WO2009106372A1

WO2009106372A1 - Diagnostic method

Info

Publication number: WO2009106372A1
Application number: PCT/EP2009/001915
Authority: WO
Inventors: Roberto Foa'; Marco Tartaglia
Original assignee: Istituto Superiore di Sanita ISS
Current assignee: Istituto Superiore di Sanita ISS
Priority date: 2008-02-29
Filing date: 2009-02-27
Publication date: 2009-09-03
Anticipated expiration: 2010-08-29

Abstract

A method of detecting the presence of, or a predisposition towards, cancer in a patient. The method comprises detecting the presence of a mutant variant of the JAKl gene in a sample obtained from the patient. The presence of the mutant variant is indicative of the presence of, or a predisposition towards, cancer in the patient.

Description

Diagnostic Method

Field of the Invention

The present invention relates to a method of detecting the presence of, or a predisposition towards, cancer in a patient; a method of assaying a product for efficacy in the treatment or prophylaxis of cancer and to a method of treatment or prophylaxis of cancer in a patient. The invention also relates to protein, polypeptide and nucleic acid sequences.

Background

Acute lymphoblastic leukemia (ALL) comprises a biologically heterogeneous group of clonal disorders that originate from the uncontrolled proliferation and expansion of immature lymphoblastic cells and are characterized by an extremely variable clinical outcome (1, 2). In recent years, substantial progress has been made towards understanding the molecular events contributing to malignant transformation. This has permitted the recognition of relevant prognostic factors and risk stratification, and has favored the implementation of therapeutic approaches based on cytogenetic and molecular lesions (2-5). Despite these accomplishments, long-term survival in adults with ALL remains largely unsatisfactory, making the design of novel anti-leukemic drugs tailored to specific biological targets a current priority (4, 6).

The four mammalian members of the JAK family, JAKl, JAK2, JAK3 and TYK2, are non-receptor tyrosine kinases functioning as signal transducers to control cellular proliferation, survival and differentiation (7, 8). JAK proteins associate constitutively with a variety of cytokine receptors lacking intrinsic kinase activity, and promote signal flow by phosphorylating tyrosyl residues of activated receptors to allow the recruitment and activation of STAT proteins. They share a complex multidomain structure characterized by a tyrosine kinase domain at the C-terminus, which is flanked by a catalytically inactive pseudokinase domain with regulatory function. Their N-terminal half contains a FERM homology domain, which is implicated in receptor binding and possibly regulates the catalytic activity of the kinase, and an adjacent SH2-like domain. In contrast to their conserved structure, increasing experimental data indicate that JAK family members preferentially associate with a diverse subset of cytokine receptors, each differentially expressed by individual cell lineages and tissues, facilitating specificity in function (9). Among them, JAKl plays an essential and non-redundant role in mediating biological responses induced by a specific subgroup of cytokines controlling lymphoid cell precursor development (10). Jakl^'1' mouse pups exhibit a thymus markedly reduced in size, which is associated with a severe decrease in cellularity. Jakl loss of function is also associated with profound abnormalities in the B- cell compartment due to a block in differentiation at the pro-B/pre-B cell transition step, resulting in a deficit in the production of mature B lymphocytes (10).

The present invention is based on the finding that somatic activating JAKl mutations occur among adults with T-cell precursors ALL, and are associated with poor response to therapy and overall prognosis. This supports the view that upregulation of JAKl signaling contributes to malignancies of the lymphoid lineage.

Summary of the Invention

According to one aspect of the present invention, there is provided a method of detecting the presence of, or a predisposition towards, cancer in a patient comprising detecting the presence of a mutant variant of the JAKl gene in a sample obtained from the patient, wherein the presence of the mutant variant is indicative of the presence of, or a predisposition towards, cancer in the patient.

The sample may be a sample of blood, tissue, bronchial lavage fluid or the like. The mutant variant of the JAKl gene may be detected directly, such as by probing for the presence of DNA having the mutant variant, or may be detected indirectly by examining the RNA in the sample transcribed from the gene or the expressed protein itself. For example, the presence of a mutant variant of the protein can be detected by using antibodies specific for the mutant variant which have reduced affinity for the wild-type protein.

Also provided in the present invention is a kit for detecting cancer, or a predisposition towards cancer, in a sample from a patient, wherein the kit comprises means to detect the presence of a mutant variant of the JAKl gene in the sample. The kit is used as described above. The means may comprise, for example: opportunely selected primer pairs to amplify the JAKl coding sequence allowing these amplified genomic fragments to be sequenced or analyzed with other screening methods to identify disease-associated mutations; a nucleic acid probe capable of hybridising with the mutant variant of the JAKl gene or an antibody capable of binding to the protein encoded by the mutant variant. The kit may also comprise a label attached to the probe or antibody and, optionally, instructions for use of the kit.

Conveniently, the JAKl gene comprises a nucleic acid sequence encoding a protein having at least 80% identity to SEQ. ID NO: 2. Preferably there is at least 90%, 95%, 99% or, most preferably 100% identity to SEQ ID NO:2.

Preferably, the mutant variant of the JAKl gene comprises a nucleic acid sequence encoding a protein having at least 80% identity to SEQ. ID NO: 2 and in which at least one of the amino acid residues at the following positions is substituted with an alternative amino acid: 204, 512, 634, 653, 724, and 879. More preferably there is at least 90%, 95%, 99% or, most preferably 100% identity to SEQ ID NO:2 (aside from the substituted amino acid).

Advantageoulsy, the mutant variant of the JAKl gene comprises a nucleic acid sequence encoding a protein having at least 80% identity to SEQ. ID NO: 2 and in which the protein has at least one amino acid substitution selected from the group consisting of: Lys204Met, Ser512Leu, Ala634Asp, Leu653Phe, Arg724His, Arg879Ser, Arg879Cys, Arg879His and combinations thereof. More preferably there is at least 90%, 95%, 99% or, most preferably 100% identity to SEQ ID NO:2 (aside from the substituted amino acid).

Conveniently, the patient is an adult (i.e. over the age of 16 years).

Preferably, the cancer is leukaemia such as acute lymphoblastic leukaemia, in particular, B-cell precursor acute lymphoblastic leukaemia or, more frequently, T-cell precursor acute lymphoblastic leukaemia.

According to another aspect of the present invention, there is provided a method of assaying a product for efficacy in the treatment or prophylaxis of cancer comprising determining the kinase activity of the mutant variant of the JAKl protein, or activation level of signalling pathways modulated by JAKl protein, in the presence and absence of the product wherein a reduced kinase activity of the mutant variant, or a reduced activation of signalling pathways modulated by the JAKl protein, in the presence of the product as compared with kinase activity in the absence of the product, or with the activation level of signalling pathways modulated by the JAKl protein, is indicative that the product is efficacious in the treatment or prophylaxis of cancer.

Conveniently, determining the kinase activity, or more generally JAKl functional upregulation, comprises determining at least one of: the level of STATl, AKT and ERK phosphorylation.

Preferably, the JAKl protein is present in a cell during the assay process.

Advantageously, the JAKl protein comprises a sequence having at least 80% identity to SEQ. ID NO: 2. Preferably there is at least 90%, 95%, 99% or, most preferably 100% identity to SEQ ID NO:2.

Conveniently, the mutant variant of the JAKl protein comprises a sequence having at least 80% identity to SEQ. ID NO: 2 and in which at least one of the amino acid residues at the following positions is substituted with an alternative amino acid: 204, 512, 634, 653, 724, and 879. More preferably there is at least 90%, 95%, 99% or, most preferably 100% identity to SEQ ID NO:2 (aside from the substituted amino acid).

Advantageously, the mutant variant of the JAKl protein comprises a sequence having at least 80% identity to SEQ. ID NO: 2 and in which the protein has at least one amino acid substitution selected from the group consisting of: Lys204Met, Ser512Leu, Ala634Asp, Leu653Phe, Arg724His, Arg879Ser, Arg879Cys, Arg879His and combinations thereof. More preferably there is at least 90%, 95%, 99% or, most preferably 100% identity to SEQ ID NO:2 (aside from the substituted amino acid).

Preferably, the cancer is acute lymphoblastic leukaemia, in particular, T-cell precursor acute lymphoblastic leukaemia or B-cell precursor acute lymphoblastic leukaemia.

According to a further aspect of the present invention, there is provided a method of treatment or prophylaxis of cancer in a patient comprising administering to the patient a therapeutically effective quantity of a product that inhibits the activity of the JAKl protein.

Conveniently, the JAKl protein comprises a sequence having at least 80% identity to SEQ. ID NO: 2. Preferably there is at least 90%, 95%, 99% or, most preferably 100% identity to SEQ ID NO:2.

Advantageously, the product inhibits the signalling activity of the JAKl protein. Alternatively, the product inhibits the kinase activity of the JAKl protein. Alternatively, the product inhibits the expression of the JAKl protein.

An exemplary product that inhibits the activity of the JAKl protein is a molecule that mimics a JAKl substrate and binds to the active site blocking the catalytic activity of the protein, or is an antibody or an antigen-binding fragment thereof that binds specifically to the protein. It is particularly preferred that the antibody is specific for an epitope of the protein that incorporates the substituted amino acid, thereby being specific for the mutant variant of the JAKl protein.

The product that inhibits the activity of the JAKl protein may comprise part of a pharmaceutical composition which also comprises a pharmaceutically acceptable carrier, diluent or excipient (see Remington's Pharmaceutical Sciences in US Pharmacopoeia, 1984, Mack Publishing Company, Easton, PA, USA). The dose required for a patient may be determined using methods known in the art, for example, by dose-response experiments. The product may be administered by a range of routes, for example, orally or parenterally.

According to another aspect of the present invention, there is provided an isolated protein comprising a sequence having at least 80% identity to SEQ. ID NO. 2 and wherein at least one of the amino acid residues at the following positions is substituted with an alternative amino acid: 204, 512, 634, 653, 724, and 879. More preferably there is at least 90%, 95%, 99% or, most preferably 100% identity to SEQ ID NO:2 (aside from the substituted amino acid).

Coveniently, the protein comprises an amino acid substitution selected from the group consisting of: Lys204Met, Ser512Leu, Ala634Asp, Leu653Phe, Arg724His, Arg879Ser, Arg879Cys, Arg879His and combinations thereof.

According to yet another aspect of the present invention, there is provided an isolated polypeptide comprising a fragment of the isolated protein of the invention, wherein the fragment is at least 8 amino acids long and includes the substituted amino acid residue. It is preferred that the fragment is at least 9, 10, 1 1, 12, 14, 16 or 20 amino acids long.

According to a further aspect of the present invention, there is provided an isolated nucleic acid sequence comprising a sequence encoding the isolated protein or the isolated polypeptide of the invention. Definitions

In this specification, the following definitions are used.

"ALL" means "acute lymphoblastic leukaemia".

"CM" means "conditional medium".

"Detecting the presence of cancer, or a predisposition towards cancer" includes: providing a diagnosis of cancer; assessing the progression of the condition after initial diagnosis; monitoring the response of the condition to a treatment; and establishing the extent of a patient's condition (i.e. staging). For example, by making a quanitative assessment of the proportion of cells in a patient's sample that contain a mutant variant of the JAKl gene, the relative advancement of the cancer may be determined. The term also includes detecting one step of the molecular events causing malignancy.

"DFS" means "disease-free survival".

"DHPLC" means "denaturing high performance liquid chromatography".

"ERK" means "extracellular signal-regulated kinase".

The percentage "identity" between two sequences is determined using the BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A.

Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997),

"Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402) using default parameters. In particular, the BLAST algorithm can be accessed on the internet using the URL http://www.ncbi.nlm.nih.gov/blast/. "Isolated" means that a product is removed from its natural surroundings such as the cellular components which would be present in vivo.

"Mutant variant" when referring to a protein or nucleic acid sequence means a protein or nucleic acid sequence having at least one amino acid or nucleotide deletion, insertion or substitution as compared with a wild type sequence.

"Prophylaxis" includes lowering the incidence of a condition as well as complete prevention thereof.

"OS" means "overall survival".

"Treatment" includes remission or reduction of symptoms and alleviation of the condition as well as complete eradication of the condition.

Brief Description of the Figures

Figure IA depicts representative electropherograms showing the occurrence of somatically acquired JAKl mutations in subjects with T-ALL. In all cases, mutations were observed at diagnosis (above), but were undetectable during remission (below).

Figure IB shows JAKl domain structure and location of affected residues. The predicted amino acid substitutions resulting from the JAKl mutations are positioned below the cartoon of the protein with its functional domains indicated (left) and shown in JAKl three-dimensional modeled structure (right).

Figure 1 C depicts electropherograms showing the occurrence of mutations in a fraction of leukemic cells of two individuals with T-ALL. In both patients, the mutant allele constituted only a portion of the amplified fragment from BM obtained at diagnosis (blasts >70% of total cells) (above). The heterozygous status of each subject for an intragenic polymorphic site (below) is shown for comparison. Figure 2 A shows STATl phosphorylation assays. Basal and IFN-γ-stimulated endogenous STATl phosphorylation in JAKl -defective U4C cells transiently transfected with wild type (WT) JAKl or selected mutants was studied. Blots are representative of three experiments performed.

Figure 2B shows the results of STATl activation assays. Basal and IFN-γ-stimulated endogenous STATl transcriptional activity in JAKl -defective U4C cells transiently cotransfected with p-GAS-Luc and phRL-TK constructs, and wild type (WT) JAKl or a mutant allele was studied. STATl -induced luciferase gene expression levels were determined by measuring the luciferase activity (counts per second, CPS) normalized to the activity of the Renilla luciferase, using a dual luciferase reporter assay system. Activity ratios are expressed as averages of three replicates ± SD.

Figure 2C shows Ba/F3 survival assays. Wild type (WT) or mutant JAKl transduced Ba/F3 cells were grown in absence of IL-3 (left) or with 0.5% or 5% WEHI-3B cell conditional medium (CM) as source of IL-3 (right). Cell numbers (average of three replicates ± SD) were counted at the indicated time points (left) or at day 3 of culture (right).

Figure 2D shows Stat5, Akt and Erk phosphorylation assays. Endogenous Stat5 Tyr694, Akt Ser473 and Erk 1/2 Thr202/Tyr204 phosphorylation levels from lysates of Ba/F3 cells transduced with wild type (WT) Jakl or a mutant form and cultured without IL-3 (left) or with 2% WEHI-3B cell CM as the source of IL-3 (right) were studied. Activation of Stat5 (pStat5/Stat5), AKT (pAkt/Akt) and Erkl/2 (pErk/Erk) is expressed as a multiple of activation in untransduced cells. Blots are representative of at least three experiments performed.

For convenience, the amino acid changes affecting residues Ala , Arg and Arg of the murine Jakl protein (encoded by the pMX-Jakl -IRES-GFP constructs used to transduce the Ba/F3 cell line) are indicated according to the homologous residues in human JAKl. Figure 3 A shows supervised hierarchical clustering of gene expression profiles performed on blasts from 16 adult T-ALL patients, with or without a JAKl mutation.

Figure 3B depicts Kaplan-Meier estimates of disease-free survival (DFS) (above) and overall survival (OS) (below) in subjects with (gray) or without (black) a JAK mutation. Multivariate analysis confirmed the statistical significance of the reduced DFS and OV among JAKl mutation-posive patients, and excluded a significant contribution of the more advanced age of these subjects.

Figure 4 shows JAKl domain structure and location of JAKl, JAK2 and JAK3 residues reported to be mutated in myeloproliferative disorders and leukemias. The predicted amino acid substitutions are positioned below the cartoon of the JAKl protein with its functional domains indicated above (JAKl, top; JAK2, middle; JAK3, bottom) (left) and in its three-dimensional modeled structure (JAKl, Ser512, Lys204, Arg724, Ala634 and Arg879; JAK2 Lys607, Leuόl l and Val617; JAK3 Prol32, Val722 and Ala572) (right).

Figure 5 shows gene expression profiles of JAKl mutation-positive and mutation- negative blasts from adult subjects with T-ALL. Unsupervised hierarchical clustering was performed on blasts from 16 adult T-ALL patients, with or without a JAKl mutation. Relative expression levels are shown according to the reported color scale diagram (below). Clustering based on 1345 probe sets grouped the expression profiles of the 5 JAKl mutation-positive patients (light gray at top of figure) into 2 distinct clusters.

Figure 6 shows GFP expression levels of purified Ba/F3 cells transduced with the pMX-JAKl -IRES-GFP bicistronic retroviral vector coding for wild type (WT) Jakl or one of the three generated mutants. Brief Description of the Sequences

SEQ. ID NO: 1 is the wild type nucleotide sequence of the JAKl gene held under GenBank Accession no. NM_002227.2. The protein coding sequence is also shown.

SEQ. ID NO: 2 is the protein sequence encoded by the nucleotide sequence of SEQ. ID NO: 1.

SEQ. ID NO: 3 is another wild type protein sequence of JAKl, which has 99.4% identity to SEQ. ID NO: 2.

Experimental

MATERIALS AND METHODS

ALL cohorts and molecular analyses. Cohorts studied included patients enrolled in the GIMEMA 0496 and 2000 (adults), and AIEOP ALL 2000 (children and adolescents) clinical trials. Written informed consent for genetic analyses was obtained from all subjects according to the declaration of Helsinki. BM mononuclear cells were isolated by density gradient centrifugation and cryopreserved. Genomic DNA was isolated in a standard fashion. The entire JAKl coding region were screened by DHPLC (Wave 2100 System, Transgenomic) and direct sequencing. Primer sequences are shown in Table IV.

Table IV List of the oligonucleotide sequences used to amplify the entire coding sequence of the JAKl gene (exons 1 to 24).

Primer Sequence Exon Amplified product lforward 5'-CTTCTCTGAAGTAGCTTTGGAAAG-S' 1 323 bp lreverse 5'-AATAGTGGTGAACATCTAGGAGAG-S ' 2forward 5'-GAGAGGTACGTATCCAATACC-S ' 2 428 bp

2reverse 5'-ACAACTCTTTCTGCCCAGCAG-S'

3forward 5'-AAGAGGATATGAGTGACCCAG-S' 3 349 bp 3reverse 5'-CAATACTTCTTGGTAAGTGACTC-S '

4forward 5' -TAAGAAGGAAAGAGATGGTGAGG-S' 4 381 bp

4reverse 5' -TCTG AGCTCTAC A ATGCCTCTC-3 '

5 forward 5' -TCTGATGCTAGAGAAACTGCC-S' 5 363 bp

5reverse 5' -TA AG A AC ATGTAGAA AC ACCACC-3 ' όforward 5' -TTGGCAACATGTGGATTCATGG-S' 6 531 bp όreverse 5' -G ACATGCAG AC AG ATGACTCC-3 '

7forward 5' -CTTTCTCTTCCTTGGACCTAGG-S ' 7 366 bp

7reverse 5' -G A ACGG A ACTCAGC AATTCTCC-3 ' δforward 5' -ATCTTCTCACTGTGCACCTCC-S' 8 339 bp δreverse 5' -ACTGGCCTG ACCTA AAC A ATG-3 '

9forward 5' -TAGCCCAGAGGTTCAAAGTCC-S' 9 309 bp

9reverse 5' -ACCC AGGCTTTTC AGTTCC AC-3 ' lOforward 5' -AGAAATGTTACAGAGATGGTGC-S ' 10 402 bp lOreverse 5' -CTTTGTTTAAGTCAGTCAGCG-3 '

1 1 forward 5' -AGACGTTGGCTGTCTGAGAGC-S' 1 1 31 1 bp

1 1 reverse 5' -CC ATC A A AGG A AAGTCTCCCTG-3 '

12forward 5' -CAGACGGTCCATCACTTCAGG-S' 12 263 bp

12reverse 5' -GTTCC ACTGGCTCCAG AA ACG-3 '

13 forward 5' -CCAGAGGATTGATGTTCAGG-S ' 13 383 bp

13reverse 5' - AGC AA ATG ACCTGCTCAGTC-3 '

14forward 5' -ACAGACCAGGTTCCAGACATGG^' 14 208 bp

14reverse 5' -GTTTCTGGTGGGACCATTATGG-S '

15forward 5' -GAGCAGCTTGGCTAAACTTGAC-S' 15 270 bp

15reverse 5' -G ACTCTCTAA A AGG AG ACC A ACC-3 '

1 όforward 5' -CTGTTCTGGCTGCAGTGACAG-S' 16 280 bp

1 όreverse 5' -C ACCTGAAAGCCCTCACTTGC-3 '

17forward 5' GGCTG AGA AGTTTGTAGGTGG-3 ' 16 and 17 504 bp

18reverse 5' ■ A ATGG AG AGC AGCTGTACGTG-3 '

19forward 5' •AAGTACAGTCCAGGTGAGAGG-3' 19 493 bp

19reverse 5' ACTTGG A AGCCTTG AGAGTGTG-3'

20forward 5' •CCTCTGTCTCATATCCTGTGC-3' 20 271 bp

20reverse 5' C A ATTACCC AGG AC AG AGTGC-3 '

21 forward 5' ■GCTTCTACTAAGAAAGTAAACGG^' 21 51 1 bp

21 reverse 5' CTTCTCTA ACCTTG AGTC AGG-3 '

22forward 5' ■TTATCCTGTAATGTCGGATGGAG-3' 22 257 bp

22reverse 5' TTAACC AAGC AG AGGGATGG AC-3 '

23 forward 5' G ACACTGGTTAGAGACAGATCAT-S' 23 496 bp

23 reverse 5' ■CTAACTGGTGGCTATCATCTAG-3 ' 24forward 5'-CAGTCAACCCTTGAGCTTCTC-S' 24 453 bp

24reverse 5'-GTACTGAAGTATGAGTTCAGTGAC-S '

Functional studies. The mutations resulting in the A634D, R724H and R879C changes were introduced by site-directed mutagenesis in a VSV-tagged JAKl cDNA cloned in pRc/CMV vector. JAKl -defective human fibrosarcoma U4C cells were maintained in DMEM supplemented with 10% heat-inactivated FCS and antibiotics. To evaluate endogenous STATl phosphorylation, following starvation (24 h), transiently transfected cells were stimulated with IFN-γ (1000 units/ml, 15 min) or left unstimulated. Lysed samples were analyzed by 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Pierce) and probed with anti-phospho-Statl (9171, Cell Signaling), anti- Statl (9172, Cell Signaling) and anti-Jakl (3332, Cell Signaling) antibodies. Endogenous STATl transcriptional activity was assessed by luciferase transactivation assays in cells co-transfected with a p-GAS-Luc construct, switched to serum-starvation medium (8 h) and then stimulated with IFN-γ (1000 units/ml, 16 h) or left unstimulated. STATl -induced luciferase expression was assessed and normalized using a dual luciferase reporter assay system (Promega) and a phRL-TK plasmid constitutively expressing the Renilla luciferase.

Each of the three leukemia-associated mutations was also introduced in the murine JAKl cDNA cloned in the bicistronic retroviral vector pMX-IRES-GFP. Constructs were transfected into Phoenix or BOSC packaging cells to produce retroviruses, and murine Ba/F3 (mantained in RPMI- 1640 medium containing 10% FCS, 1% L- glutamine and 10% WEHI-3B cell CM) cells were infected with retroviral supernatants. GFP-positive populations were purified by flow-cytometric sorting and then expanded. Equal GFP expression levels of transduced cells were confirmed by FACS analysis (Figure 6). Transduced Ba/F3 cells were cultured in the absence or presence of IL-3 (0.5% or 5% WEHI-3B cell CM) for assaying cytokine independence and response, and viable cells were counted by trypan-blue exclusion. For Western blot studies, Ba/F3 cells were starved in RPMI- 1640 medium containing 1% BSA (5 h) and then stimulated with 2% WEHI-3B cell CM (30 min) or left unstimulated. Evaluation of Jakl, Akt, Stat3, Stat5 and Erkl/2 expression and phosphorylation levels was performed using anti-phospho-Jakl (#3331), anti-Jakl (#3332), anti-phospho-Akt (#9271), anti-Akt (#9272), anti-phospho-Stat3 (#9138), anti-Stat3 (#9132), anti-phospho-Stat5 (#9351), anti-Stat5 (#9310), anti-phospho-p44/42 Erk (#9106) and anti-p44/42 Erk (#9102) antibodies (Cell Signaling).

Statistical analysis. Confidence intervals of proportions (at 95% level) were calculated based on the binomial distribution. The probabilities of OS and DFS were estimated using the Kaplan-Meier method. The log-rank test was used to compare treatment effect and risk factor categories. All tests were two-sided, accepting P<0.05 as indicating a statistically significant difference. Cox proportional hazard models, including age and JAKl as variables, were used in order to perform multivariate analyses for OS and DFS. The SAS software (SAS Institute) was used for the analysis.

Molecular modeling. Structural models of the kinase, pseudokinase, SH2 and FERM domains of JAKl were obtained by employing the DeepView software and the Swiss Model server (29, 30), after manual optimization of the alignments. The kinase domain (residues 876-1 153) was modeled by homology to the crystallographic structures of the following kinase domains obtained from the Protein Data Bank (pdb): JAK2 (pdb code 2b7a, identity 54%), JAK3 (pdb code lyvj, identity 51%), FGFRl (pdb code lfgi, identity 35%) and FGFR2 (pdb code loec, identity 34%). Residues 604-852 of the pseudokinase domain (residues 583-855) were modeled by homology to the structures of the kinase domains of RET (pdb code 2ivt, identity 26%) and ABL (pdb code lfpu, identity 24%). The SH2 domain (residues 439-544) was modeled by homology to the structures of the following SH2 domains: GRBlO (pdb code lnrv, identity 23%), LCK (pdb code llkk, identity 21%), HCK (pdb code lqcf, identity 20%) and SHP-2 (pdb code 2shp, C-terminal SH2 domain, identity 16%). The FERM domain (residues 34- 420) was modeled by homology to the structures of the FERM domains of the following proteins: ezrin (pdb code Ini2, identity 12%), radixin (pdb code Ij 19, identity 14%), moesin (pdb code Ie5w, identity 12%) and focal adhesion kinase (pdb code 2al6, identity 15%). A possible relative orientation of the four domains was obtained by superimposing them on a complete model of JAK2 (31). Despite the fact that the modeled JAKl and JAK2 structures were obtained with different protocols and using different substrates for homology modeling, conformations of JAKl and JAK2 domains were well superimposed, with Ca root mean square deviations of 1.1 A (kinase), 1.2 A (pseudokinase), 1.3 A (SH2) and 1.6 A (FERM), supporting the reliability of both models. Molecular graphics were created with the program MOLMOL (32).

Gene expression profiling. Thawed or freshly isolated cells (>90% blasts) were homogenized and total RNA was extracted either using the Trizol reagent (Life Technologies) and further purified with the SV total isolation system (Promega) or using the RNeasy mini kit (Qiagen). RNA quality was checked by agarose gel electrophoresis and spectrophotometry. HGU 133 Plus 2.0 gene chips (Affymetrix) were used to determine gene expression profiles. The detailed protocol for sample preparation and microarray processing is available from the. Oligonucleotide microarray analysis and gene expression data were performed using the dChip software (www.dchip.org) (33), which utilizes an invariant set normalization method where the array with median overall intensity is chosen as the baseline for normalization. Model-based expressions were computed for each array and probe set using only perfect match probes. For unsupervised analysis, non-specific criteria included the requirement for individual gene expression levels to be higher than 100 in at least 20% of samples, and for the ratio of SD to the mean expression across samples to be included between 0.8 and 1000. Analysis of Variance (ANOVA) with .P-value <0.001 was performed to compare profiles obtained from T-ALL patients with or without a JAKl mutation.

RESULTS AND DISCUSSION JAKl mutation analysis in ALL

To explore possible contributions of somatic JAKl gene mutations in ALL, genomic DNA samples from BM aspirates of adult subjects with B-cell precursor ALL (B-ALL)

(n=88) and T-ALL (n=38) obtained at diagnosis and prior to therapy were screened for mutations in the entire JAKl coding region using denaturing high performance liquid chromatography (DHPLC). Direct sequencing of variant elution profiles allowed the identification of 37 intronic and exonic changes, including 9 non-synonymous variants observed in 14 individuals (Table I and II).

Table I. List of non-synonymous JAKl changes identified in subjects with acute lymphoblastic leukemia.

Cohort # of Nucleotide Exon Amino Acid Domain Mutation/ and Cases Substitution Substitution Polymorphism

Lineage

Adult

ALL

B-cell 88

1 184A>G 2 Ile62Val FERM Polymorphism⁸

1 611A>T 5 Lys204Met FERM Mutation^b

1 1901OA 13 Ala634Asp Pseudokinase Mutation

1 2171G>A 15 Arg724His Pseudokinase Mutation^d

T-cell 38

1 184A>G 2 Ile62Val FERM Polymorphism⁸

1 1078OT 7 Arg360Trp Polymorphism⁰

1 1535OT 10 Ser512Leu SH2 Mutation^d

1 1901C>A 13 Ala634Asp Pseudokinase Mutation^d

3 2171G>A 15 Arg724His Pseudokinase Mutation⁶

1 2635OA 18 Arg879Ser Kinase Mutation^b>f

1 2635OT 18 Arg879Cys Kinase Mutation^b

1 2636G>A 18 Arg879His Kinase Mutation*⁵

Childhood

ALL

B-cell 85

T-cell 49

1 1957OT 13 Leu653Phe Pseudokinase Mutation^d

^aThis change was observed in unaffected individuals. bThis change was not present among 335 population-matching control individuals. cThis change was present at remission. This change was not present at remission. eThis change was not present at remission in the one individual analyzed. fThis subject carried a concomitant 2171G-»A change. Table II. List of intronic or synonymous JAKl changes identified in the study.

Exon Nucleotide Amino Acid

Substitution

1 -50A>T^a

+1 18OC

+165OT

+194A>G

2 +21A>G

+57A/T

3 228OT^a Asn76

4 414OT^a Tyrl38

5 546A>G^a GIy 182

579T>C^a Alal93

6 +39C>T

7 -27OT

8 -25G>A

+49G>A

9 +84A>T

10 -38OT

-7OT

1590C>T Ile530

13 -9OT

1977OT Arg659

+92OT

14 2049OT Ser683

15 2199A>G Pro733

20 2877G>C Leu959

21 3078A>G LyslO26

3096G>A LyslO32

23 -49T>C

24 -141A>G

¹ Position referred to the A of the ATG start codon. Among the missense defects, genotyping of genomic DNAs from BM obtained during remission demonstrated the somatic origin of the 1535OT (Ser512Leu), 1901C>A (Ala634Asp) and 2171G>A (Arg724His) changes in the leukemic clones (Fig. IA and Table I). To verify that the non- synonymous substitutions identified in patients for whom non-leukemic DNA was not available were not gene variants occurring in the population, 335 population-matching control individuals were analyzed and none harbored the 61 1A>T (Lys204Met), 2635OA (Arg879Ser), 2635OT (Arg879Cys) and 2636G>A (Arg879His) changes or other defects altering those codons. While the T-ALL-restricted occurrence of three distinct substitutions affecting Arg⁸⁷⁹ (3/38 vs 0/335, Fisher's exact probability< 0.001) further supported the relevance of the substitution of this residue, we could not exclude that the 611A>T change might represent a private neutral variant. The two remaining missense changes, 184A>G (Ile62Val) and 1078C>T (Arg360Trp), were deemed non-pathogenic variants, as they were observed in non-leukemic cells of affected patients or in unaffected control subjects.

All mutations occurred as heterozygous changes and affected conserved residues within the FERM, SH2, pseudokinase and kinase domains (Fig. IB). In two cases, DHPLC profiles and electropherograms indicated that the mutant allele might be present in only a fraction of leukemic cells, suggesting that these lesions did not represent early events during leukeniogenesis but were acquired during disease progression (Fig. 1C). Remarkably, mutations were relatively common among individuals with T-ALL (18.4% of cases, 95% CI= 7.7-34.3%), while they occurred in only three patients with B-ALL (3.4% of cases, 95% CI=0.7-9.6%) (Table I). Such a difference in mutation prevalence between cohorts was statistically significant (Fisher's exact probability= 0.003). DHPLC screening performed on affected exons by using pooled DNAs excluded loss of the normal allele and a homozygous condition for a gene variant due to mitotic recombination in all cases.

To investigate the prevalence of JAKl mutations among pediatric ALL cases, genomic DNA from BM obtained at diagnosis was scanned for mutations in affected exons. No lesion was observed within the B-ALL cohort (N= 85), while a non-synonymous 1957C>T transition (Leu653Phe) was identified in one of 49 subjects with T-ALL (2.0% of cases, 95% CI= 0.05-10.9%). This mutation was not observed in the BM obtained from the patient at the time of remission, indicating that it was somatically acquired in the leukemic cells.

Overall, these results indicated that JAKl gene mutations occur in ALL and are more frequently observed among adult individuals with involvement of the T-cell lineage.

Functional consequences of somatic JAKl mutations To examine the effects of the identified mutations on protein function, wild type JAKl or a mutant form (A634D, R724H and R879C) was expressed transiently in JAKl- defective human fibrosarcoma U4C cells, and endogenous STATl phosphorylation was compared basally and following stimulation with IFN-γ (Fig. 2A). Consistent with previous studies (10, 11), untransfected cells lacking functional JAKl did not exhibit STATl phosphorylation in response to IFN-γ. All JAKl mutants promoted an enhanced response to the ligand compared to wild type JAKl . Of note, basal STATl phosphorylation was observed in cells expressing the A634D mutant, suggesting ligand-independent upregulation of the kinase. Consistent with this, expression of the A634D mutant resulted in an essentially constitutive STATl transcriptional activation, while a statistically significant increase in STATl activity was observed in U4C cells expressing both the R724H and R879C mutants, basally and following stimulation (Fig. 2B).

To assess the ability of mutations to upregulate signal flow further, we transduced Ba/F3 cells with wild type JAKl or each of the selected mutants to evaluate whether their expression induced autonomous growth of cytokine-dependent cells. GFP- expressing Ba/F3 cells were selected by flow cytometry, cultured in 5% or 0.5%

WEHI-3B cell conditional medium (CM) as a source of IL-3 as well as in absence of the cytokine, and counted to assess proliferation (Fig. 2C). Three independent experiments indicated that expression of the A634D and, with less efficiency, R724H

Jakl mutants conferred IL-3 -independent growth to cells, whereas cells expressing wild type Jakl or the R879C Jakl mutant retained dependence on the cytokine for survival. Of note, cells expressing each of the three mutants exhibited enhanced growth in response to IL-3. Consistent with these findings, Ba/F3 cells expressing the A634D Jakl mutant exhibited enhanced Stat5, Akt and Erk phosphorylation basally and following stimulation, while a higher phosphorylation level of these signal transducers in cells expressing the R724H Jakl protein was observed in cultures maintained in presence of IL-3 (Fig. 2D).

Overall, these data indicated that the three selected leukemia-associated JAKl mutants are hypermorphs, with A634D Jakl having a seemingly stronger effect, and that different mechanisms are likely to be involved in their cell context-related gain of function.

Molecular modeling of JAKl and location of affected residues

To look at the structural causes resulting in JAKl functional upregulation, we generated a model of JAKl structure since no crystallographic information was available for this protein. Energy-minimized models of each of the four domains were generated separately by homology to available crystallographic structures of proteins with similar sequences and overall fold. The quaternary arrangement of the four domains was then determined by superimposing the models on a predicted three- dimensional structure of JAK2 (14). According to the superimposed structure, Ala and Leu ⁵³ are placed on the surface of the pseudokinase domain involved in the interaction with the kinase domain (Fig. IB). Based on the evidence supporting a negative regulatory role of the pseudokinase domain on catalytic function of JAK proteins (15-17), the pathogenetic mechanism of the A634D and L653F changes is predicted to involve a looser interdomain interaction, relaxing inhibitory control on the kinase activity. Consistent with this hypothesis, substitution of two residues located in the pseudokinase domain at the interface with the kinase domain in JAK2 (VaI⁶¹⁷) and JAK3 (Ala⁵⁷²) promote increased catalytic activity basally (18-20) (Figure 4). Our model also predicts that residues Lys²⁰⁴ and Ser⁵¹² would perturb the SH2/FERM interdomain interaction since they are located at the interface between these domains, approximately facing each other. This finding is noteworthy since it has been proposed that JAKl ' s SH2 domain does not function as phosphotyrosyl-binding domain but rather plays a structural role in stabilizing the conformation of the FERM domain (21), which mediates its association to cytokine receptors and exerts an as-yet- uncharacterized restraint on catalytic function (22, 23). The molecular mechanism through which these mutations affect JAKl function remains to be explained. Structural and functional consequences were not obvious for the activating changes affecting residues Arg 724 and Arg ,879

Gene expression profile analysis in blasts with a mutated JAKl allele

Total RNA was available from blasts of five JAKl mutation-positive (S512L, A634D

10 and R724H) and eleven mutation-negative subjects of the T-ALL cohort. Unsupervised clustering based on 1345 probe sets selected by non-specific filtering clustered expression profiles of mutation-positive blasts into two clusters, suggesting contribution of JAKl mutations to distinct major mechanisms of deregulation (Figure 5). Notably, supervised gene expression analysis revealed a distinctive expression

15 signature shared by leukemic cells with a mutated JAKl gene (Fig. 3A) based on the expression of 133 differentially expressed probe sets, consisting of 112 differentially expressed genes, the majority of them being overexpressed in JAKl mutation-positive samples (Table III). Among the upregulated genes, those whose transcription was known to be positively modulated by JAK/STAT signaling, including IRFl, SOCS3,

20 ISG15, ISGF3G, IFI44L and IRFl, were overrepresented in all the JAKl mutation- positive subjects, further supporting the gain of function role of the ALL-associated JAKl lesions.

Table III. Differentially expressed genes in JAKl mutation-positive vs mutation- 25 negative adult subjects with T-ALL.

Chromosome

Probe set GenBank Gene ID Gene Symbol P Value Fold Change Location GO Annotation

204439_at NM_006820 10964 IFI44L Ip31 1 Immune response 0 000302 13 99

207677_s_at NMJ) 13416 4689 NCF4 22ql3 1 Immune response 4 52E-06 10 17

20₅147_x_at NM 000631 4689 NCF4 22ql3 1 Immune response 1 49 E-06 9 38

20253 l_at NM_002198 36₅9 IRFl 5q31 1 Immune repsonse 2 83E-05 7 04

205483_s_at NMJ)O₅IOl 9636 ISGl₅ 1 p36 33 Immune response 0 000132 6 7962₅9

212775_at A1978623 23363 OBSLl 2q3₅ Unknown 4 78 E-06 6 73

202086 at NM 002462 4₅99 MXl 21q22 3 Immune response 0 00011 5 328979 Chromosome

Small GTPase mediated signal l₅5S630_a_at AF327350 83871 RAB34 17ql l 2 0 000381 5 01698 transduction

208436_s_at NM_004030 3665 IRF7 I lpl 5 5 Immune response 0 000209 ₅ 02

238725_at AW3925₅1 Unknown Unknown Unknown Unknown 0 000667 4 86

228617_at AA 142842 ₅4739 BIRC4BP 17pl3 2 Unknown 7 12E-05 4 731248

Small GTPase mediated signal

224710_at AF3273₅₀ 83871 RAB34 17ql l 2 0000274 4681032 transduction

203₅₀₅_at AF285167 19 ABCAl 9q31 1 Lipid metabolism 9 91E-07 4 594248

227697_at AI244908 9021 SOCS3 17q25 3 Immune response 0 0006 4 38

203882_at NM_006084 10379 1SGF3G 14ql l 2 Immune response 4 51E-06 4 34215

238365_s_at AI638342 339541 MGC33556 Ip34 1 Unknown 2 56E-05 4 29

I₅59263_s_at BG397809 340152 ZC3H12D 6q25 1 Unknown 0 000198 4 03

227609_at AA633203 94240 EPSTI l 13ql3 3 Unknown 2 30E-05 3 865121

38671_at AB014₅20 23129 PLXNDI 3q21 3 Development 2 48E-05 3 81

210439_at AB023135 29851 ICOS 2q33 Immune response 4 79E-05 3 76

212235_at AL57₅403 23129 PLXNDl 3q21 3 Development 5 26E-O₅ 3 62

206133_at AA 142842 54739 BIRC4BP 17pl3 2 Unknown 8 15E-O₅ 3 590951

227336_at NM_017523 1840 DTXl 12q24 13 Notch signaling pathway 0 000784 3 547231

218986_s_at NM O 17631 556Of FLJ20035 4q32 3 Unknown 0 000983 3 48366

214453_s_at BE049439 10561 1FI44 Ip31 1 Immune response 0 000349 3 39

203504_s_^at NM_005502 19 ABCAl 9q31 1 Lipid metabolism 8 36E-05 3 38

244050_at AI804932 401494 PTPLAD2 9p21 3 Unknown 3 72E-05 3 33

200923_at NM_005₅67 3959 LGALS3BP 17q25 Cell adhesion 0 000202 3 28

23₅643_at BE88622₅ 219285 SAMD9L 7q21 2-q21 3 Unknown 0 000779 3 186075

212776_s_at AI978623 23363 OBSLl 2q35 Unknown 0 004127 3 14

228116_at AW 167298 441552 Unknown Unknown Unknown 0 000191 3 13

Small GTPase mediated signal

228708_at BF438386 5874 RAB27B 18q21 2 0 000327 3 102189 transduction

219383_at NM_024841 79899 FLJ14213 I lpl3-pl2 Unknown 0000536 3 033726

Regulation of transcription,

20286 l_at NM_002616 5187 PERl 17pl3 1-I 7pl2 0 000976 3 016825 DNA-dependent

2140₅9_at BE049439 10561 1FI44 Ip31 1 Immune response 1 99E-05 2 96

218928_s_at NM O 18964 54020 SLC37A1 21q22 3 Transport 4 48E-05 2 95 ATP synthesis coupled proton

₅937₅_at AI82₅877 80022 MYO15B 17q25 1 0 000546 2 83 transport ATP synthesis coupled proton

219173_at NM_024957 80022 MYO15B 17q25 1 0 000832 2 72 transport

201641_at NM 004335 684 BST2 19pl3 2 Cell proliferation 0 000316 2 68

2281₅2_s_at AK023743 91351 FLJ31033 4q32 3 Unknown 0 0007 2 590295

226068_at BF₅93625 6850 SYK 9q22 Signal transduction 0 000832 2 58

20328 l_s_at NM_003335 7318 UBEl L 3p21 Ubiquitin cycle 1 74E-05 2 55

214366_s_at AA99₅910 240 ALOX5 1OqI l 2 electron transport 0 000529 2 536541

Protein amino acid ADP-

218₅43_s_at NM_022750 64761 PARP 12 7q34 6 00E-05 2 51 rybosylation

213294_at AV755522 253635 Unknown Unknown Unknown 8 85E-05 2 51

201601_x_at NM_003641 8519 IFITM l I lpl5 5 Immune response 0 000728 2 4

217983_s_at NMJJ03730 8635 RNASET2 6q27 RNA catabolism 0 000364 2 39

203927_at NM_004556 4794 NFKBIE 6p21 1 Apoptosis 0 000613 2 38

214022_s_at AA749101 8519 IFlTMl I lpl5 5 Immune response 0 000451 2 36

22187₅_x_at AW₅14210 3134 HLA-F 6p21 3 Antigen presentation 0 000618 2 33

217984_at NM_003730 8635 RNASET2 6q27 RNA catabolism 5 27E-05 2 32

223600_s_at AL136867 80726 KIAA1683 19pl3 1 Unknown 0 000345 2 31

Regulation of progression

202934_at AI761561 3099 HK2 2pl3 0 000721 2 31 through cell cycle

212203_x_at BF338947 10410 IFITM3 I lpl 5 5 Immune response 0 000637 23 Probe set GenBank Gene ID Gene Symbol

Location GO Annotation P Value Fold Change

1294_at L13852 7318 UBEl L 3p21 Ubiqurlin cycle 3 62E-07 2 29

Negative regulation of cell

238509_at A1628926 8454 CULl 7q36 l 0 000317 2 27 proliferation

Regulation of transcription,

2247Cl_at AA056548 54625 PARP 14 3q21 1 2 20E-06 2 26 DNA-dependent

Negative regulation of cell

207₆l 4_s_at NM_003592 8454 CULl 7q36 1 0 000797 2 25 proliferation

Protein amino acid

20₅418_at NM_002005 2242 FES 15q26 1 6 04E-05 2 21 phosphorylation

209269_s_at AW450910 6850 SYK 9q22 Signal transduction 0 000549 22

1556643_at AK055623 93343 LOC93343 19pl3 1 1 Unknown 0 000971 2 19

204806_x_at NM_018950 3134 HLA-F 6p21 3 Antigen presentation 0 00073 2 18

218322_s_at NM_016234 51703 ACSL5 10q25 l-q25 2 Lipid metabolism 0 000468 2 16

218019_s_at NM_021941 8566 PDXK 21q22 3 Unknown 0 000749 2 15 regulation of transcription, DNA

202864_s_at NM_003113 6672 SPIOO 2q37 1 0000683 2 14 dependent

202771_at NM_014745 9780 FAM38A I 6q24 3 Unknown 3 66E-05 2 13

Insulin receptor signaling

203627_at A1830698 3480 IGFl R 15q26 3 0 00078 2 12 pathway

225929_s_at AA233374 57674 C17orf27 17q25 3 Unknown 0 000635 2 1

227₅74_at BF446688 23363 OBSLl 2q35 Unknown 7 68E-05 2 06

201819_at NM_005505 949 SCARB l 12q24 31 Cholesterol metabolism 0 000117 2 05

211323_s_at L38019 3708 ITPRl 3p26-p25 Signal transduction 0 000434 2 02

Regulation of transcription,

2424₆3_x_at AI620827 162966 ZNF600 19q 13 41 0 000189 2 02 DNA-dependent

15₅2256_a_at NM_005505 949 SCARBl 12q24 31 Cholesterol metabolism 4 54E-05 2 03

22₅861_at AWOO 1250 84331 C16orfl4 16pl3 3 Unknown 0 000628 1 97

217914_at NM_017901 53373 TPCNl 12q24 13 Ion transport 7 20E-05 1 97

210571_s_at AF074480 8418 CMAH 6p21 32 Unknown 0 000609 1 95

22₅883_at AK024423 89849 ATG16L2 I lql3 4 Unknown 7 49E-05 1 92

219452_at NM_0223₅5 64174 DPEP2 16q22 1 Proteolysis 0 000495 1 91

Negative regulation of

224473_x_at BC006212 84445 LZTS2 10q24 0 000406 1 89 progression through cell cycle

202104_s_at NM_0031 19 6687 SPG7 16q24 3 Regulation of cell adhesion 0 000948 1 89

222487_s_at BC003667 51065 RPS27L 15q22 2 Protein biosynthesis 0 000964 1 86

2227₅6_s_at BC003636 408 ARRBl I lql3 Signal transduction 0 00037 1 82

21341 l_at AW242701 53616 Unknown Unknown Unknwon 0000553 1 79

2007₅2_s_at NMJ)O₅186 823 CAPNl I lql3 Proteolysis 0 000197 1 79

233880_at AL161961 57674 C17orf27 17q25 3 Ubiquitin cycle 0 00061 1 72

228410_at AA495984 139716 GAB3 Xq28 Unknown 0 000288 1 71

207131_x_at NM_013430 2678 GGTl 22ql l 23 Amino acid metabolism 0 000786 1 70

202100_at BG 169673 5899 RALB 2cen-ql3 Signal transduction 0000367 1 67

209919_x_at L20490 2678 GGTl 22ql l 23 Amino acid metabolism 0 000914 1 67

₆3₅_s_at L42374 5526 PPP2R5B 1 1 q 12 -q 13 Signal transduction 0 000253 1 66

217891_al NM_022744 64755 C16orf58 16pl l 2 Unknown 0 000497 1 66

22₅272_al AA128261 1 12483 SAT2 17pl3 1 Unknown 0 000465 1 66

223313_s_at BCOO 1207 653210 MAGED4 XpI l 22 Unknown 0 000296 1 64

208284_x_at NM_013421 2678 GGTl 22ql l 23 Amino acid metabolism 0 00053 1 64

241899_at AA524418 6583 LOC553103 Unknown Unknown 0 000911 1 62

209₅22_s_at BC000723 1384 CRAT 9q34 Lipid metabolism 0 000643 1 57

20359S_s_at N47725 24138 IF1T5 10q23 31 Immune response 0 000856 1 56

22₅874_at BE382898 124402 FAMlOOA 16p 13 3 Unknown 0 00018 1 56

21₅749_s_at AKOO 1574 64689 GORASPl 3p22-p21 33 Protein transport 0 00077 1 52

203412_at NM_006767 8216 LZTRl 22ql l 21 Morphogenesis 0 000708 1 51

201556 s at BC002737 6844 VAMP2 17pl3 1 Vesicle-mediated transport 0 000859 1 5 Chromosome

224191_x_at AF303889 ₅4763 ROPN l 3q21 1 Signal transduction 0 00084 -1 54 Protein amino acid

215161_at AWO 16039 57172 CAMK lG Iq32-q41 0 000837 -1 55 phosphorylation

216349_at AL136527 341651 LOC34165 I 13ql4 1 1 Unknown 0 000771 -1 59

206197_at NM_0035₅ l 8382 NME5 5q31 Anti-apoptosis 0 000321 -1 6 Protein amino acid

216752 at AK025026 30849 PIK3R4 3q22 1 0 000465 -1 69 phosphorylation

Clinical relevance of somatic JAKl mutations

5 The clinical relevance of JAKl mutations within the adult T-ALL cohort was investigated. While no statistically significant difference was observed in white blood cell counts, gender distribution or association with specific chromosomal rearrangements, patients with a mutated JAKl allele tended to have a more advanced age at diagnosis (median= 40.6 vs 24.2; P<0.0l), which was consistent with the lower

10 prevalence of mutations identified among children and adolescents with T-ALL included in the study. Comparison of the response to therapy between JAKl mutation- positive and mutation-negative patients indicated a higher percentage of cases exhibiting resistance to induction therapy in the former (43% vs 20%), although this difference did not reach statistical significance due to the relatively small size of the

15 study cohort. Consistent with that finding, a statistically significant reduced disease-free survival (DFS) (median=8.7 vs 20.5 months; P=0.01) and overall survival (OS) (median=10.6 vs 32.5; P<0.01) was observed among JAKl mutation-positive patients (Fig. 3B). Multivariate analysis confirmed the statistical significance of these associations (DFS: HR= 6.20, 95% CI = 1.32-29.09, P= 0.02; OS: HR= 2.82, 95% CI=

20 1.07-7.48, P= 0.04), and excluded a significant contribution of patients' age (DFS: HR= 0.99, 95% CI = 0.93-1.05, P= 0.64; OS: HR= 1.01, 95% CI= 0.97-1.05, P= 0.60).

Aberrant JAKl function and leukemogenesis

25 Functional upregulation of two members of the JAK family, JAK2 and JAK3, has recently been discovered in myeloproliferative disorders and other malignancies of the myeloid lineage (18-20, 24). The JAK2 V617F amino acid change occurs in the majority of polycythemia vera cases and in approximately 50% of individuals with essential thrombocythemia or idiopathic myelofibrosis. The available data support the view that this recurrent change, which affects the pseudokinase domain of the protein, induces constitutive activation of the kinase and hypersensitivity to cytokines. Similarly, three JAK3 hypermorphic alleles promoting cytokine independence in Ba/F3 cells have been identified in acute megakaryoblastic myeloid leukemia. Here, we showed that somatically acquired activating JAKl mutations occur in ALL, particularly in adults, further emphasizing the importance of JAK-mediated signaling dysregulation in leukemogenesis and extending the spectrum of hematologic malignancies associated with aberrant activation of this signal transduction pathway. Even though the molecular mechanisms by which individual JAKl mutations promote gain of function are likely to be diverse and remain to be fully characterized, modeling and biochemical data are consistent with the view that, similar to what has been observed for somatic leukemia- associated JAK2 and JAK3 defects, most mutations would interfere with the autoinhibitory control on the catalytic activity. For most mutations, this effect would be achieved by triggering local structural rearrangements in regions involved in interdomain interactions between the pseudokinase and kinase domains or the FERM and SH2 domains (Figure 4).

JAKl is expressed widely and participates in intracellular signaling elicited by class II cytokine receptors and receptors that utilize the gpl30 or γ_c receptor subunit. While the hematopoietic defects in Jakl^'1' mice were restricted to the lymphoid cell compartment as a result of an impaired response to IL-7 (10) and the present findings indicate a cell- context dependence of somatically acquired JAKl mutations' contribution to leukemogenesis, this kinase may be involved in other malignancies. A concomitant genetic event, including a mutation affecting other members of the JAK family, may synergize with the JAKl defect to promote aberrant cell proliferation and/or survival in a cell-specific context.

While 70-80% of pediatric patients with either B- or T-cell ALL have excellent long- term response to intensive combination chemotherapy, adult patients exhibit a less favorable outcome (4, 25). In B-lineage ALL, such a poor prognosis has been associated in part to the presence of BCRIABL or ALLlI AF4 gene rearrangements. In contrast, the unfavorable outcome of adult patients with T-ALL has not conclusively been attributed to any cytogenetic lesion, albeit the prognostic relevance of aberrant ERG and TLXl gene expression and NOTCHl mutations has recently been reported (26-28). The experimental data disclosed herein provide the first evidence that JAKl gene defects are associated with a poor response to therapy, frequent relapse of the disease and reduced overall survival, identifying such mutations as a novel informative prognostic marker occurring in a sizable proportion of adult T-ALL. The data also enable the development of therapeutic approaches tailored at interfering with JAKl signaling, expression and kinase activity.

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Claims

CLAIMS:

1. A method of detecting the presence of, or a predisposition towards, cancer in a patient comprising detecting the presence of a mutant variant of the JAKl gene in a sample obtained from the patient, wherein the presence of the mutant variant is indicative of the presence of, or a predisposition towards, cancer in the patient.

2. A method according to claim 1 wherein the JAKl gene comprises a nucleic acid sequence encoding a protein having at least 80% identity to SEQ. ID NO: 2.

3. A method according to claim 1 wherein the mutant variant of the JAKl gene comprises a nucleic acid sequence encoding a protein having at least 80% identity to SEQ. ID NO: 2 and in which at least one of the amino acid residues at the following positions is substituted with an alternative amino acid: 204, 512, 634, 653, 724, and 879.

4. A method according to claim 3 wherein the mutant variant of the JAKl gene comprises a nucleic acid sequence encoding a protein having at least 80% identity to SEQ. ID NO: 2 and in which the protein has at least one amino acid substitution selected from the group consisting of: Lys204Met, Ser512Leu, Ala634Asp, Leu653Phe, Arg724His, Arg879Ser, Arg879Cys, Arg879His and combinations thereof.

5. A method according to claim 1 wherein the patient is an adult.

6. A method according to claim 1 wherein the cancer is acute lymphoblastic leukaemia.

7. A method according to claim 6 wherein the acute lymphoblastic leukaemia is selected from the group consisting of B-cell precursor acute lymphoblastic leukaemia and T-cell precursor acute lymphoblastic leukaemia.

8. A method of assaying a product for efficacy in the treatment or prophylaxis of cancer comprising determining the kinase activity of the mutant variant of the JAKl protein, or the activation level of JAKl -mediated downstream signalling pathways, in the presence and absence of the product wherein a reduced kinase activity of the mutant variant, or reduced activation levels of downstream signalling transducers whose function is controlled by JAKl, in the presence of the product as compared with kinase activity, or the activation levels of the downstream signalling transducers, in the absence of the product is indicative that the product is efficacious in the treatment or prophylaxis of cancer.

9. A method according to claim 8 wherein the determining the kinase activity, or upregulation of JAKl function in general, comprises determining at least one of: the level of STATl, STAT5, AKT and ERK phosphorylation.

10. A method according to claim 8 wherein the JAKl protein is present in a cell.

11. A method according to claim 8 wherein the JAKl protein comprises a sequence having at least 80% identity to SEQ. ID NO: 2.

12. A method according to claim 8 wherein the mutant variant of the JAKl protein comprises a sequence having at least 80% identity to SEQ. ID NO: 2 and in which at least one of the amino acid residues at the following positions is substituted with an alternative amino acid: 204, 512, 634, 653, 724, and 879.

13. A method according to claim 8 wherein the mutant variant of the JAKl protein comprises a sequence having at least 80% identity to SEQ. ID NO: 2 and in which the protein has at least one amino acid substitution selected from the group consisting of: Lys204Met, Ser512Leu, Ala634Asp, Leu653Phe, Arg724His, Arg879Ser, Arg879Cys, Arg879His and combinations thereof.

14. A method according to claim 8 wherein the cancer is acute lymphoblastic leukaemia.

15. A method according to claim 14 wherein the acute lymphoblastic leukaemia is selected from the group consisting of B-cell precursor acute lymphoblastic leukaemia and T-cell precursor acute lymphoblastic leukaemia.

16. A method of treatment or prophylaxis of cancer in a patient comprising administering to the patient a therapeutically effective quantity of a product that inhibits the activity of the JAKl protein.

17. A method according to claim 16 wherein the JAKl protein comprises a sequence having at least 80% identity to SEQ. ID NO: 2.

18. A method according to claim 16 wherein the product inhibits the signalling activity of the JAKl protein.

19. A method according to claim 16 wherein the product inhibits the kinase activity of the JAKl protein.

20. A method according to claim 16 wherein the product inhibits the expression of the JAKl protein.

21. A method according to claim 16 wherein the cancer is acute lymphoblastic leukaemia.

22. A method according to claim 21 wherein the acute lymphoblastic leukaemia is selected from the group consisting of B-cell precursor acute lymphoblastic leukaemia and T-cell precursor acute lymphoblastic leukaemia.

23. An isolated protein comprising a sequence having at least 80% identity to SEQ. ID NO. 2 and wherein at least one of the amino acid residues at the following positions is substituted with an alternative amino acid: 204, 512, 634, 653, 724, and 879.

24. An isolated protein according to claim 23, wherein the protein comprises an amino acid substitution selected from the group consisting of: Lys204Met, Ser512Leu, Ala634Asp, Leu653Phe, Arg724His, Arg879Ser, Arg879Cys, Arg879His and combinations thereof.

25. An isolated polypeptide comprising a fragment of the isolated protein according to claim 23, wherein the fragment is at least 8 amino acids long and includes the substituted amino acid residue.

26. An isolated nucleic acid sequence comprising a sequence encoding the isolated polypeptide according to claim 25.

27. A kit for detecting cancer, or a predisposition towards cancer, in a sample from a patient, wherein the kit comprises means to detect the presence of a mutant variant of the JAKl gene in the sample.

28. The kit according to claim 27 wherein the means to detect the presence of a mutant variant of the JAKl gene comprises an nucleic acid probe capable of hybridising with the mutant variant of the JAKl gene.

29. The kit according to claim 27 wherein the means to detect the presence of a mutant variant of the JAKl gene comprises an antibody that binds to the protein encoded by the mutant variant of the JAKl gene.