WO2016042137A1 - Method for diagnosing myeloproliferative chronic myelomonocytic leukemia or unclassified myeloproliferative myelodysplastic neoplasm - Google Patents

Abstract

The present invention relates to a method for diagnosing a myeloproliferative disease such as myeloproliferative chronic myelomonocytic leukaemia (MP-CMML) or unclassified- myeloproliferative myelodysplastic neoplasm (U-MPN/MDS).

Description

Method for Diagnosing Myeloproliferative Chronic Myelomonocytic Leukemia or Unclassified Myeloproliferative Myelodysplastic Neoplasm

Related Application

The present application claims priority to European Patent Application No. EP 14 306 448 filed on September 19, 2014. The European patent application is incorporated herein by reference in its entirety.

Field of the Invention

The present invention relates to a method for diagnosing a myeloproliferative disease such as myeloproliferative chronic myelomonocytic leukaemia (MP-CMML) and unclassified-myeloproliferative myelodysplastic neoplasm (U-MPN/MDS).

Background of the Invention

The RAS gene family produces RAS proteins that are involved in cell signalling. When RAS proteins are altered by a mutation in the gene, cells divide uncontrollably and evade signals to die. More than 30% of all human cancers, including a high percentage of pancreatic, lung, and colon cancers, are driven by mutations and possibly amplification of RAS genes.

Downstream of tyrosine kinases (DOK) proteins are substrates of protein tyrosine kinases, acting as negative regulators of cell signaling pathways. Loss of DOKl gene expression has been detected in human lung adenocarcinomas, and mice with a DOKl haploinsufficiency develop lung cancers. Mice lacking both DOKl and DOKl genes (the two first described DOK gene family members) present a myeloproliferative chronic myelogenous leukaemia (CML)-like syndrome. Moreover, the genetic ablation of Dok genes in a BCR/ABL transgenic background accelerates the apparition of the blastic crisis and leukaemia induced by the fusion oncoprotein, BCR-ABL. DOKl and DOK2 adaptor proteins attenuate RAS/ERK and PI3K/AKT dependent- signaling pathways involved in myeloid cell proliferation. Based on these studies, on animal models and data from some solid tumors, DOK genes are now considered as tumor suppressors. However, the mutation status of DOKl and DOKl genes in patients with a chronic myeloproliferative neoplasm (MPN) remains to be defined. Mutations in cell signaling genes have been reported in myeloproliferative neoplasms. Chronic myelomonocytic leukaemia (CMML) belongs to the MPN class.

Upon white blood cells (WBC) count, CMML has been subdivided in a myelodysplatic (MD-CMML) and a myeloproliferative (MP-CMML) subtype. These two subtypes are associated with different gene expression profiles.

Summary of the Invention

The present inventors have found that myeloproliferative disease, such as myeloproliferative chronic myelomonocytic leukaemia (MP-CMML) and unclassified- myeloproliferative myelodysplasia neoplasm (U-MPN/MDS), is associated with a high rate of RAS mutations. More particularly, mutations were found in genes involved in the negative regulation of RAS signaling pathway, DOKl and DOKl genes (DOK for Downstream of tyrosine kinase).

DOKl and DOK2 were originally identified as substrates of the oncogenic tyrosine kinase, BCR-ABL. The present inventors have found that DOKl point mutations occur in a functional protein-protein interaction domain and abolish the binding capacity of this domain. Furthermore, the expression of a DOK2 mutant induces an increase of cell proliferation.

The search of these mutations could therefore be added with benefit to the panel of gene mutations used to classify myeloproliferative neoplasms (MPN). Accordingly, the present invention relates to a method for diagnosing myeloproliferative disease, preferably myeloproliferative chronic myelomonocytic leukaemia (MP-CMML) or unclassified-myeloproliferative myelodysplasia neoplasm (U-MPN/MDS), in a subject, said method comprising a step of detecting a mutation in the DOKl gene in a biological sample obtained from said subject, wherein the presence of a mutation, preferably a mutation present in coding region of exons 4-5 of a DOKl gene, more preferably a L238P mutation, is indicative of myeloproliferative neoplasm.

The present invention also relates to a method for predicting a risk of a subject to transmit a myeloproliferative disease, such as myeloproliferative chronic myelomonocytic leukaemia (MP-CMML) or unclassified- myeloproliferative myelodysplasia neoplasm (U-MPN/MDS), to his progeny, said method comprising a step of detecting a mutation, preferably a mutation present in coding region of exons 4-5 of a DOK2 gene, more preferably a L238P mutation in the DOK2 gene in a biological sample obtained from said subject, wherein the presence of a mutation in the DOK2 gene is indicative of a risk of transmitting myeloproliferative neoplasm.

The present invention also relates to a method for detecting a subject carrying a defective DOK2 gene, wherein said method comprises step of detecting a mutation, preferably a mutation present in coding region of exons 4-5 of a DOK2 gene, more preferably a L238P mutation, in the DOK2 gene in a biological sample obtained from said subject.

The present invention further relates to a DOK2- encoding polynucleotide encoding a functional DOK2 protein for use in therapy, in particular for treating a myeloproliferative chronic myelomonocytic leukaemia or unclassified-myeloproliferative myelodysplasia neoplasm.

Detailed Description of the Invention

Definitions

A "coding sequence" or a sequence "encoding" an expression product, such as a

RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon. The term "gene" means a DNA sequence that encodes or corresponds to a particular sequence of amino acids which comprises all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may be intended for the genomic sequence encoding a protein, i.e. a sequence comprising regulator, promoter, intron and exon sequences.

As used herein, the term "DOK2 gene" denotes the DOK2 gene of any species, especially human, but of also other mammals or vertebrates to which the methods of the invention can be applied. The human DOK2 gene located at 8p21.3 covers a genomic region of 4,988 bases and is composed of 5 exons (5 coding). The transcript is 1870 bp long (NM_003974.2), with a coding sequence of 1239 bps. The encoded DOK2 protein is 412 amino-acid long (NP 003965.2). The DOK2 protein contains a carboxy-terminal part with proline -rich sequences and tyrosine residues that are phosphorylated by a wide range of activated protein tyrosine kinases, a central phosphotyro sine-binding (PTB) domain important for dimerization, and an amino-terminal pleckstrin homology (PH) domain.

The phosphotyrosine-binding (PTB) domain of the DOK2 protein is encoded by the corresponding DNA from nucleotide 445 to nucleotide 738. The corresponding protein is from amino acid 149 to amino acid 246.

By convention, in the present document, the A of the start codon (ATG - position 94 to 96) of the cDNA sequence of DOK2 (NCBI Reference Sequence: NM_003974.2) has been numbered nucleotide 1.

A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et ah, 1989).

The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm (melting temperature) of 55°C, can be used, e.g., 5x SSC, 0.1 % SDS, 0.25 % milk, and no formamide ; or 30 % formamide, 5x SSC, 0.5 % SDS). Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40 % formamide, with 5x or 6x SCC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et ah, 1989, 9.50-9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et ah, 1989 11.7-11.8). A minimum length for a hybridizable nucleic acid is at least about 10 nucleotides, preferably at least about 15 nucleotides, and more preferably the length is at least about 20 nucleotides.

In a specific embodiment, the term "standard hybridization conditions" refers to a Tm of 55°C, and utilizes conditions as set forth above. In a preferred embodiment, the Tm is 60°C. In a more preferred embodiment, the Tm is 65°C. In a specific embodiment, "high stringency" refers to hybridization and/or washing conditions at 68°C in 0.2 X SSC, at 42°C in 50 % formamide, 4 X SSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions. As used herein, an amplification primer is an oligonucleotide for amplification of a target sequence by extension of the oligonucleotide after hybridization to the target sequence or by ligation of multiple oligonucleotides which are adjacent when hybridized to the target sequence. At least a portion of the amplification primer hybridizes to the target. This portion is referred to as the target binding sequence and it determines the target- specificity of the primer. In addition to the target binding sequence, certain amplification methods require specialized non-target binding sequences in the amplification primer. These specialized sequences are necessary for the amplification reaction to proceed and typically serve to append the specialized sequence to the target. For example, the amplification primers used in Strand Displacement Amplification (SDA) include a restriction endonuclease recognition site 5' to the target binding sequence (US Patent No. 5,455,166 and US Patent No. 5,270,184). Nucleic Acid Based Amplification (NASBA), self-sustaining sequence replication (3SR) and transcription based amplification primers require an RNA polymerase promoter linked to the target binding sequence of the primer. Linking such specialized sequences to a target binding sequence for use in a selected amplification reaction is routine in the art. In contrast, amplification methods such as PCR which do not require specialized sequences at the ends of the target, generally employ amplification primers consisting of only target binding sequence. As used herein, the terms "primer" and "probe" refer to the function of the oligonucleotide. A primer is typically extended by polymerase or ligation following hybridization to the target but a probe typically is not. A hybridized oligonucleotide may function as a probe if it is used to capture or detect a target sequence, and the same oligonucleotide may function as a primer when it is employed as a target binding sequence in an amplification primer. It will therefore be appreciated that any of the target binding sequences disclosed herein for amplification, detection or quantification of DOK2 may be used either as hybridization probes or as target binding sequences in primers for detection or amplification, optionally linked to a specialized sequence required by the selected amplification reaction or to facilitate detection.

The terms "mutant" and "mutation" mean any detectable change in genetic material, e.g. DNA, RNA, cDNA, or any process, mechanism, or result of such a change. This includes gene mutations, in which the structure {e.g. DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product {e.g. protein or enzyme) expressed by a modified gene or DNA sequence. Generally a mutation is identified in a subject by comparing the sequence of a nucleic acid or polypeptide expressed by said subject with the corresponding nucleic acid or polypeptide expressed in a control population. A mutation in the genetic material may also be "silent", i.e. the mutation does not result in an alteration of the amino acid sequence of the expression product.

In the context of the instant application, mutations identified in a DOK2 gene are designated according to the nomenclature of Dunnen and Antonarakis Hum. Mutat. (2000).

As defined by Dunnen and Antonarakis, to avoid confusion in the description of a sequence change, preceed the description with a letter indicating the type of reference sequence used: "g." for a genomic sequence {e.g., g.76A>T), "c." for a cDNA sequence {e.g., c.76A>T), "m." for a mitochondrial sequence {e.g., m.76A>T), "r." for an RNA sequence {e.g., r.76a>u), "p." for a protein sequence {e.g., p.K76A).

Substitutions are designated by a ">"-character: - 76A>C denotes that at nucleotide 76 an A is changed to a C

- 88+lG>T denotes the G to T substitution at nucleotide +1 of intron 2, relative to the cDNA positioned between nucleotides 88 and 89 Deletions are designated by "del" after the nucleotide(s) flanking the deletion site :

- 76_78del (alternatively 76_78delACT) denotes a ACT deletion from nucleotides 76 to 78

Duplications are designated by "dup" after the first and last nucleotide affected by the duplication :

- 77-79dup (or 77_79dupCTG) denotes that the nucleotides 77 to 79 were duplicated

Thus, for example, "c. l990G>A" denotes that at nucleotide 1990 of the cDNA sequence a G is changed to a A. The expression "homozygous DOK2 mutation", as used herein, refers to a subject whose two alleles of the DOK2 gene are mutated. The expression thus encompasses both homozygous mutations strico sensu (wherein the same mutation is present on both alleles) and compound heterozygous mutation (wherein each allele presents a different mutation). In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. A "therapeutically effective amount" is intended for a minimal amount of active agent {e.g., DOK2 encoding polynucleotide) which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a mammal is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder.

Diagnostic methods of the invention:

The present invention relates to a method for diagnosing myeloproliferative disease, preferably myeloproliferative chronic myelomonocytic leukaemia (MP-CMML) or unclassified-myeloproliferative myelodysplasia neoplasm (U-MPN/MDS), in a subject, said method comprising a step of detecting a mutation in the DOK2 gene in a biological sample obtained from said subject, wherein the presence of a mutation is indicative of a myeloproliferative chronic myelomonocytic leukaemia or unclassified-myeloproliferative myelodysplasia neoplasm (U-MPN/MDS). Indeed, the inventors have shown that a mutation found in the DOK2 gene is associated with myeloproliferative disease, such as myeloproliferative chronic myelomonocytic leukaemia or unclassified-myeloproliferative myelodysplasia neoplasm. Defective DOK2 is involved in the pathological process of myeloproliferative chronic myelomonocytic leukaemia and of unclassified- myeloproliferative myelodysplasia neoplasm.

The mutation is in the DOK2 gene coding the phosphotyrosine-binding (PTB) domain of a DOK2 protein, said mutation preferably resulting in loss of stable binding of the DOK2 protein to phosphotyrosyl peptides. The mutation is preferably a mutation present in coding region of exons 4-5 of a

DOK2 gene.

The mutation is particularly a mutation of one or more of residues R200, R201, L238, preferably the latter.

Loss of stable binding of the DOK2 protein to phosphotyrosyl peptides may easily be evidenced. For example, Example 4 hereafter entitled "Study of the three-dimensional structure of DOK2" shows how a three-dimensional (3D) structure model can be generated. An in silico assay allows visualising of the alteration of stable binding between DOK2 point-mutated PTB and phosphotyrosyl peptides. Alternatively, plasmid constructs may be prepared and transfected into myeloid cells such as KG- 1 cells such as shown in Example 1.

The present invention more particularly relates to a method for diagnosing myeloproliferative disease such as myeloproliferative chronic myelomonocytic leukaemia (MP-CMML) or unclassified-myeloproliferative myelodysplasia neoplasm (U-MPN/MDS) in a subject, said method comprising a step of detecting a mutation in the DOK2 gene in a biological sample obtained from said subject, wherein the presence of a L238P mutation is indicative of myeloproliferative chronic myelomonocytic leukaemia (MP-CMML) or unclassified-myeloproliferative myelodysplasia neoplasm (U- MPN/MDS).

The terms "biological sample" means any biological sample derived from a subject. Examples of such samples include fluids, tissues, cell samples, tissue biopsies, etc. Preferred biological samples are a cell or tissue sample.

Preferred biological samples are whole blood, serum or plasma. Typically, in the case where the subject is a foetus, the sample may be an amniocentesis sample.

Preferably, the subject according to the invention is a human. The subject may be an adult, a teenager, a child, an infant, a baby or a foetus. The method of the invention may be used in a prenatal method for diagnosing or predicting myeloproliferative chronic myelomonocytic leukaemia or unclassified- myeloproliferative myelodysplasia neoplasm.

The present invention also relates to a method for predicting a risk of a subject to transmit myeloproliferative disease such as myeloproliferative chronic myelomonocytic leukaemia or unclassified-myeloproliferative myelodysplasia neoplasm to his progeny, said method comprising a step of detecting a mutation, preferably a mutation present in the coding region of exons 4-5 of a DOK2 gene, more preferably a L238P mutation in the DOK2 gene, in a biological sample obtained from said subject, wherein the presence of a mutation in the DOK2 gene is indicative of a risk of transmitting a myeloproliferative disease such as myeloproliferative chronic myelomonocytic leukaemia or unclassified- myeloproliferative myelodysplasia neoplasm.

The present invention also relates to a method for detecting a subject carrying a defective DOK2 gene, wherein said method comprises a step of detecting a mutation, preferably a mutation present in the coding region of exons 4-5 of a DOK2 gene, more preferably a L238P mutation, in the DOK2 gene in a biological sample obtained from said subject.

Preferably, the mutation, particularly a L238P mutation, is a mutation which results in DOK2 defective gene.

A DOK2 defective gene results in a defect in the phosphotyrosine-binding (PTB) domain of the DOK2 protein, resulting in a loss of stable binding to phosphotyrosyl peptides. The Defective DOK2 proteins can no longer bind via their PTB domain to tyrosine phosphorylated DOK1 proteins.

In an embodiment, the DOK2 mutation is a mutation which results in a truncated DOK2 protein, a DOK2 mislocalization or in a reduction of DOK2 expression. Typically, the DOK2 mutation is a missense mutation, a donor splice mutation or a truncating mutation. Preferably, the DOK2 mutation is a L238P mutation.

In an embodiment, the DOK2 mutation is heterozygous.

In a preferred embodiment, the DOK2 mutation is homozygous.

In a more preferred embodiment, the DOK2 mutation is homozygous and is a L238P mutation.

In a preferred embodiment, the mutation is a homozygous mutation stricto sensu, wherein both alleles of the DOK2 gene present a L238P mutation.

DOK2 mutations may be detected by analyzing a DOK2 nucleic acid molecule. In the context of the invention, DOK2 nucleic acid molecules include mRNA, genomic DNA and cDNA derived from mRNA. DNA or RNA can be single stranded or double stranded.

DNA may be extracted using any methods known in the art, such as described in Sambrook et al., 1989.

RNA may also be isolated, for instance from tissue biopsy, using standard methods well known to the one skilled in the art such as guanidium thiocyanate-phenol- chloroform extraction.

DOK2 mutations may be detected in a RNA or DNA sample, preferably after amplification.

For instance, the isolated RNA may be subjected to coupled reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a mutated site or that enable amplification of a region containing the mutated site. According to a first alternative, conditions for primer annealing may be selected to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of a particular DOK2 mutation. Otherwise, RNA may be reverse-transcribed and amplified, or DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art. For instance, a cDNA obtained from RNA may be cloned and sequenced to identify a mutation in DOK2 sequence. Actually numerous strategies for genotype analysis are available (Antonarakis et al. , 1989; Cooper et ah, 1991; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site. When a base substitution mutation creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR test for the mutation. Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele- specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele- specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single- stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et ah, 1997). Direct sequencing may be accomplished using any method, including, without limitation, chemical sequencing, using the Maxam-Gilbert method; enzymatic sequencing, using the Sanger method; mass spectrometry sequencing; sequencing using a chip-based technology; and real-time quantitative PCR. Preferably, DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. However several other methods are available, allowing DNA to be studied independently of PCR, such as the rolling circle amplification (RCA), the InvaderTMassay, or oligonucleotide ligation assay (OLA). OLA may be used for revealing base substitution mutations. According to this method, two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the mutation. DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized. Therefore, useful nucleic acid molecules, in particular oligonucleotide probes or primers, according to the present invention include those which specifically hybridize the regions where the mutations are located.

Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides. Preferred probes or primers contain from 10 to 200, particularly from 15 to 100 nucleotides. According to a further embodiment said mutation in the DOK2 gene may be detected at the protein level.

Such a mutation may be detected according to any appropriate method known in the art. In particular a biological sample obtained from a subject may be contacted with antibodies specific of the mutated form of DOK2, i.e. antibodies that are capable of distinguishing between a mutated form of DOK2 and the wild-type protein (or any other protein), to determine the presence or absence of a DOK2 specified by the antibody.

Antibodies that specifically recognize a mutated DOK2 are also encompassed by the invention. The antibodies are specific of mutated DOK2, i.e. they do not cross-react with the wild-type DOK2.

The antibodies of the present invention may be monoclonal or polyclonal antibodies, single chain or double chain, chimeric antibodies, humanized antibodies, or portions of an immunoglobulin molecule, including those portions known in the art as antigen binding fragments Fab, Fab', F(ab')2 and F(v). They can also be immunoconjugated, e.g. with a toxin, or labelled antibodies.

Whereas polyclonal antibodies may be used, monoclonal antibodies are preferred for they are more reproducible in the long run.

Procedures for raising "polyclonal antibodies" are also well known. Polyclonal antibodies can be obtained from serum of an animal immunized against the appropriate antigen, which may be produced by genetic engineering for example using standard methods well-known by one skilled in the art. Typically, such antibodies can be raised by administering mutated DOK2 subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 μΐ per site at six different sites. Each injected material may contain adjuvants with or without pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. This and other procedures for raising polyclonal antibodies are disclosed by Harlow et al. (1988) which is hereby incorporated in the references. A "monoclonal antibody" in its various grammatical forms refers to a population of antibody molecules that contains only one species of antibody combining site capable of immunoreacting with a particular epitope. A monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immuno specific for a different epitope, e.g. a bispecific monoclonal antibody. Although historically a monoclonal antibody was produced by immortalization of a clonally pure immunoglobulin secreting cell line, a monoclonally pure population of antibody molecules can also be prepared by the methods of the present invention.

Laboratory methods for preparing monoclonal antibodies are well known in the art (see, for example, Harlow et al., 1988). Monoclonal antibodies (mAbs) may be prepared by immunizing purified mutated DOK2 into a mammal, e.g. a mouse, rat, human and the like mammals. The antibody-producing cells in the immunized mammal are isolated and fused with myeloma or heteromyeloma cells to produce hybrid cells (hybridoma). The hybridoma cells producing the monoclonal antibodies are utilized as a source of the desired monoclonal antibody. This standard method of hybridoma culture is described in Kohler and Milstein (1975).

While mAbs can be produced by hybridoma culture the invention is not to be so limited. Also contemplated is the use of mAbs produced by an expressing nucleic acid cloned from a hybridoma of this invention. That is, the nucleic acid expressing the molecules secreted by a hybridoma of this invention can be transferred into another cell line to produce a transformant. The transformant is genotypically distinct from the original hybridoma but is also capable of producing antibody molecules of this invention, including immunologically active fragments of whole antibody molecules, corresponding to those secreted by the hybridoma. See, for example, U.S. Pat. No. 4,642,334 to Reading; PCT Publication No.; European Patent Publications No. 0239400 to Winter et al. and No. 0125023 to Cabilly et al.

Antibody generation techniques not involving immunisation are also contemplated such as for example using phage display technology to examine naive libraries (from non-immunised animals); see Barbas et al. (1992), and Waterhouse et al. (1993). Antibodies raised against mutated DOK2 may be cross reactive with wild-type DOK2. Accordingly a selection of antibodies specific for mutated DOK2 is required. This may be achieved by depleting the pool of antibodies from those that are reactive with the wild-type DOK2, for instance by submitting the raised antibodies to an affinity chromatography against wild-type DOK2.

Alternatively, binding agents other than antibodies may be used for the purpose of the invention. These may be for instance aptamers, which are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity . Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consist of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et ah, 1996). Probes, primers, aptamers or antibodies used in the invention may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

The term "labelled", with regard to the probes, primers, aptamers or antibodies of the invention, is intended to encompass direct labelling of the probe, primers, aptamers or antibodies of the invention by coupling {i.e., physically linking) a detectable substance to the probe, primers, aptamers or antibodies of the invention, as well as indirect labeling of the probe, primers, aptamers or antibodies of the invention by reactivity with another reagent that is directly labelled. Other examples of detectable substances include but are not limited to radioactive agents or a fluorophore {e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)). Green Fluorescent Protein (GFP) can also be cited. Examples of indirect labelling include detection of a primary antibody using a fluorescently labelled secondary antibody and end-labelling of a DNA probe with biotin such that it can be detected with fluorescently labelled streptavidin. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl l l, Rel86, Rel88. Kits of the invention

According to another aspect of the invention, the mutation, preferably a mutation present in the coding region of exons 4-5 of a DOK2 gene, more preferably a L238P mutation, is detected by contacting the DNA of the subject with a nucleic acid probe, which is optionally labelled.

Primers may also be useful to amplify or sequence the portion of the DOK2 gene containing the mutated positions of interest.

Such probes or primers are nucleic acids that are capable of specifically hybridizing with a portion of the DOK2 gene sequence containing the mutated positions of interest. That means that they are sequences that hybridize with the portion mutated DOK2 nucleic acid sequence to which they relate under conditions of high stringency.

The present invention further provides kits suitable for determining at least one of the mutations of a DOK2 gene.

The kits may include the following components:

(i) a probe, usually made of DNA, and that may be pre-labelled. Alternatively, the probe may be unlabelled and the ingredients for labelling may be included in the kit in separate containers; and

(ii) hybridization reagents: the kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid- phase matrices, if applicable, and standards.

In another embodiment, the kits may include:

(i) sequence determination or amplification primers: sequencing primers may be pre-labelled or may contain an affinity purification or attachment moiety ; and

(ii) sequence determination or amplification reagents: the kit may also contain other suitably packaged reagents and materials needed for the particular sequencing amplification protocol. In one preferred embodiment, the kit comprises a panel of sequencing or amplification primers, whose sequences correspond to sequences adjacent to at least one of the polymorphic positions, as well as a means for detecting the presence of each polymorphic sequence. In a particular embodiment, it is provided a kit which comprises a pair of nucleotide primers specific for amplifying all or part of the DOK2 gene comprising at least one of mutations that are identified herein, especially a L238P DOK2 mutation.

Preferred primers are those cited hereunder.

Alternatively, the kit of the invention may comprise a labelled compound or agent capable of detecting the mutated polypeptide of the invention {e.g. , an antibody or aptamers as described above which binds the polypeptide). For example, the kit may comprise (1) a first antibody {e.g., attached to a solid support) which binds to a polypeptide comprising a mutation of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

The kit can also comprise, e.g. , a buffering agent, a preservative, or a protein stabilizing agent. The kit can also comprise components necessary for detecting the detectable agent {e.g. , an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instructions for observing whether the tested subject is suffering from or is at risk of developing a myeloproliferative chronic myelomonocytic leukaemia. The present invention also relates to a DOK2- encoding polynucleotide encoding a functional DOK2 protein for use in therapy, particularly for treating a myeloproliferative chronic myelomonocytic leukaemia. A functional DOK2 protein is able to stably binding to phosphotyrosyl peptides. Such a polynucleotide is preferably for use in a patient wherein said patient has been subjected to a method for diagnosing a MP-CMML or U- MPN/MDS in a subject as defined above.

The invention will further be illustrated in view of the following figures and examples.

Figure 1 represents results of phospho-ERK Western-blots of different Green Fluorescent Protein (GFP)-tagged DOK2 constructs in KG-1 myeloid cells.

Figure 2 represents the absolute cell number of WT mouse embryonic fibroblasts as a function of time obtained by flow cytometry after infection by different retroviruses expressing DOK2 expression constructs.

Figure 3 represents the absolute cell number of Dokl-Dok2 double KO mouse embryonic fibroblasts as a function of time obtained by flow cytometry after infection by different retroviruses expressing DOK2 expression constructs.

Figure 4 represents the DOK2 L238P mutation.

Top view of Figure 5 represents the linear structure of the DOK2 molecule and bottom view represents a 3D structural model of the DOK2 PTB domain interacting with DOK1 EMLENLpY phospho-peptide complex.

Examples

Example 1. Effect of L238P mutant DOK2 protein on pervanadate-induced ERK- 1/2 phosphorylation in KG-1 myeloid cells

Culture of KG-1 myeloid cells. KG-1 myeloid cells were cultured in IMDM (Iscove's Modified Dulbecco's Medium; Gibco) with 20% heat-inactivated fetal bovine serum (Eurobio) supplemented with lOOU/ml penicillin (Gibco), 10C^g/ml streptomycin (Gibco).

KG-1 cells were starved in IMDM containing 5% FCS during 2h. Then KG-1 cells were treated by 1 μΜ of sodium pervanadate as previously described by Firaguay G, Nunes JA. Analysis of signaling events by dynamic phosphoflow cytometry. Sci Signal 2009; 2(86): pl3., for 5 minutes at 37°C.

To test the capacity of a L238P DOK2 mutant to attenuate Extracellular signal- Regulated Kinases- 1/2 (ERK-1/2) activation, the KG-1 cells were transfected with different Green Fluorescent Protein (GFP)-tagged DOK2 constructs. These cells were then treated or not with sodium pervanadate (pV), and ERK-1/2 activation was detected by phospho-ERK Western-blot.

More precisely, KG-1 myeloid cells were transfected by nucleofection with plasmids respectively encoding for GFP-tagged wild-type (WT) DOK2, a control DOK2 mutant with a loss-of-function in the PTB domain (RR200-201AA, RR), a L238P point mutant (L238P) or GFP alone (Mock).

2 μg of the previously described plasmids were used by nucleofection using the Amaxa Technology (Solution R, program V-001; Lonza Cologne AG). Transfection efficiency was quantified by flow cytometry (>50 GFP positive cells for all constructs). KG-1 cells were treated or not with sodium pervanadate (pV) at 1 μΜ for 5 minutes at 37°C. After stimulation, cells were submitted to lysis.

For immunoprecipitation, post-nuclear lysates were incubated 10 μg of anti-Dok-1 Ab (Ab8112, Abeam) coupled to protein A-Sepharose for 2 h at 4°C. Western Blots were performed as described in Gerard A, Ghiotto M, Fos C, Guittard G, Compagno D, Galy A, et al. Dok-4 is a novel negative regulator of T cell activation. J Immunol 2009 Jun 15; 182(12): 7681-7689.

Whole cell lysates (WCL) were separated by SDS-PAGE and subsequently immunoblotted for phospho-ERK- 1/2 (p-ERKl/2 WB). The blots were re-probed for β- tubulin expression as a loading control and GFP expression to detect the presence of GFP-DOK2 fusion proteins. Molecular weight markers were reported in the left size of the blots.

Detections were performed with the following antibodies: anti-phospho ERK1/2 (Thr202/Thr204) (#4377, Cell Signaling Technology), anti-ERKl/2 (#9101, Cell Signaling Technology), anti-GFP (#11814450001, Roche), anti-P-tubulin (#2128, Cell Signaling Technology) and anti-Dokl (Ab8112, Abeam). Results

The results are represented in Figure 1. This panel shows representative blots of two independent experiments.

The results show that DOK2 WT overexpression reduced pV-induced ERK phosphorylation in comparison with the control DOK2 mutant RR200-201AA, RR, the L238P point mutant (L238P) and GFP.

The DOK2 L238P mutant was unable to inhibit ERK activation. Accordingly, the DOK2 L238P mutant is not functional.

Conclusion

DOK2 protein was found to be able to block an effector of RAS signaling, ERK activation whereas DOK2 PTB-mutants loose this property to inhibit RAS activation. RAS signaling pathway is frequently up-regulated in myeloproliferative disorders. Since DOK2 PTB mutations inhibit the RAS inhibition, detection of DOK2 mutations allows detection of myeloproliferative cancers. Example 2. Expression of L238P mutant Dok2 molecule increases cell proliferation

Preparation of plasmid constructs. Constructs encoding GFP-tagged full-length DOK2 were described by Gerard A, Favre C, Garcon F, Nemorin JG, Duplay P, Pastor S, et al. Functional interaction of RasGAP-binding proteins Dok-1 and Dok-2 with the Tec protein tyrosine kinase. Oncogene 2004 Feb 26; 23(8): 1594-1598. and Guittard G, Gerard A, Dupuis-Coronas S, Tronchere H, Mortier E, Favre C, et al. Cutting edge: Dok- 1 and Dok-2 adaptor molecules are regulated by phosphatidylinositol 5-phosphate production in T cells. J Immunol 2009 Apr 1; 182(7): 3974-3978.

Standard site-directed mutagenesis techniques (QuickChange Site Directed Mutagenesis Kit, Stratagene) were used to generate DOK2 RR200-201AA (DOK2 RR) and DOK2 L238P from plasmid constructs coding for GFP- or HA-tagged wild-type (WT) DOK2. The Inventors generated DOK2 RR200-201AA introducing base pair changes: c860g, g861c, c863g and g964c coding for two Alanine (A) codons (GCG- GCC) instead of the two Arginine (R) codons (CGG-CGC) in the DOK2 open reading frame (ORF). DOK2 L238P was constructed by introducing a t972c change in the DOK2 ORF. HA-tagged DOK2 WT and mutant sequences were then subcloned into the EcoRI and the BamHI restriction sites of pMSCV-IRES-GFP (pMIG) vector for retroviral infections also known as pFBMoSALF containing the env gene. All sequences were confirmed by direct sequencing before expression in Cos-1 cells and retrovirus generation.

Preparation of mouse embryonic fibroblasts . Dok ^A Dok2^'A double knockout (DKO) mouse strain in 129/Sv genetic background was described previously and wild- type 129/Sv mouse strain was purchased from Charles River Laboratories France (L'Arbresle, France). Pregnant mice were sacrificed at 13.5 days post-coitus. Embryos were dissected from the uterus, and extra embryonic membranes and visceras were subsequently removed. Embryos were washed in PBS, and then head and liver were removed. Tissue were dissociated on a 70 μιη filter, cells were then washed and seed edon culture dishes. MEF were cultured in DMEM (Dulbecco's modified Eagles 's medium; Gibco) with 10% heat-inactivated fetal bovine serum (Eurobio) supplemented with 50 nM p2-mercapto ethanol (Gibco), ImM sodium pyruvate, 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco), and 2mM L-Glutamine (Gibco) and nonessential amino acids (Gibco). The major characteristic of myeloproliferative chronic myelomonocytic leukaemia

(MP-CMML) is an abnormally high rate of cell proliferation.

A cell proliferation assay was designed using the above WT and DOK1/DOK2- deficient mouse embryonic fibroblasts (MEF).

Mouse embryonic fibroblasts (MEFs) from wild- type or Dokl-Dok2 double KO mice were infected by the above retroviruses expressing DOK2 expression constructs (pMIG alone, pMIG Dok2 wt; pMIG Dok2 RR for RR200-201AA and pMIG Dok2 L238P) and were sorted for positive GFP expression.

Infection was implemented as follows. 293T cells were cultured in DMEM (Dulbecco's modified Eagles' s medium; Invitrogen) with 10% fetal bovine serum (Eurobio) were used for expression of HA-tagged wild-type DOK2 and mutants (Dok2 RR, Dok2 L238P) in pMIG vectors. Cells were transfected with Lipofectamine 2000 (Invitrogen™, Life Technologies) according to the manufacturer's instructions. For generation of DOK2 constructs retrovirus, supernatants from 293T cells transfected with previously described pMIG-DOK2 constructs were collected after 24 and 48 hours, filtered and applied to MEFs by 2 rounds of centrifugation. MEFs subsequently infected were at passage 2 to passage 4. For generating MEF cell lines with stable expression of DOK2 WT or mutants (Dok2 RR, Dok2 L238P), cells were grown for 7 to 14 days following retroviral transduction and GFP positive cells were sorted by Cell Sorter.

Stable expression of HA-tagged Dok2 WT and mutants (Dok2 RR, Dok2 L238P) was confirmed by Western blot analysis. Absolute cell counts were performed by flow cytometry. MEFs expressing wild- type or DOK2 mutants were seeded in triplicate and each day trypsinized, washed and counted with CountBright™ absolutes counting beads (Life Technologies), by flow cytometry during 6 days. Flow cytometry analysis was performed on a BD LSR II flow cytometer and cell sorting was executed on a BD FACS Aria III or a BD FACS Aria II. FACS data were analyzed with BD-DIVA Version 6.1.2 software (BD Biosciences).

Results

The results are represented in Figures 2 and 3. (fig. 2: MEFs wt) or Dokl-Dok2 double KO (fig. 3: MEFs Dok DKO) * P<0.05; *** P<0.005 (two-way ANOVA test). The data are representative of 3 independent experiments. Only DOK2 PTB loss-of-function mutants (L238P and RR) induce an increase of cell proliferation in MEFs wt compared to mock- infected cells. DOK2 wt is acting as a cell proliferation attenuator in the absence of endogenous DOK proteins (MEFs Dok DKO).

Conclusion:

When the PTB domain of DOK2 protein is not functional (no binding), the expression of this point-mutated DOK2 protein induces a hyperproliferation in cellular models.

Detecting a mutation in a DOK2 gene, particularly a L238P mutation from a subject, is therefore indicative of a myeloproliferative disease such as a myeloproliferative chronic myelomonocytic leukaemia.

Example 3. Identification of gene mutations in bone marrow samples of MD- CMML, MP-CMML, and U-MPN/MDS patients

Mutations in cell signaling genes have been reported in chronic myeloproliferative neoplasms (MPNs). Chronic myelomonocytic leukemia (CMML) belongs to the MPN class. Upon white blood cell count, CMML has been subdivided in myelodysplasia (MD-CMML) and myeloproliferative (MP-CMML) subtypes. These two subtypes are associated with different gene expression profiles.

Gene mutations in bone marrow samples from 30 MD-CMML (i.e., myelodysplasia form of chronic myelomonocytic leukemia) patients, 36 MP-CMML (i.e., myeloproliferative form of chronic myelomonocytic leukemia) patients and 2 U- MPN/MDS (i.e., unclassified myeloproliferative myelodysplasia neoplasm) patients were analyzed.

A total of 68 samples, including 66 cases with CMML (30 MD-CMML and 36 MP- CMML patients) and 2 U-MPN/MDS patients were analyzed. Bone marrow samples were obtained at diagnosis or after diagnosis free of disease.

Nucleic acid extraction was done as previously described by Gelsi-Boyer V, Trouplin V, Adelaide J, Bonansea J, Cervera N, Carbuccia N, et al. Mutations of polycomb-associated gene ASXL1 in myelodysplasia syndromes and chronic myelomonocytic leukaemia. Br J Haematol 2009 Jun; 145(6): 788-800. Genes were selected for their known involvement in leukemo genesis. DNA

Sanger-sequencing of exon-coding sequences of CBL (exons 8, 9), JAK2 (exon 14), NRAS (exons 1, 2), KRAS (exons 1, 2), DOKl (exons 1-5), DOK2 (exons 1-5), FLT3 (exons 14,15,20), NF1 (all exons), PTPN11 (exons 3,11), RUNX1 (exons 1-8), TET2 (exons 3-11), IDH1/2 (exon 4), DNMT3A (exons 15-23), ASXL1 (exon 12), EZH2 (all exons), SUZ12 (exons 14-18), SRSF2 (exon 2), SF3B1 (exons 12-16), U2AF1 (exons 2-6) and ZPvSR2 (exon 2).

Primers used were previously described by Gelsi-Boyer V, Trouplin V, Adelaide J, Bonansea J, Cervera N, Carbuccia N, et al. Mutations of polycomb-associated gene ASXL1 in myelodysplasia syndromes and chronic myelomonocytic leukaemia. Br J Haematol 2009 Jun; 145(6): 788-800 and Rocquain J, Carbuccia N, Trouplin V, Raynaud S, Murati A, Nezri M, et al. Combined mutations of ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 and WT1 genes in myelodysplasia syndromes and acute myeloid leukemias. BMC Cancer 2010; 10: 401.. The DOKl and DOK2 primers used for amplified DNA sequencing are described in Table 1 below. Table 1. Primers used for DOK1 and DOK2 gene sequencing

DNAs were amplified in a total volume of 25 μΐ_^ PCR mix containing at least 5 ng template DNA, Taq buffer, 200 μιηοΐ of each deoxynucleotide triphosphate, 20 pmol of each primer, and 1 U of Hot Star Taq (Qiagen). PCR conditions were 95 °C 10 minutes; 95°C 30 seconds, variable temperature 30 seconds, 72°C 30 secconds to 1 minute depending on PCR product length for 35 cycles, 72°C 10 minutes. PCR products were purified using Millipore plate MSNU030. Two μL· of the purified PCR products were used for sequencing using the Big Dye terminator vl.l kit (Applied Biosystems) including the forward or reverse primer. After G50 purification, sequences were loaded on ABI 3130x1 and 3730 automats (Applied Biosystems). The sequence data files were analyzed using SeqScape software. The genetic alterations identified were cross- referenced to information from the Ensembl Genome Browser (http://www.ensembl.org) and all mutations were confirmed on an independent PCR product.

Mutations previously reported in CMML patients (such as NRAS, CBL, PTPN11, FLT3, JAK2, NF1 genes) were present in the above MP-CMML cohort.

Somatic mutations in DOK1 and DOK2 genes were analyzed. Genomic DNA from bone marrow (BM) cells was amplified with 6 primer pairs covering the entire coding region (exon 1-5) of each DOK gene. Point mutations were identified in both DOK genes.

For DOK1, two variants were found; L60Q in exon 1 coding for a functional protein-lipid interaction domain, a pleckstrin homology domain, 10 and D263E in exon 5.

For DOK2, four variants (2 x R201H, L238P and R215H) were found in 3 out of the 66 CMML patients and 1 out of 2 unclassified myeloproliferative myelodysplasia neoplasm patients. These DOK2 point mutations are located in exon 4 and the 5' end of exon 5 that code for the phosphotyrosine binding (PTB) domain of the DOK protein. Sorted CD3+ lymphocytes of peripheral blood from the MP-CMML patient with a DOK2 L238P mutation were available.

The DOK2 L238P mutation was present in myeloid cells but not in lymphoid cells. The results are represented in Figure 4. The DOK2 L238P mutation is provided by a c.713T>C substitution.

Example 4. Study of the three-dimensional structure of DOK2

A three-dimensional (3D) structure model was generated as follows: A 3D model of the DOK2 PTB - EMLENLpY phospho-peptide complex was generated by 3D homology. The 1UEF (PDB code) structure of the DOK1 PTB - RET complex was used as representative 3D structure of the complex. Chains B and D from the 1UEF structure were deleted (with their water molecules associated) using SYBYL XI.3 and the resulting DOK1 PTB - RET structure was aligned with the 2DLW structure of the PTB DOK2 protein (rmsd of 4.7A for the two monomers). The RET sequence TWIENKLpY (SEQ ID 25) was then mutated by the DOK1 PH-PTB phospho-peptide EMLENLpY (SEQ ID 26) sequence and energy minimize the resulting model (using the torsion module from SYBYL XI .3).

The three-dimensional (3D) structure model revealed that the P238L substitution alters the structure of the DOK2 PTB domain, resulting in a loss of stable binding to phosphotyrosyl peptides. The results are represented in Figure 5.

The top view represents the linear structure of the DOK2 molecule, containing an N-terminal PH domain, a central PTB domain and several phosphorylable tyrosines (Y) residues in the C-terminal part. The L238P point mutation (asterisk) is located in the PTB functional domain. The bottom view represents a 3D structural model of the DOK2 PTB domain (large ribbon) interacting with DOKl EMLENLpY phospho-peptide (short thin ribbon having a form of letter L in the middle of the figure) complex.

The L238P single mutation affects the 3D conformation of the protein, bending the helix a2, thus preventing the clamp between the C-terminal part of the DOK2 protein and the phospho-peptide.

In cancer cells, dysregulated cell signaling and proliferation may occur through overexpression or mutation of proto-oncogenes. RAS signaling pathway is frequently up-regulated in cancer cells leading to a hyperproliferation. DOK2 is a member of the panel of genes that are involved in the regulation of the RAS proteins. Mutations in the coding region for the PTB domain of DOK2 affect RAS signaling and cell proliferation. Detecting a DOK2 gene mutation, particularly a PTB mutation from a subject, is therefore indicative of a myeloproliferative disease such as a myeloproliferative chronic myelomonocytic leukaemia.

Web Resources

The URLs for data presented herein are as follows: Align DGVD, Polyphen-2,

SIFT, SpliceSiteFinder-like, MaxEntScan, NNSPLICE and Human Splicing Finder available through the Alamut Interpretation Software 2.0, http://alamut.interactive- biosoftware.com

Genome browser, http://genome.ucsc.edu

Online Mendelian Inheritance in Man (OMIM), http://www.omim.org 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. 1. Mashima R, Hishida Y, Tezuka T, Yamanashi Y. The roles of Dok family adapters in immunoreceptor signaling. Immunological reviews 2009 Nov; 232(1): 273- 285.

2. Berger AH, Niki M, Morotti A, Taylor BS, Socci ND, Viale A, et al. Identification of DOK genes as lung tumor suppressors. Nature genetics 2010 Mar; 42(3): 216-223.

3. Niki M, Di Cristofano A, Zhao M, Honda H, Hirai H, Van Aelst L, et al. Role of Dok-1 and Dok-2 in leukaemia suppression. The Journal of experimental medicine 2004 Dec 20; 200(12): 1689-1695.

4. Yasuda T, Shirakata M, Iwama A, Ishii A, Ebihara Y, Osawa M, et al. Role of Dok-1 and Dok-2 in myeloid homeostasis and suppression of leukaemia. The Journal of experimental medicine 2004 Dec 20; 200(12): 1681-1687.

5. Suzu S, Tanaka-Douzono M, Nomaguchi K, Yamada M, Hayasawa H, Kimura F, et al. p56(dok-2) as a cytokine-inducible inhibitor of cell proliferation and signal transduction. The EMBO journal 2000 Oct 2; 19(19): 5114-5122. 6. An CH, Kim MS, Yoo NJ, Lee SH. Mutational and expressional analysis of a haploinsufficient tumor suppressor gene DOK2 in gastric and colorectal cancers. APMIS : acta pathologica, microbiologica, et immunologica Scandinavica 2011 Aug; 119(8): 562-564.

7. Tefferi A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1.

Leukaemia 2010 Jun; 24(6): 1128-1138.

8. Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukaemia: rationale and important changes. Blood 2009 Jul 30; 114(5): 937-951. 9. Gelsi-Boyer V, Cervera N, Bertucci F, Trouplin V, Remy V, Olschwang S, et al. Gene expression profiling separates chronic myelomonocytic leukaemia in two molecular subtypes. Leukaemia 2007 Nov; 21(11): 2359-2362.

10. Favre C, Gerard A, Clauzier E, Pontarotti P, Olive D, Nunes JA. DOK4 and DOK5: new Dok-related genes expressed in human T cells. Genes Immun 2003 Jan; 4(1):

40-45.

11. Boulay I, Nemorin JG, Duplay P. Phosphotyrosine binding-mediated oligomerization of downstream of tyrosine kinase (Dok)-l and Dok-2 is involved in CD2-induced Dok phosphorylation. J Immunol 2005 Oct 1; 175(7): 4483-4489. 12. Mihrshahi R, Brown MH. Downstream of tyrosine kinase 1 and 2 play opposing roles in CD200 receptor signaling. J Immunol 2010 Dec 15; 185(12): 7216-7222.

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Claims

Claims

1. A method for diagnosing a myeloproliferative disease in a subject, said method comprising a step of detecting a mutation in a DOK2 gene in a biological sample obtained from said subject, wherein the presence of a mutation is indicative of a myeloproliferative disease.

2. A method of claim 1, wherein the mutation is in the DOK2 gene coding the phosphotyrosine-binding (PTB) domain of a DOK2 protein, said mutation resulting in loss of stable binding of the DOK2 protein to phosphotyrosyl peptides.

3. The method according to claim 1 or claim 2 wherein the mutation is present in coding region of exons 4-5 of the DOK2 gene.

4. The method according to any one of claims 1 to 3, wherein the mutation is a L238P mutation.

5. The method according to any one of claims 1 to 4, wherein the mutation is homozygous.

6. A method for predicting a risk of a subject of transmitting a myeloproliferative disease to his progeny, said method comprising detecting a mutation as defined in one of claims 1 to 5 in a DOK2 gene in a biological sample obtained from said subject, wherein the presence of a mutation is indicative of a risk of transmitting a myeloproliferative disease.

7. The method according to any one of claims 1 to 6, wherein the myeloproliferative disease is myeloproliferative chronic myelomonocytic leukaemia or unclassified-myeloproliferative myelodysplasia neoplasm.

8. A method for detecting a subject carrying a defective DOK2 gene, wherein said method comprises a step of detecting a mutation as defined in any one of claims 1 to 5 in a DOK2 gene in a biological sample obtained from said subject.

9. A polynucleotide coding for a DOK2 protein able to stably binding to phosphotyrosyl peptides, for use in a method of treatment of a myeloproliferative disease. A polynucleotide according to claim 9 for use in a patient wherein the patient has been subjected to a method according to one of claims 1 to 5.

A polynucleotide according to claim 9 or 10 wherein the myeloproliferative disease is myeloproliferative chronic myelomonocytic leukaemia or unclassified-myeloproliferative myelodysplasia neoplasm.