WO2019008408A1 - Methods for determining whether a patient suffering from a myeloproliferative neoplasm is at risk of thrombosis - Google Patents

Abstract

Myeloproliferative neoplasms (MPNs) are acquired clonal hematopoietic stem cell disorders, characterized by an increase in one or more myeloid lineages. More than 90% of patients with PV and half of those with ET and PMF carry a mutation in the Janus kinase 2 (JAK2) gene, ie. JAK2V617F. Thrombosis reveals MPN in about 30% of patients and is a major cause of morbidity and mortality. The mechanisms underlying the MPN thrombotic diathesis are still largely elusive. In MPN patients, the inventors found: 1) increased neutrophils CD1 lb expression, 2) increased neutrophils TF expression in patients with history of thrombosis, 3) increased plasma levels of free DNA in all patients and increased plasma levels of MPO-DNA complex in patients with history of thrombosis. Accordingly, the present invention relates to a method of identifying whether a patient suffering from a myeloproliferative neoplasm is at risk of thrombosis comprising determining in a blood sample obtained from the patient whether neutrophils exhibit a hyperactivated state (e.g. by quantifying plasma levels of MPO-DNA by ELISA). The present invention also relates to a method of treating thrombosis in a patient suffering from a myeloproliferative neoplasm comprising administering to the patient a therapeutically effective amount of an anti-NET compound.

Description

METHODS FOR DETERMINING WHETHER A PATIENT SUFFERING FROM A MYELOPROLIFERATIVE NEOPLASM IS AT RISK OF THROMBOSIS FIELD OF THE INVENTION:

The present invention relates to methods and kits for determining whether a patient suffering from a myeloproliferative neoplasm is at risk of thrombosis.

BACKGROUND OF THE INVENTION:

Myeloproliferative neoplasms (MPNs) are acquired clonal hematopoietic stem cell disorders, characterized by an increase in one or more myeloid lineages. The Philadelphia chromosome-negative (Ph-) MPNs include polycythemia vera (PV) with an excess of red blood cells, essential thrombocythemia (ET) with an increase of platelets and primary myelofibrosis (PMF) (Vardiman et al. 2002). More than 90% of patients with PV and half of those with ET and PMF carry a mutation in the Janus kinase 2 (JAK2) gene, ie. JAK2V611F (James et al. 2005, Baxter et al. 2005, Kralovics et al. 2005, Levine et al. 2005). JAK2 is a tyrosine kinase that initiates intracellular signaling of various type 1 cytokine receptors, such as erythropoietin and thrombopoietin receptors (Oh et al. 2010). The JAK2V611F mutation is responsible for a constitutive activation of the JAK2 kinase, resulting in subsequent activation of its downstream signaling pathways, ultimately leading to overproduction of myeloid cells.

Thrombosis reveals MPN in about 30% of patients and is a major cause of morbidity and mortality. The mechanisms underlying the MPN thrombotic diathesis are still largely elusive: platelets were shown to be activated, leading to treatment with antiplatelet treatment (such as aspirin). Nevertheless aspirin-treated patients still develop thrombosis and recent evidences suggest that platelets are not key actors in the pathogenesis of thrombosis in MPN. There is thus a need to target the right actor(s) in order to avoid thrombosis.

Results from large clinical studies identified increased leukocytosis as a risk factor for thrombosis in PV and ET, suggesting that neutrophils may be involved in the pathogenesis of thrombosis. Besides, these last ten years have revealed that neutrophils, in certain settings, can dismantle and expel nuclear DNA, thus forming Neutrophil Extracellular Traps (NETs). These NETs not only trap bacteria but also promote activation of platelets, extrinsic and intrinsic pathways of coagulation and inhibit fibrinolysis, thus promoting thrombosis. Increased NETosis has been reported in various diseases, such as antiphospholipid syndrome (Yalavarthi, 2015), lupus, small vessel vasculitis (Kessenbrock, 2009), thrombotic microangiopathies (Fuchs, Blood, 2012) and even recently cancer (Demers, PNAS, 2012). Using mouse models of cancer, Demers et al demonstrated that cancer prone neutrophils to release NET that contribute to cancer-associated thrombosis.

SUMMARY OF THE INVENTION:

The present invention relates to methods and kits for determining whether a patient suffering from a myeloproliferative neoplasm is at risk of thrombosis. In particular the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The objective of the inventors was to assess if JAK2V617F neutrophils are more activated than JAK2WT neutrophils, emit more NET, thus promoting thrombosis. In MPN patients, they found: 1) increased neutrophils CDl lb expression, 2) increased neutrophils TF expression in patients with history of thrombosis, 3) increased plasma levels of free DNA in all patients and increased plasma levels of MPO-DNA complex in patients with history of thrombosis. In PF4-iCreERT2;JAK2^v617F/WT mice, they observed : 1) proliferation of all hematopoietic lineage, secondary to presence of JAK2V617F in neutrophils, platelets and red blood cells, 2) increased NET formation after neutrophils activation, 3) increased plasma level of free DNA and MMP9, 4) increased pulmonary thrombus formation. Their results show that neutrophils are hyperactivated during MPN, in a patient cohort, and in a mouse model with JAK2V617F expression in neutrophils. Increased NET formation in PF4- iCreERT2;JAK2^v617F/WT model, associated with increased thrombus formation suggests an important role of neutrophils in thrombus formation during JAK2V617F positive MPN.

Accordingly, the first object of the present invention relates to a method of identifying whether a patient suffering from a myeloproliferative neoplasm is at risk of thrombosis comprising i) determining in a blood sample obtained from the patient whether neutrophils exhibit a hyperactivated state and ii) concluding that the patient is at risk of thrombosis when it is determined that neutrophils exhibit a hyperactivated state.

MPNs typically include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). They are a diverse but inter-related group of clonal disorders of pluripotent hematopoietic stem cells that share a range of biological, pathological and clinical features including the relative overproduction of one or more cell types from myeloid origin with growth factor independency/hypersensitivity, marrow hypercellularity, extramedullary hematopoiesis, spleno- and hepatomegaly, and thrombotic and/or hemorrhagic diathesis. An international working group for myeloproliferative neoplasms research and treatment (IWG- MRT) has been established to delineate and define these conditions (see for instance Vannucchi et al, CA Cancer J. Clin., 2009, 59: 171-191), and those disease definitions are to be applied for purposes of this specification.

In some embodiments, the patient harbours one mutation in JAK2. As used herein the term "JAK2" has its general meaning in the art and refers to the Janus Kinase 2 protein. The amino acid sequence of human JAK2 is well known in the art. Human JAK2 sequences are, for example, represented in the NCBI database (www.ncbi.orgwww.ncbi.nlm.nih.gov/), for example, under accession number NP 004963. Typical MPD associated mutation is the JAK2V617F mutation which refers to the point mutation (1849 G for T) in exon 14, which causes the substitution of phenylalanine for valine at codon 617 in the JAK homology JH2 domain. Other examples of JAK2 mutations include exon 12 mutations which can be substitutions, deletions, insertions and duplications, and all occur within a 44 nucleotide region in the JAK2 gene which encompasses amino acids 533-547 at the protein level. The most commonly reported mutations are small in- frame deletions of 3-12 nucleotides with a six nucleotide deletion being the most frequent. Complex mutations are present in one-third of cases with some mutations occurring outside this hotspot region. The N542-E543del is the most common mutation (23-30%), the K537L, E543-D544del and F537-K39delinsL represent 10- 14%, and R541-E543delinsK comprise less than 10% of these mutations. JAK2 exon 12 mutations are located in a region close to the pseudo-kinase domain which acts as a linker between this domain and the Src homology 2 domain of JAK2.

As used herein, the term "thrombosis" has its general meaning in the art and is the process by which an unwanted blood clot forms in a blood vessel. It can occur in a vein or in an artery. Arterial thrombosis is the cause of almost all cases of myocardial infarction and the majority of strokes, collectively the most common cause of deaths in the developed world. Deep vein thrombosis and pulmonary embolism are referred to as venous thromboembolism, which is currently the third leading cause of cardiovascular-associated death. Thus the term "thrombosis" includes inter alia atrophic thrombosis, arterial thrombosis, cardiac thrombosis, coronary thrombosis, creeping thrombosis, mesenteric thrombosis, placental thrombosis, propagating thrombosis, traumatic thrombosis and venous thrombosis.

As used herein, the term "risk" relates to the probability that an event will occur over a specific time period, as in the conversion to thrombosis, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. Alternative continuous measures which may be assessed in the context of the present invention include time to thrombosis conversion and therapeutic thrombosis conversion risk reduction ratios. "Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to thrombosis or to one at risk of developing thrombosis. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of thrombosis, such as alcohol consumption or cigarette smoking, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to thrombosis, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for thrombosis. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for thrombosis. In some embodiments, the present invention may be used so as to discriminate those at risk for developing thrombosis from those having thrombosis, or those having thrombosis from normal.

As used herein the term "blood sample" means a whole blood, serum, or plasma sample obtained from the patient. Preferably the blood sample according to the invention is a plasma sample. A plasma sample may be obtained using methods well known in the art. For example, blood may be drawn from the patient following standard venipuncture procedure on tri-sodium citrate buffer. Plasma may then be obtained from the blood sample following standard procedures including but not limited to, centrifuging the blood sample at about l,500*g for about 15-20 minutes (room temperature), followed by pipeting of the plasma layer. Platelet- free plasma (PFP) will be obtained following centrifugation at about 13,000*g for 5 min. In order to collect or discard the microparticles, the plasma sample may be centrifuged in a range of from about 15,000 to about 20,000*g. Preferably, the plasma sample is ultra-centrifuged at around 17,570*g at a temperature of about 4°C. Different buffers may be considered appropriate for resuspending the pelleted cellular debris, which contains the microparticles. Such buffers include reagent grade (distilled or deionized) water and phosphate buffered saline (PBS) pH 7.4. Preferably, PBS buffer (Sheath fluid) is used. More preferably, the blood sample obtained from the patient is a platelet free platelet sample (PFP) sample. PFP may be separated from 10 ml citrated whole blood drawn from the fistula- free arm, 72 hours after the last dialysis. PFP may be obtained after citrate blood centrifugation at 1500*g (15 min), followed by 13000*g centrifugation (5 min, room temperature).

In some embodiments, the determination of determining in the blood sample obtained from the patient whether neutrophils exhibit a hyperactivated state can be performed by any method well known in the art. For instance, said methods can include or combine measurement of CD1 lb expression level, tissue factor (TF) expression, quantification of circulating cell free DNA level, quantification of DNA-histone complexes, or quantification of DNA-MPO complexes.

As used herein, the term "CD1 lb" has its general meaning in the art and refers to the integrin alpha M (ITGAM) (NCBI amino acid reference sequence: NP 000623).

As used herein, the term "tissue factor" or "TF" has its general meaning in the art and refers to the protein encoded by the F3 gene. The term is also known as CD 142 (NCBI amino acid reference sequence: NP 001171567 or NP 001984).

The expression of level of CD1 lb or TF is determined by any method well known in the art and typically involves flow cytometry. For example, fluorescence activated cell sorting (FACS) may be therefore used to separate in the blood sample the activated neutrophils with a set of antibodies wherein among them are specific for CD1 lb or TF. In another embodiment, magnetic beads may be used to isolate neutrophils (MACS). For instance, beads labelled with monoclonal specific antibodies may be used for the positive selection of neutrophils. Other methods can include the isolation of neutrophils by depletion of non-neutrophils components (negative selection). Accordingly, in a specific embodiment, the method of the invention comprises the steps of obtaining a blood sample as above described; putting said prepared sample into a container; adding both labeled antibodies against surface markers that are specific to neutrophils of interest including antibodies specific for CD l ib or TF, and a known concentration of fluorescent solid surfaces; performing a FACS analysis on the prepared sample in order to calculate the absolute number of activated neutrophils therein. Accordingly, in some embodiments, the method of the present invention comprises i) determining the expression level of CD1 lb or TF in neutrophils, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the patient is at risk of having thrombosis when the expression level determined at step i) is higher than its predetermined reference value.

As used herein, the term "circulating cell-free DNA" has its general meaning in the art and refers to the DNA released by the cell and present in the patient's blood stream. It is easy and routine for one of ordinary skill in the art to determining the level of circulating cell-free DNA in a blood sample obtained from the patient. In particular, the assay described in the EXAMPLE is particularly suitable for determining the level of circulating cell-free DNA. Briefly, circulating cell-free DNA may be quantified by colorimetric or fluorometric assays which are typically performed by adding reagents to the patient's blood sample, which produces a color change, the degree of which correlates with the level of circulating cell-free DNA. Other assays include hemagglutinin inhibition, complement fixation, and diffusion in agarose. Other assays involve RNA-DNA hybridization, RIA, and counter Immunoelectrophoresis assays that allow quantification of nanogram amounts of circulating DNA. With real-time PCR and PicoGreen double-stranded DNA quantification assays, picogram amounts of free DNA can be quantified. Those skilled in the art will readily appreciate various methods to determine the level of circulating cell-free DNA; the methods suggested are merely for purposes of example. Accordingly, in some embodiments, the method of the present invention comprises i) determining the level of circulating cell free DNA ii) ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the patient is at risk of having thrombosis when the level determined at step i) is higher than its predetermined reference value.

As used herein, the term "DNA-histone complexes" has its general meaning in the art and refers to fibrillary networks of DNA, nuclear proteins including histones and granular proteins that are released from neutrophils during a complex cell death signaling pathway termed NETosis. As used herein, the term "DNA-MPO complexes" has its general meaning in the art and refers to fibrillary networks of DNA with myeloperoxidase (MPO). Standard methods for determining the level of these complexes are ELISA (enzyme-linked immunosorbent assay) methods, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize histones or MPO. The blood sample is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled anti-DNA is added. The anti-DNA is allowed to react with any captured sample marker protein, the plate washed and the presence of anti-DNA detected using methods well known in the art. Accordingly, in some embodiments, the method of the present invention comprises i) determining the level of DNA-histone complexes ii) ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the patient is at risk of having thrombosis when the level determined at step i) is higher than its predetermined reference value. In some embodiments, the method of the present invention also comprises i) determining the level of DNA-MPO complexes ii) ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the patient is at risk of having thrombosis when the level determined at step i) is higher than its predetermined reference value.

Typically, the predetermined reference value is a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the marker (e.g. CD1 lb, circulating cell free DNA, DNA-histone complex or DNA-MPO complex) in properly banked historical patient samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after calculating the marker in a group of reference, one can use algorithmic analysis for the statistic treatment of the determined levels in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is Receiver Operator Characteristic Curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc. In some embodiments, high statistical significance values (e.g. low P values) are obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value. Thus, in some embodiments, instead of using a definite predetermined reference value, a range of values is provided. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P value) are retained, so that a range of quantification values is provided. This range of quantification values includes a "cut-off value as described above. For example, according to this specific embodiment of a "cut-off value, the patient's state can be determined by comparing the capacity or level with the range of values which are identified. In some embodiments, a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum P value which is found). For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6. For example, a patient may be assessed by comparing values obtained by determining capacities or levels, where values greater than 5 reveal that the patient is at risk of thrombosis and values less than 5 reveal that the patient is not at risk of thrombosis. In some embodiments, a patient may be assessed by comparing values obtained by measuring capacities or levels and comparing the values on a scale, where values above the range of 4-6 indicate that the patient is at risk of thrombosis and values below the range of 4-6 indicate that the patient is not at risk of thrombosis, with values falling within the range of 4-6 indicate that further investigation is needed for concluding that the patient is at risk of thrombosis.

The further object of the present invention to a method of treating thrombosis in a patient suffering from a myeloproliferative neoplasm comprising administering to the patient a therapeutically effective amount of an anti-NET compound.

In some embodiments, the patient has been considered at risk of thrombosis by the method of the present invention.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

In some embodiments, the anti-NET compound of the present invention is particularly suitable for the prophylactic treatment of thrombosis.

As used herein, the term "anti-NET compound" refer to any compound that degrades or targets for degradation any component of a NET and/or prevents the formation of NETs (e.g. PAD4 inhibitors). In some embodiments the anti-NET compound can be, but is not limited to; DNase, an antibody (i.e. an antibody to histones or to a particular histone), a histone degrading enzyme (i.e. mast cell proteinase 1 (Gene ID: 1215)), plasmin (Gene ID: 5340), cathepsin D (Gene ID: 1509) or activated protein C (Gene ID: 5624)) or an inhibitor of chromatin decondensation (i.e. staurosporine, HDAC inhibitors (i.e. M344), PAD4 inhibitors, or elastase inhibitors (i.e. Gelin®)). In some embodiments, the anti-NET agent is a PAD4 inhibitor. As used herein, "PAD4" refers to peptidylarginine deiminase 4, an enzyme that converts protein arginine residues to citrulline through a deimination reaction. Small molecule inhibitors of PAD4 are known in the art (see, for example, Luo et al. Biochemistry 2006; U.S. Patent 7,964.636; and U.S. Patent Publications 2007/0276040 and 2011/0142868; each of which is incorporated by reference herein in its entirety). In some embodiments, the anti-NET compound is a DNA-hydrolysing antibody such as described in Kozyr AV, Gabibov AG. DNA- hydrolyzing Ab: is catalytic activity a clue for physiological significance? Autoimmunity. 2009 May;42(4):359-61. In some embodiments, the anti-NET compound is a DNAse. Any suitable DNase may be used in the present invention. The DNase will most preferably be a DNase I (EC 3.1.21.1). It may, however, in some embodiments be a DNase II (EC 3.1.21.1). DNases occur in a number of species and any DNase capable of cleaving DNA may be used in the invention. The DNase may be from an animal source such as of bovine or porcine origin. It may be of plant, fungal, or microbial origin. However, typically and most preferably the DNase is of human origin and is preferably a recombinant human DNase. Commercially available DNase preparations such as Dornase™ and Pulmozyme™ may be used in embodiments of the invention. In some embodiments, the DNase has DNA hydrolytic activity, for example in the case of DNase I it may hydro lyse DNA to give 5 '-phosphate nucleotides and in the case of DNase II it may hydro lyse DNA to give 3' phosphate nucleotides. A fluorescence-based assay using, for example, Hoechst Stain may be used such as that which was detected in Labarce & Paiden, 1980, Anal. Biochem., 102:344-352 to assay for DNA hydrolysis. Hydrolytic activity may be assessed in a variety of ways known in the art including analytical polyacrylamide and agarose gel electrophoresis, hyperchromicity assay (Kunitz, J. Gen. Physiol. 33:349-362 (1950); Kunitz, J. Gen. Physiol. 33:363-377 (1950)) or methyl green assay (Kurnick, Arch. Biochem. 29:41-53 (1950); Sinicropi, et al, Anal. Biochem. 222:351-358 (1994)). The breakdown of DNA molecules from high to lower molecular weight forms may be monitored preferably by techniques such as agarose gel electrophoresis. Control experiments in the absence of the enzyme and/or in the presence of a protein known to possess DNase activity may be performed. DNases are known to be prone to deamidation. Asparagine residues and in particular the asparagine at amino acid positions 7 and 74 of the mature human DNase I are prone to deamidation. This process converts the asparagine residues in question to aspartic acid or iso-aspartate residues. Deamidation reduces the activity of the enzyme and this is particularly the case for deamidation at the asparagine at amino acid position 74 of mature human DNase I. Techniques are available for removing deamidated forms of the enzymes to leave the amidated forms and these may be employed to prepare DNases for use in the invention. These techniques may include tentacle cation exchange (TCX) or affinity purification using DNA to purify the amidated forms of the enzyme which are still capable of binding DNA. A DNase preparation for use in the invention may typically comprise from 85 to 100%, preferably from 90 to 100%), more preferably from 95 to 100% and even more preferably from 99 to 100%) amidated, or partially amidated, enzyme by weight. In particular these figures refer to the amount of wholly amidated enzyme i.e. with all of the residues which are naturally amidated being amidated. They will typically have more than 95%>, preferably more than 99%> and even more preferably more than 99.9% of the DNase in an amidated form. In particular, these values refer to values at the time of production or to their values from one month to a year, preferably from two to six months and more preferably from three to four months after production. They may refer to the value during any point of the shelf-life of the product. Variants of naturally occurring or known DNases may be used in the invention. Thus the term DNase encompasses such variants. The term "variants" refers to polypeptides which have the same essential character or basic biological functionality as DNase. The essential character of a DNase is phosphodiesterase activity and the ability to hydrolyse DNA. Assays for measuring DNA cleavage are described herein and these may be used to determine whether a variant has hydrolytic activity. The sequence of the DNase may be modified so that it has extended half- life. For example the sequence of the enzyme may be changed to remove recognition sequences for certain proteases and in particular those derived from inflammatory cells. The DNase employed in the invention may also be chemically modified to alter its properties such as, for example, its biological half-life. To achieve this covalent modifications may be introduced by reacting targeted amino acid residues of the native or variant DNase with an organic derivatising agent that is capable of reacting with selected amino acid side-chains or N- or C-terminal residues. Suitable derivatising agents and methods are well known in the art. Residues which in particular may be derivatised include cysteinyl residues (most commonly by reaction with a- haloacetates), histidyl residues (by reaction with diethylpyrocarbonate at pH 5.5-7.0), lysinyl and amino terminal residues (by reaction with succinic or other carboxylic acid anhydrides), arginyl residues (by reaction with reagents such as phenylglyoxal, 2,3-butanedione, 1,2- cyclohexanedione, and ninhydrin). Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides or may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. The covalent attachment of agents such as polyethylene glycol (PEG) or human serum albumin to the DNases may reduce their immunogenicity and/or toxicity of the variant and/or prolong its half-life and hence may be used in the invention. In some embodiments of the invention the DNase may be directly conjugated to the glycosaminoglycan or joined through an intermediate molecule.

As used herein, the term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the anti-NET compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the anti-NET compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the anti-NET compound are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the anti- NET compound depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of anti-NET compound employed in the pharmaceutical composition at levels lower than that required achieving the desired therapeutic effect and gradually increasing the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound, which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. An exemplary, non-limiting range for a therapeutically effective amount of an anti-NET compound of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1- 20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.

Typically, the anti-NET compound is administered to the patient in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. For use in administration to a patient, the composition will be formulated for administration to the patient. The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. The compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include, e.g., lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. For example, an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5. An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg/m² and 500 mg/m². However, it will be appreciated that these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials. A pharmaceutical composition of the invention for injection (e.g., intramuscular, i.v.) could be prepared to contain sterile buffered water (e.g. 1 ml for intramuscular), and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of the inhibitor of the invention. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: MPN patients have increased neutrophils activation. A. Neutrophils

CD1 lb expression in MPN patients and in control. B. Neutrophils CD1 lb expression between patients with history of thrombosis and those with not. C. TF expression in MPN patients versus controls. D. TF expression in patients with history of thrombosis and in patients with no history of thrombosis.

Figure 2. MPN patients have increased markers of NETosis. A. DNA concentration in MPN patients and in controls. B. DNA concentration in patients with thrombosis history and in those with not. C. DNA concentration in patients with JAK2V6\1V allelic load higher than 50% and in patients with JAK2V617F allelic load between 20 and 50% and in patients with JAK2V611F allelic load lower than 20%. D. Levels of MPO-DNA complexes in patients and in controls. E. MPO-DNA complexes concentration in patients with history of thrombosis and in patients without.

EXAMPLE:

Material & Methods

Generation and characterization of PF4-iCreERT2;JAK2^{v617F WT} mice

The conditional flexed JAK2 (JAK2V617F/WT) mice were generously provided by J.L

Villeval and have been previously described (Marty et al, 2010). The double-heterozygous PF4-iCreERT2;JAK2^v617F/WT mice were generated by crossing JAK2^V617F/WT mice with PF4- iCreERT2 mice allowing constitutive expression of JAK2^V617F under dependence of PF4 promoter. Littermate PF4-iCreERT2-negative;JAK2^v617F/WT mice were used as controls. Ears of adult mice were genotyped by PCR using the P1/P2 primer set for the wild-type allele and for the JAK2 Flex allele. Haematocrit, hemoglobin level, platelet, and white cells count were determined using an automated counter (scil Vet abc Plus+) on blood collected from the sublingual vein in EDTA containing tubes. For blood and bone marrow flow cytometry analysis in Cre;mT/mG mice, cells were stained with TER-119 APC (BD Biosciences, Ter-119 Clone), CD42-APC (BioLegend), and Ly6-APC (BD Biosciences, RB6-8C5 clone). FACs analysis was performed using an Accuri C6 flow cytometer (BD Biosciences). Data were interpreted using BD Accuri C6 Analysis Software.

Histopathology analysis Spleens were fixed in 4% neutral buffered formalin and embedded in paraffin. 7 μηι- thick spleen sections were stained with hematoxylin, eosin and safran and images were taken using a Nikon Microphot-FXA microscope with a xlO objective, captured with a Axio Cam HR digital camera (Zeiss, Germany), and analyzed using ZEN imaging software (Zeiss).

Mouse model of thrombus formation

10 weeks-old PF4-iCreERT2;JAK2V617F^v617F/WT mice and PF4-iCreERT2- negative;JAK2WT^v617F/WT mice were utilised for experiments. We studied spontaneous thrombus formation. Mice were anesthetized with isoflurane and blood was obtained by sublingual sampling in polypropylene Eppendorf tubes containing 5μί of EDTA in order to perform blood count. After euthanasia, an incision was performed in thoracic wall to expose mice heart and lungs were washed with intra-cardiac perfusion of PBS without CaC12 and MgC12 (Gibco ThermoFisher Scientific) during 3 minutes. Lungs were fixed with secondary three-minutes injection of 10% formalin and collected before formalin fixation and paraffin embedding.

Thrombus formation quantification

To quantify thrombus formation in mice, Carstair's staining was performed. Slides were hydrated in xylol and ethanol to distilled water, followed by incubation in 5% ferric ammonium sulfate for 5 min, washing, and staining by Mayer hematoxylin for 5 min, washing, and Picric Acid-orange G solution for 1 hour, and washing, 1% phosphotungstic acid for 10 min. After washing, slides were stained by Ponceau Fuchsin solution for 7 min, washing, 1% phosphotungstic acid for 10 minutes, Anilin blue solution for 30 min, and rinsed in distilled water. Slides were dehydrated covered with a coverslip using mounting medium. Thrombus quantification was performed using an optical microscope and pulmonary area was quantitated to perform the following ratio: thrombi number / pulmonary area (number of thrombi/cm2). Three slides by mice were analysed by two independent observers, blindly.

ELISA for MMP9 quantification

Mice were anesthetized, and blood was obtained by retro-orbital venous plexus sampling in polypropylene Eppendorf tubes containing heparin. Plasma was prepared by centrifugation of the blood at 2000 g for 30 min at room temperature. Enzyme-linked immunoabsorbent assay (ELISA) was performed according to manufacturer's instructions (Biosensis).

Plasma free DNA quantification in mice and patients

To perform mice DNA quantification, mice were anesthetized, and blood was obtained by retro-orbital venous plexus sampling in polypropylene Eppendorf tubes containing citrate. Plasma was prepared by two centrifugation of the blood at 2400 g for 10 min at room temperature. DNA level was determinate using fluorescent probe and detection of fluorescence at 520nm after excitation at 480 nm (Quant-It PicoGreen dsDNA, Fisher Scientific). 50μί of plasma per mice was used, in duplicate. Method was similar with patients, except using 150μί of plasma.

MPO-DNA complexes quantification in mice and patients

We performed MPO-DNA complexes quantification using homemade double-sandwich ELISA. Mice were anesthetized, and blood was obtained by retro-orbital venous plexus sampling in polypropylene Eppendorf tubes containing citrate. Before deposit of plasma, high affinity 96 wells plate was coated with anti-MPO antibody (AbD Serotec, 4A4 clone) overnight at +4°c, before washing and saturation using DuoSet ELISA Ancillary Reagent kit (RD Systems). Then, we added 40 of plasma in association with blocking reagent during 2 hours at room temperature. We next added anti-DNA antibody (Roche, MCA-33 clone) coupled to peroxydase during 2 hours following manufacturer's instructions (Cell Death Detection ELISA Plus, Sigma-Aldrich). Finally, tetramethylbenzidine (TMB), peroxydase substrate, was added for 15 minutes before stopping reaction with sulfuric acid. We next measured absorbance at 450 nm. All samples were analyzed in duplicate.

NETs quantification in mice

Mice were anesthetized, and blood was obtained by retro-orbital venous plexus sampling in polypropylene Eppendorf tubes containing EDTA. Blood Neutrophils isolation was performed using EasySep Mouse Neutrophil Enrichment Kit (StemCell), following manufacturer's instructions. Isolated neutrophils were allowed to adhere and incubated in presence or absence of phorbol 12-myristate 13 -acetate (PMA, Sigma-Aldrich) at the concentration of 50nM during 15 hours at 37°C. After fixation with paraformaldehide (PFA) during 1 hour and saturation with PBS containing 5% of bovine serum albumin (BSA), NETs were stained with primary antibody anti DNA-Histone HI (EMD Millipore, IgG2A isotype, reference: MAB3864) resolved with Alexa Fluor 588 conjugated secondary antibody (Invitrogen, Carlsbad, California). Cells were mounted in Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, California), imaged with a fluorescent microscope (AxioObserver, Zeiss) and analysed by ZEN imaging software (Zeiss). Number of neutrophils emitting NETs were analysed and a ratio between neutrophils emitting NETs and total number of neutrophils was performed.

Neutrophils flow cytometry analysis in patient samples Blood samples were collected and deposited in polypropylene Eppendorf tubes containing EDTA. After lysis of red blood cells, neutrophils were stained using PE-Mouse anti- Human CD1 lb (BD Pharmingen, ICRF44 clone) and PE-Mouse anti-Human Tissue Factor.

Results

V//' V patients have increased neutrophils activation

To study neutrophils activation in a cohort of patients with MPN, we first analyzed membrane bound CD1 lb at their surface, using flow cytometry. CD1 lb, from beta-2-integrin family, is expressed at the surface of neutrophils, monocytes and natural killer (NK) cells. Playing a role in leukocyte adhesion, migration, oxy dative metabolism, CD l ib protein expression is associated with cell activation. We first showed that neutrophils CD l ib expression was increased in MPN patients than in control (Figure 1A). We did not find any difference of neutrophils CD l ib expression between patients with history of thrombosis and those with not (Figure IB). We also analyzed expression of tissue factor (TF) at the surface of neutrophils using flow cytometry. TF is principally expressed at the surface of platelets, but also at the surface of endothelial cells, monocytes and neutrophils and is the main activator of coagulation extrinsic pathway. We showed that TF expression was not increased in MPN patients versus control (Figure 1C), but was significantly higher in patients with history of thrombosis than in patients with no history of thrombosis (Figure ID).

V//' V patients have increased markers ofNETosis

Circulating free DNA is associated with neutrophils extracellular traps (NETs) formation (Hamaguchi, 2015). To evaluate NETosis phenomenon in MPN patients, we first quantified free DNA concentration in plasmas. We observed that DNA concentration was significantly increased in MPN patients than in controls (Figure 2A) with no difference between patients with thrombosis history and those with not (Figure 2B). Moreover, DNA concentration was increased in patients with JAK2V6\1V allelic load higher than 50% than in patients with JAK2V617F allelic load between 20 and 50% and patients with JAK2 ^r617F allelic load lower than 20%) (Figure 2C). Circulating DNA possibly being reflect of necrosis or apoptosis, we needed a specific marker of NET formation in MPN patients. Myeloperoxydase (MPO) is a specific neutrophil enzyme, secreted during exocytosis of granules and associated with DNA during NET emission. Using detection of MPO-DNA complexes in patient plasmas, we showed that MPN patients do not have increased levels of MPO-DNA complexes than controls (Figure 2D) but, interestingly, MPO-DNA complexes concentration was significantly increased in patients with history of thrombosis than patients without (Figure 2E). Those data indicate that patients with MPN have neutrophils activation associated with increased NETosis. PF4-iCre2;JAK2^v617F/WT mice is a reliable model to investigate consequences of JAK2V617F mutation in neutrophils

To analyze consequences of presence of JAK2V 617F mutation in neutrophils, we chose to cross PF4-iCre mice with conditional flexed JAK2 ( JAK2 V617F/WT) mice. PF4-iCreERT2 mice is a mouse model using Cre-loxP technology, with constitutive expression of Cre under dependence of platelet factor 4 promoter (Tiedt, Blood, 2007), initially used for megakaryocytes and platelets studies. But, as previously described (Calaminus et al), using PF4-iCreERT2;JAK2^v617F/WT mice, we observed apparition of hematopoietic proliferation 10 weeks after birth, not only concerning platelets, but also red blood cells, leukocytes and neutrophils. These multi-lineage proliferation was in agreement with apparition of splenomegaly and spleen architecture disturbances in PF4-iCre;JAK2^v617F/WT mice. To analyze more precisely Cre-recombination in hematopoietic cells and assure lineage tracing of this recombination, we crossed PF4-iCre;JAK2^v617F/WT mice with mT/mG mice to generate PF4- iCre;mT/mG;JAK2^v617F/WT mice, permitting coexpression of JAK2V 617F and GFP in cells expressing the Cre recombinase. We first performed flow cytometry analysis of blood compartment and showed that GFP was, as expected, expressed in CD42 positive cells (platelets) with no difference between control mice and PF4-iCre;JAK2^v617F/WT mice. GFP was also expressed in Terl 19 positive cells (red blood cells) with a significant difference between control mice and PF4-iCre;JAK2^v617F/WT ice. Finally, GFP was expressed in Ly6 positive cells (neutrophils) in control mice (11,9 +/- 2,7%) and PF4-iCre;JAK2^v617F/wr mice (67+/-l l%), testifying of presence of JAK2V611F in all hematopoietic lineages. Then, we analyzed bone marrow compartment and showed that GFP was also expressed in megakaryocytes (CD42 positive cells), erythroblasts (Terl 19 positive cells) and granulocytic precursors (Ly6 positive cells) in control mice and PF4-iCre;JAK2^v617F/WT mice. Finally, we wanted to analyze if Cre- recombination occurred in early primitive progenitors or hematopoietic stem cells (HSCs). We showed that a weak proportion of cells was GFP positive in compartment containing early progenitors: Lin-Sca+cKit+ (LSK) compartment and CD150+CD244-CD48- compartment (Signaling Lymphocyte Activation Molecule (SLAM) family markers), indicates that recombination occurs in hematopoietic stem cells in P F4-iC re ;JAK2^V617F/WT mice. Moreover, GFP positive cells were present in megakaryocyte-erythrocyte progenitor (MEP) cell populations, common myeloid progenitor (CMP) cells population and granulocyte myeloid progenitor (GMP) cell populations.

In conclusion, we demonstrate that in PF4-iCre;JAK2^v617F/WT mice, Cre-recombination is not restricted to the megakaryocytic/platelet lineage but is also present in HSCs, primitive progenitor cell compartments and circulating cells. More, we show that PF4- iCreERT2;JAK2^v617F/WI mice is a good model to study consequences of JAK2N6\1F mutation in neutrophils, with mild-severe myeloproliferative disease mimicking disease found in patients and the presence of JAK2N6\1F mutation in neutrophils.

PF4-iCre2;JAK2^v617F/WT mice model have neutrophils activation and increased NETs formation

We then investigated neutrophils activation status in PF4-iCre;JAK2^v617F/WT mice. We analyzed matrix metallopeptidase 9 (MMP9) neutrophils membrane expression using flow cytometry and found increased MMP9 expression in PF4-iCre;JAK2^v617F/WT mice than in controls. As performed in MPN patients, we next analyzed DNA concentration in plasmas of mice, in order to indirectly evaluate NET formation. We found that DNA concentration was increased in PF4-iCre;JAK2^v617F/WT mice than in control mice. To rule out the possibility of neutrophils activation by other JAK2V611F mutated hematopoietic cells, we performed neutrophils extracellular traps (NETs) quantification in neutrophils isolated from control and VF4-iCre;JAK2^v617F/WTmice. In absence of neutrophils activation by phorbol myristate acetate (PMA), NETs emission was similar between VF4-iCre;JAK2^v617F/WTmice and control. But, after neutrophils activation, we showed that NET emission was increased in neutrophils isolated from VF4-iCreERT2; JAK2^v617F/WTmice. Finally, we analyzed spontaneous pulmonary thrombus formation. Small spontaneously formed thrombi were observed in the lungs of PF4- iCreERT2; JAK2^V617F/WT mice but not in control mice. Altogether, these results demonstrated that neutrophils are activated in PF4-iCreERT2; JAK2^V617F/WT mice, emit more NETs and have a prothrombotic phenotype.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

1. A method of identifying whether a patient suffering from a myeloproliferative neoplasm is at risk of thrombosis comprising i) determining in a blood sample obtained from the patient whether neutrophils exhibit a hyperactivated state and ii) concluding that the patient is at risk of thrombosis when it is determined that neutrophils exhibit a hyperactivated state.

2. The method of claim 1 wherein the patient harbours one mutation in JAK2

3. The method of claim 1 the patient harbours the JAK2V617F mutation.

4. The method of claim 1 which includes or combines measurement of CD1 lb expression level, tissue factor (TF) expression, quantification of circulating cell free DNA level, quantification of DNA-histone complexes, or quantification of DNA-MPO complexes.

5. The method of claim 1 which comprises i) determining the expression level of CD1 lb or TF in neutrophils, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the patient is at risk of having thrombosis when the expression level determined at step i) is higher than its predetermined reference value.

6. The method of claim 5 wherein the expression of level of CD1 lb or TF is determined by flow cytometry.

7. The method of claim 1 which comprises i) determining the level of circulating cell free DNA ii) ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the patient is at risk of having thrombosis when the level determined at step i) is higher than its predetermined reference value.

8. The method of claim 7 wherein the circulating cell-free DNA is quantified by colorimetric assays, fluorometric assays or RT-PCR.

9. The method of claim 1 which comprises i) determining the level of DNA-histone complexes ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the patient is at risk of having thrombosis when the level determined at step i) is higher than its predetermined reference value.

10. The method of claim 10 wherein the level of DNA-histone complexes is determined by ELISA.

11. The method of claim 1 which comprises i) determining the level of DNA-MPO complexes ii) ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the patient is at risk of having thrombosis when the level determined at step i) is higher than its predetermined reference value.

12. The method of claim 11 wherein the level of DNA-MPO complexes is determined by ELISA.

13. A method of treating thrombosis in a patient suffering from a myeloproliferative neoplasm comprising administering to the patient a therapeutically effective amount of an anti-NET compound.

14. The method of claim 13 the patient has been considered at risk of thrombosis by the method of claim 1.

15. The method of claim 13 wherein the anti-NET compound is administered for the prophylactic treatment of thrombosis.

16. The method of claim 13 wherein the anti-NET compound is a PAD4 inhibitor

17. The method of claim 13 wherein the anti-NET compound is a DNAse.