WO2017020128A1 - Markers for acute myeloid leukemias with core binding factor rearrangements and other genetic subtypes and uses thereof - Google Patents

Markers for acute myeloid leukemias with core binding factor rearrangements and other genetic subtypes and uses thereof Download PDF

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WO2017020128A1
WO2017020128A1 PCT/CA2016/050909 CA2016050909W WO2017020128A1 WO 2017020128 A1 WO2017020128 A1 WO 2017020128A1 CA 2016050909 W CA2016050909 W CA 2016050909W WO 2017020128 A1 WO2017020128 A1 WO 2017020128A1
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aml
gpcr
receptor
expression
subject
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PCT/CA2016/050909
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French (fr)
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Josée HÉBERT
Guy Sauvageau
Vincent-Philippe LAVALLÉE
Bernhard LEHNERTZ
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Université de Montréal
Rsem, Limited Partnership
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention generally relates to acute myeloid leukemias (AMLs), and more particularly to the characterization and diagnosis of AML subtypes including core-binding factor- (CBF-) AMLs and other cytogenetic groups of AML.
  • AMLs acute myeloid leukemias
  • CBF- core-binding factor-
  • AML Acute Myeloid Leukemia
  • AML is a particularly lethal form of cancer, with most patients dying within two years of diagnosis. It is one of the leading causes of death among young adults.
  • AML is a collection of neoplasms with heterogeneous pathophysiology, genetics and prognosis.
  • AML patients are presently classified into groups or subsets of AML with markedly contrasting prognosis.
  • RUNX1 also known as AML1 or CBFA2
  • CBFB encode respectively the alpha and beta subunits of a heterodimeric core binding transcription factor complex involved in the development of normal hematopoiesis.
  • AMLs with chromosomal rearrangements t(8;21)/ RUNX1-RUNX1T1 and inv(16)/t(16; 16)/CSFS- YW 7, commonly designated as core binding factor- (CBF-) AMLs are classified under "AML with recurrent genetic abnormalities" in the 4 th edition of the World Health Organization (WHO) classification, and account for about 12 to 15 percent of AML cases in adults.
  • WHO World Health Organization
  • a method for treating a subject suffering from Core-Binding Factor Acute Myeloid Leukemia comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is ADRA2C (UniProt P18825), GPR153 (UniProt Q6NV75), LPAR5 (UniProt Q9H1 C0), LPAR6 (UniProt P43657), PTGIR (UniProt P431 19), CCR2 (UniProt P41597), CXCR7/ACKR3 (UniProt P25106), FPR1 (UniProt P21462), GPR183 (UniProt P32249) and/or RXFP1 (UniProt Q9HBX9).
  • GPCR G-protein coupled receptor
  • GPR183 GPR183, RXFP1, LPAR6 and/or PTGIR.
  • a method for treating a subject suffering from Acute Myeloid Leukemia with Mixed Lineage Leukemia chromosomal rearrangement comprising administering to said subject an effective amount of an agent targeting the G-protein coupled receptor (GPCR) GPR126 (UniProt Q86SQ4).
  • GPCR G-protein coupled receptor
  • GPCR G-protein coupled receptor
  • NK-AML is NK-AML with FL73-ITD and/or NPM1 mutations.
  • a method for treating a subject suffering from myelomonocytic acute myeloid leukemia comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is FPR1 (UniProt P21462), CCR2 (UniProt P41597), C5AR1 (UniProt P21730), GPR183 (UniProt P32249), P2RY13 (UniProt Q9BPV8), PTAFR (UniProt P25105), CCR1 (UniProt P32246) and/or VIPR1 (UniProt P32241).
  • GPCR G-protein coupled receptor
  • a method for treating a subject suffering from monocytic acute myeloid leukemia comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CCR1 (UniProt P32246), PTAFR (UniProt P25105), GABBR1 (UniProt Q9UBS5), CX3CR1 (UniProt P49238), P2RY2 (UniProt P41231), NMUR1 (UniProt Q9HB89) and/or HTR7 (UniProt P34969).
  • GPCR G-protein coupled receptor
  • GPCR G-protein coupled receptor
  • GPCR G-protein coupled receptor
  • GPCR G-protein coupled receptor
  • GPCR G-protein coupled receptor
  • GPCR Myeloid Leukemia
  • GPCR G-protein coupled receptor
  • GPCR G-protein coupled receptor
  • MLL-AML Mixed Lineage Leukemia chromosomal rearrangement
  • GPCR G-protein coupled receptor
  • GPCR G-protein coupled receptor
  • NK-AML is NK-AML with FLT3- ⁇ TD and/or NPM1 mutations.
  • GPCR is CYSLTR2.
  • GPCR G-protein coupled receptor
  • GPCR G-protein coupled receptor
  • GPCR G-protein coupled receptor
  • GPCR is CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 and/or HTR7.
  • GPCR G-protein coupled receptor
  • a method for determining whether a test agent may be useful for treating Core Binding Factor Acute Myeloid Leukemia comprising: (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and RXFP1 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one of more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating CBF-AML.
  • GPCRs G-protein coupled receptors
  • a method for determining whether a test agent may be useful for treating Acute Myeloid Leukemia with Mixed Lineage Leukemia chromosomal rearrangement (i) contacting a cell expressing the G-protein coupled receptor (GPCR) GPR126 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said GPCR, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating MLL-AML.
  • MLL-AML Acute Myeloid Leukemia with Mixed Lineage Leukemia chromosomal rearrangement
  • GPCRs G-protein coupled receptors
  • NK-AML is NK-AML with FL73-ITD and/or NPM1 mutations.
  • a method for determining whether a test agent may be useful for treating myelomonocytic acute myeloid leukemia comprising (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 or VIPR1 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one or more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating M4-AML.
  • GPCRs G-protein coupled receptors
  • a method for determining whether a test agent may be useful for treating monocytic acute myeloid leukemia comprising (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 or HTR7 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one or more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating M5-AML.
  • GPCRs G-protein coupled receptors
  • a method for determining the likelihood that a subject suffers from Core Binding Factor Acute Myeloid Leukemia comprising: determining the level of expression of at least one of the genes depicted in bold in Tables 8A and 8B in a leukemia cell sample from said subject, wherein a higher expression of said at least one genes depicted in bold in Table 8A, and/or a lower expression of said at least one genes depicted in bold in Table 8B, in said sample relative to a control non-CBF-AML sample is indicative that said subject has a high likelihood of suffering from CBF-AML.
  • CBF-AML Core Binding Factor Acute Myeloid Leukemia
  • 61 The method of item 60, wherein said method comprises determining the level of expression of at least one of ADARB2-AS1 and LINC00958, wherein a higher expression of said at least one genes in said sample relative to a control non-t(8;21) AML sample is indicative that said subject has a high likelihood of suffering from t(8;21) AML.
  • CBF-AML Core Binding Factor Acute Mye
  • RNA sequencing RNA-seq
  • RT-PCR reverse transcription polymerase chain reaction
  • a method for determining the likelihood that a subject suffers from AML comprising: determining the presence of at least one of a RUNX1-USP2 fusion and a RUNX1- ERG fusion in a sample comprising leukemic cells from the subject, wherein the presence of said at least one RUNX1 fusion is indicative that the subject has a likelihood of suffering from AML.
  • kits for carrying out the methods of any one of items 56-74 comprising reagents for determining the level of expression of said at least one gene and/or the presence of said one or more mutations or fusions.
  • kit of item 77 wherein said reagents comprise one or more oligonucleotides comprising a sequence complementary to a sequence of said at least one genes.
  • kit of item 77 or 78 wherein said reagents comprise reagents for nucleic acid amplification.
  • FIGs. 1A-1 I show the mutational landscape of CBF-AMLs.
  • FIG. 1A Mutational, morphological, cytogenetic and clinical information of CBF-AML. Each column represents a patient sample.
  • FIG. 1 B Variant allele frequency (VAF) of mutations in activated signaling genes across t(8;21) and inv(16) genetic groups. Each bar represents a patient sample. Stars identify samples with 2 or more mutations. Note that for each mutation in a given specimen VAF are stacked with a no co-occurrence presumption to facilitate presentation of data.
  • FIG. 1 C Activated signaling mutations in a sample at diagnosis and relapse. FIGs.
  • FIG. 1 D-F Primary structures of ZBTB7A, ASXL2 and SMC1A proteins, respectively, with corresponding positions of mutations.
  • FIG. 1 G Number of non-activated signaling mutations in CBF AML subgroups.
  • FIG. 11 confirmation of acquired ZBTB7A mutations in t(8;21) AML in samples 05H184, 05H1 18, and 07H137. Statistics are based on Fisher's exact test. MS: missense; NS: nonsense; FS: frameshift; NA: Not available; dx: diagnosis; rel: relapse; ASXN: Additional sex combs (ASX) N-terminal; ASXH: ASX Homology; PHD: plant homeodomain.
  • FIGs. 2A-2H show the transcriptome of CBF-AMLs compared to that of other AML subtypes.
  • FIGs. 2A-2C Comparative analyses of expressed genes in t(8;21) (FIG. 2A), inv(16) (FIG. 2B) and CBF (FIG. 2C) AML subgroups. Diamonds correspond to differentially expressed genes (difference > 1 or ⁇ -1) listed in Tables 5-7 for panel FIGs. 2A-2C, respectively.
  • FIG. 2D IRX1 expression in different leukemia genetic subgroups.
  • FIGs. 2E-2G Differential expression of HOXA9 (FIG. 2E), HOXB5 (FIG.
  • FIG. 2F Comparative analyses of expressed genes in t(8;21) (RUNX1-RUNX1T1) vs. inv(16) (CBFB-MYH11) AMLs. For all the comparisons, in order to log 10 transform expression values, a small constant of 0.0001 was added.
  • CBF Core-binding factor
  • AML Acute myeloid leukemia.
  • FIGs. 3A-3F show the transcriptomic similarities between RUNX1-RUNX1T1 and RUNX1-CBFA2T3 AML.
  • FIG. 1A Principal component analysis
  • FIG. 3C Characterization of samples with RUNX1 in frame fusions other than RUNX1-RUNX1T1. Each fusion was identified using RNA-sequencing and confirmed by targeted sequencing. The RUNX1-USP42 fusion was previously described Giguere A and Hebert J. Genes, Chromosomes and Cancer. 201 1 ;50(4):228-238).
  • FIG. 3D Primary structure of the predicted RUNX1 -ERG fusion protein.
  • FIG. 3E Expression of ERG and USP2 in the entire cohort. Sample 12H 149, harboring RUNX1- ERG and RUNX1-USP2 fusions, is in the top right corner.
  • RUNX1T1 and CBFA2T3 fusion partners
  • Medians are indicated by horizontal bars.
  • PC principal component
  • CBF Core-binding factor
  • 5'-UTR 5'-untranslated region.
  • FIGs. 4A-40 show cufflinks isoform expression of MYH11_iso_14 (FIG. 4A); MYH11_iso_6 (FIG. 4B), APLN_iso_4 (FIG. 4C), ST18_iso_19 (FIG. 4D), ST18_iso_20 (FIG. 4E), ST18_iso_2 (FIG. 4F), ST18_iso_17 (FIG. 4G), ST18_iso_1 (FIG. 4H), MSLN_iso_2 (FIG. 4I), NT5E_iso_1 (FIG. 4J), MSLN_iso_8 (FIG.
  • FIGs. 5A-50 show cufflinks isoform expression of RUNX1T1_iso_21 (FIG. 5A); RUNX1T1_iso_15 (FIG. 5B), ZNF598_iso_6 (FIG. 5C), RUNX1T1_iso_18 (FIG. 5D), RAE1_iso_9 (FIG. 5E), CWC15_iso_15 (FIG. 5F), RUNX1T1_iso_22 (FIG. 5G), SETD5- AS1_iso_35 (FIG. 5H), RNF13_iso_15 (FIG. 5I), AP1B1_iso_8 (FIG.
  • FIG. 6A shows GPCR classification in ligand groups.
  • the GPCR classification in ligand groups was based on the IUPHAR database. To complete and subdivide the class A group, the GRAFS phylogenetic classification of GPCRs was used. Taste 2 receptors, which are not included in the IUPHAR database, have also been added as well as vomeronasal receptors, opsins and 3 orphan GPCRs (GPR137B, TAPT1, XPR1). GPCRs have been classified in 18 ligand groups of receptors.
  • FIGs. 6B and 6C show the threshold definition for highly expressed GPCRs (FIG. 6A) and GPCRs with low variability in their expression (FIG. 6B).
  • the mean expression level or the coefficient of variation (CV) (X axis) is represented for each of the 772 GPCRs.
  • Each dot represents a GPCR after classification by decreasing median level of expression (FIG. 6A) increasing CV (Y axis) (FIG. 6B).
  • Highly expressed GPCRs were selected using a threshold of 3.5 IRPKM.
  • the threshold for GPCRs with low variability in their expression was established at a CV of 50%.
  • FIG. 7 is a schematic representation of the correlation between the results obtained from the qPCR experiments (Delta CT, Y axis) and the RNA-Seq analysis (IRPKM, X axis) was 0.94 (p ⁇ 0.0001). Each dot represents a mean of 2 independent experiments. The Pearson's R correlation coefficient varies from -0.79 to -0.98, supporting a robust correlation between the two datasets.
  • FIG. 8 shows the validation of antibodies for flow cytometry analysis of selected GPCRs.
  • HEK293 cells were transfected with CXCR4, CD97, LTB4R, FPR1 or C5AR1 cDNA.
  • Transfected or untransfected cells were stained with the respective antibodies for flow cytometry analysis. Positive population of transfected cells was compared to the untransfected cells using their respective unstained cells as controls. For the five receptors tested, a clear difference of staining between these cells confirms that the antibodies target the expected proteins.
  • FIG. 9A and 9B show flow cytometry analysis of selected highly expressed GPCRs. The protein expression of 5 selected GPCR members was assessed in 20 AML samples with of different morphologic subtypes by flow cytometry analysis.
  • FIG. 9A Representative histogram of one AML sample (03H041) following staining with antibodies for CD97, LTB4R, CXCR4, C5AR1 or FPR1. Red histograms represent unstained AML cells used as controls
  • FIG. 9B Dot plot representing the percentage of protein-expressing cells observed in each AML specimen stained.
  • CD97 shows a strong expression in all cells of each of the 20 studied AML samples (92.5 to 99.6 % positive cells).
  • FIG. 10 shows the correlation between GPCR expression in AML and in normal CD34 + cells.
  • the median gene expression level (in IRPKM) of the 772 GPCRs in normal CD34 + cells (X axis) is represented against their expression in AML cells (Y axis).
  • the 30 upregulated GPCRs in AML (upper portion) have a difference of median expression level between AML and normal CD34 + cells greater than 1 .
  • the 19 downregulated GPCRs in AML lower portion have a difference of median expression less than -1 .
  • GPCRs represented in black dots (between the two parallel lines) are not differentially expressed between AML and normal cord blood-derived CD34 + cells.
  • RNA-Seq data were transformed to IRPKM (log2(RPKM+ 1)).
  • FIGs. 11A and 11 B show ligand family distribution of up- and down-regulated GPCRs in AML.
  • FIG. 11 A the proportion of genes up- (left bars) or down-regulated (middle bars) in AMLs, and all GPCRs (right bars) into the different families of GPCRs (adhesion, amine, chemokine, etc.) is shown. The p-values are indicated for the significant families by Fisher's exact tests.
  • FIG. 11 B Individual GPCRs of the enriched groups are shown. The values indicated in the second and third columns correspond to the receptor mean expression level in AML and in CD34 + cells.
  • GPCR genes have mean expression levels in AML significantly different from their mean in normal CD34 + cells, p-values ⁇ 0.005.
  • the Fisher's exact test was performed between the up- or downregulated group and the remaining GPCRs, i.e. all the GPCRs excluding differentially expressed members.
  • FIGs. 12A-12C show GPCR expression level analysis in AMLs of different genetic subgroups. Expression (in IRPKM) of the deregulated GPCR members in AML samples with t(8;21), inv(16) and MLL translocations (FIG. 12A) and normal karyotype with FLT3-ITD or NPM1 mutations (FIG. 12B).
  • the differentially expressed GPCRs were identified in the Leucegene cohort and are defined as having a difference of mean expression higher or equal to 1 .5 IRPKM between samples with (+) and without (-) the genetic abnormality and a significant student's t test (p ⁇ 0.05). Data are expressed as individual sample expression value and means +/- 1 SEM for all the samples.
  • FIG. 12C GPCR members with a significant difference of mean expression level in AML of the representative genetic groups in the Leucegene and TCGA cohorts. RNA-Seq data were transformed to RPKM (log2(RPKM+1)). SEM: standard error of the mean.
  • FIGs. 13A and 13B show GPCRs with deregulated expression in at least one frequent genetic subgroup. Expression (in IRPKM) of the deregulated GPCR members in AML samples with t(8;21), inv(16), normal karyotype (NK) and MLL translocations and normal karyotype with DNMT3A, FLT3- ⁇ TD or NPM1 mutations (FIG. 13B).
  • the differentially expressed GPCRs were identified in the Leucegene cohort and are defined as having a difference of mean expression higher or equal to 1 .5 IRPKM between samples with (+) and without (-) the genetic abnormality and a significant student's t test (p ⁇ 0.05).
  • RNA-Seq values were transformed to IRPKM ( ⁇ og2(RPKM+1)).
  • FIG. 14 shows GPCR expression level analysis in M4 and M5 AML morphologic subtypes. Expression levels of the 772 GPCRs were analyzed in AML samples with a monocytic component (FAB M4 and M5 subtypes) and compared to their expression in all other AML samples of the Leucegene cohort. Overexpressed GPCRs in AML M4 or M5 are defined as having a difference of mean expression higher or equal to 1 .5 IRPKM between AML M4 or M5 samples and other AML samples and a significant student's t test (p ⁇ 0.05). Results obtained for the GPCRs identified in the table have been validated in the TCGA dataset. Receptors in bold are also overexpressed in AML with inv(16).
  • FIG. 15 shows GPR126 expression in AML samples with different MLL translocation partners.
  • GPR126 expression levels values in transformed log2(RPKM+1), IRPKM
  • RPKM+1 transformed log2(RPKM+1)
  • IRPKM different translocation partners of the Leucegene AML samples with MLL translocations
  • Data are expressed as individual sample expression value and means +/- 1 SEM for all the samples.
  • FIGs. 16A and 16B show the expression levels of the GPCRs overexpressed in specific AML genetic subgroups compared to normal blood and bone marrow cell populations.
  • FIG. 16A Expression levels of the GPCRs previously identified as overexpressed in specific genetic subgroups of AML are compared to their expression in normal blood and bone marrow cell populations.
  • NK Normal Karyotype
  • WBC White Blood Cells
  • nBM normal Bone Marrow
  • Ery-I Immature proerythroblasts CD34+CD71+Gpa-
  • Ery-ll Early erythroblasts CD34-CD71+Gpa-
  • Ery-lll Erythroblasts CD34- CD71+Gpa+
  • Ery-IV Mature erythroblasts CD34-CD71-Gpa+
  • Pre-B-I Early Pre-B precursors
  • Pre-B-ll Pre-B precursors
  • Gran-I Myeloid precursors
  • Gran-ll Promyelocytes
  • Gran-Ill Myelocytes
  • Gran-IV Metamyelocytes
  • Gran-V Granulocytes.
  • the term “about” has its ordinary meaning.
  • the term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% or 5% of the recited values (or range of values).
  • the terms "subject” and “patient” are used interchangeably herein, and refer to an animal, preferably a mammal, most preferably a human.
  • the AML patient is less than 60 years old.
  • the AML patient is 60 years old or older.
  • the AML patient is a pediatric AML patient.
  • next generation RNA sequencing analyses of 415 primary AML specimens that were selected to represent AML genetic diversity and which include 20 samples with t(8;21) rearrangements and 28 samples with inv(16)/t(16; 16) rearrangements.
  • CBF-AMLs exhibit distinct mutational and transcriptional signatures relative to other AML subtypes, which may be useful for the characterization, diagnosis and prognosis of CBF-AMLs. They have also identified points of convergence and divergence in the genetic/mutational and transcriptional signatures of these two CBF-AML subgroups, and established the phenotypes of the non-classical fusions identified in the cohort studied.
  • GPCRs are expressed in this cohort of human AML.
  • 30 are overexpressed in AML samples compared to normal CD34 + cells.
  • Upregulated GPCRs are enriched in chemokine (including CCR1, CXCR4, CCR2, CX3CR1, CCR7 and CCRL2), adhesion (including CD97, EMR1, EMR2 and GPR114) and purine (including P2RY2 and P2RY13) receptor families.
  • some GPCRs were differentially expressed in normal karyotype AML with NPM1 or FLT3-ITD mutation, and in specimens with Core Binding Factor and MLL rearrangements.
  • inv(16) and t(8;21) AMLs exhibit distinct mutational signatures which may be useful for the characterization, classification and diagnosis of AMLs and AMLs subtypes (e.g., inv(16) and t(8;21)) AMLs (FIG. 1A and Table 5).
  • the present invention relates to a method for determining the likelihood that a subject suffers from CBF-AML subtype, said method comprising: determining the presence of one or more of the mutations depicted in FIG. 1A and/or Table 5 in a cell (e.g., blood cell, a leukemic cell) sample from the subject, wherein the presence of the one or more mutations is indicative that said subject suffers or is likely to suffer from a CBF-AML, and wherein the absence of said one or more mutations is indicative that the subject does not suffer or has a low likelihood of suffering from an a CBF-AML.
  • the cell sample is from a subject already diagnosed from AML or from CBF-AML.
  • the cell sample is from a subject already diagnosed with AML.
  • the at least one mutation is a mutation in one or more of ZBTB7A, ASXL2, SMC1A, TET2, and DNMT3A.
  • the method is for determining the likelihood that a subject suffers from CBF-AML with t(8;2 ⁇ /RUNX1-RUNX1T1 chromosomal rearrangement (t(8;21) AML).
  • the mutation is a missense mutation.
  • the mutation is a nonsense mutation.
  • the mutation is a frameshift mutation.
  • the mutation is an inframe mutation.
  • the present invention relates to a method for determining the likelihood that a subject suffers from Core Binding Factor Acute Myeloid Leukemia (CBF-AML) with t(8;2 ⁇ /RUNX1-RUNX1T1 chromosomal rearrangement (t(8;21) AML), said method comprising: determining the presence of one or more mutations in at least one of the following genes: ZBTB7A, ASXL2, SMC1A, TET2, and DNMT3A in a sample comprising leukemic cells from the subject, wherein the presence of the one or more mutations in said at least one genes is indicative that the subject has a high likelihood of suffering from t(8;21) AML.
  • the cell sample is from a subject already diagnosed with AML or CBF-AML.
  • the mutation is in ZBTB7A, for example at a position corresponding to residue 122 or 174 of ZBTB7A.
  • the mutation is a V to G substitution at residue 122 of ZBTB7A.
  • the mutation is a frameshift mutation at residue 174 of ZBTB7A (introducing a premature stop codon).
  • the mutation is in SMC1A, for example at a position corresponding to residue 96 or 71 1 of SMC1A.
  • the mutation is an R to H substitution at residue 96 of SMC1A.
  • the mutation is an R to Q substitution at residue 71 1 of SMC1A
  • the at least one mutation is a RUNX1 fusion.
  • the RUNX1 fusion is a RUNX1-USP2 fusion or a RUNX1-ERG fusion.
  • the at least one mutation is a mutation in one or more of ZBTB7A, ASXL2, and SMC1A.
  • the at least one mutation is a frameshift mutation in ASXL2.
  • the at least one frameshift mutation is at position 523, 587, 594 and/or 614 of ASLX2, leading to a premature stop codon.
  • the at least one mutation is a mutation in TET2 and/or DNMT3A.
  • the mutation in TET2 is at position 330, 731 or 735.
  • the mutation is an F to Y substitution at position 731 .
  • the mutation is a premature stop codon at position 330 or 735.
  • the mutation in DNMT3A is at position 635, 1050 or 1501 .
  • the mutation is a premature stop codon at position 635 or 1050.
  • the mutation is a frameshift mutation at position 1501 .
  • the present invention encompasses the detection of any mutation or any combination/sub-combination of the mutations defined herein, for example the detection of a single mutation, or of 2, 3, 4, 5 or more of the mutations defined herein, within a gene or a combination of genes defined herein.
  • the term "high likelihood” means that the individual is more likely to have the disorder or disease (CBF-AML) than an individual without the mutation(s), or that the sample is more likely to be an CBF-AML leukemia sample than an AML sample without the mutation(s).
  • the determination of the presence (or absence) of the mutation(s) in the sample may be performed using any suitable methods (see, e.g., Syvanen, Nat Rev Genet. 2001 Dec;2(12):930-42).
  • the presence of the mutation(s) may be detected at the genomic DNA, transcript (RNA or cDNA) or protein level.
  • RNA sequencing RNA-seq
  • hybridization of a nucleic acid probe capable of specifically hybridizing to a nucleic acid sequence comprising the mutation(s) and not to (or to a lesser extent to) a corresponding nucleic acid sequence that does not comprises the mutation(s) under comparable hybridization conditions, such as stringent hybridization conditions) (e.g., molecular beacons); restriction fragment length polymorphism analysis (RFLP); Amplified fragment length polymorphism PCR (AFLP-PCR); amplification of a nucleic acid fragment comprising the mutation(s) using a primer specifically hybridizing to a nucleic acid sequence comprising the mutation(s), wherein the primer produces an ampl
  • the determination of the presence of the mutation(s) may also be achieved at the polypeptide/protein level.
  • suitable methods for detecting alterations at the polypeptide level include sequencing of the encoded polypeptide; digestion of the encoded polypeptide followed by mass spectrometry or HPLC analysis of the peptide fragments, wherein the mutated polypeptide results in an altered mass spectrometry or HPLC spectrum as compared to the unmutated polypeptide; and immunodetection using an immunological reagent (e.g., an antibody, a ligand) which exhibits altered immunoreactivity with a mutated polypeptide relative to a corresponding unmutated polypeptide.
  • an immunological reagent e.g., an antibody, a ligand
  • Immunodetection can measure the amount of binding between a polypeptide molecule and an anti-protein antibody by the use of enzymatic, chromodynamic, radioactive, magnetic, or luminescent labels which are attached to either the anti-protein antibody or a secondary antibody which binds the anti-protein antibody.
  • other high affinity ligands may be used.
  • Immunoassays which can be used include e.g. ELISAs, Western blots, and other techniques known to those of ordinary skill in the art (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999 and Edwards R, Immunodiagnostics: A Practical Approach, Oxford University Press, Oxford; England, 1999). Methods to generate antibodies exhibiting altered immunoreactivity with a mutated polypeptide relative to a corresponding unmutated polypeptide are described in more detail below.
  • microarrays e.g., SNP microarrays
  • protein-arrays e.g., SNP microarrays
  • antibody microarrays e.g., antibody microarrays
  • tissue microarrays e.g., tissue microarrays
  • electronic biochip or protein-chip based technologies see Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, Mass., 2000.
  • nucleic acid-containing sequences may be amplified prior to or in conjunction with the detection methods noted herein.
  • the design of various primers for such amplification is known in the art.
  • a nucleic acid (RNA, cDNA, genomic DNA) comprising the mutation(s) may be amplified using primers hybridizing to sequences located on each side of the mutation(s).
  • Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8: 14-25. Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill.
  • Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the ⁇ replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1 173-1 177; Lizardi et al., 1988, BioTechnology 6: 1 197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., supra).
  • amplification will be carried out using PCR.
  • PCR Polymerase chain reaction
  • PCR involves, a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected.
  • An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith.
  • the extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers.
  • the sample is analyzed to assess whether the mutation(s) to be detected is/are present. Detection of the amplified sequence may be carried out by visualization following Ethidium Bromide (EtBr) staining of the DNA following gel electrophoresis, or using a detectable label in accordance with known techniques, and the like.
  • EtBr Ethidium Bromide
  • Ligase chain reaction is carried out in accordance with known techniques (Weiss, 1991 , Science 254: 1292). Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill. Strand displacement amplification (SDA) is also carried out in accordance with known techniques or adaptations thereof to meet the particular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA 89:392-396; and 1992, Nucleic Acids Res. 20: 1691 -1696).
  • SDA Strand displacement amplification
  • Nucleic acid hybridization refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure. Examples of hybridization conditions can be found in laboratory manuals (e.g., Green and Sambrook, supra) and are commonly known in the art. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1 % SDS at 42°C.
  • SDS sodium dodecyl sulfate
  • hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0.1 % SDS at 68°C.
  • a nitrocellulose filter can be incubated overnight at 65°C with a labeled probe specific to one or the other two alleles in a solution containing 50% formamide, high salt (5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1 % SDS, and 100 ⁇ g/ml denatured carrier DNA (i.e. salmon sperm DNA).
  • the non-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0.1 % SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42°C (moderate stringency) or 65°C (high stringency).
  • Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest.
  • the selected temperature is based on the melting temperature (Tm) of the DNA hybrid.
  • Tm melting temperature
  • stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • AMLs By comparing the transcriptomic GPCR signature of AMLs with normal CD34 + cells, the present inventors have identified GPCR genes which are differentially expressed in AMLs.
  • the present invention relates to a method for determining the likelihood that a subject suffers from Acute Myeloid Leukemia (AML), the method comprising, determining the level of expression (i.e., the amount) of at least one of the GPCR genes showing differential expression in AMLs vs. normal CD34 + cells listed in Table 12 in a cell sample (e.g., blood, bone marrow, leukemic cell sample) from the subject and comparing the level of expression of the at least one gene to that in a control sample (or to a reference value derived therefrom), wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from AML.
  • AML Acute Myeloid Leukemia
  • a higher level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference level derived therefrom) is indicative that the subject suffers or is likely to suffer from AML.
  • a lower or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from AML.
  • a lower level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject suffers or is likely to suffer from AML.
  • a higher or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from AML.
  • the above methods comprise the determination of the level of expression of at least one of the following GPCR genes: CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 or FPR2.
  • GPCR genes CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7,
  • the above methods comprise the determination of the level of expression of at least one of the following GPCR genes: CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1 or C5AR1. In a further embodiment, the above methods comprise the determination of the level of expression of at least one of the following GPCR genes: CXCR4, CD97, PTGER4, GPR183, PTGER2, C3AR1 or EMR2.
  • CBF-AMLs By comparing the transcriptomic signature of CBF-AMLs with other AMLs described in the studies presented herein, the present inventors have identified genes that are differentially expressed in CBF-AMLs (see, e.g., Tables 8A and 8B).
  • the present invention relates to a method for determining the likelihood that a subject suffers from a Core-Binding Factor acute myeloid leukemia (CBF-AML), the method comprising determining the level of expression (i.e., the amount) of at least one of the genes identified in bold in Tables 8A and/or 8B in a cell sample from the subject and comparing the level of expression of the at least one gene to that in a control sample (or to a reference value derived therefrom), wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from a CBF-AML.
  • CBF-AML Core-Binding Factor acute myeloid leukemia
  • the above method comprises the determination of the level of expression of a combination of at least two, three, four or five of the genes listed in Tables 8A and/or 8B.
  • the above method comprises the determination of the level of expression of at least one of ARC, CACNA1H and LOC200772. In an embodiment, the above method comprises the determination of the level of expression of ARC. In an embodiment, the above method comprises the determination of the level of expression of CACNA1H. In an embodiment, the above method comprises the determination of the level of expression of LOC200772.
  • the present invention relates to a method for determining the likelihood that a subject suffers from CBF-AML, the method comprising determining the level of expression (i.e., the amount) of at least one of the following GPCR genes: ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and RXFP1 in a cell sample from the subject and comparing the level of expression of the at least one GPCR gene to that in a control sample (or to a reference value derived therefrom), wherein a higher expression of the at least one gene is indicative that the subject suffers or is likely to suffer from a CBF-AML.
  • the level of expression i.e., the amount
  • inv16 AMLs. Comparative analysis of genes and transcripts expressed in inv(16)/t(16; 16)/CSFS- YW 7 myeloid leukemia (inv(16) AML) relative to other AML subtypes enabled the identification of a set of differentially expressed genes/transcripts (See Tables 7A- 7B and 10), which may be useful for the characterization, classification, diagnosis and treatment of inv(16) AMLs.
  • the present invention concerns a method for determining the likelihood that a subject suffers from an inv(16) AML, the method comprising, determining the level of expression (i.e., the amount) of at least one of the genes identified in bold in Tables 7A-7B, or at least one of the transcripts identified in Table 10 (SEQ ID NOs:21 - 35) in a cell sample from the subject and comparing the level of expression of the at least one gene to that in a control sample (or to a reference value derived therefrom), wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from an inv(16) AML.
  • the cell sample is a leukemia cell sample from a subject diagnosed with AML or CBF-AML.
  • the above method comprises the determination of the level of expression of a combination of at least two, three, four or five of the genes/transcripts listed in Tables 7A, 7B and/or 10.
  • the method comprises the determination of the level of expression of a at least one of MEGF10 and APLN.
  • the above methods comprise the determination of the level of expression of at least one of the following GPCR genes: C5AR1, CCR2, ACKR3/CXCR7, FPR1, GPR183, LPAR6, PTGIR and RXFP1, in a further embodiment at least one of CXCR7/ACKR3 and RXFP1.
  • GPCR genes C5AR1, CCR2, ACKR3/CXCR7, FPR1, GPR183, LPAR6, PTGIR and RXFP1, in a further embodiment at least one of CXCR7/ACKR3 and RXFP1.
  • the present invention concerns a method for determining the likelihood that a subject suffers from a t(8;21) AML, the method comprising, determining the level of expression (i.e., the amount) of at least one of the genes identified in bold in Tables 6A-6B, or at least one of the transcripts identified in Table 11 (SEQ ID NOs:36- 50) in a sample from the subject and comparing the level of expression of the at least one gene to that in a control sample (or to a reference value derived therefrom), wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from a t(8;21) AML.
  • the cell sample is a leukemia cell sample from a subject diagnosed with AML or CBF-AML.
  • a lower level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject is likely to suffer from a t(8;21) AML.
  • a higher or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from a t(8;21) AML.
  • the above method comprises the determination of the level of expression of a combination of at least two, three, four or five of the genes/transcripts listed in Tables 6A, 6B and/or 10.
  • the above method comprises the determination of the level of expression of at least one of LINC00958 and ADARB2-AS1.
  • the above method comprises the determination of the level of expression of at least one of the following GPCR genes: ADRA2C, GPR153, LPAR5, LPAR6 and PTGIR. In an embodiment, the above method comprises the determination of the level of expression of at least one of the following GPCR genes: ADRA2C and GPR153.
  • MLL-rearranged AMLs Comparative analysis of GPCR genes expressed in MLL myeloid leukemia (MLL-AMLs) further enabled the identification differentially expressed GPCR genes which may be useful for the characterization, classification, diagnosis and treatment of MLL-rearranged AMLs (see FIG. 12A).
  • the present invention concerns a method for determining the likelihood that a subject suffers from a MLL-rearranged AML, the method comprising, determining the level of expression (i.e., the amount) of at least one of the genes identified FIG. 12A in a sample from the subject and comparing the level of expression of the at least one gene to that in a control sample (or to a reference value derived therefrom), wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from a MLL-rearranged AML.
  • the cell sample is a leukemia cell sample from a subject diagnosed with AML.
  • a higher level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject suffers or is likely to suffer from a MLL-rearranged AML.
  • a lower or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from a MLL-rearranged AML.
  • a lower level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject is likely to suffer from a MLL-rearranged AML.
  • a higher or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from a MLL-rearranged AML.
  • the above method comprises the determination of the level of expression of GPR126.
  • the MLL-rearranged AML is MLL-MLLT4, MLL-ELL or MLL-SEPT9.
  • Normal karyotype AML Comparative analysis of GPCR genes expressed in normal karyotype (NK) AMLs with internal tandem duplication in FLT3 (FLT3-ITD) and mutated NPM1 show distinct transcriptional profiles with respect to each other and with respect to non-NK AML samples.
  • the present invention provides a method for determining the likelihood that a subject suffers from FLT3-ITD AML, the method comprising determining the level of expression of at least one of the differentially expressed genes identified and listed in FIG. 13B in a cell sample from the subject and comparing the level of expression of the at least one gene to a control sample or reference level derived therefrom, wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from FLT3- ⁇ TD AML.
  • the above method comprises determining the level of at least one of the following genes: i) CYSLTR2, GPR114, and GPR156, wherein an increased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from FLT3- ⁇ TD AML; and/or ii) P2RY13; FRP1 and C5AR1, wherein a decreased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from FLT3- ⁇ TD AML.
  • the present invention further provides a method for determining the likelihood that a subject suffers from an AML subtype with a mutated NPM1 (NPM1m), the method comprising, determining the level of expression of at least one of the differentially expressed genes identified and listed in FIG. 13B in a cell sample from the subject and comparing the level of expression of the at least one gene to a control sample or reference level derived therefrom, wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from NPM1m AML.
  • NPM1m mutated NPM1
  • the above method comprises determining the level of i) GPR56 and/or CYSLTR2, wherein an increased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from a NPM1m AML; and/or ii) FPR1 and/or C5AR1, wherein a decreased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from a NPM1 AML.
  • M4 and M5 AMLs Analysis of GPCRs expression fingerprints also revealed distinct gene expression profiles between M4 and M5 AMLs and other AML subtypes.
  • M4 (acute myelomonocytic leukemia) and M5 (acute monocytic leukemia) refer to AML subtypes according to the French-American-British (FAB) classification system (Bennett JM et al., Ann Intern Med. 1985; 103(4):620).
  • FAB French-American-British
  • the present invention further relates to a method for determining the likelihood that a subject suffers from a M4 AML subtype, the method comprising, determining the level of expression of at least one of the differentially expressed genes identified and listed in FIG. 14 in a cell sample from the subject and comparing the level of expression of the at least one gene to a control sample or reference level derived therefrom, wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from M4 AML subtype.
  • the above method comprises determining the level of at least one of the following genes: FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 and VIPR1, wherein an increased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from M4 AML subtype.
  • the present invention concerns a method for determining the likelihood that a subject suffers from a M5 AML subtype, the method comprising, determining the level of expression of at least one of the differentially expressed genes identified and listed in FIG. 14 in a cell sample from the subject and comparing the level of expression of the at least one gene to a control sample or reference level derived therefrom, wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from M5 AML subtype.
  • the above method comprises determining the level of at least one of the following genes i) CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1, and HTR7, wherein an increased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from a M5 AML: and/or ii) GPR174, GPR56, NMUR1, SUCNR1, OR2W3 and/or GPR97, wherein a decreased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from a M5 AML.
  • the present invention concerns a method for distinguishing between inv(16) AML and t(8;21) AML in a leukemia cell sample from a subject, the method comprising, determining the level of expression (i.e., the amount) of at least one of the genes identified and listed in FIG. 2H and/or Table 8C in the leukemia cell sample from the subject and comparing the level of expression of the at least one gene to that in a control sample (or to a reference value derived therefrom), wherein the level of expression of the at least one gene enables to distinguish between inv(16) AML and t(8;21) AML.
  • the level of expression i.e., the amount
  • any gene or combination of gene listed in FIG. 2H and/or Table 8C may be used in accordance with the present invention to distinguish between inv(16) and t(8;21) AMLs. Genes identified as being overexpressed in t(8;21) in FIG. 2H and/or Table 8C are correspondingly underexpressed in inv(16) and vice versa.
  • a higher level of expression of the at least one gene in a cell sample from a subject relative to that in an inv(16) control sample (or relative to a reference value derived therefrom) is indicative that the subject suffers or is likely to suffer from a t(8;21) AML.
  • a lower or equal level of expression of the at least one gene in the cell sample from the subject relative to that in an inv(16) control sample (or relative to a reference value derived therefrom) is indicative that the subject suffers from an inv(16) AML or is likely to suffer from an inv(16) AML.
  • a lower level of expression of the at least one gene in the cell sample from the subject relative to that in a inv(16) control sample (or relative to a reference value derived therefrom) is indicative that the subject is likely to suffer from a t(8;21) AML.
  • a higher or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a inv(16) control sample (or relative to a reference value derived therefrom) is indicative that the subject suffers from an inv(16) AML or is likely to suffer from an inv(16) AML.
  • the method comprises the determination of the level of expression of a combination of at least one or two of the following genes: RUNX1T1, POU4F1, MEIS1, CD1E, TM4SF1, KCNK17, RXFP1, ENHO, MYBPH, C3orf80, CLEC10A, SPINK2, SHD, COLEC12, AGR2, PRTFDC1, ASGR2, CPVL, CD1C, HOXB-AS1, LOC283683, CES1, NPTX2, LGALS2, TREM2, MEGF10, RHBDL3, SULF2, HOXB4 and MSR1.
  • the method comprises the determination of the level of expression of a combination of at least two of the following genes: RUNX1T1, POU4F1, MEIS1 and CD1E.
  • the method comprises the determination of the level of expression of at least one gene identified in FIG. 2H. In another embodiment, the method comprises the determination of the level of expression of at least one gene identified in Table 8C.
  • all methods described herein encompasses the determination of the level of expression of any gene or any combination/sub-combination of the genes defined herein (e.g., those identified in Table 12 for AMLs; Tables 8A-8B for CBF-AMLs; Tables 7A-7B and 10 for inv(16) AMLs, Tables 6A-6B and 11 for t(8;21) AMLs; FIG. 12A for MLL-AMLs; FIG. 13B for NK-AMLs with DNMT3A, FLT3-ID or NPM1 mutations, etc.).
  • methods described herein may comprise the determination of the level of expression of a single gene, or of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125 or more of the differentially expressed genes identified herein.
  • the at least one gene has a mean differential expression value of +/- 1 .2, 1 .25, 1 .3, 1 .35, 1 .5, 2.75, 2, 2.25, 2.35, 2.4, 2.5, 2.6, 2.8, or 3.
  • the determination of the expression of the one or more genes or encoded gene products (e.g., mRNA, protein) listed above may be performed using any known methods to detect nucleic acids or proteins.
  • the expression is compared to a control or reference level (e.g., the level obtained a sample from one or more non-CBF, inv(16) and/or t(8;21) AML sample(s), and/or a cell sample enriched in CD34 + cells) to assess the subject's likelihood of suffering from a CBF-AML or CBF-AML subtype (e.g., inv(16) AML or t(8:21) AML), or the likelihood that the AML sample is CBF- AML or CBF-AML subtype (e.g., inv(16) AML or t(8:21) AML) sample.
  • a control or reference level e.g., the level obtained a sample from one or more non-CBF, inv(16) and/or t(
  • nucleic acid amplification methods can be used to detect the level of expression of the one or more genes.
  • the oligonucleotide primers and probes may be used in amplification and detection methods that use nucleic acid substrates isolated by any of a variety of well-known and established methodologies, such as those described above.
  • the above-mentioned method comprises a step of amplification.
  • the nucleic acid or amplification product may be detected or quantified by hybridizing a probe (e.g., a labeled probe) to a portion of the nucleic acid or amplified product.
  • the probe may be labelled with a detectable group that may be, for example, a fluorescent moiety, chemiluminescent moiety, radioisotope, biotin, avidin, enzyme, enzyme substrate, or other reactive group.
  • Other well-known detection techniques include, for example, gel filtration, gel electrophoresis and visualization of the amplicons, and High Performance Liquid Chromatography (HPLC).
  • HPLC High Performance Liquid Chromatography
  • the level of amplified product is detected as the product accumulates.
  • the above-mentioned method comprises a step of detection or quantification with a probe.
  • the level of expression of any differentially expressed gene disclosed herein is measured and the method comprises detecting or quantifying the nucleic acid or amplified product with a probe.
  • the probe comprises from about 7-8 to about 100, 90, 80, 70, 60 or 50 nucleotides, in further embodiments from about 10 to about 50, 45 or 40 nucleotides, from about 10 to about 35 nucleotides, from about 10 to about 35, 34, 33, 32, 31 or 30 nucleotides, from about 15 to about 25 nucleotides or from about 16 to about 24 nucleotides.
  • the probe comprises about 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.
  • the above-mentioned method comprises a step of normalizing the gene expression levels, i.e. normalization of the measured levels of the above-noted genes against a stably expressed control gene (or housekeeping gene) to facilitate the comparison between different samples.
  • "Normalizing” or “normalization” as used herein refers to the correction of raw gene expression values/data between different samples for sample to sample variations, to take into account differences in "extrinsic” parameters such as cellular input, nucleic acid (RNA) or protein quality, efficiency of reverse transcription (RT), amplification, labeling, purification, etc. , i.e. differences not due to actual "intrinsic” variations in gene expression by the cells in the samples.
  • Such normalization is performed by correcting the raw gene expression values/data for a test gene (or gene of interest) based on the gene expression values/data measured for one or more "housekeeping" or “control” genes, i.e. whose expressions are known to be constant (i.e. to show relatively low variability) between the cells of different tissues and under different experimental conditions.
  • the above-mentioned method further comprises measuring the level of expression of a housekeeping gene in the biological sample. Suitable housekeeping genes are known in the art and several examples are described in WO 2014/134728.
  • the method further comprises measuring the level of expression of one or more housekeeping genes in a biological sample from the subject.
  • Suitable housekeeping genes are known in the art and several examples are described in WO 2014/134728, including those depicted in Table 1 below.
  • Table 1 Examples of housekeeping genes
  • Other commonly used housekeeping genes include TBP, YWHAZ, PGK1, LDHA, ALDOA, HPRT1, SDHA, UBC, GAPDH, ACTB, G6PD, VIM, TUBA1A, PFKP, B2M, GUSB, PGAM1 and HMBS.
  • the method further comprises measuring the level of expression of one or more housekeeping genes in a biological sample from the subject.
  • the level of expression of the housekeeping gene is measured and the method comprises amplifying a housekeeping gene nucleic acid using a suitable pair of primers.
  • one or more of the primers and/or probe is/are detectably labelled, i.e. comprises a detectable label attached thereto.
  • detectable label refers to a moiety emitting a signal (e.g., light) that may be detected using an appropriate detection system. Any suitable detectable label may be used in the method described herein. Detectable labels include, for example, enzyme or enzyme substrates, reactive groups, chromophores such as dyes or colored particles, luminescent moieties including a bioluminescent, phosphorescent or chemiluminescent moieties, and fluorescent moieties. In an embodiment, the detectable label is a fluorescent moiety.
  • Fluorophores that are commonly used include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'- dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), ⁇ , ⁇ , ⁇ ', ⁇ '- tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'- dimethylaminophenylazo) benzoic acid (DABCYL), and 5-(2'-aminoethyl)aminonaphthalene-l - sulfonic acid (EDANS).
  • FAM 5-carboxyfluorescein
  • JE 2'7'-dimethoxy-4'5'- dichloro-6-carboxyfluorescein
  • rhodamine 6-carboxyrhodamine
  • the fluorophore may be any fluorophore known in the art, including, but not limited to: FAM, TET, HEX, Cy3, TMR, ROX, Texas Red®, LC red 640, Cy5, and LC red 705.
  • Fluorophores for use in the methods and compositions provided herein may be obtained commercially, for example, from Biosearch Technologies (Novato, CA.), Life Technologies (Carlsbad, CA), GE Healthcare (Piscataway NJ), Integrated DNA Technologies (Coralville, Iowa) and Roche Applied Science (Indianapolis, IN).
  • the fluorophore is chosen to be usable with a specific detector, such as a specific spectrophotometric thermal cycler, depending on the light source of the instrument.
  • two or more different fluorophores may be chosen with absorption and emission wavelengths that are well separated from each other (i.e., have minimal spectral overlap).
  • the fluorophore is chosen to work well with one or more specific quenchers.
  • Covalent attachment of detectable label and/or quencher to primer and/or probe can be accomplished according to standard methodology well known in the art as discussed, for example in Sambrook and Green, supra, Ausubel et al., supra, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckerman et al., Nucleic Acids Research, 15: 5305-5321 (1987) (3' thiol group on oligonucleotide); Sharma et al., Nucleic Acids Research, 19:3019 (1991) (3' sulfhydryl); Giusti et al., PCR Methods and Applications, 2:223-227 (1993) and Fung et al, U.S. Pat. No.
  • the expression of the one or more genes or encoded gene products is measured at the protein level.
  • Methods to measure the amount/level of proteins are well known in the art. Protein levels may be detected directly using a ligand binding specifically to the protein, such as an antibody or a fragment thereof.
  • a binding molecule or reagent e.g., antibody
  • is labeled/conjugated e.g., radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled to facilitate detection and quantification of the complex (direct detection).
  • protein levels may be detected indirectly, using a binding molecule or reagent, followed by the detection of the (protein/ binding molecule or reagent) complex using a second ligand (or second binding molecule) specifically recognizing the binding molecule or reagent (indirect detection).
  • a second ligand may be radiolabeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled to facilitate detection and quantification of the complex.
  • Enzymes used for labeling antibodies for immunoassays are known in the art, and the most widely used are horseradish peroxidase (HRP) and alkaline phosphatase (AP).
  • binding molecules or reagents include antibodies (monoclonal or polyclonal), natural or synthetic ligands, and the like.
  • Examples of methods to measure the amount/level of protein in a sample include, but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbent assay (ELISA), "sandwich” immunoassays, radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance (SPR), chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical (IHC) analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, antibody array, microscopy (e.g., electron microscopy), flow cytometry, proteomic-based assays, and assays based on a property or activity of the protein including but not limited to ligand binding or interaction with other protein partners, enzymatic activity, fluorescence.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • SPR surface plasmon
  • the level or activity of the protein of interest may be determined by the measuring the level of phosphorylation of the target in the presence of the test compound. If the protein of interest is a transcription factor known to induce the expression of one or more given target gene(s), the level or activity of the protein of interest may be determined by the measuring the level of expression of the target gene(s).
  • Control level or “reference level” “reference value” or “standard level” are used interchangeably herein and broadly refers to a separate baseline level measured in a comparable “control” sample, which is generally from a subject not suffering, or not likely to suffer, from the target disease (e.g., not suffering from AML, CBF-AML, inv(16)-/A L and/or t(8;21)-/A L), for example an AML sample from another AML subtype (or a mixture of other AML subtypes) or a sample enriched in CD34 + cells from a subject not suffering from AML such as a healthy subject not suffering from leukemia).
  • the target disease e.g., not suffering from AML, CBF-AML, inv(16)-/A L and/or t(8;21)-/A L
  • AML sample from another AML subtype or a mixture of other AML subtypes
  • a sample enriched in CD34 + cells from a subject not suffering from AML such
  • the corresponding control level may be a level corresponding to an average or median level calculated based on the levels measured in several reference or control subjects (e.g., a pre-determined or established standard level or reference value).
  • the control level may be a pre-determined "cut-off value recognized in the art or established based on levels measured in samples from one or a group of control subjects, i.e., subjects not suffering from CBF-AML, t(8;21) AML and/or inv(16) AML).
  • the "threshold reference level” of may be a level corresponding to the minimal level of expression (cut-off) of a differentially expressed gene disclosed herein (or combination thereof), that permits to distinguish in a statistically significant manner AML patients having the target disease (e.g., a CBF-AML or CSF-AML subtype) from those having another form of AML or from healthy subjects, which may be determined using samples from such subjects, for example.
  • the "threshold reference level” of may be a level corresponding the level of expression (cut-off) of a differentially expressed gene disclosed herein, that permits to best or optimally distinguish in a statistically significant manner between AML patients (e.g., AML types and subtypes).
  • control level may be adjusted or normalized for age, gender, race, or other parameters.
  • the "control level” can thus be a single number/value, equally applicable to every patient individually, or the control level can vary, according to specific subpopulations of patients. Thus, for example, older subjects might have a different control level than younger subjects, and women (female subjects) might have a different control level than men (male subjects).
  • the predetermined standard level can be arranged, for example, where a tested population is divided equally (or unequally) into groups, such as a low- risk group, a medium-risk group and/or a high-risk group or into quadrants or quintiles, the lowest quadrant or quintile being individuals with the lowest risk (i.e., lowest level of expression of the one or more genes) and the highest quadrant or quintile being individuals with the highest risk (i.e., highest level of expression of the one or more genes).
  • the control levels according to the invention may be, in addition to predetermined levels or standards, levels measured in other samples (e.g. from healthy/normal subjects, or AML patients) tested in parallel with the experimental sample.
  • the reference or control levels may correspond to normalized levels, i.e. reference or control values subjected to normalization based on the expression of a housekeeping gene.
  • a corresponding threshold reference level which would define a similar threshold value for one or more differentially expressed gene(s) disclosed herein, expression levels, may be calculated based, for example, on the expression of another housekeeping gene or using another method of calculation.
  • Higher expression refers to significantly (i) higher expression of the one or more of the above-mentioned genes (protein and/or mRNA) in one or more given cells present in the sample (relative to the control) and/or (ii) higher amount of cells expressing the one or more genes in the sample (relative to the control).
  • “Lower expression”, “lower level of expression”, “underexpression”, “underexpressed”, or “decreased level of expression” as used herein refers to significantly (i) lower expression of the one or more genes (protein and/or mRNA) in one or more given cells present in the sample (relative to the control) and/or (ii) lower amount of cells expressing the one or more genes in the sample (relative to the control).
  • higher or lower refers to a level of expression that is above or below the control level (e.g., the predetermined cut-off value or reference value).
  • higher or lower refers to a level of expression that is at least one standard deviation above or below the control level (e.g., the predetermined cut-off value) (e.g. that is statistically significant as determined using a suitable statistical analysis), and a "similar expression” or “similar level of expression” refers to a level of expression that is less than one standard deviation above or below the control level (e.g., the predetermined cut-off value) (e.g. that is not statistically significant as determined using a suitable statistical analysis).
  • higher or lower refers to a level of expression that is at least 1 .5, 2, 2.5, 3, 4 or 5 standard deviations above or below the control level (e.g., the predetermined cut-off value.
  • “higher expression” refers to an expression that is at least 10, 20, 30, 40, 45 or 50% higher in the test sample relative to the control level.
  • “lower expression” refers to an expression that is at least 10, 20, 25, 30, 35, 40, 45, or 50% lower in the test sample relative to the control level.
  • higher or lower refers to a level of expression that is at least 1 .5, 2-, 5-, 10-, 25-, or 50-fold higher or lower in the test sample relative to the control sample.
  • methods described herein further comprise obtaining or collecting a biological sample from a subject.
  • the sample can be from any source that contains biological material suitable for the detection of the mutation(s), such as genomic DNA, RNA (cDNA), and/or proteins, for example a tissue or cell sample from the subject (blood cells, immune cells (e.g., lymphocytes), bone marrow cells, etc. that comprises leukemic cells (AML cells).
  • the sample may be subjected to cell purification/enrichment techniques to obtain a cell population enriched in a specific cell subpopulation or cell type(s).
  • the sample may be subjected to commonly used isolation and/or purification techniques for enrichment in nucleic acids (genomic DNA, cDNA, mRNA) and/or proteins. Accordingly, in an embodiment, the method may be performed on an isolated nucleic acid and/or protein sample, such as isolated genomic DNA.
  • the biological sample may be collected using any methods for collection of biological fluid, tissue or cell sample, such as venous puncture for collection of blood cell samples.
  • the biological sample comprising leukemic cells comprises nucleic acids (RNA, cDNA) obtained or isolated from leukemic cells or bone marrow cells.
  • methods of diagnosis described herein may be at least partly, or wholly, performed in vitro. In a further embodiment, the method is wholly performed in vitro.
  • the present invention provides performing any combinations of the steps/methods described herein on biological samples from subjects for the diagnosis/prognosis of AML, CBF-AML, t(8;21) AML or inv(16) AML, or other AML genetic subtypes described herein for example detecting one or more of the mutations described herein, detecting the levels of expression of one or more differentially expressed genes described herein, etc.
  • the above-mentioned method further comprises selecting and/or administering a course of therapy or prophylaxis to said subject in accordance with the diagnostic results obtained using any of the above-mentioned methods. For example, if it is determined that the subject has a high likelihood of suffering from a particular AML subtype associated with a poor prognosis, a more aggressive or a treatment regimen adapted for treatment of AML may be used, such as for example a more aggressive chemotherapy regimen (e.g., high-dose chemotherapy, longer administration schedule, etc.) and/or stem cell/bone marrow transplantation (e.g., allogeneic transplantation).
  • a more aggressive chemotherapy regimen e.g., high-dose chemotherapy, longer administration schedule, etc.
  • stem cell/bone marrow transplantation e.g., allogeneic transplantation
  • the method further comprises subjecting the subject to a suitable anti-leukemia therapy (e.g., bone marrow or hematopoietic stem cell transplantation, chemotherapy, etc.) in accordance with the diagnostic/prognostic result.
  • a suitable anti-leukemia therapy e.g., bone marrow or hematopoietic stem cell transplantation, chemotherapy, etc.
  • the present invention thus provides a method of treating a subject suffering from AML diagnosed based on any of the methods described herein, said method comprising administering to said subject a suitable anti-leukemia therapy in accordance with or adapted to the diagnosis.
  • the present invention provides an assay mixture for the assessment of AML (e.g., for the classification and diagnosis of CBF-AML, inv(16) AML or t(8;21) AML, or other AML genetic subtypes described herein), the assay mixture comprising: (i) a biological sample from a subject suffering from AML (or suspected of suffering from AML); and (ii) one or more assays to determine the level of expression of of at least one of the differentially expressed genes disclosed herein, and/or for detecting the mutations disclosed herein.
  • AML e.g., for the classification and diagnosis of CBF-AML, inv(16) AML or t(8;21) AML, or other AML genetic subtypes described herein
  • the assay mixture comprising: (i) a biological sample from a subject suffering from AML (or suspected of suffering from AML); and (ii) one or more assays to determine the level of expression of of at least one of the differentially expressed genes disclosed herein,
  • the present invention provides a system for the assessment of AML (e.g., for the classification and diagnosis of CBF-AML, inv(16) AML or t(8;21) AML, or other AML genetic subtypes described herein) in a subject, the system comprising: (i) a biological sample from a subject suffering from AML (or suspected of suffering from AML); and (ii) one or more reagents for determining/measuring the level of expression of at least one of the differentially expressed genes disclosed herein, and/or for detecting the mutations disclosed herein.
  • AML e.g., for the classification and diagnosis of CBF-AML, inv(16) AML or t(8;21) AML, or other AML genetic subtypes described herein
  • the system comprising: (i) a biological sample from a subject suffering from AML (or suspected of suffering from AML); and (ii) one or more reagents for determining/measuring the level of expression of at
  • the present invention provides a system for the assessment of AML (e.g., for the classification and diagnosis of CBF-AML, inv(16) AML or t(8;21) AML, or other AML genetic subtypes described herein) in a subject, comprising: a sample analyzer configured to produce a signal for at least one of the differentially expressed genes disclosed herein, and/or for one or more of the mutations disclosed herein, in a biological sample of the subject; and a computer sub-system programmed to calculate, based on the at least one differentially expressed genes or mutation, whether the signal is higher or lower than a reference value or whether the mutation is present in the sample.
  • the system further comprises the biological sample.
  • the present invention further provides a kit for the assessment of AML (e.g., for the diagnosis of CBF-AML, inv(16) AML or t(8;21) AML, or other AML genetic subtypes described herein), the kit comprising: (i) one or more reagents for detecting one or more of the mutations set forth in FIG. 1 and/or Table 5 in a biological sample.
  • the kit comprises reagents for detecting at least 2, 3, 4, or 5 of the mutations set forth in FIG. 1 and/or Table 5 in a biological sample.
  • the present invention further provides a kit for the assessment of AML (e.g., for the diagnosis of CBF-AML, inv(16) AML or t(8;21) AML, or other AML genetic subtypes described herein), the kit comprising: (i) one or more reagents for determining/measuring the level of expression of at least one of the differentially expressed genes disclosed herein (e.g., listed in Tables 6A-B, 7A-B, 8A-B, 10 and 11) in a biological sample.
  • the kit comprises reagents for detecting the level of expression of at least 2, 3, 4, 5, 10, 12, 15, 20, 30, 50, 75, 50, 100 or more differentially expressed genes disclosed herein, in a biological sample.
  • the one or more reagents present in the assay mixture or kit comprise, for example, primer(s), probe(s), antibody(ies), solution(s), buffer(s), nucleic acid amplification reagent(s) (e.g., DNA polymerase, DNA polymerase cofactor, dNTPs), nucleic acid hybridization/detection reagent(s), and/or reagents for detecting antigen-antibody complexes, etc.
  • the assay mixture or kit comprises one or more pairs of primers for amplifying one or more nucleic acids corresponding to the differentially expressed or mutated gene(s) disclosed herein (e.g., genes listed in FIG.
  • the assay mixture or kit comprises one or more probes for detecting one or more nucleic acids correspond to the mutated or differentially expressed gene(s) disclosed herein.
  • the assay mixture or kit further comprises one or more reagents for determining/measuring the level of expression of at least one normalization/housekeeping gene (e.g., ABL1) in the sample.
  • the kit may be divided into separate packages or compartments containing the respective reagent components explained above.
  • such a kit may optionally comprise one or more of the following: (1) instructions for using the reagents for the diagnosis and/or prognosis of A L/CBF-/4 L/t(8;21), /A L/inv(16) AML, other AML genetic subtypes described herein or any combination of these applications; (2) one or more containers; and/or (3) appropriate controls/standards.
  • Such a kit can include reagents for collecting a biological sample from a subject and reagents for processing the biological sample.
  • the kits featured herein can also include an instruction sheet describing how to perform the assays for measuring gene expression or the presence of mutations.
  • the instruction sheet can also include instructions for how to determine a reference cohort (control subject population), including how to determine expression levels of genes in the reference cohort and how to assemble the expression data to establish a reference for comparison to a test subject.
  • the instruction sheet can also include instructions for assaying gene expression in a test subject and for comparing the expression level with the expression in the reference cohort to subsequently determine the appropriate treatment regimen for the test subject.
  • Informational material included in the kits can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the reagents for the methods described herein.
  • the informational material of the kit can contain contact information, e.g. , a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about performing a gene expression analysis and interpreting the results, particularly as they apply to an AML patient's likelihood of having the AML genetic subtype.
  • kits featured herein can also contain software necessary to infer a patient's likelihood of having the AML genetic subtype from the gene expression data.
  • GPCRs are differently expressed by AML cells, notably in frequent AML genetic subgroups, relative to normal hematopoietic cells, which may be exploited for the treatment of AMLs.
  • GPCRs are targets for approximately 30% of all marketed drugs, and several modulators of GPCRs are known.
  • the present invention provides a method for treating a subject suffering from Acute Myeloid Leukemia (AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 and/or FPR2.
  • GPCR G-protein coupled receptor
  • the present invention provides an agent targeting at least one GPCR for treating a subject suffering from AML, wherein said GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 and/or FPR2.
  • GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, C
  • the present invention provides a method for treating a subject suffering from Core Binding Factor Acute Myeloid Leukemia (CBF-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and/or RXFP1.
  • GPCR G-protein coupled receptor
  • the present invention provides an agent targeting at least one GPCR for treating a subject suffering from CBF-AML, wherein said at least one GPCR is ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and/or RXFP1.
  • the present invention provides a method for treating a subject suffering from Acute Myeloid Leukemia with Mixed Lineage Leukemia chromosomal rearrangement (MLL-AML), said method comprising administering to said subject an effective amount of an agent targeting the G-protein coupled receptor (GPCR) GPR126.
  • GPCR G-protein coupled receptor
  • the present invention provides an agent targeting the GPCR GPR126 for treating a subject suffering from MLL-AML,
  • the present invention provides a method for treating a subject suffering from normal karyotype Acute Myeloid Leukemia (NK-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CYSLTR2, GPR114 and/or GPR56.
  • GPCR G-protein coupled receptor
  • the present invention provides an agent targeting a GPCR for treating a subject suffering from NK-AML, wherein said GPCR is CYSLTR2, GPR114 and/or GPR56.
  • the present invention provides a method for treating a subject suffering from myelomonocytic acute myeloid leukemia (M4-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 and/or VIPR1.
  • GPCR G-protein coupled receptor
  • the present invention provides an agent targeting at least one GPCR for treating a subject suffering from M4-AML, wherein said at least one GPCR is FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 and/or VIPR1.
  • the present invention provides a method for treating a subject suffering from monocytic acute myeloid leukemia (M5-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 and/or HTR7.
  • GPCR G-protein coupled receptor
  • the present invention provides an agent targeting at least one GPCR for treating a subject suffering from M5-AML, wherein said at least one GPCR is CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 and/or HTR7.
  • the term "agent targeting a G-protein coupled receptor (GPCR)” includes any compound able to directly or indirectly affect the regulation of a GPCR by reducing for example the expression of the GPCR (i.e. , transcription and/or the translation), or an activity of the GPCR, since as to reduce AML cell proliferation, dissemination, and/or induce AML cell death. It includes intracellular as well as extracellular agents. Without being so limited, such inhibitors include siRNA, antisense molecules, proteins, peptides, small molecules and antibodies, etc.
  • Agents that target GPCRs are described in the IUPHAR/BPS database (http://www.quidetopharmacoloqv.org/; Alexander SPH et al., The Concise Guide to PHARMACOLOGY 2015/16. Br J Pharmacol. 172: 5729-5743).
  • the agent may be an agonist or an antagonist of the GPCR, and/or an agent that specifically binds to the GPCR (e.g, antibody or antigen-binding fragment thereof).
  • the agent is an antagonist of the GPCR.
  • GPCR antagonists are known in the art. Natural and synthetic antagonists of GPCRs are described in the IUPHAR/BPS database.
  • CXCR4 antagonists include plerixafor, TG-0054, AMD070, AMD3465 and BL-8040 (see, for example, Debnath et al. Theranostics 2013; 3(1):47-75).
  • GPCR ADRA2C GPCR ADRA2C
  • FDA-approved drugs such as the antihypertensive drug clonidine and the antidepressant Mirtazapine, as well as others such as JP1302, lisuride, terguride, rauwolscine, spiroxatrine, yohimbine, WB 4101 , roxindole, RX821002, lurasidone, phentolamine, all-frans-4-oxo-retinoic acid, cabergoline, bromocriptine, ARC-239, apomorphine, prazosin, piribedil, chlorpromazine, BRL 44408 and tolazoline.
  • CYSLTR2 a receptor for the inflammatory mediators cysteinyl leukotrienes
  • NMLTC4 Selective CYSLTR2 agonist
  • HAMI3379 Wunder F, et al. British Journal of Pharmacology. 2010; 160(2):399-409
  • BayCysLT2 BayCysLT2
  • chemokine receptor CCR2 selective antagonists of the chemokine receptor CCR2, such as CCX140-B, studied in diabetic mice and tested in clinical trials for patients with diabetic nephropathy (www.clinicaltrials.gov, NCT01447147 and NCT01440257) may be used for treating AMLs expressing this GPCR (Sullivan T, et al. American Journal of Physiology - Renal Physiology. 2013;305(9):F1288-97).
  • Other antagonists of CCR2 include TAK-779, SB-282241 , RS-504393, N-[(3R)-1-[(2,4-dimethylphenyl)methyl]pyrrolidin-3-yl]-2- ⁇ [3-
  • Antagonists of CCR1 include CCX721 and CCX354-C (Dairaghi DJ et al., Blood. 2012; 120(7): 1449-57; Tak PP et al., Annals of the rheumatic diseases.
  • Selective antagonists of PTGER2 include TG4-155 and TG6-10-1 (Jiang J et al., PNAS 2013; 1 10(9):3591 -6).
  • Examples of antagonists of PTGER4 include ER-819762, AH-23848, CJ- 023423, GW-627368, L-161982, ONO-AE3-208 and grapiprant.
  • Examples of antagonists of FPR1 include cyclosporin H, 3570-0208 (PMID: 19807662), chenodeoxycholic acid, deoxycholic acid.
  • Antagonists of C3AR include SB290157, FLTChaAR and (2S)-5- (diaminomethylideneamino)-2-[[2-(2,2-diphenylethylsulfanyl)acetyl]amino]pentanoic acid (PubChem CID: 91827355).
  • Antagonists of C5AR1 include CHIPS, W5401 1 , NDT9520492, AcPhe-Orn-Pro-D-Cha- Trp-Arg, PMX205, PMX53, N-methyl-Phe-Lys-Pro-D-Cha-Trp-D-Arg-COOH, ⁇ 8 ⁇ 71"73 , JPE1375, C089, RPR121 154 and L-156,602.
  • Antagonists of PAFR include PCA 4248, foropafant, apafant, ABT-299, ABT-491 , RP- 52770, L659989, 10-OBn-7a-F-ginkgolide B, 7a-CI-ginkgolide B, 10-OBn-ginkgolide B, BN 50739, 7a-N3-ginkgolide B, 10-OBn-epi-ginkgolide C, 7a-NHMe-ginkgolide B, ginkgolide B, 7a- F-ginkgolide B, 10-OBn-ginkgolide C, 7a-NHEt-ginkgolide B, ginkgolide A, 7a-OCOCH 2 Ph- ginkgolide B, 7-epi-ginkgolide C, 7a-NH2-ginkgolide B, 7a-OAc-ginkgolide B, ginkgolide J, ginkgolide C, israpafant, CV-6209, SDZ
  • Antagonists of ADRB2 include carazolol, timolol, carvedilol, CGP 12177, ICI 1 18551 , SR59230A, propranolol, levobunolol, alprenolol, bupranolol, labetalol, nadolol, NIP, levobetaxolol, propafenone, betaxolol, sotalol, metoprolol, cicloprolol, NIHP, atenolol and LK 204-545.
  • Antagonists of SUCNR1 include compound 5g [PMID: 21571530], compound 5g [PMID: 21571530] and compound 7e [PMID: 21571530] (Bhuniya D et al., Bioorg. Med. Chem. Lett., 21 (12): 3596-602).
  • Antagonists of P2RY2 include AR-C126313, AR-C1 18925XX, suramin, reactive blue-2 and 1 -amino-4-phenylamino-2-sulfoanthraquinone derivatives (Weyler S et al., Bioorg Med Chem Lett, 18: 223-227).
  • Antagonists of P2RY13 include cangrelor, ApA MRS2603, MRS221 1 , reactive blue-2, suramin, 2MeSAMP and PPADS.
  • Antagonists of HRH2 include iodoaminopotentidine, tiotidine, ranitidine, cimetidine, JNJ-39758979, metiamide, burimamide, INCB-38579, ABT-239, clobenpropit, ciproxifan, MK- 0249, conessine, pitolisant and famotidine.
  • Antagonists of PTH2R include [His 4 , Tyr 5 , Trp 6 , His 7 ]TIP39, PTHrP-(1 -21)/PTH-(22-34), TIP39-(7-39), [Ne 5 ,Trp 23 ]PTHrP-(5-36) and PTHrP-(7-34).
  • Antagonists of PTGIR include R01 138452, R03244794, R03244794, R01 138452, R03244794 and BAY-73-1449 (Bley KR et al., 2006. Br J Pharmacol , 147: 335-345).
  • Antagonists of GPR65 include psychosine and psychosine-related lysoslipids.
  • Antagonists of FPR2 include quin-C7, isopropylureido-FLFLF, compound 1754-31 [PMID: 23788657], WRWWWW, t-Boc-FLFLF and FPRL1 -inhibitor protein.
  • Antagonists of FPR1 include CHIPS, 3570-0208 [PMID: 19807662], cyclosporin H, t- Boc-FLFLF, chenodeoxycholic acid, deoxycholic acid, cyclosporin A, BVT173187, i-Boc-Met- Leu-Phe, diamide 7, methionine benzimidazole 6, group E 1682-2106 [PMID: 161 18363], sulfinpyrazone and spinorphin.
  • Antagonists of RXFP1 include B-R13/17K H2 relaxin and RXFPi-truncate.
  • Antagonists of VIPR1 include PG 97-269 and N-stearyl-[Nle 17 ] neurotensin-(6-1 1)/VIP-
  • Antagonists of GABBR1 include CGP 56999A, CGP 62349, CGP 54626A, CGP 64213, CGP 71872, SCH 5091 1 , CGP 35348, saclofen and 2-hydroxy-saclofen.
  • the agent is an antibody or an antigen-binding fragment thereof.
  • Antibodies or antigen-binding fragments thereof may interfere with or modulate the activity of the GPCRs in tumor cells, e.g., by blocking the interaction with a ligand, thereby modulating the signaling within the tumor cells (which may in turn affect tumor cell proliferation, survival and/or migration).
  • Some of the GPCRs overexpressed in AMLs are adhesion GPCRs (CD97, EMR2/ADGRE2, GPR56 and GPR114), which may be targeted by antibodies to modulate AML cell migration, for example.
  • the antibody may alternatively or further be conjugated to an anti-tumor agent (chemotherapeutic agent, toxin) so as to deliver the anti-tumor agent to the tumor AML cells that expressed the GPCRs, i.e. using the antibody or antigen-binding fragment thereof as a targeting agent for tumor AML cells.
  • an anti-tumor agent chemotherapeutic agent, toxin
  • the antibody or antigen-binding fragment thereof induces the killing of the AML cells.
  • Antibodies or antigen-binding fragments thereof that are specific for GPCRs that are primarily or exclusively present on AMLs cells can be injected into AML patients and will then bind the cognate protein epitopes on the tumor cells. Once the antibody binds the tumor protein (i.e. GPCR primarily present on AMLs cells), the non-variable base of the antibody (also called the Fc region) can then be recognized by cells of the human immune system. Specific immune cells (such as natural killer cells or NK cells) have receptors (i.e. CD16) which recognize the Fc portion of antibodies.
  • NK cells and/or other cytotoxic cells such as macrophages, monocytes and/or eosinophils
  • ADCC Antibody-Dependent Cell-Mediated Cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • the C1 q complex of the complement system binds the antibody and this binding triggers the complement cascade which leads to the formation of the membrane attack complex (MAC) (C5b to C9) at the surface of the target AML cell, as a result of the classical pathway complement activation.
  • MAC membrane attack complex
  • GPCRs proteins that are specifically expressed on the surface of tumor cells, where they are accessible to the antibodies.
  • ADCC antibody-based cell cytotoxicity
  • This approach has been used successfully to target ERBB2 receptor in certain breast cancers (with Trastuzumab; Nat Med. 2000 Apr;6(4):443-6), the EGF receptor in metastatic colorectal cancer and head and neck cancer (Cetuximab; Lancet Oncol. 2010 Jan; 1 1 (1):21 -8. Epub 2009 Nov 10.), the CD20 protein in lymphomas and leukemias (e.g., with Rituximab; Blood.
  • Antibodies against chemokine receptors have also been shown to be effective for the treatment of certain cancers in animal models and humans (see, Vela et al., Front Immunol. 2015; 6: 12), for example the CCR4-specific antibody mogamulizumab in patients with relapsed or refractory CCR4 + adult T-cell leukemia (ATL), CXCR4-specific antibody ulocuplumab for the treatment of multiple myeloma, CCR2-specific antibody MLN1202 for the treatment of bone metastases, and a CCR7-specific antibody in a murine model of human mantle cell lymphoma (MCL).
  • ATL relapsed or refractory CCR4 + adult T-cell leukemia
  • CXCR4-specific antibody ulocuplumab for the treatment of multiple myeloma
  • CCR2-specific antibody MLN1202 for the treatment of bone metastases
  • CCR7-specific antibody in a murine model of
  • the antibody or antigen/binding fragment thereof binds to an extracellular domain of the GPCR.
  • Extracellular domains of the GPCRs are known in the art and may be found, for example, in the UniProtKB database.
  • the extracellular domains correspond to residues 1 -83, 139-157, 223- 236, and 347-367.
  • One or more of these extracellular domains may be used as an antigen to raise antibodies.
  • antibody or antigen-binding fragment thereof refers to any type of antibody/antibody fragment including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, humanized antibodies, CDR-grafted antibodies, chimeric antibodies and antibody fragments so long as they exhibit the desired antigenic specificity/binding activity.
  • Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single- chain antibody molecules, single domain antibodies (e.g. , from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments.
  • Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, V H regions (V H , V H -V H ), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies.
  • the antibody is a monoclonal antibody.
  • the above-mentioned agent may be present in a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers and/or excipients.
  • Such compositions may be prepared in a manner well known in the pharmaceutical art.
  • Supplementary active compounds can also be incorporated into the compositions.
  • the carrier/excipient can be suitable, for example, for intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g. , aerosol) administration (see Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro, 2003, 21 th edition, Mack Publishing Company).
  • an "excipient,” as used herein, has its normal meaning in the art and is any ingredient that is not an active ingredient (drug) itself. Excipients include for example binders, lubricants, diluents, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizing agent, release-delaying agents and other components. "Pharmaceutically acceptable excipient” as used herein refers to any excipient that does not interfere with effectiveness of the biological activity of the active ingredients and that is not toxic to the subject, i.e. , is a type of excipient and/or is for use in an amount which is not toxic to the subject.
  • Excipients are well known in the art, and the present system is not limited in these respects. Excipients, include, for example and without limitation, binders (binding agents), thickening agents, surfactants, diluents, release-delaying agents, colorants, flavoring agents, fillers, disintegrants/dissolution promoting agents, lubricants, plasticizers, silica flow conditioners, glidants, anti-caking agents, anti-tacking agents, stabilizing agents, anti-static agents, swelling agents and any combinations thereof. As those of skill would recognize, a single excipient can fulfill more than two functions at once, e.g. , can act as both a binding agent and a thickening agent. As those of skill will also recognize, these terms are not necessarily mutually exclusive.
  • any suitable amount of the agent or pharmaceutical composition may be administered to a subject.
  • the dosages will depend on many factors including the mode of administration, the age, weight, condition of the patient etc.
  • the amount of the agent or pharmaceutical composition contained within a single dose will be an amount that effectively prevents, delays or treats AML without inducing significant toxicity.
  • the appropriate dosage of the compound/composition will depend on the severity and course of the disease or condition, whether the agent/composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound/composition, and the discretion of the attending physician.
  • the compound/composition is suitably administered to the patient at one time or over a series of treatments.
  • the present invention provides dosages for the compounds and compositions comprising same.
  • the effective dose may be 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/ 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000 mg/kg, or may range between any two of the foregoing values.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired relief or suppression of disease symptoms occurs.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the present invention provides a method for determining whether a test compound may be useful for treating AML or a specific AML subtype of interest, comprising contacting a cell expressing a GPCR normally expressed by AML cells (or cells of the AML subtype of interest) with the test compound; and determining whether the test compound modulates (inhibits or increases) the expression and/or activity of the GPCR, wherein said modulation is indicative that the test compound may be useful for treating the AML or the specific AML subtype of interest.
  • the method comprises determining whether the test compound inhibits the expression and/or activity of the GPCR.
  • test compounds may be obtained from any number of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Screening assay systems may comprise a variety of means to enable and optimize useful assay conditions.
  • Such means may include but are not limited to: suitable buffer solutions, for example, for the control of pH and ionic strength and to provide any necessary components for optimal activity and stability (e.g. , protease inhibitors), temperature control means for optimal activity and/or stability, of the GPCR, and detection means to enable the detection of its activity.
  • suitable buffer solutions for example, for the control of pH and ionic strength and to provide any necessary components for optimal activity and stability (e.g. , protease inhibitors), temperature control means for optimal activity and/or stability, of the GPCR, and detection means to enable the detection of its activity.
  • detection means may be used, including but not limited to one or a combination of the following: radiolabelling, antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g., generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g., horseradish peroxidase, green fluorescent protein), specific binding reagents (e.g., biotin/(strept)avidin), and others.
  • radiolabelling e.g., antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g., generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g., horseradish peroxidase, green fluorescent protein), specific binding reagents (e.g., biotin/(strept)avidin), and others.
  • GPCR activity may be assessed using methods well known in the art, for example by measuring the activation of signaling pathways, for example increase in cAMP, phosphorylation of proteins, etc.
  • GPCR activity may be assessed using biosensors, for example biosensors based on FRET/BRET technology (e.g., as described in US Patents Nos. 8,883,485 and 9,029,097).
  • Table 2 Characteristics of CBF and comparative AML cohorts.
  • WBC white blood cells
  • FAB French-American-British classification
  • RNA and DNA isolation were isolated from primary AML cells using TRIzol® reagent according to the manufacturer ' s instructions (lnvitrogen®/Life Technologies®) with an additional purification on RNeasy® mini columns (Qiagen®) to obtain high quality RNA. DNA was isolated and purified using DNeasy® protocols (Qiagen®). Integrity verification of isolated RNA was performed on a Bioanalyzer® 2100 with a RIN > 8 deemed acceptable. For sequencing experiments, libraries were constructed with the TruSeq® RNA Sample Preparation Kit (lllumina®) according to manufacturer's protocols.
  • SNP single nucleotide polymorphisms
  • dbSNP dbSNP, version 137
  • All variants reported have a variant allelic frequency (VAF) > 20%, >8 variant reads, > 20 total reads and a quality score > 20 except for FLT3, NRAS, KRAS, PTPN11 and KIT for which a VAF of > 5% was required.
  • VAF variant allelic frequency
  • All recurrent mutations identified by next-generation sequencing (NGS) in the CBL-AML cohort have been validated by Sanger sequencing of tumoral DNA or cDNA.
  • Transcript levels are given as Reads Per Kilobase per Million mapped reads (RPKM) and genes are annotated according to RefSeq annotations (UCSC, April 16 th , 2014).
  • TCGA Cancer Genome Atlas
  • Table 3 Sorting strategy for normal bone marrow cell populations
  • Peripheral blood was collected from healthy donors, subjected to red blood cell lysis, and subsequently sorted based on the following sorting strategy: Granulocytes (SSC hlQh , CD33 + ), B cells (Lymphocyte gate FSC
  • SSC hlQh , CD33 + Granulocytes (SSC hlQh , CD33 + ), B cells (Lymphocyte gate FSC
  • RNA-Seq RNA- Sequencing
  • RNA-Seq was performed on the AML and cord blood samples using an lllumina® HiSeq 2000 instrument. Libraries were prepared according to the manufacturer's recommendations (lllumina). RefSeq annotations were based on the UCSC January 27th 201 1 version. The alignment to reference genome (hg19) was done using the CASAVA 1 .8.2 package and Eland v2 mapping software and bioinformatic analyses were performed as described previously (Lavallee VP et al. Blood. 2015; 125(1): 140-3).
  • RNA-Seq data in RPKM Reads Per Kilobase of transcript per Million mapped reads
  • IRPKM log 2 (RPKM+1)
  • + 1 was added to avoid excessive variations due to very small values.
  • Log transformation was performed to avoid overrepresentation of extreme values.
  • Highly expressed GPCRs were selected using a threshold of 3.5 IRPKM (or 10.35 RPKM) (FIG. 6B).
  • the variability of expression between samples was determined by calculating the coefficient of variation (CV), a ratio between the standard deviation and the mean expression value. Genes with a CV smaller than (50%,) will be considered as GPCRs with low variability in their expression (FIG. 6C).
  • Up- and downregulated GPCRs were described as those having a difference of median expression between AML and normal CD34+ cells greater than 1 and less than -1 , respectively.
  • GPCR subfamily enrichment analysis Grouping of GPCR subfamilies was based on the International Union of Basic and Clinical Pharmacology (lUPHAR) database classification (http://www.guidetopharmacology.org/). To complete and subdivide the class A group, the GRAFS phylogenetic classification of GPCRs was used (Fredriksson R et a/., Molecular Pharmacology. 2003;63(6): 1256-72). Taste 2 receptors, which are not included in the lUPHAR database, have also been added as well as vomeronasal receptors, opsins and 3 orphan GPCRs (GPR137B, TAPT1, XPR1). Overall GPCRs were classified in distinct ligand groups (FIG. 6A).
  • the GPCR subfamily enrichment in the up- or down-regulated groups was performed using a Fisher's exact test and significance (two-tailed p-value) was calculated using the function FET of the add-in Fisher's exact downloaded from http://www.obertfamily.com/software/fisherexact.html.
  • the GPCR members associated to a specific AML genetic subgroup were selected by calculating the ratio between the mean GPCR expression level (RPKM values) in samples with and without the genetic abnormality. Further statistical analyses were performed by calculating a difference of mean expression level (IRPKM) between samples with and without the genetic abnormality. An arbitrary difference of 1.5 IRPKM and a significant Student's t-test (p-value ⁇ 0.05) were used as cut-off levels to identify differentially expressed GPCRs in the Leucegene cohort.
  • HEK293 cell line which has a low expression level of LTB4R, FPR1 and C5AR1 was used for antibody validation. Antibodies were validated by comparing their staining pattern between untransfected cells and cells transfected with a vector containing the corresponding cDNA.
  • HEK293 cells were cultured in 6-well plates in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) FBS at 37°C with 5% C0 2 . At 80% of confluence, cells were transfected with ⁇ g of plasmid DNA encoding a GPCR by using LipofectamineTM 2000 (Invitrogen). Cell were harvested at 48h after transfection, and suspended in 100 ⁇ _ PBS buffer before staining with the antibodies.
  • DMEM Dulbecco's Modified Eagle's Medium
  • CD34 FITC (BD 555821), CD71 SPC (BD 334108), Gpa PerCP eFluor710 (eBioscience® 46-9987-42), CD10 BV421 (BD 562902), CD15 FITC (BD 555401), CD33 PE (BD 555450), CD34 APC (BD 555824), CD1 1 b PE-Cy5 (BD 555389), CD16 Pacific Blue (BD 558122), CD13 APC-Cy7 (BioLegend® 301710), CD3 FITC (BD 555332), CD14 APC- Cy7 (BD 560919), CD19 APC-Cy7 (BioLegend® 302218), CD19 PE-Cy7 (BD 557835). Cells were stained for 30 minutes at 4°C. Fc-blocking reagent (BD 565821), CD71 SPC (BD 334108), Gpa PerCP eFluor710 (eBioscience® 46-9987-42), CD10 BV421
  • the amplification was processed with the following protocol: 3min at 95°C, followed by 40 cycles at 95°C 5sec and 60°C 30sec on a thermal cycler 7900HT (Applied Biosystems®). Data were analyzed using the SDS 2.4 and Data Assist software. Delta CTs were obtained from Ct gene - Ct reference using 4 control genes (GAPDH, ACTB, EIF4H, HNRNPL).
  • High frequency of activated signaling gene mutations defines CBF leukemia subclones. Characterization of mutations present in 415 primary AML specimens (48 CBF-AML, including 28 samples with inv(16) and 20 with t(8;21), and 367 control AML specimens) revealed that mutational landscapes of RUNX1-RUNX1T1 and CBFB-MYH11 AML are distinct (FIG. 1A). Genes mutated in t(8;21) and inv(16) cohorts are detailed in Table 5. The most frequent mutations in both CBF subgroups were found in activated signaling genes (FIG. 1A).
  • the activated signaling genes (14/20, 70%) were the most frequently mutated, followed by chromatin modifier (10/20, 50%), cohesin (5/20, 25% each) and DNA methylation (4/20, 20%) genes (FIG. 1A). No association was found between mutations and additional cytogenetic aberrations and clinical or laboratory characteristics (FIG. 1A). An association was observed between t(8;21) and del(9q) or -Y.
  • ZBTB7A is recurrently mutated in t(8;21) AML.
  • Three of 20 t(8;21) AML samples contained novel acquired mutations in ZBTB7A. Mutation in this gene occurred in only one other specimen, suggesting that it is specific to t(8;21) AML (3/20 vs. 1/395, p 0.0004, Fisher's exact test, FIGs. 1 D and 1 H).
  • ZBTB7A expression was the lowest in specimens with frameshift mutations suggesting that nonsense mediated decay is at play (FIG. 11).
  • Chromatin modifier, cohesin and DNA methylation genes are recurrently mutated in t(8;21) AML.
  • ASXL2 mutations were also largely restricted to t(8;21) AML subgroup (5/20 vs. 3/395, p ⁇ 0.0001 , Fisher's exact test, FIG. 1 E).
  • the 3 ASXL2 mutated samples in the non t(8;21) cohort harbored typical frameshift mutations, and they occurred in samples with intermediate or adverse risk cytogenetics.
  • 3 SMC1A acquired mutations occurred at position R96 (FIG. 1 F).
  • LGALS2 1.59E- ⁇ 08 5.66 4.50 1 .15
  • Fusion partner genes RUNX1T1 and MYH11, remain among the single most specifically and differentially expressed genes in t(8;21) and inv(16) groups respectively.
  • Previously reported candidates such as POU4F1 (t(8;21)) and ST18 (inv(16)) were among the most discriminatory genes identified by the analysis.
  • Other CBF microarray datasets were readily enriched in GSEA studies (Tables 6 and 7). Importantly, about 80% of the genes identified in the CBF AML signatures have not been previously described in those datasets (Tables 6 and 7). For example, ADARB2-AS1 and LINC00958 were typical for t(8;21) AML and MEGF10 and APLN for inv(16) specimens.
  • CBF AML Overlap in gene expression pattern between CBF AML subgroups. Although certain genes are specifically expressed in either inv(16) or t(8;21) AML, a defined expression signature also distinguishes CBF AMLs from other AML subgroups (FIG. 2C and Tables 8A and 8B). CBF AML express high levels of the IRX1 homeobox gene which is also found in t(15; 17) (FIG. 2D). Other genes preferentially expressed in both CBF subgroups include FLT4, MSLN, ST18, LCN6, TRH, CD34 and LOC200772 (FIG. 2C).
  • HOXA, HOXB and MEIS1 genes are expressed at low levels in CBF AMLs (FIG. 2C). Unexpectedly, it was found that these genes are expressed at lower levels in t(8;21) AML when compared to inv(16) AML samples (FIG. 2E-G). Table 8C and FIG. 2H show the list of most differentially expressed genes between t(8;21) and inv(16) AML.
  • PROSER2-AS1 1.58E- ⁇ 16 4.26 3.07 1.19
  • Table 8C Genes differentially expressed in t(8:21) vs. inv(16) AML
  • NCAM1 2.48E-06 4.38 2.91 ACP6 2.80E-09 4.78 3.73
  • CD 163 4.10E-05 4.09 5.20 CECR6 3.85E-1 1 3.09 4.40
  • FCGR2C 1.00E-10 3.26 4.64 RFX8 6.18E-1 1 3.15 4.94
  • the in frame fusions include RUNX1-USP42 (previously characterized, Giguere A and Hebert J. Genes, Chromosomes and Cancer. 201 1 ;50(4):228-238), and a novel RUNX1-ERG.
  • This last fusion transcript leads to ERG overexpression (FIG. 3E) and was found in a sample which had the following karyotype: 48,XX,+9,t(1 1 ;21)(q2?4;q22),?add(21)(q22),+22[23] (FIG.
  • RUNX1-USP2 A second fusion involving RUNX1 (RUNX1-USP2) was also present in this last specimen, leading to USP2 overexpression (FIG. 3E). In contrast to the RUNX1-CBFA2T3 sample discussed above, none of these 2 samples showed a transcriptional profile similar to that of RUNX1-RUNX1T1 (FIG. 3B).
  • Splice isoforms were identified with Tophat 2.0.7 and Cufflinks 2.1.1., and are expressed in Fragments Per Kilobase of exon per Million fragments mapped (FPKM)). The results are presented in Tables 10-11 , FIGs. 4A-40 and FIGs. 5A-50.
  • Table 10 Gene isoforms expression for inv(16) AML subtype.
  • MYH11 iso chr16: 15489610-
  • MYH11 iso chr16: 15489610-
  • APLN iso chrX: 128779239-

Abstract

Genes exhibiting specific mutational and/or transcriptional patterns in AMLs and various AML subtypes such as CBF-AMLs, t(8;21) AMLs, inv(16) AMLs; MLL-rearranged AMLs and other subtypes relative to other types of AMLs and/or normal CD34+ cells are disclosed. The use of these mutational and/or transcriptional patterns for the classification, diagnosis and treatment of AMLs is also disclosed.

Description

MARKERS FOR ACUTE MYELOID LEUKEMIAS WITH CORE BINDING FACTOR REARRANGEMENTS AND OTHER GENETIC SUBTYPES AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefits of U.S. provisional application No. 62/200,406 filed August 3, 2015, which is incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention generally relates to acute myeloid leukemias (AMLs), and more particularly to the characterization and diagnosis of AML subtypes including core-binding factor- (CBF-) AMLs and other cytogenetic groups of AML. BACKGROUND ART
[0003] Acute Myeloid Leukemia (AML) is a particularly lethal form of cancer, with most patients dying within two years of diagnosis. It is one of the leading causes of death among young adults. AML is a collection of neoplasms with heterogeneous pathophysiology, genetics and prognosis. Mainly based on cytogenetics and molecular analysis, AML patients are presently classified into groups or subsets of AML with markedly contrasting prognosis.
[0004] RUNX1 (also known as AML1 or CBFA2) and CBFB encode respectively the alpha and beta subunits of a heterodimeric core binding transcription factor complex involved in the development of normal hematopoiesis. AMLs with chromosomal rearrangements t(8;21)/ RUNX1-RUNX1T1 and inv(16)/t(16; 16)/CSFS- YW 7, commonly designated as core binding factor- (CBF-) AMLs, are classified under "AML with recurrent genetic abnormalities" in the 4th edition of the World Health Organization (WHO) classification, and account for about 12 to 15 percent of AML cases in adults. Both subgroups share a relatively favorable outcome, a partial overlap in gene expression such as low HOXA/HOXB/MEIS expression, and frequent mutations in KIT, NRAS, KRAS and FLT3. With the exception of the TCGA study, which included a limited number of CBF-AML, the full genetic/mutational and transcriptional signatures of CBF-AML have not been explored, notably by next generation sequencing.
[0005] Thus, there is a need for a better characterization of the genetic/mutational and transcriptional signature of AMLs such as CBF-AMLs, and for the identification of markers useful for the diagnosis, characterization and treatment of different AMLs subtypes.
[0006] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
[0007] More specifically, in accordance with the present invention, there is provided the following items: 1 . A method for treating a subject suffering from Acute Myeloid Leukemia (AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CXCR4 (UniProt P61073), CD97 (UniProt P48960), PTGER4 (UniProt P35408), PTGER2 (UniProt P431 16), EMR2 (UniProt Q7Z618), GPR183 (UniProt P32249), FPR1 (UniProt P21462), C3AR1 (UniProt Q16581), C5AR1 (UniProt P21730), EMR1/ADGRE1 (UniProt Q14246), GPR114/ADGRE5 (UniProt Q8IZF4), PTAFR (UniProt P25105), GPR160 (UniProt Q9UJ42), ADRB2 (UniProt P07550), CCR1 (UniProt P32246), GPR109B/HCAR3 (UniProt P49019), SUCNR1 (UniProt Q9BXA5), GPR109A/HCAR2 (UniProt Q8TDS4), P2RY2 (UniProt P41231), P2RY13 (UniProt Q9BPV8), GPR27 (UniProt Q9NS67), HRH2 (UniProt P25021), PTH2R (UniProt P49190), CCRL2 (UniProt 000421), PTGIR (UniProt P431 19), GPR65 (UniProt Q8IYL9), CX3CR1 (UniProt P49238), CCR7 (UniProt P32248), CCR2 (UniProt P41597) and/or FPR2 (UniProt P25090).
2. The method of item 1 , wherein said at least one GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1 and/or C5AR1.
3. The method of item 2, wherein said at least one GPCR is CXCR4, CD97, PTGER4, GPR183, PTGER2, C3AR1 and/or EMR2.
4. A method for treating a subject suffering from Core-Binding Factor Acute Myeloid Leukemia (CBF-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is ADRA2C (UniProt P18825), GPR153 (UniProt Q6NV75), LPAR5 (UniProt Q9H1 C0), LPAR6 (UniProt P43657), PTGIR (UniProt P431 19), CCR2 (UniProt P41597), CXCR7/ACKR3 (UniProt P25106), FPR1 (UniProt P21462), GPR183 (UniProt P32249) and/or RXFP1 (UniProt Q9HBX9).
5. The method of item 4, wherein said CBF-AML is AML with t(8;21) chromosomal rearrangement, and said at least one GPCR is ADRA2C, GPR153, LPAR5, LPAR6 and/or PTGIR.
6. The method of item 5, wherein said at least one GPCR is ADRA2C and/or GPR153.
7. The method of item 4, wherein said CBF-AML is AML with inv(16) chromosomal rearrangement, and said at least one GPCR is C5AR1, CCR2, CXCR7/ACKR3, FPR1,
GPR183, RXFP1, LPAR6 and/or PTGIR.
8. The method of item 7, wherein said at least one GPCR is CXCR7/ACKR3 and/or RXFP1.
9. A method for treating a subject suffering from Acute Myeloid Leukemia with Mixed Lineage Leukemia chromosomal rearrangement (MLL-AML), said method comprising administering to said subject an effective amount of an agent targeting the G-protein coupled receptor (GPCR) GPR126 (UniProt Q86SQ4). 10. The method of item 9, wherein said MLL-AML is MLL-MLLT4, MLL-ELL or MLL-SEPT9.
1 1 . A method for treating a subject suffering from normal karyotype Acute Myeloid Leukemia (NK-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CYSLTR2 (UniProt Q9NS75), GPR114/ADGRG5 (UniProt Q8IZF4) and/or GPR56/ADGRG1 (UniProt Q9Y653).
12. The method of item 1 1 , wherein said NK-AML is NK-AML with FL73-ITD and/or NPM1 mutations.
13. The method of item 1 1 or 12, wherein said GPCR is CYSLTR2.
14. A method for treating a subject suffering from myelomonocytic acute myeloid leukemia (M4-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is FPR1 (UniProt P21462), CCR2 (UniProt P41597), C5AR1 (UniProt P21730), GPR183 (UniProt P32249), P2RY13 (UniProt Q9BPV8), PTAFR (UniProt P25105), CCR1 (UniProt P32246) and/or VIPR1 (UniProt P32241).
15. A method for treating a subject suffering from monocytic acute myeloid leukemia (M5- AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CCR1 (UniProt P32246), PTAFR (UniProt P25105), GABBR1 (UniProt Q9UBS5), CX3CR1 (UniProt P49238), P2RY2 (UniProt P41231), NMUR1 (UniProt Q9HB89) and/or HTR7 (UniProt P34969).
16. The method of any one of items 1 to 15, wherein said agent is an antagonist of said at least one GPCR.
17. The method of any one of items 1 to 16, wherein said agent is an antibody directed against said at least one GPCR, or an antigen-binding fragment thereof.
18. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for treating a subject suffering from Acute Myeloid Leukemia (AML), wherein said at least one GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 and/or FPR2.
19. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for the manufacture of a medicament for treating a subject suffering from Acute Myeloid Leukemia (AML), wherein said at least one GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 and/or FPR2. 20. The use of item 18 or 19, wherein said at least one GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1 and/or C5AR1.
21 . The use of item 20, wherein said at least one GPCR is CXCR4, CD97, PTGER4, GPR183, PTGER2, C3AR1 and/or EMR2.
22. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for treating a subject suffering from Core-Binding Factor Acute Myeloid Leukemia (CBF-AML), wherein said at least one GPCR is ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and/or RXFP1.
23. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for the manufacture of a medicament for treating a subject suffering from Core-Binding Factor Acute
Myeloid Leukemia (CBF-AML), wherein said GPCR is ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and/or RXFP1.
24. The use of item 22 or 23, wherein said CBF-AML is AML with t(8;21) chromosomal rearrangement, and said at least one GPCR is ADRA2C, GPR153, LPAR5, LPAR6 and/or PTGIR.
25. The use of item 24, wherein said at least one GPCR is ADRA2C or GPR153.
26. The use of item 22 or 23, wherein said CBF-AML is AML with inv(16) chromosomal rearrangement, and said at least one GPCR is C5AR1, CCR2, CXCR7/ACKR3, FPR1, GPR183, RXFP1, LPAR6 and/or PTGIR.
27. The use of item 26, wherein said at least one GPCR is CXCR7/ACKR3 and/or RXFP1.
28. Use of an agent targeting the G-protein coupled receptor (GPCR) GPR126 for treating a subject suffering from Acute Myeloid Leukemia with Mixed Lineage Leukemia chromosomal rearrangement (MLL-AML).
29. Use of an agent targeting the G-protein coupled receptor (GPCR) GPR126 for the manufacture of a medicament for treating a subject suffering from Acute Myeloid Leukemia with
Mixed Lineage Leukemia chromosomal rearrangement (MLL-AML).
30. The use of item 28 or 29, wherein said MLL-AML is MLL-MLLT4, MLL-ELL or MLL- SEPT9.
31 . Use of an agent targeting at least one G-protein coupled receptor (GPCR) for treating a subject suffering from normal karyotype Acute Myeloid Leukemia (NK-AML), wherein said at least one GPCR is CYSLTR2, GPR114 and/or GPR56.
32. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for the manufacture of a medicament for treating a subject suffering from normal karyotype Acute Myeloid Leukemia (NK-AML), wherein said at least one GPCR is CYSLTR2, GPR114 and/or GPR56.
33. The use of item 31 or 32, wherein said NK-AML is NK-AML with FLT3-\TD and/or NPM1 mutations. 34. The use of any one of items 31 to 33, wherein said GPCR is CYSLTR2.
35. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for treating a subject suffering from myelomonocytic acute myeloid leukemia (M4-AML), wherein said at least one GPCR is FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 and/or VIPR1.
36. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for the manufacture of a medicament for treating a subject suffering from myelomonocytic acute myeloid leukemia (M4-AML), wherein said at least one GPCR is FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 and/or VIPR1.
37. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for treating a subject suffering from monocytic acute myeloid leukemia (M5-AML), wherein said at least one
GPCR is CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 and/or HTR7.
38. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for the manufacture of a medicament for treating a subject suffering from monocytic acute myeloid leukemia (M5-AML), wherein said at least one GPCR is CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 and/or HTR7.
39. The use of any one of items 18 to 38, wherein said agent is an antagonist of said GPCR.
40. The use of any one of items 18 to 39, wherein said agent is an antibody directed against said GPCR, or an antigen-binding fragment thereof.
41 . A method for determining whether a test agent may be useful for treating Acute Myeloid Leukemia (AML), said method comprising (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 and FPR2 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one of more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating AML
42. The method of item 41 , wherein said one or more GPCRs is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1 and/or C5AR1.
43. The method of item 42, wherein said one or more GPCRs is CXCR4, CD97, PTGER4, GPR183, PTGER2, C3AR1 and/or EMR2.
44. A method for determining whether a test agent may be useful for treating Core Binding Factor Acute Myeloid Leukemia (CBF-AML), said method comprising: (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and RXFP1 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one of more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating CBF-AML.
45. The method of item 44, wherein said CBF-AML is AML with t(8;21) chromosomal rearrangement, and said one or more GPCRs is ADRA2C, GPR153, LPAR5, LPAR6 and/or PTGIR.
46. The method of item 45, wherein said one or more GPCRs is ADRA2C and/or GPR153.
47. The method of item 44, wherein said CBF-AML is AML with inv(16) chromosomal rearrangement, and said one or more GPCRs is C5AR1, CCR2, CXCR7/ACKR3, FPR1, GPR183, RXFP1, LPAR6 and/or PTGIR.
48. The method of item 47, wherein said one or more GPCRs is CXCR7/ACKR3 and/or RXFP1.
49. A method for determining whether a test agent may be useful for treating Acute Myeloid Leukemia with Mixed Lineage Leukemia chromosomal rearrangement (MLL-AML), (i) contacting a cell expressing the G-protein coupled receptor (GPCR) GPR126 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said GPCR, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating MLL-AML.
50. The method of item 49, wherein said MLL-AML is MLL-MLLT4, MLL-ELL or MLL-SEPT9.
51 . A method for determining whether a test agent may be useful for treating normal karyotype Acute Myeloid Leukemia (NK-AML), said method comprising: (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): CYSLTR2, GPR114 or GPR56 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one or more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating NK-AML.
52. The method of item 51 , wherein said NK-AML is NK-AML with FL73-ITD and/or NPM1 mutations.
53. The method of item 51 or 52, wherein said one or more GPCRs is CYSLTR2.
54. A method for determining whether a test agent may be useful for treating myelomonocytic acute myeloid leukemia (M4-AML), said method comprising (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 or VIPR1 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one or more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating M4-AML.
55. A method for determining whether a test agent may be useful for treating monocytic acute myeloid leukemia (M5-AML), said method comprising (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 or HTR7 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one or more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating M5-AML.
56. The method of any one of items 41 to 55, wherein said method comprises determining whether said test agent inhibits the activity of said one or more GPCRs.
57. The method of any one of items 41 to 56, wherein said agent is an antibody.
58. A method for determining the likelihood that a subject suffers from Core Binding Factor Acute Myeloid Leukemia (CBF-AML), said method comprising: determining the level of expression of at least one of the genes depicted in bold in Tables 8A and 8B in a leukemia cell sample from said subject, wherein a higher expression of said at least one genes depicted in bold in Table 8A, and/or a lower expression of said at least one genes depicted in bold in Table 8B, in said sample relative to a control non-CBF-AML sample is indicative that said subject has a high likelihood of suffering from CBF-AML.
59. The method of item 58, wherein said method comprises determining the level of expression of at least one of ARC, CACNA1H and LOC200772, wherein a higher expression of said at least one genes in said sample relative to a control non-CBF-AML sample is indicative that said subject has a high likelihood of suffering from CBF-AML.
60. A method for determining the likelihood that a subject suffers from Core Binding Factor Acute Myeloid Leukemia (CBF-AML) with t(8;21)/ RUNX1-RUNX1T1 chromosomal rearrangement (t(8;21) AML), said method comprising: determining the level of expression of (i) at least one of the genes depicted in bold in Tables 6A and 6B; and/or (ii) at least one of the transcripts depicted in Table 11 , in a leukemia cell sample from said subject, wherein a higher expression of said at least one genes depicted in bold in Table 6A and/or of said at least one transcripts depicted in Table 11 , and/or a lower expression of said at least one genes depicted in bold in Table 6B, in said sample relative to a control non-t(8;21) AML sample is indicative that said subject has a high likelihood of suffering from t(8;21) AML.
61 . The method of item 60, wherein said method comprises determining the level of expression of at least one of ADARB2-AS1 and LINC00958, wherein a higher expression of said at least one genes in said sample relative to a control non-t(8;21) AML sample is indicative that said subject has a high likelihood of suffering from t(8;21) AML.
62. A method for determining the likelihood that a subject suffers from Core Binding Factor Acute Myeloid Leukemia (CBF-AML) with inv(16)/t(16; 16)/CSFS- YW 7 chromosomal rearrangement (inv(16) AML), said method comprising: determining the level of expression of (i) at least one of the genes depicted in bold in Tables 7A and 7B; and/or (ii) at least one of the transcripts depicted in Table 10, in a leukemia cell sample from said subject, wherein a higher expression of said at least one genes depicted in bold in Table 7A and/or said at least one transcripts depicted in Table 10, and/or a lower expression of said at least one genes depicted in bold in Table 7B, in said sample relative to a control non-inv(16) AML sample is indicative that said subject has a high likelihood of suffering from inv(16) AML.
63. The method of item 62, wherein said method comprises determining the level of expression of at least one of MEGF10 and APLN, wherein a higher expression of said at least one genes in said sample relative to a control non-inv(16) AML sample is indicative that said subject has a high likelihood of suffering from inv(16) AML.
64. The method of any one of items 58 to 63, wherein said level of expression is measured at the nucleic acid level.
65. The method of item 64, wherein said level of expression is measured by RNA sequencing (RNA-seq) or reverse transcription polymerase chain reaction (RT-PCR).
66. The method of any one of items 56-63, wherein the level of expression of at least two genes is determined.
67. The method of any one of items 56-63, wherein the level of expression of at least three genes is determined.
68. The method of any one of items 56-63, wherein the level of expression of at least four genes is determined.
69. A method for determining the likelihood that a subject suffers from Core Binding Factor Acute Myeloid Leukemia (CBF-AML) with t(8;21)/ RUNX1-RUNX1T1 chromosomal rearrangement (t(8;21) AML), said method comprising: determining the presence of one or more mutations in at least one of the following genes: ZBTB7A, ASXL2, SMC1A, TET2, and DNMT3A in a sample comprising leukemic cells from the subject, wherein the presence of the one or more mutations in said at least one genes is indicative that the subject has a high likelihood of suffering from t(8;21) AML.
70. The method of item 69, wherein the mutation is at a position corresponding to residue 122 and/or 174 of ZBTB7A.
71. The method of item 70, wherein the mutation is a V to G substitution at residue 122 of ZBTB7A.
72. The method of item 70, wherein the mutation is a frameshift mutation at residue 174 of ZBTB7A.
73. The method of item 69, wherein the mutation is at a position corresponding to residue 96 and/or 71 1 of SMC1A.
74. The method of item 73, wherein the mutation is an R to H substitution at residue 96 of SMC1A.
75. The method of item 73, wherein the mutation is an R to Q substitution at residue 71 1 of SMC1A.
76. A method for determining the likelihood that a subject suffers from AML, said method comprising: determining the presence of at least one of a RUNX1-USP2 fusion and a RUNX1- ERG fusion in a sample comprising leukemic cells from the subject, wherein the presence of said at least one RUNX1 fusion is indicative that the subject has a likelihood of suffering from AML.
77. A kit for carrying out the methods of any one of items 56-74 comprising reagents for determining the level of expression of said at least one gene and/or the presence of said one or more mutations or fusions.
78. The kit of item 77, wherein said reagents comprise one or more oligonucleotides comprising a sequence complementary to a sequence of said at least one genes.
79. The kit of item 77 or 78, wherein said reagents comprise reagents for nucleic acid amplification.
[0008] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
[0009] In the appended drawings:
[0010] FIGs. 1A-1 I show the mutational landscape of CBF-AMLs. FIG. 1A: Mutational, morphological, cytogenetic and clinical information of CBF-AML. Each column represents a patient sample. FIG. 1 B: Variant allele frequency (VAF) of mutations in activated signaling genes across t(8;21) and inv(16) genetic groups. Each bar represents a patient sample. Stars identify samples with 2 or more mutations. Note that for each mutation in a given specimen VAF are stacked with a no co-occurrence presumption to facilitate presentation of data. FIG. 1 C: Activated signaling mutations in a sample at diagnosis and relapse. FIGs. 1 D-F: Primary structures of ZBTB7A, ASXL2 and SMC1A proteins, respectively, with corresponding positions of mutations. FIG. 1 G: Number of non-activated signaling mutations in CBF AML subgroups. FIG. 11: confirmation of acquired ZBTB7A mutations in t(8;21) AML in samples 05H184, 05H1 18, and 07H137. Statistics are based on Fisher's exact test. MS: missense; NS: nonsense; FS: frameshift; NA: Not available; dx: diagnosis; rel: relapse; ASXN: Additional sex combs (ASX) N-terminal; ASXH: ASX Homology; PHD: plant homeodomain.
[0011] FIGs. 2A-2H show the transcriptome of CBF-AMLs compared to that of other AML subtypes. FIGs. 2A-2C: Comparative analyses of expressed genes in t(8;21) (FIG. 2A), inv(16) (FIG. 2B) and CBF (FIG. 2C) AML subgroups. Diamonds correspond to differentially expressed genes (difference > 1 or < -1) listed in Tables 5-7 for panel FIGs. 2A-2C, respectively. FIG. 2D: IRX1 expression in different leukemia genetic subgroups. FIGs. 2E-2G: Differential expression of HOXA9 (FIG. 2E), HOXB5 (FIG. 2F) and MEIS1 (FIG. 2G) in t(8;21) and inv(16) AML. FIG. 2H: Comparative analyses of expressed genes in t(8;21) (RUNX1-RUNX1T1) vs. inv(16) (CBFB-MYH11) AMLs. For all the comparisons, in order to log 10 transform expression values, a small constant of 0.0001 was added. CBF: Core-binding factor; AML: Acute myeloid leukemia. [0012] FIGs. 3A-3F show the transcriptomic similarities between RUNX1-RUNX1T1 and RUNX1-CBFA2T3 AML. FIG. 3A: Principal component analysis (PCA) performed in the Leucegene cohort (n= 415) using the 127-gene signature that characterizes AML with inv(16)/CBFB-MYH11 (light blue dots). FIG. 3B: PCA performed in the Leucegene cohort (n= 415) using the 145-gene signature that characterizes AML with t(8;21)/ RUNX1-RUNX1T1 (dark blue dots). One sample with a t^ 6;2^/RUNX1-CBFA2T3 (orange dot) clustered with t(8;21) AML whereas samples with other RUNX1 fusions (red dots) did not. FIG. 3C: Characterization of samples with RUNX1 in frame fusions other than RUNX1-RUNX1T1. Each fusion was identified using RNA-sequencing and confirmed by targeted sequencing. The RUNX1-USP42 fusion was previously described Giguere A and Hebert J. Genes, Chromosomes and Cancer. 201 1 ;50(4):228-238). FIG. 3D: Primary structure of the predicted RUNX1 -ERG fusion protein. FIG. 3E: Expression of ERG and USP2 in the entire cohort. Sample 12H 149, harboring RUNX1- ERG and RUNX1-USP2 fusions, is in the top right corner. FIG. 3F: similarity in differentially expressed genes in AML with RUNX1-RUNX1T1 (n=20, middle dots) and RUNX1-CBFA2T3 (n=1 , right dots) compared to other AML samples (n=394, left dots). In selected genes, only the expression levels of RUNX1T1 and CBFA2T3 (fusion partners) differ between RUNX1- RUNX1T1 and RUNX1-CBFA2T3 AML. Medians are indicated by horizontal bars. PC: principal component; CBF: Core-binding factor. 5'-UTR: 5'-untranslated region.
[0013] FIGs. 4A-40 show cufflinks isoform expression of MYH11_iso_14 (FIG. 4A); MYH11_iso_6 (FIG. 4B), APLN_iso_4 (FIG. 4C), ST18_iso_19 (FIG. 4D), ST18_iso_20 (FIG. 4E), ST18_iso_2 (FIG. 4F), ST18_iso_17 (FIG. 4G), ST18_iso_1 (FIG. 4H), MSLN_iso_2 (FIG. 4I), NT5E_iso_1 (FIG. 4J), MSLN_iso_8 (FIG. 4K), CLIP3_iso_3 (FIG. 4L), MSLN_iso_4 (FIG. 4M), CLEC10A_iso_18 (FIG. 4N), and AC104809.4_iso_35 (FIG. 40) for inv(16) AML subtype and normal hematopoietic populations. Ab initio transcriptome assembly based on raw sequence data using Tophat/Cufflinks identifies the isoforms listed in Table 10 and noted above. Summary dotplot of various AML genetic subtypes and normal hematopoietic populations from bone marrow, cord blood, and peripheral blood show the indicated transcripts as robustly expressed in the majority of inv(16) AMLs. Each dot represents FPKM expression for one specimen and median values are indicated by a horizontal line. A straight line demarcates an FPKM of 1 .0. A value of 0.0001 was added to all FPKM values in order to apply Iog10 transformation.
[0014] FIGs. 5A-50 show cufflinks isoform expression of RUNX1T1_iso_21 (FIG. 5A); RUNX1T1_iso_15 (FIG. 5B), ZNF598_iso_6 (FIG. 5C), RUNX1T1_iso_18 (FIG. 5D), RAE1_iso_9 (FIG. 5E), CWC15_iso_15 (FIG. 5F), RUNX1T1_iso_22 (FIG. 5G), SETD5- AS1_iso_35 (FIG. 5H), RNF13_iso_15 (FIG. 5I), AP1B1_iso_8 (FIG. 5J), RUNX1T1_iso_11 (FIG. 5K), CLIP3_iso_3 (FIG. 5L), TXNRD1 _iso_13 (FIG. 5M), MORC3_iso_10 (FIG. 5N), and GEMIN4_iso_8 (FIG. 50) for t(8;21) AML subtype and normal hematopoietic populations. Ab initio transcriptome assembly based on raw sequence data using Tophat/Cufflinks identifies the isoforms listed in Table 11 and noted above. Summary dotplot of various AML genetic subtypes and normal hematopoietic populations from bone marrow, cord blood, and peripheral blood show the indicated transcripts as robustly expressed in the majority of t(8;21) AMLs. Each dot represents FPKM expression for one specimen and median values are indicated by a horizontal line. A straight line demarcates an FPKM of 1 .0. A value of 0.0001 was added to all FPKM values in order to apply Iog10 transformation.
[0015] FIG. 6A shows GPCR classification in ligand groups. The GPCR classification in ligand groups was based on the IUPHAR database. To complete and subdivide the class A group, the GRAFS phylogenetic classification of GPCRs was used. Taste 2 receptors, which are not included in the IUPHAR database, have also been added as well as vomeronasal receptors, opsins and 3 orphan GPCRs (GPR137B, TAPT1, XPR1). GPCRs have been classified in 18 ligand groups of receptors.
[0016] FIGs. 6B and 6C show the threshold definition for highly expressed GPCRs (FIG. 6A) and GPCRs with low variability in their expression (FIG. 6B). The mean expression level or the coefficient of variation (CV) (X axis) is represented for each of the 772 GPCRs. Each dot represents a GPCR after classification by decreasing median level of expression (FIG. 6A) increasing CV (Y axis) (FIG. 6B). The grey area delimited the GPCRs considered to be highly expressed (FIG. 6A) or to have a low variability in their expression (FIG. 6B) among AML samples. Highly expressed GPCRs were selected using a threshold of 3.5 IRPKM. The threshold for GPCRs with low variability in their expression was established at a CV of 50%.
[0017] FIG. 7 is a schematic representation of the correlation between the results obtained from the qPCR experiments (Delta CT, Y axis) and the RNA-Seq analysis (IRPKM, X axis) was 0.94 (p<0.0001). Each dot represents a mean of 2 independent experiments. The Pearson's R correlation coefficient varies from -0.79 to -0.98, supporting a robust correlation between the two datasets.
[0018] FIG. 8 shows the validation of antibodies for flow cytometry analysis of selected GPCRs. HEK293 cells were transfected with CXCR4, CD97, LTB4R, FPR1 or C5AR1 cDNA. Transfected or untransfected cells were stained with the respective antibodies for flow cytometry analysis. Positive population of transfected cells was compared to the untransfected cells using their respective unstained cells as controls. For the five receptors tested, a clear difference of staining between these cells confirms that the antibodies target the expected proteins.
[0019] FIG. 9A and 9B show flow cytometry analysis of selected highly expressed GPCRs. The protein expression of 5 selected GPCR members was assessed in 20 AML samples with of different morphologic subtypes by flow cytometry analysis. FIG. 9A: Representative histogram of one AML sample (03H041) following staining with antibodies for CD97, LTB4R, CXCR4, C5AR1 or FPR1. Red histograms represent unstained AML cells used as controls FIG. 9B: Dot plot representing the percentage of protein-expressing cells observed in each AML specimen stained. Variable protein expression for CXCR4 (10 to 84% positive cells), LTB4R (0 to 95% positive cells), FPR1 (0 to 69% positive cells) and C5AR1 (1 to 95% positive cells) is demonstrated. CD97 shows a strong expression in all cells of each of the 20 studied AML samples (92.5 to 99.6 % positive cells).
[0020] FIG. 10 shows the correlation between GPCR expression in AML and in normal CD34+ cells. The median gene expression level (in IRPKM) of the 772 GPCRs in normal CD34+ cells (X axis) is represented against their expression in AML cells (Y axis). The 30 upregulated GPCRs in AML (upper portion) have a difference of median expression level between AML and normal CD34+ cells greater than 1 . The 19 downregulated GPCRs in AML (lower portion) have a difference of median expression less than -1 . GPCRs represented in black dots (between the two parallel lines) are not differentially expressed between AML and normal cord blood-derived CD34+ cells. RNA-Seq data were transformed to IRPKM (log2(RPKM+ 1)).
[0021] FIGs. 11A and 11 B show ligand family distribution of up- and down-regulated GPCRs in AML. FIG. 11 A: the proportion of genes up- (left bars) or down-regulated (middle bars) in AMLs, and all GPCRs (right bars) into the different families of GPCRs (adhesion, amine, chemokine, etc.) is shown. The p-values are indicated for the significant families by Fisher's exact tests. FIG. 11 B: Individual GPCRs of the enriched groups are shown. The values indicated in the second and third columns correspond to the receptor mean expression level in AML and in CD34+ cells. All the represented GPCR genes have mean expression levels in AML significantly different from their mean in normal CD34+ cells, p-values <0.005. The Fisher's exact test was performed between the up- or downregulated group and the remaining GPCRs, i.e. all the GPCRs excluding differentially expressed members.
[0022] FIGs. 12A-12C show GPCR expression level analysis in AMLs of different genetic subgroups. Expression (in IRPKM) of the deregulated GPCR members in AML samples with t(8;21), inv(16) and MLL translocations (FIG. 12A) and normal karyotype with FLT3-ITD or NPM1 mutations (FIG. 12B). The differentially expressed GPCRs were identified in the Leucegene cohort and are defined as having a difference of mean expression higher or equal to 1 .5 IRPKM between samples with (+) and without (-) the genetic abnormality and a significant student's t test (p < 0.05). Data are expressed as individual sample expression value and means +/- 1 SEM for all the samples. Mean expression of the GPCR member in the normal CD34+ cells is illustrated with a green dashed line. FIG. 12C: GPCR members with a significant difference of mean expression level in AML of the representative genetic groups in the Leucegene and TCGA cohorts. RNA-Seq data were transformed to RPKM (log2(RPKM+1)). SEM: standard error of the mean.
[0023] FIGs. 13A and 13B show GPCRs with deregulated expression in at least one frequent genetic subgroup. Expression (in IRPKM) of the deregulated GPCR members in AML samples with t(8;21), inv(16), normal karyotype (NK) and MLL translocations and normal karyotype with DNMT3A, FLT3-\TD or NPM1 mutations (FIG. 13B). The differentially expressed GPCRs were identified in the Leucegene cohort and are defined as having a difference of mean expression higher or equal to 1 .5 IRPKM between samples with (+) and without (-) the genetic abnormality and a significant student's t test (p<0.05). The GPCR members are identified in dark grey bars if they are validated in the TCGA dataset and in light grey bars with asterisks if not confirmed in the TCGA cohort. The remaining grey bars (no asterisk) represent the differences of expression that are not significant. RNA-Seq values were transformed to IRPKM (\og2(RPKM+1)).
[0024] FIG. 14 shows GPCR expression level analysis in M4 and M5 AML morphologic subtypes. Expression levels of the 772 GPCRs were analyzed in AML samples with a monocytic component (FAB M4 and M5 subtypes) and compared to their expression in all other AML samples of the Leucegene cohort. Overexpressed GPCRs in AML M4 or M5 are defined as having a difference of mean expression higher or equal to 1 .5 IRPKM between AML M4 or M5 samples and other AML samples and a significant student's t test (p < 0.05). Results obtained for the GPCRs identified in the table have been validated in the TCGA dataset. Receptors in bold are also overexpressed in AML with inv(16).
[0025] FIG. 15 shows GPR126 expression in AML samples with different MLL translocation partners. GPR126 expression levels (values in transformed log2(RPKM+1), IRPKM) are represented in the Y axis and the different translocation partners of the Leucegene AML samples with MLL translocations are represented in the X axis. Data are expressed as individual sample expression value and means +/- 1 SEM for all the samples.
[0026] FIGs. 16A and 16B show the expression levels of the GPCRs overexpressed in specific AML genetic subgroups compared to normal blood and bone marrow cell populations. FIG. 16A: Expression levels of the GPCRs previously identified as overexpressed in specific genetic subgroups of AML are compared to their expression in normal blood and bone marrow cell populations. The represented GPCRs, ADRA2C in AML with t(8;21), GPR126 in AML with MLL translocations, CYSLTR2 in normal karyotype AML with NPM1 and FLT3-ITD mutation and RXFP1 in AML with inv(16), show a significant difference of mean expression between the specific AML genetic subgroup and each normal cell population identified (student's t test (p < 0.05)). FIG. 16B: Expression levels of GPR153 and CXCR7/ACKR3 in specific AML genetic subgroups compared to normal blood and bone marrow cell populations show a significant difference of mean expression between AML samples with t(8;21) or with inv(16) respectively, and each normal cell sample identified (Student's t test (p < 0.05)). AML samples are represented in the first 7 columns from the left (X-axis) and normal cell samples in the remaining columns. Data are expressed as individual sample expression value and means for all the samples. RNA-Seq data were transformed to IRPKM (\og2(RPKM+1)). NK: Normal Karyotype, WBC: White Blood Cells, nBM: normal Bone Marrow, Ery-I: Immature proerythroblasts CD34+CD71+Gpa-, Ery-ll: Early erythroblasts CD34-CD71+Gpa-, Ery-lll: Erythroblasts CD34- CD71+Gpa+, Ery-IV: Mature erythroblasts CD34-CD71-Gpa+, Pre-B-I: Early Pre-B precursors, Pre-B-ll: Pre-B precursors, Gran-I: Myeloid precursors, Gran-ll: Promyelocytes, Gran-Ill: Myelocytes, Gran-IV: Metamyelocytes, Gran-V: Granulocytes.
DISCLOSURE OF THE INVENTION
[0027] Terms and symbols of genetics, molecular biology, biochemistry and nucleic acid used herein follow those of standard treatises and texts in the field, e.g. Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, 2012 (Cold Spring Harbor Laboratory Press); Ausubel et a/., Current Protocols in Molecular Biology (2001); Kornberg and Baker, DNA Replication, Second Edition (W University Science Books, 2005); Lehninger, Biochemistry, sixth Edition (W H Freeman & Co (Sd), New York, 2012); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984), and later updates thereto; and the like. All terms are to be understood with their typical meanings established in the relevant art.
[0028] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise. By way of example, "an element" means one element or more than one element. Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" are used as open-ended terms (substantially equivalent to the phrase "including, but not limited to") and will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
[0029] The information, including the nucleotide and amino acid sequences, corresponding to the Genbank, RefSeq, UniProt, NCBI and/or Ensembl accession numbers referred to in the present specification is incorporated herein by reference.
[0030] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
[0031] The use of any and all examples, or exemplary language ("e.g.", "such as") provided herein, is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.
[0032] Herein, the term "about" has its ordinary meaning. The term "about" is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% or 5% of the recited values (or range of values).
[0033] Any and all combinations and subcombinations of the embodiments and features disclosed herein are encompassed by the present invention. For example, the expression of any combination of 2, 3, 4, 5 or more of the genes identified herein may be used in the methods described herein.
[0034] The terms "subject" and "patient" are used interchangeably herein, and refer to an animal, preferably a mammal, most preferably a human. In an embodiment, the AML patient is less than 60 years old. In another embodiment, the AML patient is 60 years old or older. In another embodiment, the AML patient is a pediatric AML patient.
[0035] In the studies described herein, the present inventors have performed next generation RNA sequencing analyses of 415 primary AML specimens that were selected to represent AML genetic diversity and which include 20 samples with t(8;21) rearrangements and 28 samples with inv(16)/t(16; 16) rearrangements.
[0036] The present inventors have shown that CBF-AMLs exhibit distinct mutational and transcriptional signatures relative to other AML subtypes, which may be useful for the characterization, diagnosis and prognosis of CBF-AMLs. They have also identified points of convergence and divergence in the genetic/mutational and transcriptional signatures of these two CBF-AML subgroups, and established the phenotypes of the non-classical fusions identified in the cohort studied.
[0037] Furthermore, using a similar approach, the present inventors have also studied the expression of 772 GPCRs in 148 AML samples and in normal blood, bone marrow and cord blood-derived CD34+ cell populations. R/VA-Sequencing data analysis revealed that 239 GPCRs are expressed in this cohort of human AML. Among these members, 30 are overexpressed in AML samples compared to normal CD34+ cells. Upregulated GPCRs are enriched in chemokine (including CCR1, CXCR4, CCR2, CX3CR1, CCR7 and CCRL2), adhesion (including CD97, EMR1, EMR2 and GPR114) and purine (including P2RY2 and P2RY13) receptor families. Interestingly, some GPCRs were differentially expressed in normal karyotype AML with NPM1 or FLT3-ITD mutation, and in specimens with Core Binding Factor and MLL rearrangements.
Detection of mutation(s)
[0038] In the studies described herein, the present inventors have identified novel mutations associated with AMLs and have shown that inv(16) and t(8;21) AMLs exhibit distinct mutational signatures which may be useful for the characterization, classification and diagnosis of AMLs and AMLs subtypes (e.g., inv(16) and t(8;21)) AMLs (FIG. 1A and Table 5).
[0039] Accordingly, the present invention relates to a method for determining the likelihood that a subject suffers from CBF-AML subtype, said method comprising: determining the presence of one or more of the mutations depicted in FIG. 1A and/or Table 5 in a cell (e.g., blood cell, a leukemic cell) sample from the subject, wherein the presence of the one or more mutations is indicative that said subject suffers or is likely to suffer from a CBF-AML, and wherein the absence of said one or more mutations is indicative that the subject does not suffer or has a low likelihood of suffering from an a CBF-AML. In an embodiment, the cell sample is from a subject already diagnosed from AML or from CBF-AML. In a further embodiment, the cell sample is from a subject already diagnosed with AML. In embodiments, the at least one mutation is a mutation in one or more of ZBTB7A, ASXL2, SMC1A, TET2, and DNMT3A. In an embodiment, the method is for determining the likelihood that a subject suffers from CBF-AML with t(8;2^/RUNX1-RUNX1T1 chromosomal rearrangement (t(8;21) AML). In an embodiment, the mutation is a missense mutation. In an embodiment, the mutation is a nonsense mutation. In another embodiment, the mutation is a frameshift mutation. In a further embodiment, the mutation is an inframe mutation.
[0040] In a further aspect, the present invention relates to a method for determining the likelihood that a subject suffers from Core Binding Factor Acute Myeloid Leukemia (CBF-AML) with t(8;2^/RUNX1-RUNX1T1 chromosomal rearrangement (t(8;21) AML), said method comprising: determining the presence of one or more mutations in at least one of the following genes: ZBTB7A, ASXL2, SMC1A, TET2, and DNMT3A in a sample comprising leukemic cells from the subject, wherein the presence of the one or more mutations in said at least one genes is indicative that the subject has a high likelihood of suffering from t(8;21) AML. In an embodiment, the cell sample is from a subject already diagnosed with AML or CBF-AML.
[0041] In an embodiment, the mutation is in ZBTB7A, for example at a position corresponding to residue 122 or 174 of ZBTB7A. In a further embodiment, the mutation is a V to G substitution at residue 122 of ZBTB7A. In another embodiment, the mutation is a frameshift mutation at residue 174 of ZBTB7A (introducing a premature stop codon). In another embodiment, the mutation is in SMC1A, for example at a position corresponding to residue 96 or 71 1 of SMC1A. In a further embodiment, the mutation is an R to H substitution at residue 96 of SMC1A. In another embodiment, the mutation is an R to Q substitution at residue 71 1 of SMC1A
[0042] In an embodiment of the above methods, the at least one mutation is a RUNX1 fusion. In an embodiment, the RUNX1 fusion is a RUNX1-USP2 fusion or a RUNX1-ERG fusion.
[0043] In embodiments, the at least one mutation is a mutation in one or more of ZBTB7A, ASXL2, and SMC1A. In an embodiment, the at least one mutation is a frameshift mutation in ASXL2. In an embodiment, the at least one frameshift mutation is at position 523, 587, 594 and/or 614 of ASLX2, leading to a premature stop codon. In embodiments, the at least one mutation is a mutation in TET2 and/or DNMT3A. In an embodiment, the mutation in TET2 is at position 330, 731 or 735. In a further embodiment, the mutation is an F to Y substitution at position 731 . In another embodiment, the mutation is a premature stop codon at position 330 or 735. In an embodiment, the mutation in DNMT3A is at position 635, 1050 or 1501 . In a further embodiment, the mutation is a premature stop codon at position 635 or 1050. In another embodiment, the mutation is a frameshift mutation at position 1501 .
[0044] The present invention encompasses the detection of any mutation or any combination/sub-combination of the mutations defined herein, for example the detection of a single mutation, or of 2, 3, 4, 5 or more of the mutations defined herein, within a gene or a combination of genes defined herein.
[0045] As used herein, the term "high likelihood" means that the individual is more likely to have the disorder or disease (CBF-AML) than an individual without the mutation(s), or that the sample is more likely to be an CBF-AML leukemia sample than an AML sample without the mutation(s).
[0046] The determination of the presence (or absence) of the mutation(s) in the sample may be performed using any suitable methods (see, e.g., Syvanen, Nat Rev Genet. 2001 Dec;2(12):930-42). For example, the presence of the mutation(s) may be detected at the genomic DNA, transcript (RNA or cDNA) or protein level. Examples of suitable methods for determining sequences and polymorphisms at the nucleic acid level include sequencing of the nucleic acid sequence encompassing the mutation(s), e.g., in the genomic DNA or transcript (cDNA), for example by "Next Generation Sequencing" methods (e.g., genome sequencing, RNA sequencing (RNA-seq)) or other sequencing methods; hybridization of a nucleic acid probe capable of specifically hybridizing to a nucleic acid sequence comprising the mutation(s) and not to (or to a lesser extent to) a corresponding nucleic acid sequence that does not comprises the mutation(s) (under comparable hybridization conditions, such as stringent hybridization conditions) (e.g., molecular beacons); restriction fragment length polymorphism analysis (RFLP); Amplified fragment length polymorphism PCR (AFLP-PCR); amplification of a nucleic acid fragment comprising the mutation(s) using a primer specifically hybridizing to a nucleic acid sequence comprising the mutation(s), wherein the primer produces an amplified product if the mutation(s) is/are present and does not produce the same amplified product when a nucleic acid sequence not comprising the mutation(s) is used as a template for amplification, nucleic acid sequence based amplification (Nasba), primer extension assay, FLAP endonuclease assay (Invader assay, Olivier M. (2005). Mutat Res. 573(1-2): 103-10), 5'-nuclease assay (McGuigan F.E. and Ralston S.H. (2002) Psychiatr Genet. 12(3): 133-6), oligonucleotide ligase assay. Other methods include in situ hybridization analyses and single-stranded conformational polymorphism analyses. Several SNP genotyping platforms are commercially available. Additional methods will be apparent to one of skill in the art.
[0047] The determination of the presence of the mutation(s) may also be achieved at the polypeptide/protein level. Examples of suitable methods for detecting alterations at the polypeptide level (including polypeptides encoded by splice variants) include sequencing of the encoded polypeptide; digestion of the encoded polypeptide followed by mass spectrometry or HPLC analysis of the peptide fragments, wherein the mutated polypeptide results in an altered mass spectrometry or HPLC spectrum as compared to the unmutated polypeptide; and immunodetection using an immunological reagent (e.g., an antibody, a ligand) which exhibits altered immunoreactivity with a mutated polypeptide relative to a corresponding unmutated polypeptide. Immunodetection can measure the amount of binding between a polypeptide molecule and an anti-protein antibody by the use of enzymatic, chromodynamic, radioactive, magnetic, or luminescent labels which are attached to either the anti-protein antibody or a secondary antibody which binds the anti-protein antibody. In addition, other high affinity ligands may be used. Immunoassays which can be used include e.g. ELISAs, Western blots, and other techniques known to those of ordinary skill in the art (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999 and Edwards R, Immunodiagnostics: A Practical Approach, Oxford University Press, Oxford; England, 1999). Methods to generate antibodies exhibiting altered immunoreactivity with a mutated polypeptide relative to a corresponding unmutated polypeptide are described in more detail below.
[0048] All these detection techniques may also be employed in the format of microarrays (e.g., SNP microarrays), protein-arrays, antibody microarrays, tissue microarrays, electronic biochip or protein-chip based technologies (see Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, Mass., 2000).
[0049] Further, nucleic acid-containing sequences may be amplified prior to or in conjunction with the detection methods noted herein. The design of various primers for such amplification is known in the art. For example, a nucleic acid (RNA, cDNA, genomic DNA) comprising the mutation(s) may be amplified using primers hybridizing to sequences located on each side of the mutation(s). Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8: 14-25. Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Οβ replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1 173-1 177; Lizardi et al., 1988, BioTechnology 6: 1 197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., supra). Preferably, amplification will be carried out using PCR.
[0050] Polymerase chain reaction (PCR) is carried out in accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683, 195; 4,683,202; 4,800, 159; and 4,965, 188. In general, PCR involves, a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected. An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith. The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers. Following a sufficient number of rounds of synthesis of extension products, the sample is analyzed to assess whether the mutation(s) to be detected is/are present. Detection of the amplified sequence may be carried out by visualization following Ethidium Bromide (EtBr) staining of the DNA following gel electrophoresis, or using a detectable label in accordance with known techniques, and the like. For a review on PCR techniques (see PCR Protocols, A Guide to Methods and Amplifications, Michael et al. Eds, Acad. Press, 1990).
[0051] Ligase chain reaction (LCR) is carried out in accordance with known techniques (Weiss, 1991 , Science 254: 1292). Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill. Strand displacement amplification (SDA) is also carried out in accordance with known techniques or adaptations thereof to meet the particular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA 89:392-396; and 1992, Nucleic Acids Res. 20: 1691 -1696).
[0052] "Nucleic acid hybridization" refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure. Examples of hybridization conditions can be found in laboratory manuals (e.g., Green and Sambrook, supra) and are commonly known in the art. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1 % SDS at 42°C. Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0.1 % SDS at 68°C. In other examples of hybridization, a nitrocellulose filter can be incubated overnight at 65°C with a labeled probe specific to one or the other two alleles in a solution containing 50% formamide, high salt (5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1 % SDS, and 100 μg/ml denatured carrier DNA (i.e. salmon sperm DNA). The non-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0.1 % SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42°C (moderate stringency) or 65°C (high stringency). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest. The selected temperature is based on the melting temperature (Tm) of the DNA hybrid. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
Transcriptional signatures
[0053] AMLs. By comparing the transcriptomic GPCR signature of AMLs with normal CD34+ cells, the present inventors have identified GPCR genes which are differentially expressed in AMLs.
[0054] Accordingly, in an aspect, the present invention relates to a method for determining the likelihood that a subject suffers from Acute Myeloid Leukemia (AML), the method comprising, determining the level of expression (i.e., the amount) of at least one of the GPCR genes showing differential expression in AMLs vs. normal CD34+ cells listed in Table 12 in a cell sample (e.g., blood, bone marrow, leukemic cell sample) from the subject and comparing the level of expression of the at least one gene to that in a control sample (or to a reference value derived therefrom), wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from AML.
[0055] For GPCR genes listed in Table 12 which are overexpressed in AML, a higher level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference level derived therefrom) is indicative that the subject suffers or is likely to suffer from AML. Likewise, a lower or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from AML.
[0056] For GPCR genes listed in Table 12 which are underexpressed in AML, a lower level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject suffers or is likely to suffer from AML. Likewise, a higher or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from AML.
[0057] In an embodiment, the above methods comprise the determination of the level of expression of at least one of the following GPCR genes: CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 or FPR2. In a further embodiment, the above methods comprise the determination of the level of expression of at least one of the following GPCR genes: CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1 or C5AR1. In a further embodiment, the above methods comprise the determination of the level of expression of at least one of the following GPCR genes: CXCR4, CD97, PTGER4, GPR183, PTGER2, C3AR1 or EMR2.
[0058] CBF-AMLs. By comparing the transcriptomic signature of CBF-AMLs with other AMLs described in the studies presented herein, the present inventors have identified genes that are differentially expressed in CBF-AMLs (see, e.g., Tables 8A and 8B). [0059] Accordingly, in an aspect, the present invention relates to a method for determining the likelihood that a subject suffers from a Core-Binding Factor acute myeloid leukemia (CBF-AML), the method comprising determining the level of expression (i.e., the amount) of at least one of the genes identified in bold in Tables 8A and/or 8B in a cell sample from the subject and comparing the level of expression of the at least one gene to that in a control sample (or to a reference value derived therefrom), wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from a CBF-AML.
[0060] For genes which are overexpressed in CBF-AML (Tables 8A), a higher level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject is likely to suffer from CBF-AML. Likewise, a lower or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from CBF-AML.
[0061] For genes which are underexpressed in CBF-AML (Tables 8B), a lower level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject is likely to suffer from CBF-AML. Likewise, a higher or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from CBF-AML.
[0062] In embodiments, the above method comprises the determination of the level of expression of a combination of at least two, three, four or five of the genes listed in Tables 8A and/or 8B.
[0063] In an embodiment, the above method comprises the determination of the level of expression of at least one of ARC, CACNA1H and LOC200772. In an embodiment, the above method comprises the determination of the level of expression of ARC. In an embodiment, the above method comprises the determination of the level of expression of CACNA1H. In an embodiment, the above method comprises the determination of the level of expression of LOC200772.
[0064] In another aspect, the present invention relates to a method for determining the likelihood that a subject suffers from CBF-AML, the method comprising determining the level of expression (i.e., the amount) of at least one of the following GPCR genes: ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and RXFP1 in a cell sample from the subject and comparing the level of expression of the at least one GPCR gene to that in a control sample (or to a reference value derived therefrom), wherein a higher expression of the at least one gene is indicative that the subject suffers or is likely to suffer from a CBF-AML. [0065] (Inv16) AMLs. Comparative analysis of genes and transcripts expressed in inv(16)/t(16; 16)/CSFS- YW 7 myeloid leukemia (inv(16) AML) relative to other AML subtypes enabled the identification of a set of differentially expressed genes/transcripts (See Tables 7A- 7B and 10), which may be useful for the characterization, classification, diagnosis and treatment of inv(16) AMLs.
[0066] Accordingly, in a further aspect, the present invention concerns a method for determining the likelihood that a subject suffers from an inv(16) AML, the method comprising, determining the level of expression (i.e., the amount) of at least one of the genes identified in bold in Tables 7A-7B, or at least one of the transcripts identified in Table 10 (SEQ ID NOs:21 - 35) in a cell sample from the subject and comparing the level of expression of the at least one gene to that in a control sample (or to a reference value derived therefrom), wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from an inv(16) AML. In an embodiment, the cell sample is a leukemia cell sample from a subject diagnosed with AML or CBF-AML.
[0067] For genes/transcripts listed in Tables 7A and 10 which are overexpressed in inv(16) AML, a higher level of expression of the at least one gene/transcript in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject suffers or is likely to suffer from inv(16) AML. Likewise, a lower or equal level of expression of the at least one gene/transcript in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from inv(16) AML.
[0068] For genes listed in Table 7B which are underexpressed in inv(16) AML, a lower level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject is likely to suffer from an inv(16) AML. Likewise, a higher or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from an inv(16) AML.
[0069] In embodiments, the above method comprises the determination of the level of expression of a combination of at least two, three, four or five of the genes/transcripts listed in Tables 7A, 7B and/or 10.
[0070] In an embodiment the method comprises the determination of the level of expression of a at least one of MEGF10 and APLN.
[0071] In an embodiment, the above methods comprise the determination of the level of expression of at least one of the following GPCR genes: C5AR1, CCR2, ACKR3/CXCR7, FPR1, GPR183, LPAR6, PTGIR and RXFP1, in a further embodiment at least one of CXCR7/ACKR3 and RXFP1. [0072] t(8;21 ) AML. Comparative analysis of genes expressed in t(8;21)/ RUNX1-RUNX1T1 myeloid leukemia (t(8;21) AML) relative to other AML subtypes enabled the identification of a set of differentially expressed genes/transcripts (see Tables 6A-6B and 11) which may be useful for the characterization, classification, diagnosis and treatment of t(8;21) AMLs.
[0073] Accordingly, in another aspect, the present invention concerns a method for determining the likelihood that a subject suffers from a t(8;21) AML, the method comprising, determining the level of expression (i.e., the amount) of at least one of the genes identified in bold in Tables 6A-6B, or at least one of the transcripts identified in Table 11 (SEQ ID NOs:36- 50) in a sample from the subject and comparing the level of expression of the at least one gene to that in a control sample (or to a reference value derived therefrom), wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from a t(8;21) AML. In an embodiment, the cell sample is a leukemia cell sample from a subject diagnosed with AML or CBF-AML.
[0074] For genes/transcripts listed in Tables 6A and 11 which are overexpressed in t(8;21) AML, a higher level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject suffers or is likely to suffer from a t(8;21) AML. Likewise, a lower or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from a t(8;21) AML.
[0075] For genes listed in Table 6B which are underexpressed in t(8;21) AMLs, a lower level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject is likely to suffer from a t(8;21) AML. Likewise, a higher or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from a t(8;21) AML.
[0076] In embodiments, the above method comprises the determination of the level of expression of a combination of at least two, three, four or five of the genes/transcripts listed in Tables 6A, 6B and/or 10.
[0077] In an embodiment, the above method comprises the determination of the level of expression of at least one of LINC00958 and ADARB2-AS1.
[0078] In an embodiment, the above method comprises the determination of the level of expression of at least one of the following GPCR genes: ADRA2C, GPR153, LPAR5, LPAR6 and PTGIR. In an embodiment, the above method comprises the determination of the level of expression of at least one of the following GPCR genes: ADRA2C and GPR153.
[0079] MLL-rearranged AMLs. Comparative analysis of GPCR genes expressed in MLL myeloid leukemia (MLL-AMLs) further enabled the identification differentially expressed GPCR genes which may be useful for the characterization, classification, diagnosis and treatment of MLL-rearranged AMLs (see FIG. 12A).
[0080] Accordingly, in another aspect, the present invention concerns a method for determining the likelihood that a subject suffers from a MLL-rearranged AML, the method comprising, determining the level of expression (i.e., the amount) of at least one of the genes identified FIG. 12A in a sample from the subject and comparing the level of expression of the at least one gene to that in a control sample (or to a reference value derived therefrom), wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from a MLL-rearranged AML. In an embodiment, the cell sample is a leukemia cell sample from a subject diagnosed with AML.
[0081] For genes listed in FIG. 12A which are overexpressed in MLL-rearranged AMLs, a higher level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject suffers or is likely to suffer from a MLL-rearranged AML. Likewise, a lower or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from a MLL-rearranged AML.
[0082] For genes listed in FIG. 12A which are underexpressed in MLL-rearranged AMLs, a lower level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject is likely to suffer from a MLL-rearranged AML. Likewise, a higher or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a control sample (or relative to a reference value derived therefrom) is indicative that the subject does not suffer or is unlikely to suffer from a MLL-rearranged AML.
[0083] In an embodiment, the above method comprises the determination of the level of expression of GPR126.
[0084] In an embodiment, the MLL-rearranged AML is MLL-MLLT4, MLL-ELL or MLL-SEPT9.
[0085] Normal karyotype AML. Comparative analysis of GPCR genes expressed in normal karyotype (NK) AMLs with internal tandem duplication in FLT3 (FLT3-ITD) and mutated NPM1 show distinct transcriptional profiles with respect to each other and with respect to non-NK AML samples.
[0086] Accordingly, the present invention provides a method for determining the likelihood that a subject suffers from FLT3-ITD AML, the method comprising determining the level of expression of at least one of the differentially expressed genes identified and listed in FIG. 13B in a cell sample from the subject and comparing the level of expression of the at least one gene to a control sample or reference level derived therefrom, wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from FLT3-\TD AML.
[0087] In an embodiment, the above method comprises determining the level of at least one of the following genes: i) CYSLTR2, GPR114, and GPR156, wherein an increased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from FLT3-\TD AML; and/or ii) P2RY13; FRP1 and C5AR1, wherein a decreased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from FLT3-\TD AML.
[0088] The present invention further provides a method for determining the likelihood that a subject suffers from an AML subtype with a mutated NPM1 (NPM1m), the method comprising, determining the level of expression of at least one of the differentially expressed genes identified and listed in FIG. 13B in a cell sample from the subject and comparing the level of expression of the at least one gene to a control sample or reference level derived therefrom, wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from NPM1m AML.
[0089] In an embodiment, the above method comprises determining the level of i) GPR56 and/or CYSLTR2, wherein an increased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from a NPM1m AML; and/or ii) FPR1 and/or C5AR1, wherein a decreased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from a NPM1 AML.
[0090] M4 and M5 AMLs. Analysis of GPCRs expression fingerprints also revealed distinct gene expression profiles between M4 and M5 AMLs and other AML subtypes. M4 (acute myelomonocytic leukemia) and M5 (acute monocytic leukemia) refer to AML subtypes according to the French-American-British (FAB) classification system (Bennett JM et al., Ann Intern Med. 1985; 103(4):620).
[0091] Accordingly, the present invention further relates to a method for determining the likelihood that a subject suffers from a M4 AML subtype, the method comprising, determining the level of expression of at least one of the differentially expressed genes identified and listed in FIG. 14 in a cell sample from the subject and comparing the level of expression of the at least one gene to a control sample or reference level derived therefrom, wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from M4 AML subtype.
[0092] In an embodiment, the above method comprises determining the level of at least one of the following genes: FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 and VIPR1, wherein an increased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from M4 AML subtype.
[0093] In another aspect, the present invention concerns a method for determining the likelihood that a subject suffers from a M5 AML subtype, the method comprising, determining the level of expression of at least one of the differentially expressed genes identified and listed in FIG. 14 in a cell sample from the subject and comparing the level of expression of the at least one gene to a control sample or reference level derived therefrom, wherein a differential expression of the at least one gene is indicative that the subject suffers or is likely to suffer from M5 AML subtype.
[0094] In an embodiment, the above method comprises determining the level of at least one of the following genes i) CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1, and HTR7, wherein an increased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from a M5 AML: and/or ii) GPR174, GPR56, NMUR1, SUCNR1, OR2W3 and/or GPR97, wherein a decreased level of expression in the cell sample relative to that in the control sample is indicative that the subject suffers or is likely to suffer from a M5 AML.
[0095] lnv(16) vs. t(8:21 ). By comparing the transcriptomic signature of inv(16) AMLs with that of t(8;21) AMLs, the present inventors have identified a set of 224 differentially expressed genes (see FIG. 2H and Table 8C) which allow to distinguish between the two subgroups of CBF- AMLs.
[0096] Thus, in yet another aspect, the present invention concerns a method for distinguishing between inv(16) AML and t(8;21) AML in a leukemia cell sample from a subject, the method comprising, determining the level of expression (i.e., the amount) of at least one of the genes identified and listed in FIG. 2H and/or Table 8C in the leukemia cell sample from the subject and comparing the level of expression of the at least one gene to that in a control sample (or to a reference value derived therefrom), wherein the level of expression of the at least one gene enables to distinguish between inv(16) AML and t(8;21) AML.
[0097] Any gene or combination of gene listed in FIG. 2H and/or Table 8C may be used in accordance with the present invention to distinguish between inv(16) and t(8;21) AMLs. Genes identified as being overexpressed in t(8;21) in FIG. 2H and/or Table 8C are correspondingly underexpressed in inv(16) and vice versa.
[0098] For example, for genes in FIG. 2H and/or Table 8C which are overexpressed in t(8;21), a higher level of expression of the at least one gene in a cell sample from a subject relative to that in an inv(16) control sample (or relative to a reference value derived therefrom) is indicative that the subject suffers or is likely to suffer from a t(8;21) AML. Conversely, a lower or equal level of expression of the at least one gene in the cell sample from the subject relative to that in an inv(16) control sample (or relative to a reference value derived therefrom) is indicative that the subject suffers from an inv(16) AML or is likely to suffer from an inv(16) AML.
[0099] Similarly, for genes listed in FIG. 2H and/or Table 8C which are underexpressed in t(8;21) AMLs, a lower level of expression of the at least one gene in the cell sample from the subject relative to that in a inv(16) control sample (or relative to a reference value derived therefrom) is indicative that the subject is likely to suffer from a t(8;21) AML. Conversely, a higher or equal level of expression of the at least one gene in the cell sample from the subject relative to that in a inv(16) control sample (or relative to a reference value derived therefrom) is indicative that the subject suffers from an inv(16) AML or is likely to suffer from an inv(16) AML.
[00100] In an embodiment, the method comprises the determination of the level of expression of a combination of at least one or two of the following genes: RUNX1T1, POU4F1, MEIS1, CD1E, TM4SF1, KCNK17, RXFP1, ENHO, MYBPH, C3orf80, CLEC10A, SPINK2, SHD, COLEC12, AGR2, PRTFDC1, ASGR2, CPVL, CD1C, HOXB-AS1, LOC283683, CES1, NPTX2, LGALS2, TREM2, MEGF10, RHBDL3, SULF2, HOXB4 and MSR1. In an embodiment the method comprises the determination of the level of expression of a combination of at least two of the following genes: RUNX1T1, POU4F1, MEIS1 and CD1E.
[00101] In an embodiment, the method comprises the determination of the level of expression of at least one gene identified in FIG. 2H. In another embodiment, the method comprises the determination of the level of expression of at least one gene identified in Table 8C.
[00102] Generally, all methods described herein encompasses the determination of the level of expression of any gene or any combination/sub-combination of the genes defined herein (e.g., those identified in Table 12 for AMLs; Tables 8A-8B for CBF-AMLs; Tables 7A-7B and 10 for inv(16) AMLs, Tables 6A-6B and 11 for t(8;21) AMLs; FIG. 12A for MLL-AMLs; FIG. 13B for NK-AMLs with DNMT3A, FLT3-ID or NPM1 mutations, etc.). For example, methods described herein may comprise the determination of the level of expression of a single gene, or of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125 or more of the differentially expressed genes identified herein. In an embodiment, the at least one gene has a mean differential expression value of +/- 1 .2, 1 .25, 1 .3, 1 .35, 1 .5, 2.75, 2, 2.25, 2.35, 2.4, 2.5, 2.6, 2.8, or 3.
[00103] The determination of the expression of the one or more genes or encoded gene products (e.g., mRNA, protein) listed above may be performed using any known methods to detect nucleic acids or proteins. In embodiments, the expression is compared to a control or reference level (e.g., the level obtained a sample from one or more non-CBF, inv(16) and/or t(8;21) AML sample(s), and/or a cell sample enriched in CD34+ cells) to assess the subject's likelihood of suffering from a CBF-AML or CBF-AML subtype (e.g., inv(16) AML or t(8:21) AML), or the likelihood that the AML sample is CBF- AML or CBF-AML subtype (e.g., inv(16) AML or t(8:21) AML) sample.
[00104] The levels of nucleic acid corresponding to the above-mentioned genes can then be evaluated according to any known methods including (but not-limited to) the methods discussed below, e.g., with or without the use of nucleic acid amplification methods. In some embodiments, nucleic acid amplification methods can be used to detect the level of expression of the one or more genes. For example, the oligonucleotide primers and probes may be used in amplification and detection methods that use nucleic acid substrates isolated by any of a variety of well-known and established methodologies, such as those described above.
[00105] Thus, in an embodiment, the above-mentioned method comprises a step of amplification.
[00106] The nucleic acid or amplification product may be detected or quantified by hybridizing a probe (e.g., a labeled probe) to a portion of the nucleic acid or amplified product. The probe may be labelled with a detectable group that may be, for example, a fluorescent moiety, chemiluminescent moiety, radioisotope, biotin, avidin, enzyme, enzyme substrate, or other reactive group. Other well-known detection techniques include, for example, gel filtration, gel electrophoresis and visualization of the amplicons, and High Performance Liquid Chromatography (HPLC). In certain embodiments, for example using real-time TMA or real-time PCR, the level of amplified product is detected as the product accumulates.
[00107] Thus, in an embodiment, the above-mentioned method comprises a step of detection or quantification with a probe. In an embodiment, the level of expression of any differentially expressed gene disclosed herein is measured and the method comprises detecting or quantifying the nucleic acid or amplified product with a probe. In an embodiment, the probe comprises from about 7-8 to about 100, 90, 80, 70, 60 or 50 nucleotides, in further embodiments from about 10 to about 50, 45 or 40 nucleotides, from about 10 to about 35 nucleotides, from about 10 to about 35, 34, 33, 32, 31 or 30 nucleotides, from about 15 to about 25 nucleotides or from about 16 to about 24 nucleotides. In an embodiment, the probe comprises about 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.
[00108] In an embodiment, the above-mentioned method comprises a step of normalizing the gene expression levels, i.e. normalization of the measured levels of the above-noted genes against a stably expressed control gene (or housekeeping gene) to facilitate the comparison between different samples. "Normalizing" or "normalization" as used herein refers to the correction of raw gene expression values/data between different samples for sample to sample variations, to take into account differences in "extrinsic" parameters such as cellular input, nucleic acid (RNA) or protein quality, efficiency of reverse transcription (RT), amplification, labeling, purification, etc. , i.e. differences not due to actual "intrinsic" variations in gene expression by the cells in the samples. Such normalization is performed by correcting the raw gene expression values/data for a test gene (or gene of interest) based on the gene expression values/data measured for one or more "housekeeping" or "control" genes, i.e. whose expressions are known to be constant (i.e. to show relatively low variability) between the cells of different tissues and under different experimental conditions. Thus, in an embodiment, the above-mentioned method further comprises measuring the level of expression of a housekeeping gene in the biological sample. Suitable housekeeping genes are known in the art and several examples are described in WO 2014/134728. In a further embodiment, the method further comprises measuring the level of expression of one or more housekeeping genes in a biological sample from the subject.
[00109] Suitable housekeeping genes are known in the art and several examples are described in WO 2014/134728, including those depicted in Table 1 below.
Table 1 : Examples of housekeeping genes
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
[00110] Other commonly used housekeeping genes include TBP, YWHAZ, PGK1, LDHA, ALDOA, HPRT1, SDHA, UBC, GAPDH, ACTB, G6PD, VIM, TUBA1A, PFKP, B2M, GUSB, PGAM1 and HMBS.
[00111] In a further embodiment, the method further comprises measuring the level of expression of one or more housekeeping genes in a biological sample from the subject. In an embodiment, the level of expression of the housekeeping gene is measured and the method comprises amplifying a housekeeping gene nucleic acid using a suitable pair of primers.
[00112] In an embodiment, one or more of the primers and/or probe is/are detectably labelled, i.e. comprises a detectable label attached thereto. As used herein, the term "detectable label" refers to a moiety emitting a signal (e.g., light) that may be detected using an appropriate detection system. Any suitable detectable label may be used in the method described herein. Detectable labels include, for example, enzyme or enzyme substrates, reactive groups, chromophores such as dyes or colored particles, luminescent moieties including a bioluminescent, phosphorescent or chemiluminescent moieties, and fluorescent moieties. In an embodiment, the detectable label is a fluorescent moiety. Fluorophores that are commonly used include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'- dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), Ν,Ν,Ν',Ν'- tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'- dimethylaminophenylazo) benzoic acid (DABCYL), and 5-(2'-aminoethyl)aminonaphthalene-l - sulfonic acid (EDANS). The fluorophore may be any fluorophore known in the art, including, but not limited to: FAM, TET, HEX, Cy3, TMR, ROX, Texas Red®, LC red 640, Cy5, and LC red 705. Fluorophores for use in the methods and compositions provided herein may be obtained commercially, for example, from Biosearch Technologies (Novato, CA.), Life Technologies (Carlsbad, CA), GE Healthcare (Piscataway NJ), Integrated DNA Technologies (Coralville, Iowa) and Roche Applied Science (Indianapolis, IN). In some embodiments, the fluorophore is chosen to be usable with a specific detector, such as a specific spectrophotometric thermal cycler, depending on the light source of the instrument. In some embodiments, if the assay is designed for the detection of two or more target nucleic acids (multiplex assays), two or more different fluorophores may be chosen with absorption and emission wavelengths that are well separated from each other (i.e., have minimal spectral overlap). In some embodiments, the fluorophore is chosen to work well with one or more specific quenchers. A representative example of a suitable combination of fluorescent label and quencher is FAM (excitation max. = 494 nm, emission max. = 520 nm), ZEN™ quencher (non-abbreviation; absorption max 532 nm), and Iowa black fluorescein quencher (IBFQ, absorption max = 531 nm) (Integrated DNA Technologies®). Covalent attachment of detectable label and/or quencher to primer and/or probe can be accomplished according to standard methodology well known in the art as discussed, for example in Sambrook and Green, supra, Ausubel et al., supra, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckerman et al., Nucleic Acids Research, 15: 5305-5321 (1987) (3' thiol group on oligonucleotide); Sharma et al., Nucleic Acids Research, 19:3019 (1991) (3' sulfhydryl); Giusti et al., PCR Methods and Applications, 2:223-227 (1993) and Fung et al, U.S. Pat. No. 4,757, 141 (5' phosphoamino group via Aminolink™ II available from Applied Biosystems®, Foster City, Calif.); Stabinsky, U.S. Pat. No. 4,739,044 (3' aminoalkylphosphoryl group); Agrawal et al., Tetrahedron Letters, 31 : 1543-1546 (1990) (attachment via phosphoramidate linkages); Sproat et al., Nucleic Acids Research, 15:4837 (1987) (5' mercapto group); Nelson et al., Nucleic Acids Research, 17:7187-7194 (1989) (3' amino group); and the like.
[00113] In another embodiment, the expression of the one or more genes or encoded gene products is measured at the protein level. Methods to measure the amount/level of proteins are well known in the art. Protein levels may be detected directly using a ligand binding specifically to the protein, such as an antibody or a fragment thereof. In embodiments, such a binding molecule or reagent (e.g., antibody) is labeled/conjugated, e.g., radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled to facilitate detection and quantification of the complex (direct detection). Alternatively, protein levels may be detected indirectly, using a binding molecule or reagent, followed by the detection of the (protein/ binding molecule or reagent) complex using a second ligand (or second binding molecule) specifically recognizing the binding molecule or reagent (indirect detection). Such a second ligand may be radiolabeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled to facilitate detection and quantification of the complex. Enzymes used for labeling antibodies for immunoassays are known in the art, and the most widely used are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Examples of binding molecules or reagents include antibodies (monoclonal or polyclonal), natural or synthetic ligands, and the like.
[00114] Examples of methods to measure the amount/level of protein in a sample include, but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbent assay (ELISA), "sandwich" immunoassays, radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance (SPR), chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical (IHC) analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, antibody array, microscopy (e.g., electron microscopy), flow cytometry, proteomic-based assays, and assays based on a property or activity of the protein including but not limited to ligand binding or interaction with other protein partners, enzymatic activity, fluorescence. For example, if the protein of interest is a kinase known to phosphorylate of given target, the level or activity of the protein of interest may be determined by the measuring the level of phosphorylation of the target in the presence of the test compound. If the protein of interest is a transcription factor known to induce the expression of one or more given target gene(s), the level or activity of the protein of interest may be determined by the measuring the level of expression of the target gene(s).
[00115] "Control level" or "reference level" "reference value" or "standard level" are used interchangeably herein and broadly refers to a separate baseline level measured in a comparable "control" sample, which is generally from a subject not suffering, or not likely to suffer, from the target disease (e.g., not suffering from AML, CBF-AML, inv(16)-/A L and/or t(8;21)-/A L), for example an AML sample from another AML subtype (or a mixture of other AML subtypes) or a sample enriched in CD34+ cells from a subject not suffering from AML such as a healthy subject not suffering from leukemia). The corresponding control level may be a level corresponding to an average or median level calculated based on the levels measured in several reference or control subjects (e.g., a pre-determined or established standard level or reference value). The control level may be a pre-determined "cut-off value recognized in the art or established based on levels measured in samples from one or a group of control subjects, i.e., subjects not suffering from CBF-AML, t(8;21) AML and/or inv(16) AML). For example, the "threshold reference level" of may be a level corresponding to the minimal level of expression (cut-off) of a differentially expressed gene disclosed herein (or combination thereof), that permits to distinguish in a statistically significant manner AML patients having the target disease (e.g., a CBF-AML or CSF-AML subtype) from those having another form of AML or from healthy subjects, which may be determined using samples from such subjects, for example. Alternatively, the "threshold reference level" of may be a level corresponding the level of expression (cut-off) of a differentially expressed gene disclosed herein, that permits to best or optimally distinguish in a statistically significant manner between AML patients (e.g., AML types and subtypes). The corresponding reference/control level may be adjusted or normalized for age, gender, race, or other parameters. The "control level" can thus be a single number/value, equally applicable to every patient individually, or the control level can vary, according to specific subpopulations of patients. Thus, for example, older subjects might have a different control level than younger subjects, and women (female subjects) might have a different control level than men (male subjects). The predetermined standard level can be arranged, for example, where a tested population is divided equally (or unequally) into groups, such as a low- risk group, a medium-risk group and/or a high-risk group or into quadrants or quintiles, the lowest quadrant or quintile being individuals with the lowest risk (i.e., lowest level of expression of the one or more genes) and the highest quadrant or quintile being individuals with the highest risk (i.e., highest level of expression of the one or more genes). It will also be understood that the control levels according to the invention may be, in addition to predetermined levels or standards, levels measured in other samples (e.g. from healthy/normal subjects, or AML patients) tested in parallel with the experimental sample. The reference or control levels may correspond to normalized levels, i.e. reference or control values subjected to normalization based on the expression of a housekeeping gene. The skilled person would understand that a corresponding threshold reference level, which would define a similar threshold value for one or more differentially expressed gene(s) disclosed herein, expression levels, may be calculated based, for example, on the expression of another housekeeping gene or using another method of calculation.
[00116] "Higher expression", "higher level of expression", "overexpression", "overexpressed", "increased level of expression" as used herein refers to significantly (i) higher expression of the one or more of the above-mentioned genes (protein and/or mRNA) in one or more given cells present in the sample (relative to the control) and/or (ii) higher amount of cells expressing the one or more genes in the sample (relative to the control).
[00117] "Lower expression", "lower level of expression", "underexpression", "underexpressed", or "decreased level of expression" as used herein refers to significantly (i) lower expression of the one or more genes (protein and/or mRNA) in one or more given cells present in the sample (relative to the control) and/or (ii) lower amount of cells expressing the one or more genes in the sample (relative to the control). [00118] In an embodiment, higher or lower refers to a level of expression that is above or below the control level (e.g., the predetermined cut-off value or reference value). In another embodiment, higher or lower refers to a level of expression that is at least one standard deviation above or below the control level (e.g., the predetermined cut-off value) (e.g. that is statistically significant as determined using a suitable statistical analysis), and a "similar expression" or "similar level of expression" refers to a level of expression that is less than one standard deviation above or below the control level (e.g., the predetermined cut-off value) (e.g. that is not statistically significant as determined using a suitable statistical analysis). In embodiments, higher or lower refers to a level of expression that is at least 1 .5, 2, 2.5, 3, 4 or 5 standard deviations above or below the control level (e.g., the predetermined cut-off value. In another embodiment, "higher expression" refers to an expression that is at least 10, 20, 30, 40, 45 or 50% higher in the test sample relative to the control level. In an embodiment, "lower expression" refers to an expression that is at least 10, 20, 25, 30, 35, 40, 45, or 50% lower in the test sample relative to the control level. In another embodiment, higher or lower refers to a level of expression that is at least 1 .5, 2-, 5-, 10-, 25-, or 50-fold higher or lower in the test sample relative to the control sample.
[00119] In another embodiment, methods described herein further comprise obtaining or collecting a biological sample from a subject. In various embodiments, the sample can be from any source that contains biological material suitable for the detection of the mutation(s), such as genomic DNA, RNA (cDNA), and/or proteins, for example a tissue or cell sample from the subject (blood cells, immune cells (e.g., lymphocytes), bone marrow cells, etc. that comprises leukemic cells (AML cells). The sample may be subjected to cell purification/enrichment techniques to obtain a cell population enriched in a specific cell subpopulation or cell type(s). The sample may be subjected to commonly used isolation and/or purification techniques for enrichment in nucleic acids (genomic DNA, cDNA, mRNA) and/or proteins. Accordingly, in an embodiment, the method may be performed on an isolated nucleic acid and/or protein sample, such as isolated genomic DNA. The biological sample may be collected using any methods for collection of biological fluid, tissue or cell sample, such as venous puncture for collection of blood cell samples. In an embodiment, the biological sample comprising leukemic cells comprises nucleic acids (RNA, cDNA) obtained or isolated from leukemic cells or bone marrow cells.
[00120] In certain embodiments, methods of diagnosis described herein may be at least partly, or wholly, performed in vitro. In a further embodiment, the method is wholly performed in vitro.
[00121] In another aspect, the present invention provides performing any combinations of the steps/methods described herein on biological samples from subjects for the diagnosis/prognosis of AML, CBF-AML, t(8;21) AML or inv(16) AML, or other AML genetic subtypes described herein for example detecting one or more of the mutations described herein, detecting the levels of expression of one or more differentially expressed genes described herein, etc.
[00122] In an embodiment, the above-mentioned method further comprises selecting and/or administering a course of therapy or prophylaxis to said subject in accordance with the diagnostic results obtained using any of the above-mentioned methods. For example, if it is determined that the subject has a high likelihood of suffering from a particular AML subtype associated with a poor prognosis, a more aggressive or a treatment regimen adapted for treatment of AML may be used, such as for example a more aggressive chemotherapy regimen (e.g., high-dose chemotherapy, longer administration schedule, etc.) and/or stem cell/bone marrow transplantation (e.g., allogeneic transplantation). Thus, the method further comprises subjecting the subject to a suitable anti-leukemia therapy (e.g., bone marrow or hematopoietic stem cell transplantation, chemotherapy, etc.) in accordance with the diagnostic/prognostic result. In another aspect, the present invention thus provides a method of treating a subject suffering from AML diagnosed based on any of the methods described herein, said method comprising administering to said subject a suitable anti-leukemia therapy in accordance with or adapted to the diagnosis.
[00123] In another aspect, the present invention provides an assay mixture for the assessment of AML (e.g., for the classification and diagnosis of CBF-AML, inv(16) AML or t(8;21) AML, or other AML genetic subtypes described herein), the assay mixture comprising: (i) a biological sample from a subject suffering from AML (or suspected of suffering from AML); and (ii) one or more assays to determine the level of expression of of at least one of the differentially expressed genes disclosed herein, and/or for detecting the mutations disclosed herein.
[00124] In another aspect, the present invention provides a system for the assessment of AML (e.g., for the classification and diagnosis of CBF-AML, inv(16) AML or t(8;21) AML, or other AML genetic subtypes described herein) in a subject, the system comprising: (i) a biological sample from a subject suffering from AML (or suspected of suffering from AML); and (ii) one or more reagents for determining/measuring the level of expression of at least one of the differentially expressed genes disclosed herein, and/or for detecting the mutations disclosed herein.
[00125] The present invention provides a system for the assessment of AML (e.g., for the classification and diagnosis of CBF-AML, inv(16) AML or t(8;21) AML, or other AML genetic subtypes described herein) in a subject, comprising: a sample analyzer configured to produce a signal for at least one of the differentially expressed genes disclosed herein, and/or for one or more of the mutations disclosed herein, in a biological sample of the subject; and a computer sub-system programmed to calculate, based on the at least one differentially expressed genes or mutation, whether the signal is higher or lower than a reference value or whether the mutation is present in the sample. In various embodiments, the system further comprises the biological sample.
[00126] In another aspect, the present invention further provides a kit for the assessment of AML (e.g., for the diagnosis of CBF-AML, inv(16) AML or t(8;21) AML, or other AML genetic subtypes described herein), the kit comprising: (i) one or more reagents for detecting one or more of the mutations set forth in FIG. 1 and/or Table 5 in a biological sample. In an embodiment, the kit comprises reagents for detecting at least 2, 3, 4, or 5 of the mutations set forth in FIG. 1 and/or Table 5 in a biological sample.
[00127] In another aspect, the present invention further provides a kit for the assessment of AML (e.g., for the diagnosis of CBF-AML, inv(16) AML or t(8;21) AML, or other AML genetic subtypes described herein), the kit comprising: (i) one or more reagents for determining/measuring the level of expression of at least one of the differentially expressed genes disclosed herein (e.g., listed in Tables 6A-B, 7A-B, 8A-B, 10 and 11) in a biological sample. In an embodiment, the kit comprises reagents for detecting the level of expression of at least 2, 3, 4, 5, 10, 12, 15, 20, 30, 50, 75, 50, 100 or more differentially expressed genes disclosed herein, in a biological sample.
[00128] In an embodiment, the one or more reagents present in the assay mixture or kit comprise, for example, primer(s), probe(s), antibody(ies), solution(s), buffer(s), nucleic acid amplification reagent(s) (e.g., DNA polymerase, DNA polymerase cofactor, dNTPs), nucleic acid hybridization/detection reagent(s), and/or reagents for detecting antigen-antibody complexes, etc. In an embodiment, the assay mixture or kit comprises one or more pairs of primers for amplifying one or more nucleic acids corresponding to the differentially expressed or mutated gene(s) disclosed herein (e.g., genes listed in FIG. 1 , Tables 5, 6A-B, 7A-B, 8A-B, 10 and 11). In an embodiment, the assay mixture or kit comprises one or more probes for detecting one or more nucleic acids correspond to the mutated or differentially expressed gene(s) disclosed herein. In an embodiment, the assay mixture or kit further comprises one or more reagents for determining/measuring the level of expression of at least one normalization/housekeeping gene (e.g., ABL1) in the sample.
[00129] Furthermore, in an embodiment, the kit may be divided into separate packages or compartments containing the respective reagent components explained above.
[00130] In addition, such a kit may optionally comprise one or more of the following: (1) instructions for using the reagents for the diagnosis and/or prognosis of A L/CBF-/4 L/t(8;21), /A L/inv(16) AML, other AML genetic subtypes described herein or any combination of these applications; (2) one or more containers; and/or (3) appropriate controls/standards. Such a kit can include reagents for collecting a biological sample from a subject and reagents for processing the biological sample. The kits featured herein can also include an instruction sheet describing how to perform the assays for measuring gene expression or the presence of mutations. The instruction sheet can also include instructions for how to determine a reference cohort (control subject population), including how to determine expression levels of genes in the reference cohort and how to assemble the expression data to establish a reference for comparison to a test subject. The instruction sheet can also include instructions for assaying gene expression in a test subject and for comparing the expression level with the expression in the reference cohort to subsequently determine the appropriate treatment regimen for the test subject.
[00131] Informational material included in the kits can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the reagents for the methods described herein. For example, the informational material of the kit can contain contact information, e.g. , a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about performing a gene expression analysis and interpreting the results, particularly as they apply to an AML patient's likelihood of having the AML genetic subtype.
[00132] The kits featured herein can also contain software necessary to infer a patient's likelihood of having the AML genetic subtype from the gene expression data.
Treatment of AMLs expressing GPCRs
[00133] The present inventors have shown that certain GPCRs are differently expressed by AML cells, notably in frequent AML genetic subgroups, relative to normal hematopoietic cells, which may be exploited for the treatment of AMLs. GPCRs are targets for approximately 30% of all marketed drugs, and several modulators of GPCRs are known.
[00134] In another aspect, the present invention provides a method for treating a subject suffering from Acute Myeloid Leukemia (AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 and/or FPR2. In another aspect, the present invention provides an agent targeting at least one GPCR for treating a subject suffering from AML, wherein said GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 and/or FPR2.
[00135] In another aspect, the present invention provides a method for treating a subject suffering from Core Binding Factor Acute Myeloid Leukemia (CBF-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and/or RXFP1. In another aspect, the present invention provides an agent targeting at least one GPCR for treating a subject suffering from CBF-AML, wherein said at least one GPCR is ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and/or RXFP1.
[00136] In another aspect, the present invention provides a method for treating a subject suffering from Acute Myeloid Leukemia with Mixed Lineage Leukemia chromosomal rearrangement (MLL-AML), said method comprising administering to said subject an effective amount of an agent targeting the G-protein coupled receptor (GPCR) GPR126. In another aspect, the present invention provides an agent targeting the GPCR GPR126 for treating a subject suffering from MLL-AML,
[00137] In another aspect, the present invention provides a method for treating a subject suffering from normal karyotype Acute Myeloid Leukemia (NK-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CYSLTR2, GPR114 and/or GPR56. In another aspect, the present invention provides an agent targeting a GPCR for treating a subject suffering from NK-AML, wherein said GPCR is CYSLTR2, GPR114 and/or GPR56.
[00138] In another aspect, the present invention provides a method for treating a subject suffering from myelomonocytic acute myeloid leukemia (M4-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 and/or VIPR1. In another aspect, the present invention provides an agent targeting at least one GPCR for treating a subject suffering from M4-AML, wherein said at least one GPCR is FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 and/or VIPR1.
[00139] In another aspect, the present invention provides a method for treating a subject suffering from monocytic acute myeloid leukemia (M5-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 and/or HTR7. In another aspect, the present invention provides an agent targeting at least one GPCR for treating a subject suffering from M5-AML, wherein said at least one GPCR is CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 and/or HTR7.
[00140] As used herein, the term "agent targeting a G-protein coupled receptor (GPCR)" includes any compound able to directly or indirectly affect the regulation of a GPCR by reducing for example the expression of the GPCR (i.e. , transcription and/or the translation), or an activity of the GPCR, since as to reduce AML cell proliferation, dissemination, and/or induce AML cell death. It includes intracellular as well as extracellular agents. Without being so limited, such inhibitors include siRNA, antisense molecules, proteins, peptides, small molecules and antibodies, etc.
[00141] Agents that target GPCRs are described in the IUPHAR/BPS database (http://www.quidetopharmacoloqv.org/; Alexander SPH et al., The Concise Guide to PHARMACOLOGY 2015/16. Br J Pharmacol. 172: 5729-5743). The agent may be an agonist or an antagonist of the GPCR, and/or an agent that specifically binds to the GPCR (e.g, antibody or antigen-binding fragment thereof).
[00142] In an embodiment, the agent is an antagonist of the GPCR. GPCR antagonists are known in the art. Natural and synthetic antagonists of GPCRs are described in the IUPHAR/BPS database.
[00143] CXCR4 antagonists include plerixafor, TG-0054, AMD070, AMD3465 and BL-8040 (see, for example, Debnath et al. Theranostics 2013; 3(1):47-75).
[00144] Many agonists and antagonists of the GPCR ADRA2C are available, including several FDA-approved drugs such as the antihypertensive drug clonidine and the antidepressant Mirtazapine, as well as others such as JP1302, lisuride, terguride, rauwolscine, spiroxatrine, yohimbine, WB 4101 , roxindole, RX821002, lurasidone, phentolamine, all-frans-4-oxo-retinoic acid, cabergoline, bromocriptine, ARC-239, apomorphine, prazosin, piribedil, chlorpromazine, BRL 44408 and tolazoline.
[00145] Similarly, CYSLTR2, a receptor for the inflammatory mediators cysteinyl leukotrienes, is also shown to be overexpressed in FL73-ITD and NPM1 mutated AML samples. Selective CYSLTR2 agonist (NMLTC4) (Yan D, Stocco R et al., Molecular pharmacology. 201 1 ;79(2):270- 8) and antagonists, such as HAMI3379 (Wunder F, et al. British Journal of Pharmacology. 2010; 160(2):399-409), BayCysLT2 (Carnini C, et al. FASEB journal, 201 1 ;25(10):3519-28), BAYu9773, pranlukast, zafirlukast, iralukast, pobilukast and CI-198615, have been developed.
[00146] Also, selective antagonists of the chemokine receptor CCR2, such as CCX140-B, studied in diabetic mice and tested in clinical trials for patients with diabetic nephropathy (www.clinicaltrials.gov, NCT01447147 and NCT01440257) may be used for treating AMLs expressing this GPCR (Sullivan T, et al. American Journal of Physiology - Renal Physiology. 2013;305(9):F1288-97). Other antagonists of CCR2 include TAK-779, SB-282241 , RS-504393, N-[(3R)-1-[(2,4-dimethylphenyl)methyl]pyrrolidin-3-yl]-2-{[3-
(trifluoromethyl)phenyl]formamido}acetamide, viral macrophage inflammatory protein-ll, MC148R, CCL26, RS-136270, INCB3284, MK-0812, INCB8761 , INCB3344, RS-102895 and CCL24.
[00147] Antagonists of CCR1 include CCX721 and CCX354-C (Dairaghi DJ et al., Blood. 2012; 120(7): 1449-57; Tak PP et al., Annals of the rheumatic diseases. 2013;72(3):337-44), as well as CP-481 ,715, BX 471 , 2-[(3S)-3-(aminomethyl)piperidine-1 -carbonyl]-N-[1 -(cyclononen-1 - ylmethyl)piperidin-4-yl]-9H-xanthene-9-carboxamide (PubChem CID: 73755059), CCL4, viral macrophage inflammatory protein-ll, UCB35625 and CCL18.
[00148] Selective antagonists of PTGER2 include TG4-155 and TG6-10-1 (Jiang J et al., PNAS 2013; 1 10(9):3591 -6).
[00149] Examples of antagonists of PTGER4 (EP4 receptor) include ER-819762, AH-23848, CJ- 023423, GW-627368, L-161982, ONO-AE3-208 and grapiprant.
[00150] Examples of antagonists of FPR1 include cyclosporin H, 3570-0208 (PMID: 19807662), chenodeoxycholic acid, deoxycholic acid.
[00151] Antagonists of C3AR include SB290157, FLTChaAR and (2S)-5- (diaminomethylideneamino)-2-[[2-(2,2-diphenylethylsulfanyl)acetyl]amino]pentanoic acid (PubChem CID: 91827355).
[00152] Antagonists of C5AR1 include CHIPS, W5401 1 , NDT9520492, AcPhe-Orn-Pro-D-Cha- Trp-Arg, PMX205, PMX53, N-methyl-Phe-Lys-Pro-D-Cha-Trp-D-Arg-COOH, Α8Δ71"73, JPE1375, C089, RPR121 154 and L-156,602.
[00153] Antagonists of PAFR include PCA 4248, foropafant, apafant, ABT-299, ABT-491 , RP- 52770, L659989, 10-OBn-7a-F-ginkgolide B, 7a-CI-ginkgolide B, 10-OBn-ginkgolide B, BN 50739, 7a-N3-ginkgolide B, 10-OBn-epi-ginkgolide C, 7a-NHMe-ginkgolide B, ginkgolide B, 7a- F-ginkgolide B, 10-OBn-ginkgolide C, 7a-NHEt-ginkgolide B, ginkgolide A, 7a-OCOCH2Ph- ginkgolide B, 7-epi-ginkgolide C, 7a-NH2-ginkgolide B, 7a-OAc-ginkgolide B, ginkgolide J, ginkgolide C, israpafant, CV-6209, SDZ 64-412, CV-3988, SCH 37370, SCH 40338, PCA 4248 and bepafant.
[00154] Antagonists of ADRB2 include carazolol, timolol, carvedilol, CGP 12177, ICI 1 18551 , SR59230A, propranolol, levobunolol, alprenolol, bupranolol, labetalol, nadolol, NIP, levobetaxolol, propafenone, betaxolol, sotalol, metoprolol, cicloprolol, NIHP, atenolol and LK 204-545.
[00155] Antagonists of SUCNR1 include compound 5g [PMID: 21571530], compound 5g [PMID: 21571530] and compound 7e [PMID: 21571530] (Bhuniya D et al., Bioorg. Med. Chem. Lett., 21 (12): 3596-602).
[00156] Antagonists of P2RY2 include AR-C126313, AR-C1 18925XX, suramin, reactive blue-2 and 1 -amino-4-phenylamino-2-sulfoanthraquinone derivatives (Weyler S et al., Bioorg Med Chem Lett, 18: 223-227).
[00157] Antagonists of P2RY13 include cangrelor, ApA MRS2603, MRS221 1 , reactive blue-2, suramin, 2MeSAMP and PPADS.
[00158] Antagonists of HRH2 include iodoaminopotentidine, tiotidine, ranitidine, cimetidine, JNJ-39758979, metiamide, burimamide, INCB-38579, ABT-239, clobenpropit, ciproxifan, MK- 0249, conessine, pitolisant and famotidine.
[00159] Antagonists of PTH2R include [His4, Tyr5, Trp6, His7]TIP39, PTHrP-(1 -21)/PTH-(22-34), TIP39-(7-39), [Ne5,Trp23]PTHrP-(5-36) and PTHrP-(7-34).
[00160] Antagonists of PTGIR include R01 138452, R03244794, R03244794, R01 138452, R03244794 and BAY-73-1449 (Bley KR et al., 2006. Br J Pharmacol , 147: 335-345).
[00161] Antagonists of GPR65 include psychosine and psychosine-related lysoslipids.
[00162] Antagonists of FPR2 include quin-C7, isopropylureido-FLFLF, compound 1754-31 [PMID: 23788657], WRWWWW, t-Boc-FLFLF and FPRL1 -inhibitor protein.
[00163] Antagonists of FPR1 include CHIPS, 3570-0208 [PMID: 19807662], cyclosporin H, t- Boc-FLFLF, chenodeoxycholic acid, deoxycholic acid, cyclosporin A, BVT173187, i-Boc-Met- Leu-Phe, diamide 7, methionine benzimidazole 6, group E 1682-2106 [PMID: 161 18363], sulfinpyrazone and spinorphin.
[00164] Antagonists of RXFP1 include B-R13/17K H2 relaxin and RXFPi-truncate.
[00165] Antagonists of VIPR1 include PG 97-269 and N-stearyl-[Nle17] neurotensin-(6-1 1)/VIP-
(7-28).
[00166] Antagonists of GABBR1 include CGP 56999A, CGP 62349, CGP 54626A, CGP 64213, CGP 71872, SCH 5091 1 , CGP 35348, saclofen and 2-hydroxy-saclofen.
[00167] In another embodiment, the agent is an antibody or an antigen-binding fragment thereof. Antibodies or antigen-binding fragments thereof may interfere with or modulate the activity of the GPCRs in tumor cells, e.g., by blocking the interaction with a ligand, thereby modulating the signaling within the tumor cells (which may in turn affect tumor cell proliferation, survival and/or migration). Some of the GPCRs overexpressed in AMLs are adhesion GPCRs (CD97, EMR2/ADGRE2, GPR56 and GPR114), which may be targeted by antibodies to modulate AML cell migration, for example.
[00168] The antibody may alternatively or further be conjugated to an anti-tumor agent (chemotherapeutic agent, toxin) so as to deliver the anti-tumor agent to the tumor AML cells that expressed the GPCRs, i.e. using the antibody or antigen-binding fragment thereof as a targeting agent for tumor AML cells.
[00169] In another embodiment, the antibody or antigen-binding fragment thereof induces the killing of the AML cells. Antibodies or antigen-binding fragments thereof that are specific for GPCRs that are primarily or exclusively present on AMLs cells can be injected into AML patients and will then bind the cognate protein epitopes on the tumor cells. Once the antibody binds the tumor protein (i.e. GPCR primarily present on AMLs cells), the non-variable base of the antibody (also called the Fc region) can then be recognized by cells of the human immune system. Specific immune cells (such as natural killer cells or NK cells) have receptors (i.e. CD16) which recognize the Fc portion of antibodies. The recognition of target cells with bound antibodies leads to the activation of NK cells (and/or other cytotoxic cells such as macrophages, monocytes and/or eosinophils) which then destroy the target AML cell through processes called Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP). The binding of the antibodies to the GPCR epitopes on the AML cells may also lead to killing of the tumor cells through a mechanism called complement-dependent cytotoxicity (CDC). In CDC, the C1 q complex of the complement system binds the antibody and this binding triggers the complement cascade which leads to the formation of the membrane attack complex (MAC) (C5b to C9) at the surface of the target AML cell, as a result of the classical pathway complement activation.
[00170] It is therefore possible, with knowledge of proteins (GPCRs) that are specifically expressed on the surface of tumor cells, where they are accessible to the antibodies, to develop targeted anti-cancer therapies which function through antibody-based cell cytotoxicity (ADCC, ADCP and CDC). This approach has been used successfully to target ERBB2 receptor in certain breast cancers (with Trastuzumab; Nat Med. 2000 Apr;6(4):443-6), the EGF receptor in metastatic colorectal cancer and head and neck cancer (Cetuximab; Lancet Oncol. 2010 Jan; 1 1 (1):21 -8. Epub 2009 Nov 10.), the CD20 protein in lymphomas and leukemias (e.g., with Rituximab; Blood. 1997 Sep 15;90(6):2188-95; Ofatumumab and others; J Hematol Oncol. 2012 Oct 1 1 ;5:64), the CD38 protein in multiple myeloma (with Daratumumab; J Immunol. 201 1 Feb 1 ; 186(3): 1840-8).
[00171] Antibodies against chemokine receptors have also been shown to be effective for the treatment of certain cancers in animal models and humans (see, Vela et al., Front Immunol. 2015; 6: 12), for example the CCR4-specific antibody mogamulizumab in patients with relapsed or refractory CCR4+ adult T-cell leukemia (ATL), CXCR4-specific antibody ulocuplumab for the treatment of multiple myeloma, CCR2-specific antibody MLN1202 for the treatment of bone metastases, and a CCR7-specific antibody in a murine model of human mantle cell lymphoma (MCL).
[00172] It is also possible to improve the effectiveness of the ADCC response through additional protein modifications to the antibody (Eur J Cancer. 2013 Oct;49(15):3344-52. Epub 2013 Jul 18).
[00173] In an embodiment, the antibody or antigen/binding fragment thereof binds to an extracellular domain of the GPCR. Extracellular domains of the GPCRs are known in the art and may be found, for example, in the UniProtKB database. For example, for CXCR4 (UniProt accession No. P61073), the extracellular domains correspond to residues 1 -83, 139-157, 223- 236, and 347-367. One or more of these extracellular domains may be used as an antigen to raise antibodies.
[00174] The term "antibody or antigen-binding fragment thereof as used herein refers to any type of antibody/antibody fragment including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, humanized antibodies, CDR-grafted antibodies, chimeric antibodies and antibody fragments so long as they exhibit the desired antigenic specificity/binding activity. Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single- chain antibody molecules, single domain antibodies (e.g. , from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. In an embodiment, the antibody is a monoclonal antibody.
[00175] The above-mentioned agent may be present in a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers and/or excipients. Such compositions may be prepared in a manner well known in the pharmaceutical art. Supplementary active compounds can also be incorporated into the compositions. The carrier/excipient can be suitable, for example, for intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g. , aerosol) administration (see Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro, 2003, 21th edition, Mack Publishing Company).
[00176] An "excipient," as used herein, has its normal meaning in the art and is any ingredient that is not an active ingredient (drug) itself. Excipients include for example binders, lubricants, diluents, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizing agent, release-delaying agents and other components. "Pharmaceutically acceptable excipient" as used herein refers to any excipient that does not interfere with effectiveness of the biological activity of the active ingredients and that is not toxic to the subject, i.e. , is a type of excipient and/or is for use in an amount which is not toxic to the subject. Excipients are well known in the art, and the present system is not limited in these respects. Excipients, include, for example and without limitation, binders (binding agents), thickening agents, surfactants, diluents, release-delaying agents, colorants, flavoring agents, fillers, disintegrants/dissolution promoting agents, lubricants, plasticizers, silica flow conditioners, glidants, anti-caking agents, anti-tacking agents, stabilizing agents, anti-static agents, swelling agents and any combinations thereof. As those of skill would recognize, a single excipient can fulfill more than two functions at once, e.g. , can act as both a binding agent and a thickening agent. As those of skill will also recognize, these terms are not necessarily mutually exclusive.
[00177] Any suitable amount of the agent or pharmaceutical composition may be administered to a subject. The dosages will depend on many factors including the mode of administration, the age, weight, condition of the patient etc. Typically, the amount of the agent or pharmaceutical composition contained within a single dose will be an amount that effectively prevents, delays or treats AML without inducing significant toxicity.
[00178] For the prevention, treatment or reduction in the severity of a given disease or condition (AML), the appropriate dosage of the compound/composition will depend on the severity and course of the disease or condition, whether the agent/composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound/composition, and the discretion of the attending physician. The compound/composition is suitably administered to the patient at one time or over a series of treatments. Preferably, it is desirable to determine the dose-response curve in vitro, and then in suitable animal models prior to testing in humans. The present invention provides dosages for the compounds and compositions comprising same. For example, depending on the type and severity of the disease, about 1 μg/kg to 1000 mg per kg (mg/kg) of body weight per day. Further, the effective dose may be 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/ 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000 mg/kg, or may range between any two of the foregoing values. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired relief or suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
[00179] The knowledge of GPCRs expressed by AML cells may be exploited in screening assays to identify compounds that target the GPCRs and modulate tumor AML cell proliferation, survival and/or migration). Thus, in other aspects, the present invention provides a method for determining whether a test compound may be useful for treating AML or a specific AML subtype of interest, comprising contacting a cell expressing a GPCR normally expressed by AML cells (or cells of the AML subtype of interest) with the test compound; and determining whether the test compound modulates (inhibits or increases) the expression and/or activity of the GPCR, wherein said modulation is indicative that the test compound may be useful for treating the AML or the specific AML subtype of interest. In an embodiment, the method comprises determining whether the test compound inhibits the expression and/or activity of the GPCR.
[00180] The above-noted screening method or assay may be applied to a single test compound or to a plurality or "library" of such compounds (e.g. , a combinatorial library). Test compounds (drug candidates) may be obtained from any number of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Screening assay systems may comprise a variety of means to enable and optimize useful assay conditions. Such means may include but are not limited to: suitable buffer solutions, for example, for the control of pH and ionic strength and to provide any necessary components for optimal activity and stability (e.g. , protease inhibitors), temperature control means for optimal activity and/or stability, of the GPCR, and detection means to enable the detection of its activity. A variety of such detection means may be used, including but not limited to one or a combination of the following: radiolabelling, antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g., generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g., horseradish peroxidase, green fluorescent protein), specific binding reagents (e.g., biotin/(strept)avidin), and others.
[00181] GPCR activity may be assessed using methods well known in the art, for example by measuring the activation of signaling pathways, for example increase in cAMP, phosphorylation of proteins, etc. GPCR activity may be assessed using biosensors, for example biosensors based on FRET/BRET technology (e.g., as described in US Patents Nos. 8,883,485 and 9,029,097).
MODE(S) FOR CARRYING OUT THE INVENTION
[00182] The present invention is illustrated in further details by the following non-limiting examples.
Example 1 : Materials and Methods
[00183] Examples 1.1-1.2:
[00184] Specimen collection, ethics and cohort characteristics. This study was approved by the Research Ethics Boards (REB) of Universite de Montreal and Maisonneuve-Rosemont Hospital. Samples were collected between 2001 and 2015 according to the procedures of the Banque de cellules leucemiques du Quebec (BCLQ). Normal paired DNAs were obtained from buccal swabs or saliva. 415 primary AML specimens were analyzed, including 20 specimens with t(8;21) and 28 specimens with inv(16)/t(16;16), as well as a cohort of 367 AMLs from other groups, and processed as previously described (Lavallee et al., Nature Genetics, 2015 47(9): 1030-7). Patient characteristics are described in Table 2.
Table 2: Characteristics of CBF and comparative AML cohorts. WBC: white blood cells, FAB: French-American-British classification, T-AML: therapy-related acute myeloid leukemia lnv(16) (n=28) t(8;21) (n=20) Non-CBF AML (n=367)
T-AML 26 (7.1 %) Gender
Male 17 (60.7%) 15 (75%) 203 (55.3%)
Female 1 1 (39.3%) 5 (25%) 164 (44.7%) Age
Median (range) 51.5 (17-75) 39.5 (18-71) 59 (19-87) WBC count(x 109/L)
Median (range) 42.8 (3.9 - 276.4) 18.8 (1.5 - 94.1) 33.5 (0.8 - 361.2) FAB subtype
M0 27 (7.4%) M1 1 (3.6%) 6 (30%) 108 (29.4%)
M2 1 (3.6%) 13 (65%) 38 (10.4%)
M3 0 0 15 (4.1 %)
M4 3 (10.7%) 0 33 (9.0%)
M4Eo 21 (75%) 0 0
M5 0 0 66 (18.0%)
M6 0 0 10 (2.7%)
M7 0 0 3 (0.8)
Not classifiable 2 (7.1 %) 1 (5%) 67 (18.3)
[00185] RNA and DNA isolation. RNA was isolated from primary AML cells using TRIzol® reagent according to the manufacturer's instructions (lnvitrogen®/Life Technologies®) with an additional purification on RNeasy® mini columns (Qiagen®) to obtain high quality RNA. DNA was isolated and purified using DNeasy® protocols (Qiagen®). Integrity verification of isolated RNA was performed on a Bioanalyzer® 2100 with a RIN > 8 deemed acceptable. For sequencing experiments, libraries were constructed with the TruSeq® RNA Sample Preparation Kit (lllumina®) according to manufacturer's protocols.
[00186] Sequencing and bioinformatics analysis. Libraries were constructed with the TruSeq® RNA Sample Preparation Kit (lllumina). Sequencing was performed using an lllumina® HiSeq 2000 with 200 cycles paired end runs. Sequence data were mapped to the reference genome hg19 using the lllumina® Casava 1.8.2 package and Elandv2 mapping software according to RefSeq annotations (UCSC, January 27th 201 1 or April 16th 2014).
[00187] Mutation identification and validation. Variants were identified using Casava 1.8.2. and fusions or larger alterations were identified with TopHat 2.0.7 and Cufflinks 2.1.1. In addition, some mutations such as FLT3-ITDs and mutations in minor clones were identified using an approach developed by our group caller k-mer (bitbucket.org/iric-soft/km). Mutations outside of the coding region were excluded, and only nonsynonymous variants (SNP or Indel) were considered. Variants identified in normal controls (representing polymorphisms or sequencing artifacts) were filtered out. Known single nucleotide polymorphisms (SNP) (dbSNP, version 137) were also removed, except for those in known leukemia "hotspots". All variants reported have a variant allelic frequency (VAF) > 20%, >8 variant reads, > 20 total reads and a quality score > 20 except for FLT3, NRAS, KRAS, PTPN11 and KIT for which a VAF of > 5% was required. All recurrent mutations identified by next-generation sequencing (NGS) in the CBL-AML cohort have been validated by Sanger sequencing of tumoral DNA or cDNA.
[00188] Expression. Transcript levels are given as Reads Per Kilobase per Million mapped reads (RPKM) and genes are annotated according to RefSeq annotations (UCSC, April 16th, 2014).
[00189] Statistics. Fisher's exact test was used in the analysis of contingency tables. Analysis of differential gene expression was performed with the Wilcoxon rank-sum test (Mann-Whitney) using the stats R package (http://cran.r-project.org/) with estimation of the False-discovery rate (FDR, q-value). In order to avoid issues with log-scale representation of RPKM equal to zero or normalized copy numbers equal to zero, a small constant (0.0001 or 0.01 , respectively) was added to all expression values when log transformation was performed. For scatterplot visualizations, averages of groups were performed on log 10 transformed values to avoid overrepresentation of extremes.
[00190] Examples 1.3-1.4:
[00191] Human primary leukemic and cord blood cells. The 148 AML samples of the Leucegene cohort used for this study were collected by the BCLQ with an informed consent and approval of the project by the Research Ethics Board of the Maisonneuve-Rosemont Hospital and Universite de Montreal. The genetic subgroups of the AML samples included in this study are listed in Table 15. Cord blood samples (n=12) were provided by Hema-Quebec and pre- enriched for CD34+ cells before being sorted to keep the CD34 APC+/CD45RA PE- cell populations as previously described (20). The Cancer Genome Atlas (TCGA) RNA-Seq AML dataset has been downloaded from the TCGA website (https://tcga- data.nci.nih.gov/tcga/tcgaDownload.jsp) in November 2013 and the associated clinical information obtained from The Cancer Genome Atlas Research Network (New England Journal of Medicine, 2013;368(22):2059-74).
[00192] Sorting of normal bone marrow and peripheral blood cell populations. Three unsorted fresh bone marrow samples from healthy donors were purchased from Lonza® (Lonza 1 M-125). Red blood cell lysis was performed prior to resuspending the cells in PBS, 0.1 % BSA, Dnase ^g/mL. Subpopulations were sorted on a BD Aria® II cell sorter based on published surface marker combinations (described by Novershtern et al., Cell. 2011 Jan 21 ;144(2):296-309; Table 3 below).
Table 3: Sorting strategy for normal bone marrow cell populations
Figure imgf000049_0001
[00193] Peripheral blood was collected from healthy donors, subjected to red blood cell lysis, and subsequently sorted based on the following sorting strategy: Granulocytes (SSChlQh, CD33+), B cells (Lymphocyte gate FSC|0W, SSC|0W, CD19+), T cells (Lymphocyte gate FSC|0W, SSC|0W, CD3+), Monocytes (Monocyte gate FSC high, sSCl0W/med, CD14bright, CD33med), total white blood cells (WBC). Sorting purity was checked on aliquots after sorting, and cells were counted, resuspended in Trizol® reagent and stored at -80°C until RNA isolation was performed according to the manufacturer's instructions. An additional purification step on RNeasy® mini columns (Qiagen® 74104) was performed to optimize RNA quality, which was subsequently tested on an Agilent® bioanalyzer 2100. A minimum of 50,000 cells was used for RNA- Sequencing (RNA-Seq), which was performed as described below.
[00194] Sequencing and RNA-Seq data analysis. RNA-Seq was performed on the AML and cord blood samples using an lllumina® HiSeq 2000 instrument. Libraries were prepared according to the manufacturer's recommendations (lllumina). RefSeq annotations were based on the UCSC January 27th 201 1 version. The alignment to reference genome (hg19) was done using the CASAVA 1 .8.2 package and Eland v2 mapping software and bioinformatic analyses were performed as described previously (Lavallee VP et al. Blood. 2015; 125(1): 140-3).
[00195] Statistical analysis. RNA-Seq data in RPKM (Reads Per Kilobase of transcript per Million mapped reads) were transformed to IRPKM (log2(RPKM+1)), where + 1 was added to avoid excessive variations due to very small values. Log transformation was performed to avoid overrepresentation of extreme values. Highly expressed GPCRs were selected using a threshold of 3.5 IRPKM (or 10.35 RPKM) (FIG. 6B). The variability of expression between samples was determined by calculating the coefficient of variation (CV), a ratio between the standard deviation and the mean expression value. Genes with a CV smaller than (50%,) will be considered as GPCRs with low variability in their expression (FIG. 6C). Up- and downregulated GPCRs were described as those having a difference of median expression between AML and normal CD34+ cells greater than 1 and less than -1 , respectively.
[00196] GPCR subfamily enrichment analysis. Grouping of GPCR subfamilies was based on the International Union of Basic and Clinical Pharmacology (lUPHAR) database classification (http://www.guidetopharmacology.org/). To complete and subdivide the class A group, the GRAFS phylogenetic classification of GPCRs was used (Fredriksson R et a/., Molecular Pharmacology. 2003;63(6): 1256-72). Taste 2 receptors, which are not included in the lUPHAR database, have also been added as well as vomeronasal receptors, opsins and 3 orphan GPCRs (GPR137B, TAPT1, XPR1). Overall GPCRs were classified in distinct ligand groups (FIG. 6A).
[00197] The GPCR subfamily enrichment in the up- or down-regulated groups was performed using a Fisher's exact test and significance (two-tailed p-value) was calculated using the function FET of the add-in Fisher's exact downloaded from http://www.obertfamily.com/software/fisherexact.html. The GPCR members associated to a specific AML genetic subgroup were selected by calculating the ratio between the mean GPCR expression level (RPKM values) in samples with and without the genetic abnormality. Further statistical analyses were performed by calculating a difference of mean expression level (IRPKM) between samples with and without the genetic abnormality. An arbitrary difference of 1.5 IRPKM and a significant Student's t-test (p-value < 0.05) were used as cut-off levels to identify differentially expressed GPCRs in the Leucegene cohort.
[00198] Plasmids. CD97 cDNA inserted in pCMV SPORT6 vector was obtained from Open Biosystems (MHS4768-99609488) and CXCR4, FPR1 or C5AR1 in pcDNA3.1 was obtained from the Missouri S&T cDNA Resource Center (www.cdna.org). LTB4R expressed in pJ3M has been previously described (Gaudreau, R. et al., Biochem J, 335 (Pt 1): 15-18).
[00199] Antibody validation, cell culture and transfection. The HEK293 cell line which has a low expression level of LTB4R, FPR1 and C5AR1 was used for antibody validation. Antibodies were validated by comparing their staining pattern between untransfected cells and cells transfected with a vector containing the corresponding cDNA. HEK293 cells were cultured in 6-well plates in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) FBS at 37°C with 5% C02. At 80% of confluence, cells were transfected with ^g of plasmid DNA encoding a GPCR by using Lipofectamine™ 2000 (Invitrogen). Cell were harvested at 48h after transfection, and suspended in 100 μΙ_ PBS buffer before staining with the antibodies.
[00200] Flow cytometry analysis of selected highly expressed GPCRs. Commercial antibodies used for GPCR detection in AML or HEK293 cells were CXCR4-PerCP (BioLegend®, #306515), CD97-PE (BioLegend®, #336307), LTB4R-PE (LS Bio®, #LS-C16203), FPR1-APC (R&D System®, #FAB3744A) and C5AR1-F\TC (LS Bio, #LS-C5674). The LSR II® equipment (BD Biosciences®) was used for flow cytometry data acquisition, and flow cytometry analysis was performed using the DIVA® or FlowJo® software.
[00201] Flow cytometry for sorting of normal bone marrow and peripheral blood populations. The following anti-human antibodies were used for sorting of normal bone marrow and peripheral blood populations: CD34 FITC (BD 555821), CD71 SPC (BD 334108), Gpa PerCP eFluor710 (eBioscience® 46-9987-42), CD10 BV421 (BD 562902), CD15 FITC (BD 555401), CD33 PE (BD 555450), CD34 APC (BD 555824), CD1 1 b PE-Cy5 (BD 555389), CD16 Pacific Blue (BD 558122), CD13 APC-Cy7 (BioLegend® 301710), CD3 FITC (BD 555332), CD14 APC- Cy7 (BD 560919), CD19 APC-Cy7 (BioLegend® 302218), CD19 PE-Cy7 (BD 557835). Cells were stained for 30 minutes at 4°C. Fc-blocking reagent (BD 564219) was used to minimize non-specific binding of antibodies.
[00202] Quantitative RT-PCR experiments. Total RNA was extracted from AML cells using TRIzol® reagent (Invitrogen®). Reverse transcription for cDNA generation using MMLV reverse transcriptase was performed according to the manufacturer's instructions (Invitrogen®). Quantitative PCR was performed in 384-well plates in ^ 0\^L· final volume using the TaqMan® Fast Advanced Master Mix of Applied Biosystems®. A mix of the forward and reverse oligonucleotides (sequences in Table 4) at 250 nM final each, 100nM of the UPL probe (Universal probe library from Roche) and the cDNA was added to 1 X of the TaqMan® Fast Mix. All reactions were done in duplicates. The amplification was processed with the following protocol: 3min at 95°C, followed by 40 cycles at 95°C 5sec and 60°C 30sec on a thermal cycler 7900HT (Applied Biosystems®). Data were analyzed using the SDS 2.4 and Data Assist software. Delta CTs were obtained from Ct gene - Ct reference using 4 control genes (GAPDH, ACTB, EIF4H, HNRNPL).
Table 4: Sequences of primers and probes used for quantitative RT-PCR
Figure imgf000052_0001
Example 1.1 : Mutational analysis of CBF leukemia
[00203] High frequency of activated signaling gene mutations defines CBF leukemia subclones. Characterization of mutations present in 415 primary AML specimens (48 CBF-AML, including 28 samples with inv(16) and 20 with t(8;21), and 367 control AML specimens) revealed that mutational landscapes of RUNX1-RUNX1T1 and CBFB-MYH11 AML are distinct (FIG. 1A). Genes mutated in t(8;21) and inv(16) cohorts are detailed in Table 5. The most frequent mutations in both CBF subgroups were found in activated signaling genes (FIG. 1A). In the t(8;21) cohort, 2/8 samples lacking typical mutations in KIT, FLT3 or N/KRAS harbored JAK2 or CSF3R mutations. Fifteen (31 %) CBF samples contained 2 to 5 mutations in activated signaling genes. In the majority of cases, the sum of VAF did not exceed ~50% (FIG. 1 B) hence suggesting that they occurred in different subclones. The loss of 2 signaling mutations and expansion of a 3rd one in a relapse specimen further support this concept (FIG. 1 C).
[00204] Mutational landscape of t(8;21) AML. Sixteen different genes were mutated in t(8;21) AML cohort: KIT (8/20, 40%), ASXL2 (5/20, 25%), FLT3, ASXL1 (4/20, 20% each), ZBTB7A, NRAS, TET2 and SMC1A (3/20, 15%), DNMT3A (2/20, 10%), KDM6A, KMT2C, SMC3, STAG2, WT1, JAK2 and CSF3R (1/20, 5% each) (FIG. 1A). The activated signaling genes (14/20, 70%) were the most frequently mutated, followed by chromatin modifier (10/20, 50%), cohesin (5/20, 25% each) and DNA methylation (4/20, 20%) genes (FIG. 1A). No association was found between mutations and additional cytogenetic aberrations and clinical or laboratory characteristics (FIG. 1A). An association was observed between t(8;21) and del(9q) or -Y.
[00205] ZBTB7A is recurrently mutated in t(8;21) AML. Three of 20 t(8;21) AML samples contained novel acquired mutations in ZBTB7A. Mutation in this gene occurred in only one other specimen, suggesting that it is specific to t(8;21) AML (3/20 vs. 1/395, p = 0.0004, Fisher's exact test, FIGs. 1 D and 1 H). Interestingly, ZBTB7A expression was the lowest in specimens with frameshift mutations suggesting that nonsense mediated decay is at play (FIG. 11).
[00206] Chromatin modifier, cohesin and DNA methylation genes are recurrently mutated in t(8;21) AML. ASXL2 mutations were also largely restricted to t(8;21) AML subgroup (5/20 vs. 3/395, p < 0.0001 , Fisher's exact test, FIG. 1 E). The 3 ASXL2 mutated samples in the non t(8;21) cohort harbored typical frameshift mutations, and they occurred in samples with intermediate or adverse risk cytogenetics. Among the mutations identified in cohesin complex genes, 3 SMC1A acquired mutations occurred at position R96 (FIG. 1 F). In addition, a germline SMC1A variant at position R71 1 was identified in another specimen. Interestingly, an acquired mutation at this same residue was previously observed in human colon cancer (R71 1 L, Muzny DM et al., Nature. 2012;487(7407)). Mutations in 2 genes encoding regulators of DNA methylation, TET2 and DNMT3A, were also detected in 4 samples with t(8;21) (FIG. 1A).
[00207] Low incidence of "non-activated signaling" mutations in inv(16) AML. A strong association between inv(16) AML and trisomy 22 was noted (FIG. 1A) (Grimwade D et al., Blood. 2010;1 16(3):354-365). Six genes were mutated in the inv(16) AML cohort: KIT and NRAS (12/28, 43% each), FLT3 (8/28, 29%), KRAS (2/28) and NF1 and BCORL1 (1/28 each). In addition, 2 germline variants of uncertain consequence were identified in GATA2 M386T and DNMT3A F731Y (FIG. 1A). Strikingly, it was found that the only recurrent mutations in this disease occurred in activated signaling genes, detected in 25/28 (89%) samples. This sharply contrasts with the high frequency of other non-activated signaling gene mutations found in t(8;21) AML (FIG. 1G).
Table 5: Mutations identified in the core-binding factor AML cohort. VAF: variant allele frequency, km: km method (https://bitbucket.org/iric-soft/km)
VAF VAF
Gene Sample Mutation Transcript (CASAVA) (km) Group Method
ASXL1 05H042 R417* NM. _015338 0.31 NA t(8;21 ) casava
ASXL1 06H035 p.G643fsX (11 G) NM. _015338 0.16 NA t(8;21 ) casava
ASXL1 08H042 p.G643fsX (11 G) NM. _015338 0.16 NA t(8;21 ) casava
ASXL1 09H040 p.G643fsX (11 G) NM. _015338 NA 0.32 t(8;21 ) kmer
ASXL2 05H184 p.T523fsX (2I CG) NM. _018263 0.47 NA t(8;21 ) casava
ASXL2 12H098 p.V587fsX (11 G) NM. _018263 0.45 NA t(8;21 ) casava
ASXL2 12H166 p.Q594fsX (1 l C) NM. _018263 0.43 NA t(8;21 ) casava
ASXL2 12H183 p.R614fsX (1 l C) NM. _018263 0.57 NA t(8;21 ) casava p.N590fsX (10l
ASXL2 08H034 CGCTTGACAG) NM. _018263 NA 0.64 t(8;21 ) kmer p.Q929fsX (1 D c, 61
BCORL1 05H099 TCCCCG) NM. _021946 0.45 NA inv(16) casava VAF VAF
Gene Sample Mutation Transcript (CASAVA) (km) Group Method p.N1380fsX (5I
BCORL1 05H099 ACAGC) NM_021946 0.17 NA inv(16) casava
CSF3R 03H065 p.S783fsX (11 G) NM_000760 0.38 NA t(8;21 ) casava
DNMT3A 11 H022 F731Y NM_022552 0.62 NA inv(16) casava
DNMT3A 03H083 Y735* NM_022552 0.26 NA t(8;21 ) casava
DNMT3A 10H119 W330* NM_022552 0.32 NA t(8;21 ) casava
FLT3 03H109 D835Y NM_004119 0.37 0.38 inv(16) casava
FLT3 05H113 D835H NM_004119 NA 0.06 inv(16) kmer
FLT3 06H020 D835V NM_004119 0.34 0.35 inv(16) casava
FLT3 07H144 D835Y NM_004119 NA 0.19 inv(16) kmer
FLT3 09H066 N676K NM_004119 0.34 NA inv(16) casava
FLT3 12H165 D835V NM_004119 NA 0.19 inv(16) kmer
FLT3 12H165 D835Y NM_004119 NA 0.12 inv(16) kmer
FLT3 03H083 D835Y NM_004119 0.44 0.45 t(8;21 ) casava
FLT3 08H034 D835Y NM_004119 NA 0.10 t(8;21 ) kmer
FLT3 12H180 D839G NM_004119 NA 0.06 t(8;21 ) kmer
FLT3JTD 03H112 p.Y597 (93I) NM_004119 NA 0.41 inv(16) kmer
FLT3JTD 11 H022 p.R595 (3I CTC) NM_004119 0.17 0.22 inv(16) kmer
FLT3JTD 08H072 p.Y597 (211) NM_004119 NA 0.45 t(8;21 ) kmer
GATA2 09H066 M386T NM_001145661 0.62 NA inv(16) casava
JAK2 12H045 V617F NM_004972 0.51 0.49 t(8;21 ) casava
KDM6A 05H184 p.S385fsX NM_001291415 0.84 NA t(8;21 ) casava
KIT 03H109 D816V NM_001093772 NA 0.09 inv(16) kmer
KIT 04H030 p.Y418 (3D ACG) NM_001093772 0.11 0.09 inv(16) casava
KIT 04H030 N822K NM_001093772 NA 0.08 inv(16) kmer p.Y418 (9I
acgacaggc,
KIT 04H061 9DCAACAGGCA) NM_00 093772 NA 0.47 inv(16) kmer
KIT 05H136 D816V NM_001093772 0.46 0.47 inv(16) casava
P.T417 (7D
KIT 07H099 cttacga, 4I A A AC) NM_001093772 0.25 0.48 inv(16) kmer p.D419 (6I
KIT 08H081 TTCTTT) NM_001093772 NA 0.15 inv(16) kmer p.L416 (10D
KIT 09H016 gacttacgac/CCCG) NM_001093772 NA 0.11 inv(16) kmer p.T417 (10D
acttacgaca, 4I
KIT 09H016 GTCC) NM_001093772 NA 0.06 inv(16) kmer p.Y418 (5D acgac
KIT 09H016 ,21 GG) NM_00 093772 NA 0.11 inv(16) kmer p.D419 (6I
KIT 11 H022 TTCTTC) NM_001093772 NA 0.07 inv(16) kmer
KIT 11 H022 N822K NM_001093772 NA 0.15 inv(16) kmer p.L.416 (8D
KIT 11 H179 gacttacg, 2I TT) NM_001093772 NA 0.22 inv(16) kmer p.T417 (7D cttacga,
KIT 12H042 11 T) NM_001093772 NA 0.56 inv(16) kmer
KIT 12H044 D816V NM_001093772 0.51 0.51 inv(16) casava p.L.416 (8D
KIT 12H165 gacttacg, 2I CT) NM_001093772 NA 0.17 inv(16) kmer
KIT 05H042 D816Y NM_001093772 0.36 0.36 t(8;21 ) casava VAF VAF
Gene Sample Mutation Transcript (CASAVA) (km) Group Method
KIT 08H034 N822K NM_001093772 0.35 0.36 t(8;21 ) casava
KIT 08H042 N822K NM_001093772 NA 0.17 t(8;21 ) kmer
KIT 08H042 N822K NM_001093772 NA 0.16 t(8;21 ) kmer p.T417 (7D
KIT 08H072 cttacga,4l GGGG) NM_001093772 NA 0.09 t(8;21 ) kmer
KIT 10H119 N822K NM_001093772 0.47 0.48 t(8;21 ) casava
KIT 11 H107 D816V NM_001093772 0.45 0.46 t(8;21 ) casava
KIT 12H098 N655K NM_001093772 0.85 NA t(8;21 ) casava
KIT 12H166 N822N NM_001093772 0.47 0.48 t(8;21 ) casava
KMT2C 11 H107 R55* NM_170606 0.37 NA t(8;21 ) casava
KRAS 09H066 G13D NM_004985 NA 0.12 inv(16) kmer
KRAS 13H120 Q61 L NM_004985 NA 0.21 inv(16) kmer
NF1 05H136 A1403V NM_001042492 0.48 NA inv(16) casava
NRAS 03H095 G13D NM_002524 0.48 0.47 inv(16) casava
NRAS 04H030 G12D NM_002524 NA 0.10 inv(16) kmer
NRAS 04H091 G13R NM_002524 0.44 0.44 inv(16) casava
NRAS 05H113 Q61 H NM_002524 0.48 0.39 inv(16) casava
NRAS 06H115 G13D NM_002524 NA 0.06 inv(16) kmer
NRAS 08H081 Q61 K NM_002524 0.45 0.36 inv(16) casava
NRAS 08H099 Q61 H NM_002524 0.39 0.33 inv(16) casava
NRAS 08H099 Q61 K NM_002524 NA 0.08 inv(16) kmer
NRAS 10H008 Q61 R NM_002524 0.49 0.38 inv(16) casava
NRAS 11 H022 Q61 K NM_002524 NA 0.09 inv(16) kmer
NRAS 11 H022 G12D NM_002524 NA 0.06 inv(16) kmer
NRAS 11 H104 G12S NM_002524 NA 0.05 inv(16) kmer
NRAS 11 H179 G12D NM_002524 NA 0.18 inv(16) kmer
NRAS 13H066 Q61 K NM_002524 0.52 0.39 inv(16) casava
NRAS 05H042 Q61 R NM_002524 0.25 0.20 t(8;21 ) casava
NRAS 06H035 Q61 K NM_002524 0.50 0.46 t(8;21 ) casava
NRAS 10H030 G12D NM_002524 NA 0.21 t(8;21 ) kmer
SMC1A 03H083 R96H NM_001281463 0.94 NA t(8;21 ) casava
SMC1A 05H042 R96H NM_001281463 0.95 NA t(8;21 ) casava
SMC1A 08H042 R711 Q NM_001281463 0.97 NA t(8;21 ) casava
SMC1A 10H030 R96H NM_001281463 0.87 NA t(8;21 ) casava
SMC3 13H169 p.E390 (3I CCC) NM_005445 0.32 NA t(8;21 ) casava
STAG2 11 H107 R305* NM_001042749 0.88 NA t(8;21 ) casava
TET2 03H065 S1050* NM_017628 0.53 NA t(8;21 ) casava
TET2 03H083 p.Q1501fsX (1 D A) NM_001127208 0.38 NA t(8;21 ) casava
TET2 07H137 Q635* NM_017628 0.94 NA t(8;21 ) casava
WT1 08H034 R250L NM_024426 0.50 NA t(8;21 ) casava
ZBTB7A 05H118 p.P174fsX (11 G) NM_015898 0.30 NA t(8;21 ) casava
ZBTB7A 05H184 p.P174fsX (11 G) NM_015898 0.84 NA t(8;21 ) casava
ZBTB7A 07H137 V122G NM 015898 0.41 NA t(8;21 ) casava
Example 1.2: Transcriptomic analysis of CBF-AML
[00208] Distinguishing inv(16) and t(8;21) subgroups by transcriptome analysis. Transcriptional analysis of 415 primary AML specimens revealed that t(8;21) and inv(16) AML transcriptomic signatures partially overlap but comprise several notable differences. Using the most minimally and most differentially expressed genes, signatures of 145 and 127 genes specific to t(8;21) and inv(16) groups were identified, respectively (FIGs.2A-B, Tables 6A-6B and 7A-7B).
Table 6A: Genes underexpressed genes in t(8:21) AML
Figure imgf000056_0001
ADARB2-AS1 4.49E-13 4.34 1.09 3.25
LINC00958 1.96E-11 4.80 1.57 3.23
RUNX1T1 2.21E-11 5.24 1.18 4.06 X X X X X
ITGB4 8.71E-11 4.87 3.31 1.56 X X X X
RASAL1 9.55E-11 4.32 1.92 2.39 X
POU4F1 9.65E-11 6.08 2.83 3.24 X X X X X
SIPA1L2 9.65E-11 5.19 3.87 1.32 X
PALM 1.11E-10 5.53 4.24 1.29 X X X
KLRG2 1.29E-10 4.21 2.59 1.63
CD19 1.37E-10 5.19 3.95 1.25 X
EVC2 1.44E-10 4.33 2.22 2.11 X
CACNA2D2 1.54E-10 4.76 3.37 1.39 X X X X
LOC285762 1.54E-10 4.06 1.92 2.13
IL5RA 1.65E-10 4.97 3.18 1.79 X X X X
ADRA2C 1.80E-10 4.91 2.66 2.24 X X
BAIAP3 2.07E-10 5.06 3.67 1.39 X X X
GYLTL1B 2.15E-10 4.15 2.93 1.22 X
RBFOX3 2.85E-10 4.38 3.36 1.02
EVC 4.46E-10 4.54 2.57 1.96
WIPF3 4.57E-10 4.06 2.07 1.99 X
PAX5 4.60E-10 4.61 3.51 1.09
MYRF 5.15E-10 4.95 3.69 1.26
LPAR5 6.57E-10 4.84 3.77 1.07
SLC05A1 8.71E-10 4.77 3.16 1.60 X
ROB01 9.33E-10 4.58 2.03 2.55 X X X X
TINAGL1 1.32E-09 4.37 2.58 1.79
CLEC2L 1.44E-09 4.08 1.66 2.42
EBI3 1.82E-09 4.26 3.22 1.04
BMP3 1.96E-09 4.17 1.71 2.46 TRH 1.99E-■09 6.09 3.97 2.12
NRP2 2.02E- ■09 4.17 2.86 1.30
PRAME 2.42E- ■09 5.75 3.38 2.37 X X X
CAV1 2.67E- ■09 4.86 3.42 1.44 X X X X
FAM166B 2.78E- ■09 4.37 3.24 1.13
PROSER2-AS1 3.44E- ■09 4.64 3.14 1.50
GPR153 3.49E- ■09 4.67 3.65 1.03
SPINK4 3.56E- ■09 4.50 3.44 1.06
CACNA1H 3.56E- ■09 4.16 2.75 1.40
C2or†66 3.73E- ■09 4.12 2.21 1.92
KCNH8 5.37E- ■09 4.21 3.12 1.10
WFDC1 5.78E- ■09 4.76 3.74 1.02 X X
ARC 5.82E- ■09 4.51 3.36 1.14
LINC00649 6.35E- ■09 4.72 3.54 1.18
MY018B 6.82E- ■09 4.38 1.96 2.42
TPSD1 8.49E- ■09 5.95 4.34 1.61
HPGDS 1.45E- ■08 5.65 4.03 1.62 X X X
CSRP2 1.45E- ■08 4.59 3.28 1.30 X
PNMT 1.48E- ■08 4.56 3.12 1.44 X
TPSB2 1.82E- ■08 5.88 4.47 1.41 X
CPNE7 1.86E- -08 4.96 3.87 1.09
LINC00189 2.63E- -08 4.13 2.17 1.96
MMP28 2.64E- -08 4.54 3.33 1.21
IRX1 2.75E- -08 4.41 1.75 2.66
FLT4 2.79E- -08 4.55 3.19 1.36
EVPL 2.85E- -08 4.87 3.62 1.24
COL14A1 3.84E- -08 4.26 3.11 1.15
TPSAB1 4.20E- ■08 5.98 4.66 1.33
C1QTNF4 4.46E- -08 5.61 4.54 1.07
DOCK6 4.89E- ■08 4.51 3.44 1.07
TKTL1 7.23E- -08 4.27 3.10 1.17
LPO 7.83E- -08 5.20 4.06 1.14
FBLN5 1.12E- ■07 4.67 3.34 1.33 X X
MAPK15 1.39E- ■07 4.01 2.81 1.20
CD34 1.62E- ■07 6.41 5.10 1.31 X X
DOC2B 1.64E- ■07 4.56 3.02 1.54
CDH4 1.81 E- ■07 4.10 3.01 1.09
SHANK1 2.26E- ■07 4.67 3.60 1.06
RARRES2 4.23E- ■07 4.23 3.21 1.02
TPPP3 4.23E- ■07 5.64 4.58 1.06
PTPRH 5.06E- ■07 4.00 2.99 1.01
PPP1R14A 7.58E- ■07 4.49 3.21 1.27
BCL6B 9.34E- ■07 4.15 3.02 1.13
MSLN 1.03E- -06 4.82 2.91 1.90
FBLN2 1.07E- -06 4.37 3.27 1.10
ST18 1.10E- -06 4.09 2.62 1.46
PTX4 1.11 E- -06 4.15 2.80 1.35 THSD7A 1 .16E-06 4.08 2.47 1 .61 X
BAALC 2.04E-06 5.73 4.54 1 .19 X X
TSPAN7 2.15E-06 4.94 3.72 1 .22 X
CYP4F2 2.68E-06 4.27 3.17 1.10
MS4A2 7.08E-06 4.57 3.41 1 .16 X
TSKS 7.12E-06 4.30 3.08 1.22
AG XT 8.83E-06 4.01 1 .77 2.24 X
BAALCOS 1.29E-05 4.05 2.70 1.35
CLEC5A 1 .31 E-05 5.31 4.30 1 .01 X X
LHX6 1.61 E-05 4.02 2.92 1.10
AADAT 1.79E-05 4.21 3.09 1.12
CA4 1.87E-05 4.17 2.91 1.26
UGT2B11 2.01 E-05 4.55 3.18 1.38
LOC200772 2.10E-05 4.67 3.32 1.34
IL1RL1 2.73E-05 4.61 3.60 1.02
SNORD116-21 4.14E-05 4.14 2.97 1.17
PGLYRP1 6.29E-05 5.68 4.50 1.18
LOC284865 8.49E-05 4.05 2.62 1.43
KCNE1L 0.000200412 4.66 3.49 1.18
LOXHD1 0.0002099 4.12 3.10 1.02
EPCAM 0.0003721 16 4.52 3.44 1 .08 X X
DNTT 0.006162861 4.68 3.62 1.06
Ross ME, Rami M, Mihaela O, et al. Gene expression profiling of pediatric acute myelogenous leukemia. Blood. 2004; 104(12):3679-3687.
2 Valk PJ, Roel GV, Beijen MA, et al. Prognostically useful gene-expression profiles in acute myeloid leukemia. The New England Journal of Medicine. 2004;350(16): 1617-1628.
3 Yagi T, Morimoto A, Eguchi M, et al. Identification of a gene expression signature associated with pediatric AML prognosis. Blood. 2003; 102(5): 1849-1856.
Hsu C-HH, Nguyen C, Yan C, et al. Transcriptome Profiling of Pediatric Core Binding Factor AML. PloS one. 2015; 10(9).
Table 6B: Genes underexpressed genes in t(8:21 ) AML
Underexpressed genes
Figure imgf000059_0001
HOXB3 9.26E-■08 2.82 4.57 - 1 .75
HOXA5 1.12E- -07 2.46 4.67 -2.21
HO
KR
NG
GFI
AS
HO
AL
PP
RH
ZNF
KR
PF4
GP
LG
Figure imgf000060_0001
MYH11 3.66E- ■15 4.78 3.63 1 .15 X X
ST18 8.04E- ■15 5.1 1 2.52 2.60 X X
MEGF10 1.45E- -14 4.27 2.68 1.59
LOC200772 1.59E-■13 5.28 3.25 2.03
SPARC 1.77E- ■13 6.05 4.93 1.12 X
TREM2 2.05E ■13 4.59 2.91 1.68
COLEC12 2.42E- ■13 4.26 2.80 1.45 X
MYBPH 2.62E ■13 4.55 2.56 1.99
TRIM71 3.77E- ■13 4.83 3.60 1.23
MMP14 4.73E- ■13 5.46 4.22 1.25
CYP2S1 4.73E- ■13 5.01 3.77 1.24
ARL5C 5.13E ■13 4.30 3.27 1.03
NT5E 7.71 E- ■13 4.88 3.39 1.49 X
GPR12 9.27E- ■13 4.38 2.50 1.88
MSLN 9.42E- ■13 5.67 2.81 2.86 X
SHANK1 1.23E ■12 4.82 3.57 1.25
LCN6 1.33E ■12 5.12 2.61 2.51
A AD AT 1.50E- ■12 4.51 3.04 1.47
MN1 1.62E- ■12 5.28 4.03 1.25 X
FLT4 1.66E- ■12 4.71 3.15 1.56
BGLAP 2.37E ■12 4.87 3.76 1.11
PMP22 4.44E- ■12 4.82 3.55 1.28
TGFBI 4.51 E- ■12 5.47 4.17 1.30 X
BAALCOS 7.25E- ■12 4.36 2.65 1.72
LCN10 9.01 E- ■12 4.69 3.25 1.44
RHBDL3 9.91 E- ■12 4.07 2.86 1.20
CYGB 1.02E- ■11 4.38 2.97 1.41
VSIG4 1.15E- ■1 1 5.46 4.20 1.26 X
SLC4A3 1.30E- ■11 4.16 3.03 1.13
PDE2A 1.41 E- ■11 4.23 3.20 1.03
CLEC14A 2.00E- ■11 4.53 2.97 1.56
LINC00865 2.27E ■11 4.66 3.29 1.37
MTMR11 2.27E- ■1 1 4.99 3.76 1.23 X
EPHB2 2.57E- ■11 4.12 2.82 1.30
DUSP27 2.60E ■11 4.31 2.63 1.68
FCER2 3.23E ■11 5.24 4.19 1.05
IRX1 4.00E- ■11 4.28 1.70 2.58
EXOC3L2 5.02E ■11 4.28 3.20 1.07
ARC 5.30E ■11 4.39 3.35 1.05
KIF26B 5.91 E- ■11 4.59 3.51 1.08
KIF17 8.28E- ■1 1 4.34 3.04 1.30
RARRES2 9.02E ■11 4.47 3.18 1.29
FAM171A1 1.08E- ■10 4.86 3.15 1.71 X
ICAM5 1.94E- -10 4.81 3.78 1.03
ALOX15 2.96E -10 4.11 2.78 1.33
TPPP3 3.25E- ■10 5.69 4.56 1.13 X
PACSIN1 3.38E -10 4.15 2.98 1.18
MMP28 3.77E- -10 4.46 3.31 1.15
KRT17 4.11 E- -10 5.04 3.63 1.41
LRP6 9.55E -10 4.47 3.22 1.25 RBP7 9.55E -10 4.64 3.57 1.07
AGR2 1.19E -09 4.23 2.37 1.86
RXFP1 1.35E -09 4.62 3.30 1.32
PTRF 1.53E -09 5.18 4.02 1.16
TM4SF1 3.69E -09 5.20 3.92 1.28
MARCO 3.82E -09 4.84 3.63 1.21
S100A16 5.35E -09 4.84 3.16 1.68
SHD 6.81 E -09 4.39 2.83 1.56
CD14 7.54E -09 6.02 4.92 1.10
PTPRM 9.23E- ■09 4.71 3.58 1 .13 x
NTRK1 9.53E -09 4.79 3.76 1.02
SLC27A6 1.02E -08 4.13 2.83 1.30
MRC1 1.26E -08 4.57 3.44 1.14
NPTX2 1.42E -08 4.95 3.44 1.52
PRG3 1.47E -08 5.26 3.27 1.99
LGALS2 1.59E- ■08 5.66 4.50 1 .15
PRTFDC1 1.62E -08 4.24 3.22 1.02
SIGLEC1 1.87E -08 4.33 3.25 1.08
EPX 2.72E -08 5.75 4.38 1.37
C5orf20 2.89E -08 4.57 3.34 1.22
PLBD1 2.99E- ■08 5.91 4.82 1 .09
UPK3A 3.17E -08 4.11 2.71 1.40
PPP1R14A 5.24E -08 4.33 3.20 1.13
TIFAB 7.72E -08 4.56 3.29 1.26
LOC284865 8.03E -08 4.13 2.59 1.54
TPSAB1 9.43E -08 5.67 4.65 1.02
CCL24 1.83E -07 4.02 3.01 1.01
PCDHGC3 4.53E -07 4.12 2.95 1.17
TRH 4.94E -07 5.28 3.99 1.30
ARHGEF10L 5.79E -07 4.67 3.62 1.05
BAALC 1.25E -06 5.57 4.52 1.05
GTSF1 1.37E -06 5.68 4.56 1.12
HPGDS 2.14E -06 5.11 4.04 1.07
TARM1 8.30E -06 4.16 3.09 1.08
RAMP1 3.40E -05 4.83 3.81 1.01
MIR6503 0.0001 05 4.01 2.94 1.07
UGT2B11 0.0002 4.18 3.17 1.01
PRSS21 0.001369 5.26 4.07 1.20
Table 7B: Genes underexpressed genes in inv(16) AML
Underexpressed genes
Figure imgf000063_0001
2.69E ■14 2.99 4.01 -1.02
DNMT3B 6.96E- ■14 3.73 4.74 -1.01
LOC285758 1.31 E- ■13 1.84 4.27 -2.43
NR6A1 2.68E- ■13 3.16 4.23 -1.07
MIR4740 1.20E- ■12 0.38 4.10 -3.72
SLC40A1 1.66E- ■12 3.86 5.13 -1.27
CD59 4.53E- ■12 3.68 4.73 -1.05 X
MIR3186 2.40E- ■11 3.19 4.21 -1.02
CPNE8 3.21 E ■11 2.91 4.58 -1.67
MARCKS 2.23E ■10 3.88 4.93 -1.05
HOXA6 2.45E ■10 1.57 4.21 -2.65
HOXA3 6.55E ■10 2.27 4.10 -1.83
HTRA3 1.14E- ■09 3.34 4.45 -1.11
HOXA5 1.23E ■09 2.66 4.70 -2.05
SLC22A15 1.98E- ■09 3.40 4.42 -1.02
NXF3 2.44E- ■09 3.02 4.14 -1.12
HOXA7 5.46E- ■09 1.93 4.15 -2.22
H2AFY2 6.91 E- ■09 2.89 4.15 -1.27
ALDH1A1 9.37E- ■09 2.88 4.12 -1.24
HOXA9 9.44E- ■09 3.37 5.00 -1.63
MDFI 1.36E -08 2.70 4.08 -1.38
PPBP 2.03E -08 3.24 4.44 -1.20
BEX1 2.09E -08 3.32 4.68 -1.36
PF4 7.01 E- -08 2.87 4.09 -1.22
HOXA10 9.03E -08 3.35 4.50 -1.15
NGFRAP1 1.02E- -07 3.78 4.78 -1.01
[00209] Fusion partner genes, RUNX1T1 and MYH11, remain among the single most specifically and differentially expressed genes in t(8;21) and inv(16) groups respectively. Previously reported candidates such as POU4F1 (t(8;21)) and ST18 (inv(16)) were among the most discriminatory genes identified by the analysis. Other CBF microarray datasets were readily enriched in GSEA studies (Tables 6 and 7). Importantly, about 80% of the genes identified in the CBF AML signatures have not been previously described in those datasets (Tables 6 and 7). For example, ADARB2-AS1 and LINC00958 were typical for t(8;21) AML and MEGF10 and APLN for inv(16) specimens. The signatures shared 50% and 25% of the most significantly overexpressed or underexpressed genes with pediatric t(8;21) and inv(16) AML cohorts, respectively (Tables 6 and 7) (Hsu et a/., PloS one. 2015; 10(9)), suggesting that similar networks are at play in pediatric and adult CBF AML.
[00210] Overlap in gene expression pattern between CBF AML subgroups. Although certain genes are specifically expressed in either inv(16) or t(8;21) AML, a defined expression signature also distinguishes CBF AMLs from other AML subgroups (FIG. 2C and Tables 8A and 8B). CBF AML express high levels of the IRX1 homeobox gene which is also found in t(15; 17) (FIG. 2D). Other genes preferentially expressed in both CBF subgroups include FLT4, MSLN, ST18, LCN6, TRH, CD34 and LOC200772 (FIG. 2C). As previously reported (Hsu et a/., supra), HOXA, HOXB and MEIS1 genes are expressed at low levels in CBF AMLs (FIG. 2C). Unexpectedly, it was found that these genes are expressed at lower levels in t(8;21) AML when compared to inv(16) AML samples (FIG. 2E-G). Table 8C and FIG. 2H show the list of most differentially expressed genes between t(8;21) and inv(16) AML.
Table 8A: Gene overexpressed in CBF AML vs. other AML subgroups
Figure imgf000064_0001
/AD/ARS2-/ASi 3.94E-28 3.92 0.90 3.02 x
ST18 2.45E-22 4.69 2.43 2.25 x
PPL 1 .69E-21 3.91 2.70 1 .21 x
FLT4 1 .69E-21 4.64 3.07 1 .57 x
ARC 6.94E-21 4.44 3.28 1.16
SHANK1 7.21 E-21 4.76 3.51 1 .24 x
CACNA1H 1.03E-20 4.04 2.66 1.38
IRX1 2.59E-20 4.33 1 .56 2.78 x
MSLN 3.14E-20 5.31 2.70 2.61 x ITGB4 4.07E-■20 4.28 3.27 1.01 X
LOC200772 2.24E- ■19 5.02 3.17 1.85
NT5E 2.60E- ■19 4.63 3.34 1.29 X
MMP28 3.02E- ■19 4.50 3.24 1.25 X
PRCD 3.39E- ■19 3.60 2.39 1.20
BAIAP3 3.94E- ■19 4.70 3.61 1.09 X
LOC101929374 8.15E- ■19 3.79 2.53 1.26
PTH1R 1.03E- ■18 3.74 2.33 1.41
RARRES2 1.44E- ■18 4.37 3.12 1.25 X
AADAT 1.93E- ■18 4.38 2.98 1.41 X
TPPP3 5.84E- ■18 5.67 4.50 1.17 X
BAALCOS 5.88E- ■18 4.23 2.57 1.66
KLRG2 4.44E- ■17 3.71 2.53 1.18
TPSG1 5.11 E- ■17 3.50 1.50 2.01
TRH 8.64E- ■17 5.62 3.87 1.75 X
PROSER2-AS1 1.58E- ■16 4.26 3.07 1.19
CYGB 2.59E- ■16 4.15 2.92 1.23
TPSAB1 2.82E- ■16 5.80 4.58 1.22 X
TRIM71 3.76E- ■16 4.63 3.56 1.07 X
LCN6 6.03E- ■16 4.62 2.54 2.08
PTPRH 6.75E- ■16 3.94 2.93 1.02 X
LRP4 1.22E- ■15 3.98 2.91 1.06 X
LPO 1.35E- ■15 5.05 4.00 1.06
GPR12 1.97E- ■15 4.08 2.44 1.64 X
PPP1R14A 2.96E- ■15 4.40 3.13 1.27
HPGDS 3.91 E- ■15 5.34 3.95 1.39 X
EVPL 4.81 E- ■15 4.68 3.55 1.12
NTRK1 6.23E- ■15 4.77 3.71 1.06
FBLN2 8.78E- ■15 4.27 3.20 1.07
DUSP27 1.84E- ■14 4.06 2.57 1.49 X
TINAGL1 6.46E- ■14 3.85 2.52 1.33 X
LINC00865 1.17E- ■13 4.37 3.26 1.12
BCL6B 2.62E- ■13 4.01 2.95 1.06
BAALC 3.18E- ■13 5.64 4.46 1.18 X
TPSB2 3.36E- ■13 5.58 4.40 1.18 X
CLEC5A 4.93E- ■13 5.25 4.23 1.03 X
KIRREL 6.04E- ■13 3.93 2.55 1.38 X
LRP6 9.77E- ■13 4.27 3.18 1.09 X
LOC284865 1.01 E- 12 4.10 2.51 1.59
AG XT 3.72E- 12 3.90 1.61 2.29
TPSD1 4.59E- 12 5.54 4.27 1.27
CD34 8.97E- ■12 6.23 5.02 1.20 X
PNMT 9.08E- 12 4.09 3.07 1.02
KIF17 9.37E- 12 4.04 3.01 1.03
MS4A2 1.12E- ■11 4.44 3.33 1.10
SLC05A1 2.17E- ■11 4.17 3.12 1.04
LCN10 2.78E- ■11 4.24 3.23 1.02 TSPAN7 2.81 E-■11 4.71 3.66 1.04
LINC00676 6.48E- ■11 3.66 2.51 1.15
APLN 7.61 E- ■11 3.92 2.79 1.13
KRT17 1.18E- ■10 4.71 3.59 1.11
IL5RA 2.45E- ■10 4.15 3.15 1 .00 x
DOC2B 3.83E- ■10 4.13 2.96 1.17
LOXHD1 5.89E- ■10 4.09 3.03 1.06
UGT2B11 1.26E- ■09 4.33 3.10 1.24
THSD7A 1.26E- ■09 3.67 2.40 1.27
GTSF1 2.20E- ■09 5.60 4.50 1.10
EPX 4.91 E- ■09 5.39 4.35 1.04
FAM171A1 8.02E- ■09 4.23 3.14 1.09
PRR15 1.00E- -08 3.69 2.65 1.04
TARM1 1.92E- -08 4.10 3.04 1.06
CA4 2.52E- ■07 3.87 2.85 1.01
S100A16 7.47E- ■07 4.31 3.14 1.17
PRAME 1 .66E- ■06 4.55 3.35 1 .20 x
PRSS21 8.53E- -06 5.18 4.02 1.16
PRG3 1.74E- ■05 4.48 3.26 1.22
Table 8B: Gene overexpressed in CBF AML vs. other AML subgroups
Underexpressed genes
CO
O
o
o
o 0) o LU
+ X. 3
(A D X LU
0) I 3 CL _l 3
I
TO o + > 9 0-)
TO
tn 3 o o 0) o _l
Q. σ> LU
Q 0) 0) >- σ> 2 c
Έ
>
2 2 TO I
LU o > > I— 3
< D
0) oo υ O Q.
m
o 5 O
w
o
GENE Q
PIEZ02 1.69E-21 1.11 3.53 -2.42
CPNE8 3.38E-21 2.81 4.69 -1 .88 X
HOXA6 4.07E-20 1.23 4.40 -3.17 X
SLC40A1 7.12E-20 4.02 5.18 -1 .16 X
HOXA-AS3 8.73E-20 0.83 3.72 -2.90 X
HOXA9 2.22E-19 2.52 5.20 -2.68 X
HOXA3 4.41 E-19 2.13 4.22 -2.09 X
COL4A5 5.46E-19 0.95 3.79 -2.84
HOXA7 2.54E-18 1.50 4.32 -2.82 X CYP7B1 4.25E-■18 1.44 3.61 -2.17 X
HOXA5 5.26E- ■18 2.57 4.82 -2.25 X
HOXA10 1.77E- ■17 2.90 4.62 -1.71 X
SYTL4 4.90E- ■17 2.17 3.76 -1.59
HOXA10-AS 8.62E ■17 1.50 4.04 -2.54
MIR4740 2.88E- ■16 1.46 4.16 -2.70 X
HOXA2 3.33E- ■16 2.61 3.63 -1.03 X
SDPR 1.05E- ■15 3.20 4.37 -1.17 X
NGFRAP1 1.23E ■15 3.67 4.85 -1.18
CLGN 3.33E ■15 1.99 3.57 -1.58
ITGB3 3.33E ■15 2.67 3.78 -1.11
SPATA6 3.71 E- ■15 2.76 3.85 -1.09
ALDH1A1 4.88E ■15 2.85 4.20 -1.35
OCLN 9.52E- ■15 2.42 3.57 -1.15 X
HTRA3 1.20E- ■14 3.44 4.50 -1.06
PPBP 1.52E ■14 3.22 4.51 -1.29
WBP5 1.65E- ■14 3.13 4.60 -1.47
HOXA4 1.67E- ■14 2.27 4.01 -1.74 X
SEPP1 1.69E- ■13 3.65 5.02 -1.37
PF4 2.36E ■13 2.81 4.16 -1.35
UGGT2 3.32E- ■13 2.16 3.60 -1.44 X
NEGRI 5.80E ■13 3.07 4.28 -1.20
ZNF334 6.53E ■13 2.48 3.74 -1.25
CXCL10 1.17E- ■12 2.56 3.66 -1.10
CASC10 3.55E ■12 2.66 3.87 -1.21
APOL4 4.27E- ■12 2.42 3.55 -1.13
CGREF1 4.56E- ■12 2.63 3.64 -1.01
SLITRK4 4.70E- ■12 2.38 3.95 -1.57
SLC16A9 5.93E ■12 2.46 3.51 -1.05
MEIS1 6.57E- ■12 3.45 4.83 -1.38
OLFML2B 1.38E- ■1 1 2.69 3.69 -1.01 X
HOXB-AS3 7.61 E- ■1 1 1.12 3.51 -2.39 X
SH3BP4 1.30E- -08 2.50 3.61 -1.10
HOXB5 8.47E- ■08 2.04 3.58 -1.54 X
LOC285758 1.25E -07 2.89 4.27 -1.37
APOC1 2.43E ■07 2.85 4.04 -1.18
SPINK2 3.69E -07 4.10 5.22 -1.13
HOXB6 7.71 E- ■07 2.44 3.72 -1.28 X
CRISP3 8.21 E -07 2.68 3.71 -1.03
SCHIP1 2.50E -06 2.33 3.64 -1.31
DOCK1 3.04E- -06 2.78 3.93 -1.16
C3orf80 1.59E- -05 3.30 4.45 -1.15
Table 8C: Genes differentially expressed in t(8:21) vs. inv(16) AML
FDR q-
Gene FDR q-Value t(8;21) inv(16) Gene t(8;21) inv(16)
Value
RUNX1T1 1.66E-07 5.24 0.84 ERVFRD-1 4.70E-09 4.06 2.81 LINC00958 1.35E-07 4.80 1.08 IGSF10 6.57E-08 4.96 3.71
POU4F1 3.85E-1 1 6.08 2.38 SCRN1 3.51 E-07 4.94 3.70
BMP3 9.74E-08 4.17 0.55 PTPRN2 3.85E-1 1 4.30 3.05
MY018B 3.02E-07 4.38 1.43 GATM 1.24E-05 4.70 3.46
ROB01 2.73E-07 4.58 1.72 TSKS 2.48E-06 4.30 3.07
LOC285758 2.42E-06 4.36 1.84 PALM 3.85E-1 1 5.53 4.30
ADRA2C 1.84E-07 4.91 2.41 CAV1 3.28E-06 4.86 3.65
EVC 3.85E-1 1 4.54 2.10 EIF5A2 1.47E-10 4.14 2.95
RASAL1 2.22E-07 4.32 1.90 TRIM32 1.58E-08 4.05 2.86
EVC2 3.85E-1 1 4.33 2.04 CD19 3.85E-1 1 5.19 4.04
C2orf66 1.46E-06 4.12 1.92 LINC00189 0.000455 4.13 2.98
PRAME 4.14E-07 5.75 3.69 COL23A1 5.43E-08 4.66 3.51
LOC285762 6.18E-1 1 4.06 2.16 PCNXL2 2.80E-09 4.48 3.35
DOCK6 3.85E-1 1 4.51 2.62 MLLT4 3.85E-1 1 4.47 3.34
LHX6 1.84E-07 4.02 2.13 NOV 2.80E-09 4.52 3.40
CLEC2L 1.01 E-05 4.08 2.30 HHIP-AS1 0.002103 4.15 3.03
VLDLR 3.60E-09 4.88 3.14 BCL9 3.85E-1 1 4.84 3.73
BEX1 0.000306 5.03 3.32 NR6A1 3.85E-1 1 4.27 3.16
WIPF3 1.25E-09 4.06 2.35 SLPI 0.0018 5.42 4.32
PXDN 0.001044 4.32 2.65 GPR153 3.85E-1 1 4.67 3.58
CSRP2 6.42E-10 4.59 3.04 RAB3IP 3.85E-1 1 4.08 2.99
SIPA1L2 3.85E-1 1 5.19 3.64 PLEKHA8 2.15E-09 4.23 3.17
EPCAM 2.02E-05 4.52 3.02 SLC5A10 9.01 E-10 4.52 3.46
CPNE7 6.42E-10 4.96 3.47 JMY 4.61 E-10 4.60 3.54
NCAM1 2.48E-06 4.38 2.91 ACP6 2.80E-09 4.78 3.73
NXF3 6.57E-08 4.49 3.02 FAM134B 4.91 E-07 4.16 3.12
PODXL2 3.85E-1 1 5.10 3.66 SLC05A1 2.39E-08 4.77 3.74
IL5RA 4.70E-09 4.97 3.57 PAX5 1.00E-10 4.61 3.58
DSC2 1.63E-06 4.11 2.72 FAM84B 5.43E-08 4.40 3.38
CDH4 1.58E-08 4.10 2.72 ENPP4 6.18E-1 1 4.91 3.89
TKTL1 1.58E-08 4.27 2.93 TANC2 9.01 E-10 4.43 3.42
COL14A1 4.43E-08 4.26 2.94 LDLRAD3 4.91 E-07 4.41 3.40
FAM213A 2.15E-10 4.18 2.86 ITGB4 1.65E-09 4.87 3.86
CACNA2D2 1.47E-10 4.76 3.46 KCNH8 1.97E-08 4.21 3.21
FBLN5 4.95E-06 4.67 3.39 GPM6B 6.18E-1 1 4.46 3.46
FGD2 7.66E-09 4.23 5.23 KLF4 1.65E-09 4.94 6.1 1
OBSL1 6.08E-09 3.68 4.69 PPM1J 3.85E-1 1 2.95 4.13
IRF8 1.25E-09 4.74 5.75 HOPX 2.80E-09 3.93 5.12
COBLL1 6.42E-10 3.33 4.34 NLRP12 4.91 E-07 3.38 4.57
STAB1 9.01 E-10 4.47 5.48 BIK 1.00E-10 3.72 4.91
CHST15 2.39E-08 3.73 4.74 LCN6 1.22E-06 3.92 5.12
MGAT3 4.70E-09 3.10 4.12 CD1D 6.08E-09 3.70 4.89
ACE 2.15E-09 3.34 4.36 LINC00877 1.00E-10 2.99 4.19
ANXA5 1.65E-09 4.99 6.01 SLC27A6 2.39E-08 2.93 4.13
SECTM1 1.36E-07 3.69 4.72 TLR7 1.47E-10 3.16 4.36
ST18 2.97E-08 4.09 5.1 1 SERPINE1 3.60E-09 3.77 4.98 NOD2 1.00E-10 3.73 4.76 CD14 1.36E-07 4.81 6.02
ZC3H12C 5.74E-05 3.13 4.16 MTMR11 1.47E-10 3.77 4.99
FAM129B 1.00E-10 4.01 5.05 LRP11 2.39E-08 2.98 4.20
CD300LB 2.80E-09 3.67 4.72 MARCO 2.18E-07 3.61 4.84
PXDC1 1.61 E-07 3.08 4.14 LILRB4 2.39E-08 3.69 4.93
C15orf38 1.47E-10 3.14 4.21 SEPP1 0.000393 2.93 4.17
MIR6503 0.000914 2.95 4.01 PLXDC2 1.25E-09 3.53 4.77
SNAI1 1.47E-10 3.75 4.82 PACSIN1 6.42E-10 2.90 4.15
LCN10 2.84E-06 3.62 4.69 RHBDF1 1.00E-10 3.31 4.56
VCAN 6.67E-07 4.63 5.70 CCR2 7.66E-09 3.64 4.91
ITGB5 6.08E-09 3.32 4.40 MAFB 6.67E-07 4.09 5.36
CD86 3.60E-09 4.12 5.21 TMEM150B 4.61 E-10 3.26 4.52
KIF26B 1.47E-10 3.51 4.59 SPP1 1.27E-08 2.90 4.17
GPR183 4.70E-09 5.02 6.10 S100A16 3.02E-07 3.57 4.84
SCIMP 3.60E-09 3.72 4.81 FCGR2B 1.65E-09 3.50 4.77
SLC7A7 1.65E-09 4.46 5.55 MGP 1.36E-07 2.85 4.12
GFI1B 1.04E-06 3.48 4.58 ALDH2 1.47E-10 4.05 5.33
VOPP1 3.85E-1 1 3.72 4.82 AGPAT9 3.85E-1 1 3.63 4.93
GCSAML 2.18E-07 3.63 4.73 OLFML3 5.27E-06 2.75 4.05
CD 163 4.10E-05 4.09 5.20 CECR6 3.85E-1 1 3.09 4.40
RBP7 2.84E-06 3.51 4.64 PCCA 3.60E-09 3.40 4.71
CUEDC1 3.85E-1 1 3.39 4.53 ARHGAP21 3.85E-1 1 3.62 4.94
CHI3L1 3.66E-05 4.11 5.26 MRC1 2.80E-09 3.25 4.57
LRP1 2.39E-08 4.16 5.32 EXOC3L2 3.85E-1 1 2.95 4.28
CLEC3B 3.51 E-07 3.23 4.39 RNASE1 1.41 E-06 3.38 4.71
OLIG1 3.85E-1 1 3.50 4.83 APLN 9.01 E-10 2.94 4.62
EFHC2 2.15E-10 3.29 4.64 MYCL 6.18E-1 1 2.39 4.08
KCTD12 3.85E-1 1 4.13 5.49 NRP1 3.85E-1 1 2.91 4.64
FZD2 3.51 E-07 2.69 4.05 ARHGEF10L 1.47E-10 2.95 4.67
GPR27 0.000636 3.07 4.43 TGFBI 1.00E-10 3.71 5.47
ADRB1 6.18E-1 1 2.98 4.35 EMILIN1 3.85E-1 1 3.51 5.28
FCGR2C 1.00E-10 3.26 4.64 RFX8 6.18E-1 1 3.15 4.94
ST8SIA6 2.56E-07 3.03 4.42 PC AT 18 1.47E-10 2.88 4.68
KIAA0125 3.85E-1 1 3.43 4.81 AK5 3.85E-1 1 3.31 5.13
CD300E 2.18E-07 4.03 5.41 SLC4A3 3.85E-1 1 2.34 4.16
BGLAP 3.85E-1 1 3.46 4.87 PTGFRN 3.85E-1 1 2.45 4.27
HSPA7 2.15E-10 3.37 4.78 LPAR1 3.85E-1 1 2.51 4.37
ARHGEF11 3.85E-1 1 3.40 4.83 PRG3 9.87E-05 3.40 5.26
TICAM2 5.66E-07 2.61 4.04 CLEC14A 1.64E-06 2.66 4.53
UPK3A 2.15E-10 2.67 4.1 1 HOXB2 3.85E-1 1 3.28 5.16
DPYSL3 7.66E-09 2.99 4.44 HOXB3 3.85E-1 1 2.82 4.70
TANC1 3.14E-10 2.84 4.30 MSR1 6.16E-07 2.18 4.06
ME1 3.85E-1 1 2.68 4.17 HOXB4 3.85E-1 1 2.71 4.64
NAPSB 3.85E-1 1 4.01 5.52 SULF2 3.85E-1 1 3.73 5.69
PMP22 3.85E-1 1 3.32 4.82 RHBDL3 3.85E-1 1 2.07 4.07
FAM171A1 2.15E-10 3.35 4.86 MEGF10 3.85E-1 1 2.26 4.27 KRT8 1 .65E-09 3.10 4.61 TREM2 2.03E-07 2.57 4.59
MPEG1 1 .00E-10 4.52 6.06 LGALS2 3.85E-1 1 3.59 5.66
LTC4S 3.85E-1 1 3.63 5.19 NPTX2 1 .61 E-07 2.84 4.95
PTRF 3.85E-1 1 3.62 5.18 CES1 1 .00E-10 2.65 4.75
DEFB1 0.002565 2.48 4.05 LOC283683 1 .84E-07 1 .93 4.04
PTPRM 1 .97E-08 3.13 4.71 HOXB-AS1 1 .84E-07 2.63 4.75
CLIP3 6.18E-1 1 3.73 5.33 CD1C 3.85E-1 1 3.01 5.13
KIAA1598 1 .00E-10 2.44 4.05 CPVL 3.85E-1 1 3.35 5.53
PLBD1 9.01 E-10 4.29 5.91 ASGR2 1 .84E-07 2.74 4.92
KRT18 1 .25E-09 3.49 5.12 PRTFDC1 3.85E-1 1 2.02 4.24
EPHB2 1 .00E-10 2.48 4.12 AGR2 2.43E-07 1 .84 4.23
TDRD9 8.28E-07 2.66 4.31 COLEC12 2.03E-07 1 .82 4.26
TNFRSF11A 3.85E-1 1 2.65 4.31 SHD 1 .84E-07 1 .95 4.39
SIGLEC1 9.01 E-10 2.66 4.33 SPINK2 3.85E-1 1 2.67 5.12
MMP14 3.85E-1 1 3.78 5.46 CLEC10A 3.85E-1 1 3.01 5.59
C3orf80 1 .84E-07 1 .67 4.46 RXFP1 1 .84E-07 1 .63 4.62
MYBPH 1 .83E-07 1 .72 4.55 KCNK17 1 .84E-07 2.06 5.10
ENHO 2.18E-07 1 .77 4.66 TM4SF1 1 .84E-07 2.01 5.20
MEIS1 1 .84E-07 1 .75 4.67 CD1E 1 .61 E-07 0.96 4.42
[00211] Transcriptomes of RUNX1-RUNX1T1 and RUNX1-CBFA2T3 AML are similar. Using the subgroup specific gene signatures from Tables 6 and 7 and performing principal component analyses (PCA), each CBF subgroups homogeneously clustered together (FIGs. 3A-B). One sample harboring a t(16;21)(q24;q22);RL//VX7-CSF/4273 unambiguously clustered with t(8;21) specimens, suggesting that the transcriptional network is shared between these 2 entities (FIGs. 3B and 3F). Interestingly and in agreement with this observation, a KIT D817V mutation was detected in the RUNX1-CBFA2T3 sample.
[00212] The transcriptomic signature associated with 3 other RUNX1 fusions detected in 2 separate specimens of our global Leucegene AML cohort was also analyzed. The in frame fusions include RUNX1-USP42 (previously characterized, Giguere A and Hebert J. Genes, Chromosomes and Cancer. 201 1 ;50(4):228-238), and a novel RUNX1-ERG. This last fusion transcript leads to ERG overexpression (FIG. 3E) and was found in a sample which had the following karyotype: 48,XX,+9,t(1 1 ;21)(q2?4;q22),?add(21)(q22),+22[23] (FIG. 3C-D and Table 9). A second fusion involving RUNX1 (RUNX1-USP2) was also present in this last specimen, leading to USP2 overexpression (FIG. 3E). In contrast to the RUNX1-CBFA2T3 sample discussed above, none of these 2 samples showed a transcriptional profile similar to that of RUNX1-RUNX1T1 (FIG. 3B).
Table 9: Clinical, laboratory and mutational characteristics of samples with other in frame
RUNX1 fusions.
status at
Sample I D Sex Age WBC sampling FAB WHO 2008 Acute myelomonocytic
13H179 M 63 204 Diagnosis AML-M4 leukaemia
AML with myelodysplasia- 04H120 M 37 10.5 Diagnosis AML-M1 related changes
AML with minimal
12H149 F 39 21.4 Diagnosis AML-MO differentiation
Sample ID Karyotype Confirmed fusion Mutations
13H179 46,XY,t(16;21)(q24;q22)[20] RUNX1-CBFA2T3 KIT D816V
46,XY,del(5)(q22q33),t(7;21)(p22; NRAS G12D, 04H120 RUNX1-USP42
q22)[20] NRAS G13D
RUNX1-ERG,
12H149 48,XX ,+9,t(1 1 ;21)(q2?4;q22),?ad
RUNX1-USP2
d(21)(q22) +22[23]
[00213] Generation of a full Leucegene-specific transcriptome annotation. Complementary to the Elandv2 mapping based on RefSeq annotations, a final "ab initio" transcriptome assembly was generated based on raw sequence data using Tophat/Cufflinks methodology. This pipeline, which had served to explore new putative non-coding genes, was refined to accommodate the simultaneous detection of new intergenic transcripts as well as novel splice isoforms of already annotated genes. This analysis utilized the Gencode (version 19) annotation, a significantly more comprehensive transcriptome annotation than Refseq, resulting in the discovery of 78,336 novel splice isoforms, 2,607 new intergenic transcribed loci (with 4,814 transcripts) and 1 ,789 new antisense transcripts. The annotation of novel genes/transcripts was merged with the Gencode (v19) annotation to obtain a complete Leucegene specific transcriptome catalog comprising roughly 51 ,000 genes and almost 280,000 transcripts. FPKM expression values on both isoform and gene levels across all Leucegene AML and control samples were computed using Cufflinks. Splice isoforms were identified with Tophat 2.0.7 and Cufflinks 2.1.1., and are expressed in Fragments Per Kilobase of exon per Million fragments mapped (FPKM)). The results are presented in Tables 10-11 , FIGs. 4A-40 and FIGs. 5A-50.
Table 10: Gene isoforms expression for inv(16) AML subtype.
Transcript SEQ locus factor_10_90 q.value Refseq ref ID ENSEMBL ref ID name ID No:
MYH11 iso chr16: 15489610-
64411.5 8.05E-79 NA ENST00000573908.1 21 14 15950890
MYH11 iso chr16: 15489610-
41494 9.53E-87 NA NA 22 6 15950890
APLN iso chrX: 128779239-
36.2472212 4.63E-17 NA ENST00000307484.6 23 4 128812532
ST18 iso chr8: 53023227-
15.8364043 1.81 E-18 NA ENST00000518053.1 24 19 53373586
ST18 iso chr8: 53023227-
12.7787934 1.20E-19 NA ENST00000522861.1 25 20 53373586
ST18 iso chr8: 53023227-
9.42250887 2.60E-17 NA NA 26 2 53373586
ST18_iso_ chr8: 53023227- 4.5038287 1.37E-16 NA NA 27 17 53373586
ST18 iso chr8: 53023227-
3.2393704 9.97E-26 NA NA 28 1 53373586
MSLN iso chr16:810761-
3.15955456 8.63E-15 NM_005823 NA 29 2 818940
NT5E iso chr6:86159808-
3.14302179 1.40E-14 NA ENST00000369646.3 30 1 86353510
MSLN iso chr16:810761-
3.10982016 1.06E-12 NA ENST00000566269.2 31 8 818940
CLIP3 iso chr19:36505405-
2.93767772 8.77E-15 NM_015526 ENST00000360535.4 32 3 36596008
MSLN iso chr16:810761-
2.82758075 2.60E-15 NM_013404 ENST00000382862.3 33 4 818940
CLEC10A_ chr17:6974941-
2.66065095 2.79E-14 NM_006344 ENST00000571664.1 34 iso 18 7019940
AC 104809. chr2:241858201-
2.55146786 2.27E-18 NA NA 35 4 iso 35 241933069
The isoform list was generated based on two parameters: (1) the ratio was calculated between the 10%ile expression value of the test group (i.e. inv(16) AMLs without relapse samples) and the 90%ile control group (all other AMLs without relapse samples). This ratio is under the column headed "factoM 0_90" (2) All transcripts without significant expression differences were removed and then ranked by the factor_10_90 value.
Table 11 : Gene isoforms expression for t(8:21 ) AML subtype.
Figure imgf000072_0001
Example 1.3: GPCRs overexpressed in human AML [00214] Using RNA-Seq, the expression of GPCRs in 148 AML samples was evaluated and compared to that observed in normal bone marrow and peripheral blood cell populations and in normal cord blood-derived CD34+CD45RA" hematopoietic stem and progenitor cells (herein called CD34+ cells). The 772 GPCRs analyzed in this study consist of all the GPCR members included in the IUPHAR database (see Example 1 above for Materials and Methods), as well as 370 olfactory, 24 taste and 4 vomeronasal receptors. Information about the receptor subfamilies and the 18 sub-groups based on their ligands is provided in FIG. 6 and Table 12. Overall 239 GPCR were expressed at >1 IRPKM (or >1 RPKM) in at least one AML sample. Expression was above 3.5 IRPKM (or above 10.35 RPKM, high) for 1 1 1 members and above 6.66 IRPKM (or above 100 RPKM, very high) for 19 genes in at least one AML sample.
[00215] The various GPCRs were ranked according to their median expression level from highest to lowest in AML and in CD34+ cells. According to these data, the most highly expressed GPCRs in AML are CXCR4, CD97, PTGER4, GPR183, PTGER2, S1PR4, FPR1, EMR2, C3AR1, LTB4R, TPRA1, C5AR1, LPAR2, LTB4R2 and GPR107. The highly expressed GPCRs in CD34+ cells are GPR56, CXCR4, S1PR4, HTR1F, F2R, TAPT1, PTGER4, CD97, GABBR1, TPRA1, LPAR2, SMO, P2RY11, LPHN1, GPR107, GPR126, highlighting major differences in GPCR expression levels between these two groups of primary samples. In addition to the different order of expression levels observed among highly expressed GPCRs, some receptors that are not found among the most highly expressed ones in normal CD34+ cells are clearly overexpressed in AML.
Table 12: Expression of GPCRs in AML and CD34+ cells
t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
CXCR4 Chemokine Receptor 7.51 7.59 5.68 5.68 0.0000
CD97 Adhesion Receptor 6.97 6.91 3.97 3.98 0.0000
PTGER4 Eicosanoids Receptor 5.85 5.99 3.98 4.03 0.0000
GPR183 Other lipid Receptor 5.28 5.45 1.10 0.98 0.0000
PTGER2 Eicosanoids Receptor 4.89 5.09 3.23 3.31 0.0000
S1PR4 Lysophospholipids Receptor 4.63 4.70 4.82 4.75 0.0945
FPR1 Peptide Binging Receptor 3.84 4.20 0.20 0.11 0.0000
EMR2 Adhesion Receptor 3.76 3.99 2.93 2.99 0.0000
C3AR1 Peptide Binging Receptor 3.98 3.98 0.24 0.17 0.0000
LTB4R Eicosanoids Receptor 3.87 3.94 3.02 3.00 0.0000
C5AR1 Peptide Binging Receptor 3.74 3.77 0.44 0.39 0.0000
TPRA 1 Other 7TM proteins 3.73 3.77 3.78 3.87 0.6996
LPAR2 Lysophospholipids Receptor 3.72 3.64 3.75 3.77 0.6795
LTB4R2 Eicosanoids Receptor 3.47 3.52 3.14 3.11 0.0104
GPR107 Other 7TM proteins 3.48 3.51 3.66 3.61 0.0077
CYSLTR1 Eicosanoids Receptor 3.39 3.42 2.57 2.55 0.0001
PTAFR Lysophospholipids Receptor 3.32 3.36 1.10 0.94 0.0000
TAPT1 Other 7TM proteins 3.46 3.36 4.14 4.10 0.0002
OPN3 Class A Orphans 3.10 3.15 3.19 3.24 0.4258
ADRB2 Amine Receptor 3.12 3.08 0.18 0.12 0.0000
G PR 160 Class A Orphans 3.05 3.06 0.97 0.96 0.0000
GPR97 Adhesion Receptor 3.00 2.94 2.18 2.31 0.0000 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
P2RY11 Purin receptor cluster Receptor 2.93 2.94 3.65 3.64 0.0000
EMR1 Adhesion Receptor 2.97 2.93 1.75 1.65 0.0000
GPR114 Adhesion Receptor 2.82 2.88 1.72 1.60 0.0000
GPR109B Purin receptor cluster Receptor 2.95 2.85 0.09 0.07 0.0000
GPR162 Class A Orphans 2.71 2.83 3.18 3.16 0.0093
CCR1 Chemokine Receptor 2.81 2.81 0.1 1 0.02 0.0000
GPR109A Purin receptor cluster Receptor 2.80 2.76 0.08 0.07 0.0000
SUCNR1 Purin receptor cluster Receptor 2.63 2.76 0.21 0.09 0.0000
GPR174 Purin receptor cluster Receptor 2.49 2.70 2.50 2.58 0.9494
LPAR6 Lysophospholipids Receptor 2.51 2.53 2.31 2.24 0.2504
GPR124 Adhesion Receptor 2.48 2.52 2.67 2.89 0.2322
P2RY1 Purin receptor cluster Receptor 2.56 2.46 2.84 2.81 0.0285
P2RY2 Purin receptor cluster Receptor 2.51 2.41 0.50 0.50 0.0000
GPR137 Other 7TM proteins 2.40 2.39 2.98 2.98 0.0000
GPR27 Class A Orphans 2.11 2.38 1.34 0.90 0.0263
P2RY13 Purin receptor cluster Receptor 2.53 2.36 0.55 0.61 0.0000
ADORA2A Adenosine receptors 2.54 2.32 1.80 1.77 0.0000
GPR132 Purin receptor cluster Receptor 2.33 2.30 1.74 1.65 0.0000
HRH2 Amine Receptor 2.12 2.28 0.80 0.86 0.0000
GPR125 Adhesion Receptor 2.17 2.18 3.41 3.44 0.0000
GNRHR2 Peptide Binging Receptor 2.09 2.12 2.37 2.30 0.0022
GPR141 Class A Orphans 2.01 2.12 2.44 2.50 0.0010
OR2W3 Olfactory Receptor 2.08 2.10 2.89 2.92 0.0000
F2R Protease-activated 2.1 1 2.05 4.10 4.11 0.0000
XPR1 Other 7TM proteins 2.00 1.99 2.14 2.11 0.0304
GPR35 Purin receptor cluster Receptor 2.02 1.94 1.53 1.57 0.0000
OPRL1 Peptide Binging Receptor 1.93 1.92 2.03 2.04 0.3459
PTGIR Eicosanoids Receptor 1.95 1.87 0.97 0.90 0.0000
PTH2R Class B Receptor (peptide) 1.62 1.86 0.04 0.04 0.0000
GPR56 Adhesion Receptor 2.45 1.84 5.61 5.68 0.0000
GABBR1 Class C 2.07 1.83 3.87 3.89 0.0000
OXER1 Eicosanoids Receptor 1.71 1.71 1.52 1.56 0.0090
CXCR7 Chemokine Receptor 1.89 1.67 1.96 1.98 0.6687
CYSLTR2 Eicosanoids Receptor 1.94 1.64 1.42 1.45 0.0002
CCRL2 Chemokine Receptor 1.61 1.63 0.67 0.61 0.0000
S1PR1 Lysophospholipids Receptor 1.87 1.47 1.36 1.45 0.0321
GPR77 Peptide Binging Receptor 1.48 1.45 0.61 0.61 0.0000
GPR65 Purin receptor cluster Receptor 1.51 1.42 0.12 0.10 0.0000
FZD1 Frizzled 1.44 1.40 1.38 1.45 0.7554
LPHN1 Adhesion Receptor 1.53 1.38 3.64 3.61 0.0000
GPR75 Class A Orphans 1.36 1.33 3.00 2.99 0.0000
GPR84 Class A Orphans 1.58 1.33 0.29 0.16 0.0000
CCR7 Chemokine Receptor 1.50 1.32 0.49 0.15 0.0045
ADORA2B Adenosine receptors 1.34 1.30 1.14 1.00 0.2736
CX3CR1 Chemokine Receptor 1.76 1.29 0.12 0.10 0.0000
GPR146 Class A Orphans 1.37 1.28 1.81 1.76 0.0027
GPR137B Other 7TM proteins 1.32 1.25 1.79 1.78 0.0000
EMR4P Adhesion Receptor 1.59 1.24 0.98 0.90 0.0019
FPR2 Peptide Binging Receptor 1.52 1.24 0.04 0.00 0.0000
CCR2 Chemokine Receptor 1.67 1.19 0.03 0.01 0.0000
TAS2R14 Taste 2 Receptor 1.14 1.18 1.80 1.85 0.0000
GPR3 Class A Orphans 1.19 1.17 1.90 1.87 0.0000
A VPR2 Peptide Binging Receptor 1.06 1.03 1.38 1.35 0.0006
GPR18 Purin receptor cluster Receptor 1.24 1.03 0.19 0.19 0.0000
LPAR5 Lysophospholipids Receptor 1.26 0.99 1.34 1.31 0.4358 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
BAH Adhesion Receptor 1.17 0.98 0.75 0.76 0.0000
VIPR1 Class B Receptor (peptide) 1.19 0.97 1.10 1.13 0.3363
TBXA2R Eicosanoids Receptor 1.08 0.94 1.06 1.01 0.8483
FZD5 Frizzled 0.98 0.89 0.78 0.71 0.0009
FFAR2 Other lipid Receptor 1.08 0.82 0.03 0.01 0.0000
P2RY10 Purin receptor cluster Receptor 0.97 0.78 0.20 0.15 0.0000
S1PR3 Lysophospholipids Receptor 1.08 0.76 0.06 0.04 0.0000
HTR1F Amine Receptor 1.28 0.75 4.56 4.56 0.0000
GPBAR1 Other lipid Receptor 1.28 0.73 0.67 0.56 0.0000
CXCR2 Chemokine Receptor 0.92 0.72 0.02 0.01 0.0000
CCR5 Chemokine Receptor 0.94 0.71 0.03 0.00 0.0000
CHRM4 Amine Receptor 0.76 0.71 0.69 0.65 0.2431
VN1R1 Pheromone receptor 0.71 0.69 1.19 1.21 0.0000
ADRB 1 Amine Receptor 1.06 0.68 0.05 0.02 0.0000
NMUR1 Peptide Binging Receptor 1.09 0.68 0.1 1 0.10 0.0000
LPAR4 Lysophospholipids Receptor 0.79 0.67 0.26 0.25 0.0000
RXFP1 Peptide Binging Receptor 1.24 0.66 0.05 0.00 0.0000
TAS2R5 Taste 2 Receptor 0.77 0.66 1.44 1.39 0.0000
FZD6 Frizzled 0.93 0.64 2.52 2.42 0.0000
P2RY14 Purin receptor cluster Receptor 0.92 0.64 0.22 0.20 0.0000
GPR135 Class A Orphans 0.73 0.63 1.66 1.61 0.0000
MC1R Peptide Binging Receptor 0.71 0.63 1.57 1.54 0.0000
ADORA3 Adenosine receptors 0.71 0.61 0.23 0.23 0.0000
F2RL1 Protease-activated 0.97 0.61 3.20 3.09 0.0000
FZD2 Frizzled 0.85 0.61 0.12 0.06 0.0000
GPRC5C Class C 0.80 0.61 1.07 1.05 0.0000
CXCR3 Chemokine Receptor 0.81 0.60 0.03 0.03 0.0000
GPR44 Eicosanoids Receptor 0.92 0.60 2.52 2.41 0.0000
GPR55 Purin receptor cluster Receptor 0.68 0.60 0.25 0.23 0.0000
CNR2 Lysophospholipids Receptor 0.83 0.59 0.71 0.66 0.1783
GPR153 Class A Orphans 1.03 0.59 0.16 0.16 0.0000
HTR7 Amine Receptor 0.97 0.56 0.06 0.02 0.0000
RXFP2 Peptide Binging Receptor 0.84 0.55 0.01 0.00 0.0000
FZD7 Frizzled 0.66 0.54 0.98 1.03 0.0103
GPR157 Other 7TM proteins 0.55 0.51 0.63 0.61 0.2879
S1PR5 Lysophospholipids Receptor 0.64 0.48 0.01 0.00 0.0000
EMR3 Adhesion Receptor 0.72 0.46 0.02 0.01 0.0000
CALCRL Class B Receptor (peptide) 0.92 0.45 2.49 2.47 0.0000
TAS2R20 Taste 2 Receptor 0.48 0.45 0.70 0.71 0.0019
CXCR5 Chemokine Receptor 0.58 0.44 0.04 0.03 0.0000
RXFP4 Peptide Binging Receptor 0.57 0.44 1.86 1.75 0.0000
CELSR3 Adhesion Receptor 0.73 0.43 1.39 1.45 0.0000
GPR113 Adhesion Receptor 0.44 0.42 0.80 0.80 0.0000
GPR34 Class A Orphans 0.87 0.42 0.04 0.03 0.0000
SMO Frizzled 0.73 0.42 3.69 3.69 0.0000
S1PR2 Lysophospholipids Receptor 0.50 0.41 0.98 0.95 0.0000
TAS2R31 Taste 2 Receptor 0.43 0.41 0.89 0.88 0.0000
LPAR1 Lysophospholipids Receptor 0.65 0.39 0.06 0.05 0.0000
NTSR1 Peptide Binging Receptor 0.65 0.38 0.13 0.09 0.0000
CCR10 Chemokine Receptor 0.49 0.37 0.96 0.88 0.0000
CELSR1 Adhesion Receptor 0.67 0.37 0.34 0.36 0.0000
MLNR Peptide Binging Receptor 0.53 0.37 0.17 0.12 0.0000
CCR4 Chemokine Receptor 0.54 0.35 0.30 0.28 0.0009
GPR68 Purin receptor cluster Receptor 0.40 0.34 1.24 0.98 0.0003
CXCR1 Chemokine Receptor 0.59 0.33 0.01 0.00 0.0000 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
OR2B 11 Olfactory Receptor 0.45 0.33 0.07 0.06 0.0000
P2RY6 Purin receptor cluster Receptor 0.50 0.33 0.04 0.03 0.0000
TAS2R19 Taste 2 Receptor 0.35 0.33 0.75 0.75 0.0000
CMKLR1 Peptide Binging Receptor 0.55 0.30 0.01 0.00 0.0000
TAS2R4 Taste 2 Receptor 0.37 0.30 0.79 0.83 0.0000
CELSR2 Adhesion Receptor 0.41 0.29 0.43 0.42 0.6541
GPR171 Purin receptor cluster Receptor 0.43 0.29 0.05 0.04 0.0000
CCR6 Chemokine Receptor 0.39 0.26 0.04 0.03 0.0000
GALR2 Peptide Binging Receptor 0.45 0.26 0.62 0.62 0.0167
GPER Other lipid Receptor 0.52 0.26 0.05 0.06 0.0000
GRM2 Class C 0.35 0.26 0.51 0.43 0.1012
DRD4 Amine Receptor 0.30 0.25 0.35 0.28 0.2961
HTR2B Amine Receptor 0.29 0.25 0.35 0.33 0.1538
OR2T8 Olfactory Receptor 0.31 0.25 0.64 0.69 0.0000
TAS1R3 Class C 0.28 0.25 0.67 0.66 0.0000
CCRL1 Chemokine Receptor 0.26 0.24 0.38 0.36 0.0220
GPR150 Class A Orphans 0.33 0.24 0.62 0.62 0.0015
CXCR6 Chemokine Receptor 0.28 0.23 0.30 0.29 0.6024
OR2L13 Olfactory Receptor 0.39 0.23 1.03 1.19 0.0002
OR52N4 Olfactory Receptor 0.31 0.23 0.52 0.48 0.0180
GIPR Class B Receptor (peptide) 0.59 0.22 0.03 0.03 0.0000
TAS2R10 Taste 2 Receptor 0.25 0.22 0.46 0.45 0.0004
HRH4 Amine Receptor 0.29 0.21 0.24 0.22 0.1656
CCR3 Chemokine Receptor 0.34 0.20 0.00 0.00 0.0000
ADRA2C Amine Receptor 0.84 0.19 0.01 0.00 0.0000
FZD8 Frizzled 0.45 0.19 0.29 0.28 0.0139
LGR6 Class A Orphans 0.35 0.19 1.19 1.26 0.0000
ADRA2A Amine Receptor 0.33 0.18 1.07 1.13 0.0000
OR2L2 Olfactory Receptor 0.29 0.18 0.71 0.68 0.0009
OXTR Peptide Binging Receptor 0.22 0.18 0.26 0.25 0.0913
P2RY12 Purin receptor cluster Receptor 0.37 0.18 0.35 0.26 0.7706
P2RY4 Purin receptor cluster Receptor 0.19 0.18 0.35 0.33 0.0008
PTGDR Eicosanoids Receptor 0.24 0.17 0.09 0.08 0.0000
GPR19 Class A Orphans 0.19 0.16 0.22 0.21 0.2317
PTGER1 Eicosanoids Receptor 0.30 0.16 0.14 0.11 0.0007
TAS2R46 Taste 2 Receptor 0.19 0.16 0.33 0.33 0.001 1
FFAR3 Other lipid Receptor 0.29 0.15 0.00 0.00 0.0000
GPR133 Adhesion Receptor 0.35 0.15 0.01 0.00 0.0000
GPR161 Purin receptor cluster Receptor 0.23 0.15 1.19 1.06 0.0000
A VPR1B Peptide Binging Receptor 0.25 0.14 0.38 0.36 0.0544
FPR3 Peptide Binging Receptor 0.25 0.14 0.05 0.02 0.0000
LGR4 Class A Orphans 0.43 0.14 1.39 1.36 0.0000
GPR88 Class A Orphans 0.26 0.13 0.1 1 0.08 0.0002
OR2B6 Olfactory Receptor 0.25 0.13 0.08 0.05 0.001 1
OR2T33 Olfactory Receptor 0.21 0.13 0.51 0.51 0.0000
TACR2 Peptide Binging Receptor 0.17 0.13 0.68 0.60 0.0000
CCR9 Chemokine Receptor 0.17 0.12 0.62 0.62 0.0000
GPR12 Class A Orphans 0.63 0.12 0.09 0.03 0.0000
GPR15 Class A Orphans 0.29 0.12 0.00 0.00 0.0000
TAS2R60 Taste 2 Receptor 0.18 0.12 0.07 0.06 0.0001
GPR152 Class A Orphans 0.23 0.11 0.26 0.22 0.5205
GPR52 Class A Orphans 0.13 0.11 0.12 0.11 0.4452
GPRC5D Class C 0.23 0.11 0.15 0.13 0.0350
OR2AK2 Olfactory Receptor 0.19 0.11 0.37 0.33 0.0097
TAS2R30 Taste 2 Receptor 0.13 0.11 0.15 0.12 0.4173 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
TAS2R50 Taste 2 Receptor 0.13 0.11 0.16 0.16 0.3436
F2RL3 Protease-activated 0.26 0.10 0.34 0.18 0.5809
GNRHR Peptide Binging Receptor 0.12 0.10 0.20 0.18 0.0018
GPR21 Class A Orphans 0.36 0.10 0.84 0.79 0.0000
GRPR Peptide Binging Receptor 0.12 0.10 0.06 0.06 0.0000
TAS2R43 Taste 2 Receptor 0.14 0.10 0.35 0.40 0.0002
DARC Chemokine Receptor 0.30 0.09 0.09 0.08 0.0000
GPR22 Class A Orphans 0.10 0.09 0.06 0.05 0.0035
GPR85 Class A Orphans 0.24 0.09 0.10 0.09 0.0000
OR2A7 Olfactory Receptor 0.20 0.09 0.50 0.47 0.0000
TAS2R3 Taste 2 Receptor 0.15 0.09 0.49 0.51 0.0001
GPR45 Class A Orphans 0.11 0.08 0.27 0.27 0.0002
PTH1R Class B Receptor (peptide) 0.38 0.08 0.05 0.03 0.0000
RRH Opsin 0.10 0.08 0.14 0.14 0.1755
CCBP2 Chemokine Receptor 0.16 0.07 0.17 0.16 0.7264
CHRM5 Amine Receptor 0.10 0.07 0.09 0.09 0.7681
FFAR1 Other lipid Receptor 0.12 0.07 0.07 0.06 0.0980
FZD4 Frizzled 0.12 0.07 0.27 0.25 0.0000
GPR142 Class A Orphans 0.13 0.07 0.26 0.27 0.0004
GPR64 Adhesion Receptor 0.08 0.07 0.05 0.05 0.0016
GPRC5B Class C 0.10 0.07 0.07 0.06 0.0413
KISS1R Peptide Binging Receptor 0.12 0.07 0.14 0.12 0.601 1
OR10AD1 Olfactory Receptor 0.11 0.07 0.33 0.34 0.0002
OR2L3 Olfactory Receptor 0.14 0.07 0.63 0.65 0.0001
TAS1R1 Class C 0.09 0.07 0.24 0.22 0.0000
TAS2R40 Taste 2 Receptor 0.10 0.07 0.01 0.00 0.0000
GALR3 Peptide Binging Receptor 0.10 0.06 0.23 0.21 0.0021
GPR81 Purin receptor cluster Receptor 0.35 0.06 0.06 0.02 0.0000
GPR83 Class A Orphans 0.08 0.06 0.17 0.11 0.0565
HCRTR1 Peptide Binging Receptor 0.1 1 0.06 0.32 0.31 0.0000
MRGPRE Class A Orphans 0.10 0.06 0.02 0.01 0.0000
ADRA2B Amine Receptor 0.14 0.05 0.23 0.10 0.3990
BAI2 Adhesion Receptor 0.07 0.05 0.13 0.13 0.0026
F2RL2 Protease-activated 0.08 0.05 0.06 0.06 0.3412
GPR176 Class A Orphans 0.09 0.05 0.03 0.02 0.0061
GPR182 Class A Orphans 0.08 0.05 0.08 0.10 0.8501
GPR82 Purin receptor cluster Receptor 0.12 0.05 0.02 0.02 0.0001
LPAR3 Lysophospholipids Receptor 0.30 0.05 0.04 0.04 0.0000
OR52N2 Olfactory Receptor 0.09 0.05 0.18 0.21 0.0062
OR56B4 Olfactory Receptor 0.07 0.05 0.00 0.00 0.0000
SSTR2 Peptide Binging Receptor 0.14 0.05 0.10 0.09 0.2036
SSTR3 Peptide Binging Receptor 0.07 0.05 0.17 0.12 0.0302
TAS2R13 Taste 2 Receptor 0.06 0.05 0.08 0.07 0.3551
TSHR Glycoprotein Receptor 0.09 0.05 0.03 0.02 0.0012
CCR8 Chemokine Receptor 0.07 0.04 0.00 0.00 0.0012
FZD3 Frizzled 0.19 0.04 0.53 0.50 0.0000
FZD9 Frizzled 0.1 1 0.04 0.10 0.07 0.8242
GPR126 Adhesion Receptor 0.83 0.04 3.44 3.54 0.0000
HRH1 Amine Receptor 0.07 0.04 0.03 0.02 0.0002
OR2AE1 Olfactory Receptor 0.05 0.04 0.05 0.04 0.9412
OR56B 1 Olfactory Receptor 0.06 0.04 0.13 0.12 0.0182
OR9A4 Olfactory Receptor 0.12 0.04 0.00 0.00 0.0000
TAS2R41 Taste 2 Receptor 0.05 0.04 0.07 0.05 0.5455
ADRA 1D Amine Receptor 0.09 0.03 0.04 0.04 0.0880
A VPR1A Peptide Binging Receptor 0.27 0.03 0.02 0.00 0.0002 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
CRHR2 Class B Receptor (peptide) 0.05 0.03 0.02 0.01 0.0021
EDNRB Peptide Binging Receptor 0.09 0.03 0.01 0.01 0.0000
GPR120 Other lipid Receptor 0.1 1 0.03 0.00 0.00 0.0000
GPR173 Class A Orphans 0.32 0.03 2.58 2.57 0.0000
GPR179 Class C 0.04 0.03 0.04 0.05 0.5900
GPR25 Class A Orphans 0.08 0.03 0.01 0.00 0.0271
GPR61 Class A Orphans 0.04 0.03 0.09 0.08 0.0188
MAS1 Class A Orphans 0.07 0.03 0.08 0.05 0.6652
MCHR1 Peptide Binging Receptor 0.05 0.03 0.02 0.02 0.0000
NPY6R Peptide Binging Receptor 0.05 0.03 0.10 0.10 0.0027
OR1K1 Olfactory Receptor 0.03 0.03 0.04 0.02 0.8268
OR52B6 Olfactory Receptor 0.05 0.03 0.08 0.07 0.1019
OR52K2 Olfactory Receptor 0.07 0.03 0.00 0.00 0.0000
OR5K2 Olfactory Receptor 0.04 0.03 0.14 0.12 0.0083
VIPR2 Class B Receptor (peptide) 0.13 0.03 0.02 0.01 0.0000
VN1R5 Pheromone receptor 0.06 0.03 0.10 0.08 0.1814
CNR1 Lysophospholipids Receptor 0.07 0.02 0.00 0.00 0.0001
GPR143 Other 7TM proteins 0.02 0.02 0.09 0.08 0.0013
GPR156 Class C 0.03 0.02 0.02 0.01 0.0764
GPR20 Class A Orphans 0.04 0.02 0.03 0.03 0.5372
GPR32 Class A Orphans 0.26 0.02 0.06 0.05 0.0007
GPR63 Class A Orphans 0.08 0.02 0.52 0.48 0.0000
GPR87 Purin receptor cluster Receptor 0.04 0.02 0.08 0.07 0.0461
GPRC5A Class C 0.08 0.02 0.02 0.02 0.0000
HTR6 Amine Receptor 0.03 0.02 0.03 0.03 0.5410
LPHN2 Adhesion Receptor 0.25 0.02 0.14 0.08 0.2126
LPHN3 Adhesion Receptor 0.22 0.02 0.01 0.01 0.0000
NPBWR1 Peptide Binging Receptor 0.14 0.02 0.00 0.00 0.0000
OR13A 1 Olfactory Receptor 0.05 0.02 0.02 0.01 0.0107
OR1L8 Olfactory Receptor 0.06 0.02 0.16 0.18 0.0005
OR2C1 Olfactory Receptor 0.03 0.02 0.09 0.10 0.0194
OR2L8 Olfactory Receptor 0.06 0.02 0.09 0.08 0.4047
OR52H1 Olfactory Receptor 0.03 0.02 0.06 0.06 0.1047
OR52W1 Olfactory Receptor 0.03 0.02 0.00 0.00 0.0000
OR5B21 Olfactory Receptor 0.03 0.02 0.01 0.00 0.0009
OR7D2 Olfactory Receptor 0.05 0.02 0.12 0.06 0.2259
PROKR1 Peptide Binging Receptor 0.03 0.02 0.1 1 0.08 0.0394
PTGER3 Eicosanoids Receptor 0.17 0.02 0.37 0.33 0.0018
TACR1 Peptide Binging Receptor 0.02 0.02 0.04 0.05 0.0087
UTS2R Peptide Binging Receptor 0.04 0.02 0.03 0.03 0.5812
ADORA 1 Adenosine receptors 0.07 0.01 0.01 0.00 0.0000
ADRA 1B Amine Receptor 0.02 0.01 0.03 0.00 0.8045
ADRB3 Amine Receptor 0.02 0.01 0.00 0.00 0.0088
CASR Class C 0.01 0.01 0.00 0.00 0.0000
CHRM3 Amine Receptor 0.06 0.01 2.25 2.22 0.0000
DRD5 Amine Receptor 0.02 0.01 0.01 0.01 0.1806
GCGR Class B Receptor (peptide) 0.06 0.01 0.04 0.03 0.5077
GLP1R Class B Receptor (peptide) 0.20 0.01 0.01 0.00 0.0000
GPR144 Adhesion Receptor 0.05 0.01 0.09 0.08 0.0497
GPR17 Purin receptor cluster Receptor 0.04 0.01 0.01 0.00 0.0055
GPR4 Purin receptor cluster Receptor 0.03 0.01 0.03 0.02 0.8187
GPR62 Class A Orphans 0.03 0.01 0.15 0.12 0.0164
GPR98 Adhesion Receptor 0.02 0.01 0.02 0.02 0.8633
GRM1 Class C 0.01 0.01 0.01 0.01 0.8398
GRM7 Class C 0.01 0.01 0.01 0.00 0.0509 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
HRH3 Amine Receptor 0.02 0.01 0.02 0.00 0.4125
HTR2A Amine Receptor 0.02 0.01 0.03 0.02 0.4280
MRGPRD Class A Orphans 0.02 0.01 0.05 0.00 0.301 1
MRGPRF Class A Orphans 0.04 0.01 0.01 0.00 0.0046
OR13C5 Olfactory Receptor 0.02 0.01 0.07 0.06 0.0158
OR2H2 Olfactory Receptor 0.03 0.01 0.24 0.22 0.0003
OR4M2 Olfactory Receptor 0.03 0.01 0.00 0.00 0.0000
OR4N4 Olfactory Receptor 0.05 0.01 0.00 0.00 0.0000
OR51I1 Olfactory Receptor 0.02 0.01 0.05 0.04 0.1879
OXGR1 Purin receptor cluster Receptor 0.04 0.01 0.06 0.04 0.3061
PTGFR Eicosanoids Receptor 0.18 0.01 0.00 0.00 0.0000
TAS2R42 Taste 2 Receptor 0.02 0.01 0.06 0.06 0.0369
TAS2R9 Taste 2 Receptor 0.02 0.01 0.05 0.03 0.1071
TRHR Peptide Binging Receptor 0.02 0.01 0.05 0.04 0.0857
XCR1 Chemokine Receptor 0.04 0.01 0.00 0.00 0.0726
ADCYAP1R1 Class B Receptor (peptide) 0.00 0.00 0.00 0.00 0.7831
ADRA 1A Amine Receptor 0.00 0.00 0.00 0.00 0.5653
AGTR1 Peptide Binging Receptor 0.01 0.00 0.05 0.03 0.0092
AGTR2 Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.0154
APLNR Peptide Binging Receptor 0.06 0.00 0.06 0.04 0.7855
BAI3 Adhesion Receptor 0.03 0.00 0.05 0.03 0.2160
BDKRB 1 Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.0000
BDKRB2 Peptide Binging Receptor 0.02 0.00 0.00 0.00 0.0005
BRS3 Peptide Binging Receptor 0.01 0.00 0.00 0.00 0.0000
CALCR Class B Receptor (peptide) 0.00 0.00 0.00 0.00 0.0000
CCKAR Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.4418
CCKBR Peptide Binging Receptor 0.01 0.00 0.00 0.00 0.0026
CHRM1 Amine Receptor 0.00 0.00 0.00 0.00 0.7138
CHRM2 Amine Receptor 0.00 0.00 0.01 0.00 0.3981
CRHR1 Class B Receptor (peptide) 0.01 0.00 0.00 0.00 0.0000
DRD1 Amine Receptor 0.00 0.00 0.00 0.00 0.0723
DRD2 Amine Receptor 0.01 0.00 0.01 0.00 0.7563
DRD3 Amine Receptor 0.00 0.00 0.00 0.00 0.0000
EDNRA Peptide Binging Receptor 0.01 0.00 0.00 0.00 0.0000
ELTD1 Adhesion Receptor 0.07 0.00 0.00 0.00 0.0000
FSHR Glycoprotein Receptor 0.00 0.00 0.00 0.00 0.0007
FZD10 Frizzled 0.00 0.00 0.00 0.00 0.0014
GABBR2 Class C 0.01 0.00 0.00 0.00 0.2438
GALR1 Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.3012
GHRHR Class B Receptor (peptide) 0.00 0.00 0.00 0.00 0.0319
GHSR Peptide Binging Receptor 0.00 0.00 0.01 0.00 0.5169
GLP2R Class B Receptor (peptide) 0.00 0.00 0.00 0.00 0.1905
GPR1 Class A Orphans 0.00 0.00 0.00 0.00 0.9177
GPR101 Purin receptor cluster Receptor 0.00 0.00 0.00 0.00 0.0003
GPR110 Adhesion Receptor 0.04 0.00 0.48 0.46 0.0004
GPR111 Adhesion Receptor 0.00 0.00 0.02 0.01 0.0902
GPR112 Adhesion Receptor 0.00 0.00 0.00 0.00 0.0072
GPR115 Adhesion Receptor 0.00 0.00 0.02 0.00 0.0798
GPR116 Adhesion Receptor 0.03 0.00 0.02 0.01 0.2186
GPR119 Class A Orphans 0.00 0.00 0.00 0.00 0.0017
GPR123 Adhesion Receptor 0.00 0.00 0.00 0.00 0.1038
GPR128 Adhesion Receptor 0.05 0.00 0.00 0.00 0.0930
GPR139 Class A Orphans 0.00 0.00 0.00 0.00 0.1584
GPR148 Class A Orphans 0.00 0.00 0.00 0.00 0.0369
GPR149 Class A Orphans 0.00 0.00 0.00 0.00 0.3873 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
GPR151 Class A Orphans 0.01 0.00 0.02 0.00 0.2022
GPR158 Class C 0.01 0.00 0.00 0.00 0.2841
GPR26 Class A Orphans 0.00 0.00 0.00 0.00 0.0380
GPR31 Class A Orphans 0.02 0.00 0.03 0.00 0.6370
GPR33 Class A Orphans 0.01 0.00 0.00 0.00 0.0000
GPR37 Class A Orphans 0.00 0.00 0.01 0.00 0.2079
GPR37L1 Class A Orphans 0.01 0.00 0.04 0.05 0.0014
GPR39 Class A Orphans 0.01 0.00 0.00 0.00 0.1829
GPR50 Class A Orphans 0.00 0.00 0.00 0.00 0.0008
GPR6 Class A Orphans 0.00 0.00 0.00 0.00 0.0027
GPR78 Class A Orphans 0.00 0.00 0.00 0.00 0.0030
GPRC6A Class C 0.01 0.00 0.00 0.00 0.1459
GRM3 Class C 0.03 0.00 0.08 0.06 0.0163
GRM4 Class C 0.01 0.00 0.02 0.00 0.8570
GRM5 Class C 0.00 0.00 0.00 0.00 0.0006
GRM6 Class C 0.01 0.00 0.04 0.03 0.0056
GRM8 Class C 0.00 0.00 0.00 0.00 0.4625
HCRTR2 Peptide Binging Receptor 0.02 0.00 0.00 0.00 0.0001
HTR1A Amine Receptor 0.00 0.00 0.00 0.00 0.1637
HTR1B Amine Receptor 0.12 0.00 0.20 0.10 0.4976
HTR1D Amine Receptor 0.00 0.00 0.00 0.00 0.7400
HTR1E Amine Receptor 0.00 0.00 0.00 0.00 0.091 1
HTR2C Amine Receptor 0.00 0.00 0.01 0.00 0.2731
HTR4 Amine Receptor 0.01 0.00 0.00 0.00 0.0050
HTR5A Amine Receptor 0.00 0.00 0.00 0.00 0.0320
LGR5 Class A Orphans 0.01 0.00 0.29 0.29 0.0000
LHCGR Glycoprotein Receptor 0.00 0.00 0.01 0.00 0.5137
MAS1L Class A Orphans 0.00 0.00 0.03 0.02 0.0282
MC2R Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.1197
MC3R Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.4218
MC4R Peptide Binging Receptor 0.05 0.00 0.00 0.00 0.2042
MC5R Peptide Binging Receptor 0.01 0.00 0.00 0.00 0.0005
MCHR2 Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.0257
MRGPRG Class A Orphans 0.00 0.00 0.00 0.00 0.0365
MRGPRX1 Class A Orphans 0.00 0.00 0.00 0.00 0.1323
MRGPRX2 Class A Orphans 0.00 0.00 0.01 0.00 0.4983
MRGPRX3 Class A Orphans 0.00 0.00 0.00 0.00 0.0002
MRGPRX4 Class A Orphans 0.00 0.00 0.00 0.00 0.0066
MTNR1A Amine Receptor 0.00 0.00 0.00 0.00 0.0096
MTNR1B Amine Receptor 0.00 0.00 0.00 0.00 0.0002
NMBR Peptide Binging Receptor 0.00 0.00 0.04 0.02 0.0177
NMUR2 Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.0643
NPBWR2 Peptide Binging Receptor 0.02 0.00 0.04 0.03 0.2287
NPFFR1 Peptide Binging Receptor 0.02 0.00 0.00 0.00 0.0029
NPFFR2 Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.5736
NPSR1 Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.01 1 1
NPY1R Peptide Binging Receptor 0.01 0.00 0.00 0.00 0.0000
NPY2R Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.1037
NPY5R Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.0021
NTSR2 Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.3590
OPN1LW Opsin 0.00 0.00 0.00 0.00 0.5954
OPN1SW Opsin 0.04 0.00 0.03 0.01 0.4530
OPN4 Opsin 0.00 0.00 0.00 0.00 0.0040
OPN5 Class A Orphans 0.00 0.00 0.00 0.00 0.5582
OPRD1 Peptide Binging Receptor 0.00 0.00 0.01 0.00 0.2889 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
OPRK1 Peptide Binging Receptor 0.06 0.00 0.00 0.00 0.0656
OPRM1 Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.0002
OR10A2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.9990
OR10A3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0916
OR10A4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.6991
OR10A5 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0064
OR10A6 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1648
OR10A 7 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR10AG1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2214
OR10C1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1606
OR10G2 Olfactory Receptor 0.01 0.00 0.00 0.00 0.0000
OR10G3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1169
OR10G4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0755
OR10G7 Olfactory Receptor 0.00 0.00 0.00 0.00 0.7337
OR10G8 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0157
OR10G9 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0041
OR10H1 Olfactory Receptor 0.06 0.00 0.03 0.00 0.0922
OR10H2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.8554
OR10H3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2904
OR10H4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0367
OR10H5 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0292
OR10J1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR10J3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0000
OR10J5 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0878
OR10K1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2056
OR10K2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1586
OR10P1 Olfactory Receptor 0.01 0.00 0.00 0.00 0.0001
OR10Q 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2170
OR10R2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR10S 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2715
OR10T2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1686
OR10V1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0129
OR10W1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0577
OR10X1 Olfactory Receptor 0.00 0.00 0.04 0.00 0.0613
OR10Z1 Olfactory Receptor 0.01 0.00 0.04 0.02 0.0335
OR11A 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.01 14
OR11 G2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0478
OR11H1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2070
OR11H12 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0185
OR11H4 Olfactory Receptor 0.01 0.00 0.00 0.00 0.0284
OR11H6 Olfactory Receptor 0.01 0.00 0.01 0.00 0.9577
OR11L1 Olfactory Receptor 0.02 0.00 0.03 0.00 0.2789
OR12D2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0342
OR12D3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0169
OR13C2 Olfactory Receptor 0.02 0.00 0.10 0.11 0.0006
OR13C3 Olfactory Receptor 0.01 0.00 0.03 0.00 0.1545
OR13C4 Olfactory Receptor 0.01 0.00 0.03 0.00 0.2480
OR13C8 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0025
OR13C9 Olfactory Receptor 0.01 0.00 0.06 0.05 0.0262
OR13D1 Olfactory Receptor 0.02 0.00 0.02 0.00 0.7417
OR13F1 Olfactory Receptor 0.02 0.00 0.04 0.01 0.1745
OR13G1 Olfactory Receptor 0.02 0.00 0.03 0.00 0.4015
OR13H1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0034
OR13J1 Olfactory Receptor 0.01 0.00 0.01 0.00 0.4746
OR14A 16 Olfactory Receptor 0.01 0.00 0.01 0.00 0.7883 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
OR14C36 Olfactory Receptor 0.00 0.00 0.03 0.00 0.0865
OR14I1 Olfactory Receptor 0.02 0.00 0.00 0.00 0.0675
OR14J1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR1A 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0324
OR1A2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3756
OR1B 1 Olfactory Receptor 0.02 0.00 0.06 0.06 0.0085
OR1C1 Olfactory Receptor 0.03 0.00 0.04 0.04 0.3313
OR1D2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0176
OR1D4 Olfactory Receptor 0.00 0.00 0.01 0.00 0.2426
OR1D5 Olfactory Receptor 0.00 0.00 0.02 0.00 0.1074
OR1E1 Olfactory Receptor 0.00 0.00 0.01 0.00 0.4599
OR1E2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0168
OR1F1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0218
OR1F2P Olfactory Receptor 0.01 0.00 0.00 0.00 0.0000
OR1G1 Olfactory Receptor 0.00 0.00 0.01 0.00 0.1695
OR1I1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1090
OR1J1 Olfactory Receptor 0.04 0.00 0.04 0.03 0.8859
OR1J2 Olfactory Receptor 0.01 0.00 0.00 0.00 0.0335
OR1J4 Olfactory Receptor 0.02 0.00 0.01 0.00 0.3965
OR1L1 Olfactory Receptor 0.00 0.00 0.02 0.00 0.0619
OR1L3 Olfactory Receptor 0.00 0.00 0.01 0.00 0.4201
OR1L4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.7664
OR1L6 Olfactory Receptor 0.00 0.00 0.00 0.00 0.6446
OR1M1 Olfactory Receptor 0.00 0.00 0.00 0.00
OR1N1 Olfactory Receptor 0.01 0.00 0.01 0.00 0.5650
OR1N2 Olfactory Receptor 0.01 0.00 0.02 0.00 0.1945
OR1Q 1 Olfactory Receptor 0.06 0.00 0.12 0.08 0.0783
OR1S1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0543
OR1S2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR2A 12 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0832
OR2A 14 Olfactory Receptor 0.00 0.00 0.01 0.00 0.2365
OR2A2 Olfactory Receptor 0.00 0.00 0.01 0.00 0.5160
OR2A25 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0108
OR2A4 Olfactory Receptor 0.02 0.00 0.02 0.00 0.7505
OR2A5 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0161
OR2AG1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.7279
OR2AG2 Olfactory Receptor 0.02 0.00 0.06 0.01 0.1231
OR2AT4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.4303
OR2B2 Olfactory Receptor 0.02 0.00 0.00 0.00 0.0001
OR2B3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1668
OR2C3 Olfactory Receptor 0.03 0.00 0.09 0.09 0.0077
OR2D2 Olfactory Receptor 0.01 0.00 0.02 0.00 0.1998
OR2D3 Olfactory Receptor 0.00 0.00 0.01 0.00 0.6812
OR2F1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0821
OR2F2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0798
OR2G2 Olfactory Receptor 0.01 0.00 0.01 0.00 0.8139
OR2G3 Olfactory Receptor 0.01 0.00 0.00 0.00 0.0002
OR2G6 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0191
OR2H1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.4300
OR2J2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0026
OR2J3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0107
OR2K2 Olfactory Receptor 0.01 0.00 0.03 0.01 0.1171
OR2M2 Olfactory Receptor 0.01 0.00 0.01 0.00 0.5179
OR2M3 Olfactory Receptor 0.01 0.00 0.06 0.03 0.0243
OR2M4 Olfactory Receptor 0.02 0.00 0.05 0.02 0.1210 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
OR2M5 Olfactory Receptor 0.00 0.00 0.02 0.00 0.0871
OR2M7 Olfactory Receptor 0.00 0.00 0.00 0.00 0.4774
OR2S2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1675
OR2T1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.101 1
OR2T10 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0002
OR2T11 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0277
OR2T12 Olfactory Receptor 0.01 0.00 0.01 0.00 0.3495
OR2T2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0032
OR2T27 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR2T29 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1021
OR2T3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0000
OR2T34 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0074
OR2T35 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1706
OR2T4 Olfactory Receptor 0.01 0.00 0.01 0.00 0.4367
OR2T5 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR2T6 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0030
OR2V2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0105
OR2W1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0407
OR2W5 Olfactory Receptor 0.01 0.00 0.01 0.00 0.4887
OR2Y1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR2Z1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0188
OR3A 1 Olfactory Receptor 0.06 0.00 0.28 0.26 0.0016
OR3A2 Olfactory Receptor 0.06 0.00 0.33 0.30 0.0026
OR3A3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0043
OR3A4 Olfactory Receptor 0.01 0.00 0.07 0.02 0.0834
OR4A 15 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0662
OR4A 16 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0294
OR4A47 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0162
OR4A5 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1127
OR4B 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0756
OR4C11 Olfactory Receptor 0.00 0.00 0.00 0.00
OR4C12 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1030
OR4C13 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1226
OR4C15 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1378
OR4C16 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1101
OR4C3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0196
OR4C45 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0368
OR4C46 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1648
OR4C6 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0748
OR4D1 Olfactory Receptor 0.01 0.00 0.01 0.00 0.7914
OR4D10 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0135
OR4D11 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1583
OR4D2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.7977
OR4D5 Olfactory Receptor 0.00 0.00 0.01 0.00 0.1963
OR4D6 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0041
OR4D9 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0571
OR4E2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0981
OR4F15 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0177
OR4F17 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1676
OR4F21 Olfactory Receptor 0.00 0.00 0.00 0.00 0.6151
OR4F4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1587
OR4F5 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0885
OR4F6 Olfactory Receptor 0.00 0.00 0.00 0.00 0.5234
OR4K1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0499
OR4K13 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1042 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
OR4K14 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0621
OR4K15 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0457
OR4K17 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR4K2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0844
OR4K5 Olfactory Receptor 0.00 0.00 0.00 0.00
OR4L1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0102
OR4M1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0055
OR4N2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0639
OR4N5 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0948
OR4P4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1133
OR4Q3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0849
OR4S1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2065
OR4S2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1580
OR4X1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1154
OR4X2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1207
OR51A2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR51A4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR51A 7 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1644
OR51B2 Olfactory Receptor 0.01 0.00 0.01 0.00 0.3742
OR51B4 Olfactory Receptor 0.02 0.00 0.09 0.09 0.0004
OR51B5 Olfactory Receptor 0.01 0.00 0.02 0.00 0.3418
OR51B6 Olfactory Receptor 0.00 0.00 0.01 0.00 0.2754
OR51D1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.5227
OR51E1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0013
OR51E2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.8812
OR51F1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1582
OR51F2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR51 G1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2123
OR51 G2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1743
OR51I2 Olfactory Receptor 0.01 0.00 0.00 0.00 0.2074
OR51L1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1677
OR51M1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0001
OR51 Q 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0170
OR51S 1 Olfactory Receptor 0.00 0.00 0.01 0.00 0.3644
OR51 T1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0413
OR51 V1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0821
OR52A 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0201
OR52A4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0392
OR52A5 Olfactory Receptor 0.01 0.00 0.00 0.00 0.0209
OR52B2 Olfactory Receptor 0.01 0.00 0.01 0.00 0.5698
OR52B4 Olfactory Receptor 0.01 0.00 0.02 0.01 0.1186
OR52D1 Olfactory Receptor 0.01 0.00 0.06 0.05 0.0123
OR52E2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1787
OR52E4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0431
OR52E6 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1623
OR52E8 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0403
OR52I1 Olfactory Receptor 0.02 0.00 0.05 0.02 0.0900
OR52I2 Olfactory Receptor 0.01 0.00 0.02 0.00 0.2674
OR52J3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2210
OR52K1 Olfactory Receptor 0.03 0.00 0.00 0.00 0.0000
OR52L1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0042
OR52M1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0584
OR52N1 Olfactory Receptor 0.03 0.00 0.13 0.16 0.0017
OR52N5 Olfactory Receptor 0.02 0.00 0.02 0.00 0.7032
OR52R1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1194 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
OR56A 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0028
OR56A3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1089
OR56A4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0510
OR56A5 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0003
OR5A 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0136
OR5A2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1056
OR5AC2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1580
OR5AK2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1859
OR5AN1 Olfactory Receptor 0.01 0.00 0.00 0.00 0.0001
OR5AP2 Olfactory Receptor 0.00 0.00 0.01 0.00 0.3687
OR5AR1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR5AS 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0567
OR5AU1 Olfactory Receptor 0.00 0.00 0.01 0.00 0.2940
OR5B 12 Olfactory Receptor 0.01 0.00 0.00 0.00 0.0000
OR5B 17 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0919
OR5B2 Olfactory Receptor 0.01 0.00 0.00 0.00 0.0000
OR5B3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0125
OR5C1 Olfactory Receptor 0.01 0.00 0.01 0.00 0.9590
OR5D13 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1089
OR5D14 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1560
OR5D16 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR5D18 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR5F1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR5H1 Olfactory Receptor 0.00 0.00 0.00 0.00
OR5H14 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1902
OR5H15 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1202
OR5H2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0837
OR5H6 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0391
OR5I1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR5J2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR5K1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.5878
OR5K3 Olfactory Receptor 0.00 0.00 0.01 0.00 0.2808
OR5K4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3784
OR5L1 Olfactory Receptor 0.00 0.00 0.00 0.00
OR5L2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1528
OR5M1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1293
OR5M10 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2285
OR5M11 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0910
OR5M3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR5M8 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR5M9 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR5P2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1378
OR5P3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2147
OR5R1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1713
OR5T1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1071
OR5T2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR5T3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2091
OR5V1 Olfactory Receptor 0.00 0.00 0.00 0.00
OR5W2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0716
OR6A2 Olfactory Receptor 0.01 0.00 0.02 0.00 0.2438
OR6B 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1786
OR6B2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.8501
OR6B3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1982
OR6C1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0332
OR6C2 Olfactory Receptor 0.01 0.00 0.00 0.00 0.0002 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
OR6C3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.5296
OR6C4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0295
OR6C6 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0554
OR6C65 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0547
OR6C68 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0071
OR6C70 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0012
OR6C74 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR6C75 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2078
OR6C76 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0128
OR6F1 Olfactory Receptor 0.02 0.00 0.05 0.06 0.0271
OR6K2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0000
OR6K3 Olfactory Receptor 0.03 0.00 0.00 0.00 0.0000
OR6K6 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0199
OR6M1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.5934
OR6N1 Olfactory Receptor 0.01 0.00 0.00 0.00 0.0000
OR6N2 Olfactory Receptor 0.01 0.00 0.00 0.00 0.1124
OR6P1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0330
OR6Q 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1837
OR6S1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0064
OR6T1 Olfactory Receptor 0.00 0.00 0.02 0.00 0.0883
OR6V1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0056
OR6X1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1238
OR6Y1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0827
OR7A 10 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1580
OR7A 17 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1602
OR7A5 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1286
OR7C1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0302
OR7C2 Olfactory Receptor 0.00 0.00 0.01 0.00 0.3667
OR7D4 Olfactory Receptor 0.00 0.00 0.01 0.00 0.2103
OR7E24 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1225
OR7G1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0931
OR7G2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0860
OR7G3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0402
OR8A 1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1093
OR8B 12 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0003
OR8B2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1679
OR8B3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1502
OR8B4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR8B8 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR8D1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0468
OR8D2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0524
OR8D4 Olfactory Receptor 0.00 0.00 0.01 0.00 0.1252
OR8G1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0012
OR8G2 Olfactory Receptor 0.01 0.00 0.00 0.00 0.0000
OR8G5 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0002
OR8H1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2541
OR8H2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1270
OR8H3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0180
OR8I2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1014
OR8J1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1735
OR8J3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2668
OR8K1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.3190
OR8K3 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1757
OR8K5 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0632
OR8S1 Olfactory Receptor 0.00 0.00 0.01 0.00 0.3319 t test
Mean Median Mean Median mean
Gene GPCR subclass
AML AML CD34+ CD34+ AML vs.
CD34
OR8U1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.2176
OR8U8 Olfactory Receptor 0.00 0.00 0.00 0.00
OR9A2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0001
OR9G4 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1209
OR9I1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.1582
OR9K2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0806
OR9Q1 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0413
OR9Q2 Olfactory Receptor 0.00 0.00 0.00 0.00 0.0971
P2RY8 Class A Orphans 0.00 0.00 0.00 0.00
PRLHR Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.4468
PROKR2 Peptide Binging Receptor 0.03 0.00 0.00 0.00 0.0002
QRFPR Peptide Binging Receptor 0.01 0.00 0.00 0.00 0.2776
RGR Opsin 0.00 0.00 0.00 0.00 0.0184
RXFP3 Peptide Binging Receptor 0.00 0.00 0.04 0.01 0.0674
SCTR Class B Receptor (peptide) 0.01 0.00 0.00 0.00 0.1436
SSTR1 Peptide Binging Receptor 0.01 0.00 0.05 0.06 0.0119
SSTR4 Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.9022
SSTR5 Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.0005
TAAR1 Amine Receptor 0.03 0.00 0.01 0.00 0.0376
TAAR2 Amine Receptor 0.00 0.00 0.00 0.00 0.1580
TAAR3 Amine Receptor 0.02 0.00 0.00 0.00 0.0528
TAAR5 Amine Receptor 0.00 0.00 0.00 0.00 0.0067
TAAR6 Amine Receptor 0.00 0.00 0.00 0.00 0.0175
TAAR8 Amine Receptor 0.00 0.00 0.00 0.00 0.2133
TAAR9 Amine Receptor 0.00 0.00 0.00 0.00 0.0325
TACR3 Peptide Binging Receptor 0.00 0.00 0.00 0.00 0.0219
TAS1R2 Class C 0.01 0.00 0.10 0.10 0.0003
TAS2R1 Taste 2 Receptor 0.00 0.00 0.00 0.00 0.0225
TAS2R16 Taste 2 Receptor 0.00 0.00 0.00 0.00
TAS2R38 Taste 2 Receptor 0.03 0.00 0.00 0.00 0.0321
TAS2R39 Taste 2 Receptor 0.00 0.00 0.01 0.00 0.4198
TAS2R7 Taste 2 Receptor 0.00 0.00 0.00 0.00 0.0007
TAS2R8 Taste 2 Receptor 0.02 0.00 0.04 0.03 0.1212
VN1R2 Pheromone receptor 0.00 0.00 0.00 0.00 0.8197
VN1R4 Pheromone receptor 0.00 0.00 0.00 0.00 0.1583
[00216] Real-time quantitative RT-PCR studies confirmed the RNA-Seq results for 10 selected GPCRs and established the robust correlation between both methods (FIG. 7; Tables 13 and 14).
Table 13: Expression of 10 selected GPCRs and control genes assessed by RNA-Seq.
Figure imgf000087_0001
09H113 10.6 10.4 5.9 6.5
10H056 11.8 10.7 6.9 7.6
10H072 11.0 11.3 6.3 6.6
10H115 10.6 9.8 6.7 7.3
11 H058 10.9 10.0 6.8 7.4
Gene expression level in log2(RPKM+1 )
10 selected GPCRs
AML
sample
id C3AR1 CD97 FPR1 GPR126 GPR183 LPAR6 LTB4R OPN3 PTGER4 TAPT1
04H024 6.5 8.3 5.7 1.3 5.4 0.2 4.3 3.0 5.5 3.1
04H112 4.2 8.6 3.5 0.8 3.3 0.4 3.8 4.0 4.7 2.9
05H050 5.5 8.0 7.1 0.1 6.3 4.4 4.4 4.3 6.2 2.2
07H160 6.5 7.6 2.1 5.6 1.9 2.1 5.1 1.4 2.8 5.7
09H083 4.9 7.1 2.6 0.9 5.6 0.4 3.4 3.1 5.4 3.8
09H113 2.8 6.4 0.2 0.4 3.1 3.0 3.4 3.3 5.4 4.2
10H056 2.3 6.5 3.3 0.1 5.5 2.1 3.0 3.2 6.3 3.3
10H072 5.4 6.8 6.9 0.3 6.6 1.3 4.7 3.2 6.1 2.8
10H115 4.6 6.8 1.3 0.1 3.9 0.6 3.6 2.9 4.1 4.0
11 H058 4.5 6.6 0.3 1.2 4.4 3.3 4.5 3.6 4.7 3.5
Table 14: Expression of 10 selected GPCRs assessed by RT-PCR
Figure imgf000088_0001
For normalization of the qPCR data, 4 endogenous control genes were used, including GAPDH and ACTB and 2 recently described control genes, EIF4H and HNRNPL (Macrae T et al., PLoS one. 2013;8(9):e72884). (B and C): Data of GPCR expression levels for the 10 selected GPCR members and the 4 control genes using RNA-Seq are transformed in IRPKM (\og2(RPKM+1)). Delta Ct represents the mean difference between the Ct of the tested gene and the mean Ct of the 4 endogenous control genes of 2 independent quantitative RT-PCR experiments. RPKM: Reads Per Kilobase of transcript per Million mapped reads.
[00217] High protein expression levels of selected GPCRs for which validated antibodies were available were confirmed using flow cytometry (FIGs. 8 and 9). Notably, with the exception of CD97, GPCR expression was distributed unequally within each patient sample highlighting the possibility of defining AML subpopulations with these markers (FIG. 9B).
[00218] When compared to normal CD34+ cells, 30 GPCRs are up-regulated in AML specimens (upper left region in FIG. 10). The most A L-specific GPCRs were CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1 and C5AR1. Likewise, 19 (lower right region in FIG. 10) GPCRs are preferentially expressed in normal CD34+ cells. GPR56, HTR1F, SMO and GPR126 are most discriminatory of normal CD34+ cells. Most GPCRs are equally expressed in the two cell types (middle region in FIG. 10).
[00219] Class enrichment analyses showed that while both AML and normal CD34+ cells were enriched for specific groups of adhesion GPCRs, it was observed that AML cells specifically expressed chemokine receptor and purine receptor cluster GPCRs and that normal CD34+ cells preferentially expressed protease-activated and Frizzled GPCRs (FIG. 11). Hence, deregulated GPCRs in AML appears to belong to specific receptor subfamilies.
Example 1.4: GPCRs are differentially expressed in distinct AML genetic subgroups
[00220] GPCR expression levels in relation to the most frequent AML genetic subgroups represented in this cohort such as AML with t(8;21)(q22;q22), inv(16)(p13.1 q22) or MLL translocations, and normal karyotype (NK) AML with NPM1, DNMT3A or FLT3-ITD mutations, were studied (Table 15).
Table 15: Cytogenetic subgroups and mutational status of studied cohort
Figure imgf000089_0001
[00221] A GPCR expression fingerprint was observed for the t(8;21), inv(16) and MLL translocations leukemia subgroups (FIGs. 12A and 13A). Using a threshold ratio of 4 and above, it was found that 5 GPCRs, namely ADRA2C, MLNR, LPAR5, GPR81 and CXCR1, are up-regulated in the AML subgroup with the t(8;21) translocation. Likewise for inv(16) AML, GPR12, LPAR1, GPR34 and RXFP1 are upregulated by 4-fold and above. Further analyses (see method section in Example 1) revealed a series of GPCRs significantly differentially expressed in these 2 leukemia subsets. Eight GPCRs were specifically up- or downregulated in the AML subgroup with the t(8;21) translocation. These included the adrenergic receptor ADRA2C, the orphan receptor GPR153, the lipid receptors LPAR5, LPAR6 and PTGIR (all upregulated) as well as the adhesion GPCRs, EMR1 and GPR114, and the lipid GPCR, GPR183 (downregulated). Overexpression of 8 other GPCRs characterized the inv(16) AML subgroup. These GPCRs are C5AR1, CCR2, ACKR3/CXCR7, FPR1, GPR183, LPAR6, PTGIR and RXFP1 (FIGs. 12A and 13A). Among these, FPR1, CCR2, C5AR1 and GPR183 are also overexpressed in AML M4 (FIG. 14). AML with MLL translocations were associated with an upregulation of GPR126 and a downregulation of GPR174, SUCNR1 and LPAR6 (FIG. 13A, right panel). Notably, GPR126 expression differed between subtypes of LL-rearranged AMLs according to the translocation partners, being overexpressed at a high level in AML samples with the MLL-MLLT4, MLL-ELL and MLL-SEPT9 fusions and not expressed in the majority of AML samples with the MLL-MLLT3 and MLL-MLLT1 fusions (FIG. 15). FLT3-ITD mutated samples revealed an upregulation of CYSLTR2, also observed in NPM1 mutated samples, and of the adhesion GPCRs, GPR114 and GPR56 (FIGs. 12B and 13B). These results were validated in an independent AML dataset of 160 samples available from The Cancer Genome Atlas (TCGA) project which includes 7 samples with t(8;21), 1 1 with inv(16), 1 1 with MLL translocations and NK AML (n=75) with FLT3-ITD (n=22), NPM1 (n=43) and DNMT3A mutations (n=30) (FIG. 12C). DNMT3A mutated samples did not reveal any significant GPCR enrichment when analyzed in the TCGA cohort. GPCR expression levels in normal CD34+ cells, for comparison, are shown as dashed lines in FIGs. 12A-B.
[00222] The upregulated genes in genetic subgroups were further analyzed to compare their expression in normal blood or bone marrow cell populations. ADRA2C, GPR126, CYSLTR2, RXFP1, GPR153 and CXCR7/ACKR3 show significant overexpression in specific AML genetic subgroups compared to the normal cell populations (FIGs. 16A and 16B).
[00223] The results revealed that 19 GPCRs are very highly expressed in human AML and 9 are more specific to AML cells compared to normal CD34+ cells. Moreover, 7 of these 9 GPCRs, i.e. CXCR4, CD97, PTGER4, GPR183, PTGER2, C3AR1 and EMR2 show little inter-specimen expression variability.
[00224] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:
1 . A method for treating a subject suffering from Acute Myeloid Leukemia (AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 and/or FPR2.
2. The method of claim 1 , wherein said at least one GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1 and/or C5AR1.
3. The method of claim 2, wherein said at least one GPCR is CXCR4, CD97, PTGER4, GPR183, PTGER2, C3AR1 and/or EMR2.
4. A method for treating a subject suffering from Core-Binding Factor Acute Myeloid Leukemia (CBF-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and/or RXFP1.
5. The method of claim 4, wherein said CBF-AML is AML with t(8;21) chromosomal rearrangement, and said at least one GPCR is ADRA2C, GPR153, LPAR5, LPAR6 and/or PTGIR.
6. The method of claim 5, wherein said at least one GPCR is ADRA2C and/or GPR153.
7. The method of claim 4, wherein said CBF-AML is AML with inv(16) chromosomal rearrangement, and said at least one GPCR is C5AR1, CCR2, CXCR7/ACKR3, FPR1, GPR183, RXFP1, LPAR6 and/or PTGIR.
8. The method of claim 7, wherein said at least one GPCR is CXCR7/ACKR3 and/or RXFP1.
9. A method for treating a subject suffering from Acute Myeloid Leukemia with Mixed Lineage Leukemia chromosomal rearrangement (MLL-AML), said method comprising administering to said subject an effective amount of an agent targeting the G-protein coupled receptor (GPCR) GPR126.
10. The method of claim 9, wherein said MLL-AML is MLL-MLLT4, MLL-ELL or MLL-SEPT9.
1 1 . A method for treating a subject suffering from normal karyotype Acute Myeloid Leukemia (NK-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CYSLTR2, GPR114 and/or GPR56.
12. The method of claim 1 1 , wherein said NK-AML is NK-AML with FLT3-ITD and/or NPM1 mutations.
13. The method of claim 1 1 or 12, wherein said GPCR is CYSLTR2.
14. A method for treating a subject suffering from myelomonocytic acute myeloid leukemia (M4-AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 and/or VIPR1.
15. A method for treating a subject suffering from monocytic acute myeloid leukemia (M5- AML), said method comprising administering to said subject an effective amount of an agent targeting at least one G-protein coupled receptor (GPCR), wherein said at least one GPCR is CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 and/or HTR7.
16. The method of any one of claims 1 to 15, wherein said agent is an antagonist of said at least one GPCR.
17. The method of any one of claims 1 to 16, wherein said agent is an antibody directed against said at least one GPCR, or an antigen-binding fragment thereof.
18. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for treating a subject suffering from Acute Myeloid Leukemia (AML), wherein said at least one GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 and/or FPR2.
19. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for the manufacture of a medicament for treating a subject suffering from Acute Myeloid Leukemia (AML), wherein said at least one GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 and/or FPR2.
20. The use of claim 18 or 19, wherein said at least one GPCR is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1 and/or C5AR1.
21 . The use of claim 20, wherein said at least one GPCR is CXCR4, CD97, PTGER4, GPR183, PTGER2, C3AR1 and/or EMR2.
22. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for treating a subject suffering from Core-Binding Factor Acute Myeloid Leukemia (CBF-AML), wherein said at least one GPCR is ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and/or RXFP1.
23. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for the manufacture of a medicament for treating a subject suffering from Core-Binding Factor Acute Myeloid Leukemia (CBF-AML), wherein said GPCR is ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and/or RXFP1.
24. The use of claim 22 or 23, wherein said CBF-AML is AML with t(8;21) chromosomal rearrangement, and said at least one GPCR is ADRA2C, GPR153, LPAR5, LPAR6 and/or PTGIR.
25. The use of claim 24, wherein said at least one GPCR is ADRA2C or GPR153.
26. The use of claim 22 or 23, wherein said CBF-AML is AML with inv(16) chromosomal rearrangement, and said at least one GPCR is C5AR1, CCR2, CXCR7/ACKR3, FPR1,
GPR183, RXFP1, LPAR6 and/or PTGIR.
27. The use of claim 26, wherein said at least one GPCR is CXCR7/ACKR3 and/or RXFP1.
28. Use of an agent targeting the G-protein coupled receptor (GPCR) GPR126 for treating a subject suffering from Acute Myeloid Leukemia with Mixed Lineage Leukemia chromosomal rearrangement (MLL-AML).
29. Use of an agent targeting the G-protein coupled receptor (GPCR) GPR126 for the manufacture of a medicament for treating a subject suffering from Acute Myeloid Leukemia with Mixed Lineage Leukemia chromosomal rearrangement (MLL-AML).
30. The use of claim 28 or 29, wherein said MLL-AML is MLL-MLLT4, MLL-ELL or MLL- SEPT9.
31 . Use of an agent targeting at least one G-protein coupled receptor (GPCR) for treating a subject suffering from normal karyotype Acute Myeloid Leukemia (NK-AML), wherein said at least one GPCR is CYSLTR2, GPR114 and/or GPR56.
32. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for the manufacture of a medicament for treating a subject suffering from normal karyotype Acute
Myeloid Leukemia (NK-AML), wherein said at least one GPCR is CYSLTR2, GPR114 and/or GPR56.
33. The use of claim 31 or 32, wherein said NK-AML is NK-AML with FLT3-\TD and/or NPM1 mutations.
34. The use of any one of claims 31 to 33, wherein said GPCR is CYSLTR2.
35. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for treating a subject suffering from myelomonocytic acute myeloid leukemia (M4-AML), wherein said at least one GPCR is FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 and/or VIPR1.
36. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for the manufacture of a medicament for treating a subject suffering from myelomonocytic acute myeloid leukemia (M4-AML), wherein said at least one GPCR is FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 and/or VIPR1.
37. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for treating a subject suffering from monocytic acute myeloid leukemia (M5-AML), wherein said at least one GPCR is CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 and/or HTR7.
38. Use of an agent targeting at least one G-protein coupled receptor (GPCR) for the manufacture of a medicament for treating a subject suffering from monocytic acute myeloid leukemia (M5-AML), wherein said at least one GPCR is CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 and/or HTR7.
39. The use of any one of claims 18 to 38, wherein said agent is an antagonist of said GPCR.
40. The use of any one of claims 18 to 39, wherein said agent is an antibody directed against said GPCR, or an antigen-binding fragment thereof.
41 . A method for determining whether a test agent may be useful for treating Acute Myeloid Leukemia (AML), said method comprising (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1, C5AR1, EMR1, GPR114, PTAFR, GPR160, ADRB2, CCR1, GPR109B, SUCNR1, GPR109A, P2RY2, P2RY13, GPR27, HRH2, PTH2R, CCRL2, PTGIR, GPR65, CX3CR1, CCR7, CCR2 and FPR2 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one of more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating AML
42. The method of claim 41 , wherein said one or more GPCRs is CXCR4, CD97, PTGER4, PTGER2, EMR2, GPR183, FPR1, C3AR1 and/or C5AR1.
43. The method of claim 42, wherein said one or more GPCRs is CXCR4, CD97, PTGER4, GPR183, PTGER2, C3AR1 and/or EMR2.
44. A method for determining whether a test agent may be useful for treating Core Binding Factor Acute Myeloid Leukemia (CBF-AML), said method comprising: (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): ADRA2C, GPR153, LPAR5, LPAR6, PTGIR, CCR2, CXCR7/ACKR3, FPR1, GPR183 and RXFP1 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one of more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating CBF-AML.
45. The method of claim 44, wherein said CBF-AML is AML with t(8;21) chromosomal rearrangement, and said one or more GPCRs is ADRA2C, GPR153, LPAR5, LPAR6 and/or PTGIR.
46. The method of claim 45, wherein said one or more GPCRs is ADRA2C and/or GPR153.
47. The method of claim 44, wherein said CBF-AML is AML with inv(16) chromosomal rearrangement, and said one or more GPCRs is C5AR1, CCR2, CXCR7/ACKR3, FPR1, GPR183, RXFP1, LPAR6 and/or PTGIR.
48. The method of claim 47, wherein said one or more GPCRs is CXCR7/ACKR3 and/or RXFP1.
49. A method for determining whether a test agent may be useful for treating Acute Myeloid Leukemia with Mixed Lineage Leukemia chromosomal rearrangement (MLL-AML), (i) contacting a cell expressing the G-protein coupled receptor (GPCR) GPR126 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said GPCR, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating MLL-AML.
50. The method of claim 49, wherein said MLL-AML is MLL-MLLT4, MLL-ELL or MLL- SEPT9.
51 . A method for determining whether a test agent may be useful for treating normal karyotype Acute Myeloid Leukemia (NK-AML), said method comprising: (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): CYSLTR2, GPR114 or GPR56 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one or more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating NK-AML.
52. The method of claim 51 , wherein said NK-AML is NK-AML with FL 73-ITD and/or NPM1 mutations.
53. The method of claim 51 or 52, wherein said one or more GPCRs is CYSLTR2.
54. A method for determining whether a test agent may be useful for treating myelomonocytic acute myeloid leukemia (M4-AML), said method comprising (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): FPR1, CCR2, C5AR1, GPR183, P2RY13, PTAFR, CCR1 or VIPR1 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one or more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating M4-AML.
55. A method for determining whether a test agent may be useful for treating monocytic acute myeloid leukemia (M5-AML), said method comprising (i) contacting a cell expressing one of more of the following G-protein coupled receptors (GPCRs): CCR1, PTAFR, GABBR1, CX3CR1, P2RY2, NMUR1 or HTR7 with said test agent; and (ii) determining whether said test agent binds to and/or modulates the activity of said one or more GPCRs, wherein said binding and/or modulation of activity is indicative that said test agent may be useful for treating M5-AML.
56. The method of any one of claims 41 to 55, wherein said method comprises determining whether said test agent inhibits the activity of said one or more GPCRs.
57. The method of any one of claims 41 to 56, wherein said agent is an antibody.
58. A method for determining the likelihood that a subject suffers from Core Binding Factor Acute Myeloid Leukemia (CBF-AML), said method comprising: determining the level of expression of at least one of the genes depicted in bold in Tables 8A and 8B in a leukemia cell sample from said subject, wherein a higher expression of said at least one genes depicted in bold in Table 8A, and/or a lower expression of said at least one genes depicted in bold in Table 8B, in said sample relative to a control non-CBF-AML sample is indicative that said subject has a high likelihood of suffering from CBF-AML.
59. The method of claim 58, wherein said method comprises determining the level of expression of at least one of ARC, CACNA1H and LOC200772, wherein a higher expression of said at least one genes in said sample relative to a control non-CBF-AML sample is indicative that said subject has a high likelihood of suffering from CBF-AML.
60. A method for determining the likelihood that a subject suffers from Core Binding Factor Acute Myeloid Leukemia (CBF-AML) with t(8;21)/ RUNX1-RUNX1T1 chromosomal rearrangement (t(8;21) AML), said method comprising: determining the level of expression of (i) at least one of the genes depicted in bold in Tables 6A and 6B; and/or (ii) at least one of the transcripts depicted in Table 11 , in a leukemia cell sample from said subject, wherein a higher expression of said at least one genes depicted in bold in Table 6A and/or of said at least one transcripts depicted in Table 11 , and/or a lower expression of said at least one genes depicted in bold in Table 6B, in said sample relative to a control non-t(8;21) AML sample is indicative that said subject has a high likelihood of suffering from t(8;21) AML.
61 . The method of claim 60, wherein said method comprises determining the level of expression of at least one of ADARB2-AS1 and LINC00958, wherein a higher expression of said at least one genes in said sample relative to a control non-t(8;21) AML sample is indicative that said subject has a high likelihood of suffering from t(8;21) AML.
62. A method for determining the likelihood that a subject suffers from Core Binding Factor Acute Myeloid Leukemia (CBF-AML) with inv(16)/t(16; 16)/CSFS- YW 7 chromosomal rearrangement (inv(16) AML), said method comprising: determining the level of expression of (i) at least one of the genes depicted in bold in Tables 7A and 7B; and/or (ii) at least one of the transcripts depicted in Table 10, in a leukemia cell sample from said subject, wherein a higher expression of said at least one genes depicted in bold in Table 7A and/or said at least one transcripts depicted in Table 10, and/or a lower expression of said at least one genes depicted in bold in Table 7B, in said sample relative to a control non-inv(16) AML sample is indicative that said subject has a high likelihood of suffering from inv(16) AML.
63. The method of claim 62, wherein said method comprises determining the level of expression of at least one of MEGF10 and APLN, wherein a higher expression of said at least one genes in said sample relative to a control non-inv(16) AML sample is indicative that said subject has a high likelihood of suffering from inv(16) AML.
64. The method of any one of claims 58 to 63, wherein said level of expression is measured at the nucleic acid level.
65. The method of claim 64, wherein said level of expression is measured by RNA sequencing (RNA-seq) or reverse transcription polymerase chain reaction (RT-PCR).
66. The method of any one of claims 56-63, wherein the level of expression of at least two genes is determined.
67. The method of any one of claims 56-63, wherein the level of expression of at least three genes is determined.
68. The method of any one of claims 56-63, wherein the level of expression of at least four genes is determined.
69. A method for determining the likelihood that a subject suffers from Core Binding Factor Acute Myeloid Leukemia (CBF-AML) with t(8;21)/ RUNX1-RUNX1T1 chromosomal rearrangement (t(8;21) AML), said method comprising: determining the presence of one or more mutations in at least one of the following genes: ZBTB7A, ASXL2, SMC1A, TET2, and DNMT3A in a sample comprising leukemic cells from the subject, wherein the presence of the one or more mutations in said at least one genes is indicative that the subject has a high likelihood of suffering from t(8;21) AML.
70. The method of claim 69, wherein the mutation is at a position corresponding to residue 122 and/or 174 of ZBTB7A.
71 . The method of claim 70, wherein the mutation is a V to G substitution at residue 122 of ZBTB7A.
72. The method of claim 70, wherein the mutation is a frameshift mutation at residue 174 of ZBTB7A.
73. The method of claim 69, wherein the mutation is at a position corresponding to residue 96 and/or 71 1 of SMC1A.
74. The method of claim 73, wherein the mutation is an R to H substitution at residue 96 of SMC1A.
75. The method of claim 73, wherein the mutation is an R to Q substitution at residue 71 1 of SMC1A.
76. A method for determining the likelihood that a subject suffers from AML, said method comprising: determining the presence of at least one of a RUNX1-USP2 fusion and a RUNX1- ERG fusion in a sample comprising leukemic cells from the subject, wherein the presence of said at least one RUNX1 fusion is indicative that the subject has a likelihood of suffering from AML.
77. A kit for carrying out the methods of any one of claims 56-74 comprising reagents for determining the level of expression of said at least one gene and/or the presence of said one or more mutations or fusions.
78. The kit of claim 77, wherein said reagents comprise one or more oligonucleotides comprising a sequence complementary to a sequence of said at least one genes.
79. The kit of claim 77 or 78, wherein said reagents comprise reagents for nucleic acid amplification.
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