Mutated alpha subunit of the Gq protein induces malignant transformation in NIH 3T3 cells.

The discovery of mutated, GTPase-deficient alpha subunits of Gs or Gi2 in certain human endocrine tumors has suggested that heterotrimeric G proteins play a role in the oncogenic process. Expression of these altered forms of G alpha s or G alpha i2 proteins in rodent fibroblasts activates or inhibits endogenous adenylyl cyclase, respectively, and causes certain alterations in cell growth. However, it is not clear whether growth abnormalities result from altered cyclic AMP synthesis. In the present study, we asked whether a recently discovered family of G proteins, Gq, which does not affect adenylyl cyclase activity, but instead mediates the activation of phosphatidylinositol-specific phospholipase C harbors transforming potential. We mutated the cDNA for the alpha subunit of murine Gq in codons corresponding to a region involved in binding and hydrolysis of GTP. Similar mutations unmask the transforming potential of p21ras or activate the alpha subunits of Gs or Gi2. Our results show that when expressed in NIH 3T3 cells, activating mutations convert G alpha q into a dominant acting oncogene.

Regulation by Gi2 proteins of v-fms-induced proliferation and transformation via Src-kinase and STAT3.

We previously showed that Gi2 proteins interfere with the transduction of CSF-1 receptor (CSF-1R) proliferation signals (Corre and Hermouet, 1995). To identify CSF-1R pathways controlled by Gi2, we transfected v-fms, the oncogenic equivalent of CSF-1R, in NIH3T3 cells in which Gi2 proteins were inactivated by stably expressing a dominant negative mutant form of the alpha subunit of Gi2 (alpha i2-G204A). Expression of alpha i2-G204A resulted in decreased Src-kinase activity, delayed activation of p42 ERK-MAPK, decreased cyclin D1 expression and reduced proliferation in response to serum. In alpha i2-G204A cells transfected with v-fms, Src-kinase activity remained deficient but p42 MAPK activity and cyclin D1 expression were similar to those of vector/v-fms cells, suggesting that v-fms bypasses Src to activate the ERK-MAPK cascade. However, DNA synthesis and focus formation were inhibited by up to 80% in alpha i2-G204A/v-fms cells compared to vector/v-fms cells. We found that tyrosine phosphorylation of STAT3, also activated by CSF-1R/v-fms, was inhibited in alpha i2-G204A/v-fms cells; in addition, expression of an 85 kDa, C-terminal truncated form of STAT3 (STAT3 delta) was constitutively increased. Both the inhibition of v-fms-induced STAT3 tyrosine phosphorylation and the increased expression of STAT3 delta were reproduced by transfecting a dominant negative mutant of Src. Last, we show that expression of STAT3 delta 55C, a mutant form of STAT3 lacking the last 55 C-terminal amino acids, is sufficient to inhibit DNA synthesis and v-fms-induced transformation in NIH3T3 cells. In summary, adequate regulation by Gi2 proteins of the activity of both Src-kinase and STAT3 is required for optimal cell proliferation in response to CSF-1R/v-fms.

Mitogenic effects of pertussis toxin-sensitive G-protein alpha subunits: the mitogenic action of alpha i2 in NIH 3T3 cells is mimicked by alpha i1, but not alpha i3.

In fibroblasts and other cell types, pertussis toxin (PTX) inhibits DNA synthesis in response to serum and certain growth factors. GTPase deficient forms of the PTX-sensitive G-protein alpha i2 subunit have been shown to induce partial transformation in fibroblasts. In order to determine whether other PTX-sensitive G-proteins can stimulate mitogenic pathways, we stably expressed constitutively activated G-protein alpha i1 and alpha i3 subunits in NIH 3T3 cells. Expression of activated alpha i1, alpha i2 or alpha i3 results in inhibition of forskolin-stimulated cAMP accumulation in intact cells. Constitutively activated alpha i1, but not alpha i3, induces a loss of contact inhibition, a loss of anchorage-dependence, a reduced serum requirement and a decreased doubling time in NIH 3T3 cells. We conclude that alpha i1 and alpha i2 are both capable of transducing mitogenic signals, but that alpha i3 is not involved in the regulation of fibroblast growth. Furthermore, adenylyl cyclase inhibition is clearly not sufficient to explain the effect of alpha i2 on fibroblast growth.

In vitro and in vivo growth inhibition of murine melanoma K-1735 cell by a dominant negative mutant alpha subunit of the Gi2 protein.

In murine and rat fibroblasts, activation of several G proteins (Gi2, Gq, G12) can stimulate cell growth or transformation, and can induce tumor formation in nude mice; contrastingly, inactivation of Gi2 inhibits fibroblast proliferation in vitro. We investigated whether it is possible to modulate malignant cell growth in vitro and in vivo through alteration of Gi2 protein function. To do so, we introduced mutated alpha subunits of Gi2 (alpha i2) in CL19 cells, a clone of the murine melanoma cell line K-1735. When we did this, a constitutively activated mutant (alpha i2-Q205L) and a dominant negative mutant (alpha i2-G204A) of alpha i2 were stably expressed in CL19 cells. We found that the in vitro motility of all alpha i2-transfected CL19 cells was increased; however, overexpression and alteration of the function of Gi2 did not increase metastasis formation by CL19 cells in nude mice. Expression of alpha i2-Q205L conferred a limited growth advantage to CL19 cells in vitro; in vivo, tumor formation and size, and overall survival of animals injected with CL19 cells expressing alpha i2-Q205L, were similar to controls. In contrast, expression of the inactive alpha i2-G204A mutant inhibited CL19 growth in vitro by at least 50% in all conditions tested, and mice injected with cells expressing the alpha i2-G204A mutant showed delayed tumor formation, reduced tumor size, and longer survival. We conclude that Gi2 proteins contribute to malignant cell growth, and more importantly, that inactivation of Gi2 proteins can inhibit proliferation of melanoma cells and possibly of other malignant cells both in vitro and in vivo.

Cholera toxin resistance associated with deficient adenylate-cyclase activity in a subclone of the rat promyelocytic leukemia (BNML).

The rat promyelocytic leukemia cell line BNML is highly sensitive to cAMP elevating agents, and to cholera toxin (CT) in particular: 99.9% of the cells are killed in less than 48 hr of toxin treatment. We described here a subclone of the same leukemia, which, in contrast, is completely resistant to CT but still sensitive to other cAMP inducers. This locates the defect responsible for CT resistance at the membrane, somewhere between surface CT receptors and adenylate cyclase. CT-resistant BNML cells (CTR-BNML) do have surface CT receptors (several thousands per cell). Adenylate cyclase activity in CTR-BNML cells is not stimulated by cholera toxin. Other GS mediated stimulation of adenylate cyclase (by PGE, isoproterenol, histamine, NaF) remains relatively high, though 25-60% lower than in CTS-BNML cells. These results suggest that a specific adenylate cyclase defect is involved in the resistance of CTR-BNML cells to cholera toxin.

Qualitative and quantitative analysis of human herpesviruses in chronic and acute B cell lymphocytic leukemia and in multiple myeloma.

Real-time quantitative polymerase chain reaction (qPCR) was used to quantify viral loads of human herpesviruses (HHVs) at diagnosis in 61 samples of malignant B cells: 21 chronic lymphocytic leukemia (B-CLL), 29 acute lymphoblastic leukemia (B-ALL) and 11 multiple myeloma (MM); control samples were blasts from 16 acute myeloid leukemia (AML) and 24 blood or bone marrow samples from healthy donors. The majority of samples from healthy donors and patients (B-ALL, B-CLL or AML, but not MM) was positive for EBV and contained <100 ebv copies/100 ng dna. ebv loads were occasionally high (>500 copies/100 ng DNA) in B-ALL (2/16) and in B-CLL (2/21) samples. The fractions of samples positive for HHV-8 and HHV-6A, less than 10% for MM patients, were high for adults with B-ALL (18.8% HHV-8+, 43.8% HHV-6A+) or B-CLL (28.6% HHV-8+, 52.4% HHV-6A+). B-ALL, B-CLL and MM samples were rarely positive for HHV-6B and HHV-7. Lastly, 75% of B-ALL samples were positive for CMV, and CMV loads were significantly higher in B-ALL samples than in MM, B-CLL or AML samples. We also used PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOP) to look for novel HHVs in B cell samples: no sequence indicative of a new HHV was detected. Altogether, the data indicate that the presence of multiple HHVs, including EBV and CMV at high loads, is not rare in B-ALL and B-CLL cell samples. Future prospective studies should determine whether patients with high EBV/CMV loads at diagnosis in tumor samples face a higher risk of delayed hematological recovery, virus-related complications or relapse.

Positive regulation of human T cell activation by Gi2 proteins and interleukin-8.

We investigated whether pertussis toxin (PT)-sensitive heterotrimeric Gi proteins (Gi1, Gi2, Gi3) are involved in the regulation of TCR-induced activation of human T cells. First, Gi proteins were inactivated by PT: pretreatment with PT of purified blood T lymphocytes before CD3 cross-linking inhibited cell proliferation (-71.1 +/- 22.0%, P < 0.001), production of interleukin-2 (IL-2; -47.3 +/- 12.6%, P = 0.008), and expression of CD25 (-24.6 +/- 11.7%, P < 0.001) and CD69 (-25.7 +/- 9.0%, P < 0.001). Then, to identify which of the three Gi was involved, Gi1, Gi2, and Gi3 proteins were specifically inactivated by stably transfecting dominant-negative mutated forms of their alpha subunit in Jurkat cells. After activation, IL-2 production and CD69 expression were inhibited only in cells expressing inactive Gi2. We then studied the effects of interleukin-8 (IL-8), a CXC-chemokine with receptors coupled to Gi2 and produced in an autocrine fashion by activated T cells. Although its effects varied among donors, exogenous IL-8 stimulated proliferation and CD25 expression (up to, respectively, 200 and 77%) of PB T lymphocytes in response to CD3 activation, in a PT-sensitive manner. IL-8 also stimulated IL-2 production (by up to 42%) and CD69 expression, although weakly (+27%). Anti-human IL-8 antibody inhibited proliferation (-43%) and CD25 up-regulation (-45%) of activated T lymphocytes. In summary, several major responses of human T lymphocytes to TCR-mediated activation are regulated by Gi2 proteins, which for this function can be activated by IL-8 in an autocrine manner.

Specific expression of heterotrimeric G proteins G12 and G16 during human myeloid differentiation.

To evaluate expression of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) in human myeloid cells, we studied expression at the protein level of their alpha subunits (G alpha, the subunits responsible for the name and specificity of G proteins) in normal human myeloid progenitors and mature blood cells. We found that G alpha(s), G alpha(i2), and G alpha(q/11) proteins were expressed at high levels at all stages of granulomonocytic and erythroid differentiation, whereas expression of G alpha12 and G alpha16 proteins in normal myeloid cells was lineage-specific. G alpha12 proteins were expressed in erythroid progenitors, monocytes, and platelets, but not in normal granulocytic cells. This lineage specificity was lost in leukemic cells: G alpha12 proteins were found in human leukemic cells of both granulocytic and erythroid lineages. G alpha16 proteins were revealed in myeloid cells as two bands (43 and 46 kD), implying that G alpha16 exist in short and long forms. The 43-kD form was predominant in normal granulomonocytic cells, whereas erythroid progenitors and platelets expressed mostly the 46-kD form. Both forms of G alpha16 proteins varied during cell differentiation: in normal hematopoietic cells, G alpha16 protein expression was high in CD34+ cells, then decreased sharply during granulocytic and erythroid differentiation. In leukemic granulocytic HL60 and NB4 cells, downregulation of G alpha16 proteins was an early event (8 hours) in the process of neutrophil differentiation; in contrast, expression of G alpha16 proteins remained high during normal monocytic differentiation and in HL60 cells differentiating into monocytes with phorbol myristate acetate (PMA) or gamma-interferon (IFNgamma). Finally, we found that primary myeloid leukemia blasts, as well as leukemic cell lines, expressed G alpha16 proteins at levels higher than those found in normal CD34+ progenitors. These observations suggest that it would be worthwhile to investigate a possible role for G alpha12 and G alpha16 proteins in the regulation of human myelopoiesis.

Interleukin-8: an autocrine/paracrine growth factor for human hematopoietic progenitors acting in synergy with colony stimulating factor-1 to promote monocyte-macrophage growth and differentiation.

We previously reported that alteration of the function of heterotrimeric Gi2 proteins altered proliferation of murine macrophages in response to colony stimulating factor-1 (CSF-1). Here we show that a Gi2 agonist, C-X-C chemokine interleukin-8 (IL-8), regulates monocyte-macrophage growth and differentiation. In the absence of serum, IL-8 (10 ng/mL) synergized with CSF-1 to stimulate murine monocyte-macrophage proliferation, enhanced proliferation of purified human CD34+ cells and increased the number and size of CSF-1-induced monocyte-macrophage colonies formed by purified CD34+ cells in semisolid medium. Next, as both CD34+ cells and monocyte-macrophages can produce IL-8, we used an anti-human IL-8 antibody to block an eventual activation of IL-8 receptors by autocrine IL-8. Preincubation with anti-human IL-8 antibody (20-40 microg/mL) inhibited the proliferation as well as the monocyte-macrophage colony clonogenicity of purified human CD34+ cells. Hence, in addition to being a powerful neutrophil chemoattractant, IL-8 also acts as an autocrine/paracrine growth factor for human hematopoietic progenitors, promoting the growth and differentiation of cells of monocytic lineage.

Clinical end points for drug treatment trials in BCR-ABL1-negative classic myeloproliferative neoplasms: consensus statements from European LeukemiaNET (ELN) and Internation Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT).

The discovery of somatic mutations, primarily JAK2V617F and CALR, in classic BCR-ABL1-negative myeloproliferative neoplasms (MPNs) has generated interest in the development of molecularly targeted therapies, whose accurate assessment requires a standardized framework. A working group, comprised of members from European LeukemiaNet (ELN) and International Working Group for MPN Research and Treatment (IWG-MRT), prepared consensus-based recommendations regarding trial design, patient selection and definition of relevant end points. Accordingly, a response able to capture the long-term effect of the drug should be selected as the end point of phase II trials aimed at developing new drugs for MPNs. A time-to-event, such as overall survival, or progression-free survival or both, as co-primary end points, should measure efficacy in phase III studies. New drugs should be tested for preventing disease progression in myelofibrosis patients with early disease in randomized studies, and a time to event, such as progression-free or event-free survival should be the primary end point. Phase III trials aimed at preventing vascular events in polycythemia vera and essential thrombocythemia should be based on a selection of the target population based on new prognostic factors, including JAK2 mutation. In conclusion, we recommended a format for clinical trials in MPNs that facilitates communication between academic investigators, regulatory agencies and drug companies.

Reduction of adenylyl cyclase activity by cholera toxin in myeloid cells. Long-term down-regulation of Gs alpha subunits by cholera toxin treatment.

In IPC-81 cells, the adenylyl-cyclase activation by cholera toxin produces an elevation of cAMP that causes a rapid cytolysis. A resistant clone with deficient cholera toxin-induced cyclase activity (yet sensitive to cAMP) showed a rapid decrease in the amount of membrane-bound Gs alpha (42-47 kDa) detectable soon after ADP-ribosylation of these proteins; pertussis toxin-sensitive G proteins (41 kDa) were not affected. Resistant cells showed a rapid decrease of Gs alpha that is consistent with the finding that cAMP did not accumulate in these cells. Cholera toxin treatment of resistant cells had long-lasting effects (several weeks) on the level of Gs alpha in the cell membrane. The duration of Gs alpha decrease does not correspond to the probable life of catalytically active cholera toxin in the cells, and suggests a regulated process more complex than a proteolytic degradation targeted on ADP-ribosylated molecules.

Stable changes in expression or activation of G protein alpha i or alpha q subunits affect the expression of both beta 1 and beta 2 subunits.

G proteins consist of three subunits: alpha, beta and gamma. Four beta subunits have been cloned: beta 1 and beta 4 (36 kDa), and beta 2 and beta 3 (35 kDa). We studied endogenous beta subunits in mouse NIH 3T3 fibroblasts stably expressing high levels of G protein alpha subunits after transfection with cDNAs encoding alpha i1, alpha i2, alpha i3 and alpha q. Immunoblots showed that NIH 3T3 cells express beta 36 and beta 35 subunits; in these cells, beta 35 subunits are four times more abundant than beta 36 subunits. We could detect beta 1 and beta 2 mRNA, but neither beta 3 nor beta 4 mRNA. We found that a stable increase in expression of wild-type alpha i1, alpha i2, alpha i3 or alpha q subunits is always accompanied by an increase in beta 1 and beta 2 mRNA and protein levels. There was no evidence of selectivity for an increase in beta 1 rather than beta 2 subunits depending on the type of alpha subunit overexpressed. However, constitutive activation or inactivation of alpha subunits induced specific changes in beta subunits. Expression of constitutively inactivated alpha i2 subunits was accompanied by an increase in mRNA and protein levels of both beta subunits. In contrast, cells expressing constitutively activated alpha i2 subunits did not show any change in the amount of beta proteins expressed in membranes, despite a significant increase in beta 1 and beta 2 mRNA. We conclude that stable changes in the levels of expression or degree of activation of G alpha subunits affect the level of expression, and possibly the turn-over, of beta subunits, without selectivity among beta 1 and beta 2 subunits.

G alpha16 protein expression is up- and down-regulated following T-cell activation: disruption of this regulation impairs activation-induced cell responses.

The role of heterotrimeric G proteins in T-cell activation is poorly understood. Here we show that in normal, mature human T-cells, expression of G alpha16, the 43 kDa alpha subunit of G16, varies widely, depending on T-cell activation status. Quiescent blood lymphocytes strongly up-regulate G alpha16 after Leuco A stimulation: protein expression of G alpha16 is maximal at day 4, then decreases. Consistently, in human T-cell clones, expression of G alpha16 is high in the first week following activation and decreases rapidly within the second week. In addition, permanent disruption of regulated G alpha16 expression in Jurkat T-cells by stable overexpression of 43 kDa G alpha16 inhibited Leuco A-induced interleukin-2 production, CD69 up-regulation and cell apoptosis (by 58%, 46% and 74%, respectively), suggesting that coordinate regulation of G alpha16 expression is necessary for optimal activation-induced T-cell responses, and that G alpha16 proteins may be involved in the negative regulation of TCR signalling.

High level expression of transfected G protein alpha i3 subunit is required for plasma membrane targeting and adenylyl cyclase inhibition in NIH 3T3 fibroblasts.

The alpha subunits of pertussis toxin-sensitive G proteins Gi1, Gi2 and Gi3 have been shown to inhibit adenylyl cyclase in transfected cells. However, Gi3 has recently been associated with protein transport and localized to the Golgi apparatus in a number of cell lines, rather than to the plasma membrane. We studied NIH 3T3 clones stably expressing different levels of a constitutively activated mutant of the alpha subunit of Gi3 (alpha i3-Q204L). Transfected alpha i3 subunits were localized to the Golgi apparatus in all NIH 3T3 clones. In clones expressing alpha i3-Q204L at high levels, alpha i3 subunits were also localized to the plasma membrane. Those clones which demonstrated expression of alpha i3 at the plasma membrane showed a 40% to 60% inhibition of forskolin-induced cAMP accumulation. Transfected NIH 3T3 clones in which plasma membrane alpha i3 was undetectable, did not show inhibition of forskolin-induced cAMP accumulation. These data suggest that, unless high expression is achieved in transfected cells, alpha i3 is targeted predominantly to the Golgi, not to the plasma membrane, and does not control adenylyl cyclase activity in NIH 3T3 fibroblasts.

On growth regulation of the rat promyelocytic leukemia (BNML): growth inhibition and eradication of clonogenic cells by cholera toxin.

Our recent establishment of several permanent in-vitro cell lines from Brown Norway rat leukemia (BNML) and the development of a clonogenic assay prompted us to undertake detailed studies on the growth control mechanism of a cell type which for several years has served as an animal model for human AML and preclinical studies. So far, these cells have no defined biological regulators but require intricate cellular interactions to sustain their growth. The effects on cell growth and clonogenicity, of agents known to modify the intracellular levels of cyclic nucleotides, were analysed. Here we report that CT binding strongly inhibited cell growth at a wide range of concentrations (10(-6)-10(-14) M) while beta chain pentameric subunits or alpha chain had no effects. Cell growth was inhibited in a dose-dependent manner. The ligand-receptor interactions mediated the alpha chain's transit through the membrane; the adenylate cyclase activation and the rise in c-AMP levels (60 min) resulted in DNA synthesis arrest (5 h), then finally ended in cell death (24-48 h). A significant decrease in the clonal ability of treated cultures was seen. A decrease of up to five logs in the clonogenic cell number was observed after 48 h of toxin treatment (10(-7) M). The growth inhibition of CT were reproduced by several agents (PGE, theophylline, isobutylmethylxanthine) known to raise intracellular c-AMP levels. Data are commented from a biochemical approach to intracellular events controlling the cell growth of this leukemia. The potential interests of c-AMP inducing agents on the eradication of this leukemia by ex-vivo marrow treatments are also considered.

Comparison of four serum-free, cytokine-free media for analysis of endogenous erythroid colony growth in polycythemia vera and essential thrombocythemia.

INTRODUCTION: The assay of endogenous erythroid colony formation (EEC), a characteristic of polycythemia vera and essential thrombocythemia, is not standardized. In this multicentric study, we tested four semisolid, serum-free, cytokine-free media based on either methylcellulose (M1, M2) or collagen (C1, C2) commercialized for the EEC assay. MATERIALS AND METHODS: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26), essential thrombocythemia (19), secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without, or with 0.01 U/ml erythropoietin (EPo). RESULTS: In all four media EEC formation was specific, as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia, nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria, respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo, the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients. CONCLUSION: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay, now part of the new criteria of polycythemia vera.

Interleukin-8 and other agonists of Gi2 proteins: autocrine paracrine growth factors for human hematopoietic progenitors acting in synergy with colony stimulating factors.

We have reviewed the current knowledge on CXC chemokine interleukin-8 (IL-8) and human hematopoiesis, and more generally on agonists of heterotrimeric Gi2 proteins as regulators of human hematopoiesis. It appears that low doses of IL-8, a Gi2-agonist produced in an autocrine fashion by normal hematopoietic progenitors, mature blood cells and leukemic cells, promotes cell survival or/and proliferation in response to hematopoietic cytokines. More importantly, inactivation of the IL-8/Gi2 pathways inhibits CD34+ cell proliferation and colony formation. Similar positive effects on hematopoiesis of other, physiological or pathological, agonists of Gi2 proteins are discussed, as well as the molecular pathways involved and the consequences of activation of other G proteins (Gq, G16) by IL-8 and other Gi2-agonists.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Anti-inflammatory cytokines hepatocyte growth factor and interleukin-11 are over-expressed in Polycythemia vera and contribute to the growth of clonal erythroblasts independently of JAK2V617F.

The V617F activating mutation of janus kinase 2 (JAK2), a kinase essential for cytokine signalling, characterizes Polycythemia vera (PV), one of the myeloproliferative neoplasms (MPN). However, not all MPNs carry mutations of JAK2, and in JAK2-mutated patients, expression of JAK2V617F does not always result in clone expansion. In the present study, we provide evidence that inflammation-linked cytokines are required for the growth of JAK2V617F-mutated erythroid progenitors. In a first series of experiments, we searched for cytokines over-expressed in PV using cytokine antibody (Ab) arrays, and enzyme-linked immunosorbent assays for analyses of serum and bone marrow (BM) plasma, and quantitative reverse transcription-PCRs for analyses of cells purified from PV patients and controls. We found that PV patients over-expressed anti-inflammatory hepatocyte growth factor (HGF) and interleukin-11 (IL-11), BM mesenchymal stromal cells (BMMSCs) and erythroblasts being the main producers. In a second series of experiments, autocrine/paracrine cytokine stimulation of erythroblasts was blocked using neutralizing Abs specific for IL-11 or c-MET, the HGF receptor. The growth of JAK2V617F-mutated HEL cells and PV erythroblasts was inhibited, indicating that JAK2-mutated cells depend on HGF and IL-11 for their growth. Additional experiments showed that transient expression of JAK2V617F in BaF-3/erythropoietin receptor cells, and invalidation of JAK2V617F in HEL cells using anti-JAK2 small interfering RNA, did not affect HGF and IL-11 expression. Thus, anti-inflammatory HGF and IL-11 are upregulated in PV and their overproduction is not a consequence of JAK2V617F. As both cytokines contribute to the proliferation of PV erythroblasts, blocking the c-MET/HGF/IL-11 pathways could be of interest as an additional therapeutic option in PV.