. Lastly, in contrast to healthy donors, MK-derived from PMF CD34+ cells showed a FL-induced migration that was reversed by addition of p38αβ inhibitors. Conclusion: Our results demonstrated an increase in the FL circulating level in PMF patients that was mainly secreted by stromal cells. This was associated with an aberrant expression of Flt3 in CD34+ and MK cells and an alteration of the MAPK pathway activation in patients, independent of their JAK2 mutational status. The persistence of Flt3-mediated MAPK activation that participates in the PMF dysmegakaryopoiesis, suggests that drugs targeting “FL/Flt3-MAPK” axis could be promising agents for rescuing the altered megakaryopoiesis observed in patients. Our demonstration that FL, a cytokine mainly produced by stromal cells, participates in the altered megakaryopoiesis in PMF patients strengthens the hypothesis highlighting the crucial role of stroma cells in the hematopoietic deregulation that characterizes the disease. Disclosures: No relevant conflicts of interest to declare.

Primitive myelofibrosis (PMF) is a lethal myeloproliferative disorder (MPD) biologically characterized by a spontaneous megakaryocytic growth and the development of bone marrow and spleen fibrosis. PMF and essential thrombocytemia (ET) differ by their clinical features despite similar oncogenic molecular events (i.e. JAK2V617F and MPLW515 mutations) and spontaneous megakaryocytic growth. We investigated whether a secondary oncogenic event re-inforcing the constitutive JAK/cytokine receptor signaling could explain phenotypic differences between ET and PMF. We hypothesized that SOCS proteins, known to inhibit JAK/STAT signaling, may be down-modulated in PMF but not in ET. Komura et al. have previously reported that SOCS1 inhibits spontaneous megakaryocytic growth in PMF (Komura, Chagraoui et al. 2003). Other groups have shown that SOCS3, SOCS1 and SOCS2 promoters are hypermethylated in PMF (Capello, Deambrogi et al. 2008; Fernandez-Mercado, Cebrian et al. 2008). We investigated whether SOCS3 promoter hypermethylation plays a role in PMF megakaryocte spontaneous growth. We first studied 40 RNA samples from MPDs. RNAs were extracted from purified neutrophils and analyzed for SOCS3 expression using RT-real time PCR (21 PMF samples: 10 JAK2V617F-positive PMF, 2 MPLW515L and K PMF and 9 JAK2- and MPL-negative PMF; 10 PV samples and 10 ET samples: 5 with JAK2V617F mutation and 5 without mutation). PMF samples harbored a reduction in SOCS3 expression (related to β-Actin) when compared with all other samples (median 13 vs. median 24, student test p epigenetic events are present in PMF, SOCS3 promoter is methylated in at least part of PMFs whatever the first oncogenic event is, and SOCS3 inhibits TPO-induced megakaryocytic proliferation but not spontaneous growth in PMF.

The MPL 515L mutation has recently been described in 10% of Idiopathic Myelofibrosis (MMM) as an essential oncogenic event. Here we report another MPL mutation in the same codon of MPL (MPL515K). The MPL515L mutation has been detected in granulocytes but has not been yet detected in patient hematopietic stem cells. This raises the question whether this molecular event occurs in a true stem cell (ie: lymphoid/myeloid progenitor cell), as it has already been shown for JAK2 V617F occurrence in MMM and PV. The aim of this work was to study the presence of the mutation and another MPL mutation (MPL 515K) in both myeloid and lymphoid lineages in JAK2 V617F negative MMM. We therefore looked for the mutation first in mature myeloid and lymphoid cells and second in lymphoid/myeloid progenitors progeny after CD34+ and CD34+CD38- cell isolation from peripheral blood. Three IMF, patients harbouring MPL mutations were enrolled in this study after informed consent. Peripheral blood granulocytes and platelets were purified by standard methods and B, T, NK cells and monocytes were solated by combined immunomagnetic and flow cytometric procedures. The same techniques were used to sort CD34+ and CD34+CD38- subpopulations from peripheral blood. Clonal B/NK/Myeloid differentiation from CD34+CD38- cells and T cell differentiation from CD34+ cells were performed respectively onto a MS5 layer in the presence of SCF, FLT3L, IL2, IL3, IL7, IL15, TPO and in Fetal Thymic Organ Cultures (FTOC). Genotyping of mature cell populations, B/NK/Myeloid clones and CD34+ derived T cells were performed by direct sequencing. Moreover, CD34+ cells were cultured in a one cell per well experiment to determine the sensitivilty of these patient’s cells to low dose TPO. The MPL mutations were present in granulocytes and platelets from all patients, and in monocytes. We detected the mutation in B and NK cells in all three cases. The MPL 515L and MPL 515K mutations could be subsequently detected in CD34+ cells and in B/NK/Myeloid and/or NK/myeloid CD34+CD38- derived clones from all IMF patients. Interestingly, MPL 515L homozygous clones were detected in B/NK/Myeloid and/or NK/Myeloid clones from 1 MMM patient. Using the FTOC assay, the mutation was also detected in all T cell fractions derived from CD34+ cells from 2 MMM patients. At least we found that MPL mutations induce a spontaneous megakaryocytic growth in cell culture but also a hypersensitivity to low dose TPO. These results demonstrate that the mutation occur in a lymphoid/myeloid progenitor cell in MPL 515L or K MMM and give rise to a hypersensitivity to TPO. Thus, these MPD take their origin in a true lymphoid/myeloid progenitor cell. In accordance with mouse model, this represent a good argument for a causative role of MPL mutations in MMM.

Myeloid Metaplasia with Myelofibrosis (MMM) is a myeloproliferative disorder (MPD) associating ineffective and extramedullary hematopoiesis with progressive splenomegaly, bone marrow fibrosis and neoangiogenesis. The myeloproliferation is characterized by an increased number of circulating CD34+ cells with the prominent amplification of dystrophic megakaryocytes (Mk). We compared the transcriptome of CD34+ and Mk cell from the peripheral blood (PB) of MMM patients and from the PB and bone-marrow (BM) of unmobilized healthy donors. Application of multivariate analyses (principal component analysis and classification by partitioning around medoids algorithm) on Gene Ontology annotation of differential genes allowed a global functionally approach of the main cellular dysregulated pathways in MMM. Each sample cRNA probe was individually and differentially hybridized to the cRNA reference probe on a Human Oligo-microarray 22K (Agilent) and data were normalized by application of the local LOWESS algorithm. Comparison of the CD34+ cell transcriptome between MMM and healthy donors revealed a global down regulation of 2/3 of the expressed genes in contrast to 1/3 of genes that are up-regulated after data filtration by significance analysis microarray (SAM) algorithm (threshold p