WO2012175481A1 - Compositions et procédés destinés au traitement de la leucémie - Google Patents

Compositions et procédés destinés au traitement de la leucémie Download PDF

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WO2012175481A1
WO2012175481A1 PCT/EP2012/061675 EP2012061675W WO2012175481A1 WO 2012175481 A1 WO2012175481 A1 WO 2012175481A1 EP 2012061675 W EP2012061675 W EP 2012061675W WO 2012175481 A1 WO2012175481 A1 WO 2012175481A1
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inhibitor
igfir
bcr
leukemia
abl
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Jacques Ghysdael
Philippe Kastner
Stéphanie MOULIN
Clémence VIRELY
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Institut Curie
Inserm (Institut National De La Sante Et De La Recherche Medicale)
Universite De Strasbourg
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the field of medicine, in particular to the treatment of cancer. It relates to compositions and methods for treating leukemia. BACKGROUND OF THE INVENTION
  • the t(9;22) chromosomal translocation leading to the expression of a BCR-ABL fusion protein with constitutive tyrosine kinase activity is the founding genetic event in chronic myeloid leukemia (CML). This translocation is also found in about 25% of de novo adult and 3% childhood B-cell acute lymphoblastic leukemia (B-ALL) cases.
  • CML chronic myeloid leukemia
  • B-ALL B-cell acute lymphoblastic leukemia
  • IKAROS has an important function in maintaining the hematopoietic stem cell, in the commitment of multipotent lymphoid-myeloid progenitors into the lymphoid lineage and in specific aspects of pre- BCR signalling (reviewed in Yoshida et al. 2010).
  • IKAROS-deficient mice are thus devoid of B cells and only develop T cells in adulthood. Partial inactivation of IKZFl (e.g. the replacement of both wild type alleles by an hypomorphic allele) induces T- ALL, indicating that IKAROS has tumor suppressive function in the T-cell lineage.
  • IKAROS tumor suppressive function of IKAROS in the T- cell lineage involves its ability to repress the Notch signaling pathway through direct competition for DNA binding with the Notch-specific transcription factor RBP-JK/CSL (Kleinmann et al., 2008).
  • RBP-JK/CSL Notch-specific transcription factor
  • IKAROS is found to be associated predominantly with subunits of NuRD, an ATP-dependent nucleosome remodeling complex implicated in transcriptional repression and activation, but also with other transcriptional regulatory complexes (Sridharan et al., 2007).
  • IKZFl deletion results in haploinsufficiency, expression of dominant negative Ikaros isoforms, or the complete loss of IKAROS expression.
  • the dominant negative isoforms lack a functional DNA binding domain but contain the carboxyterminal zinc fingers that mediate their heterodimerization with full-length IKAROS and other Ikaros family members (e.g. Helios and Aiolos), driving the latter into non functional complexes.
  • IKZFl is found to be mutated in more than 80% of BCR-ABL B-ALL cases and CML lymphoid blast crisis (Mullighan et al. 2008). Furthermore, recent studies showed that the loss of a single wild-type IKZFl allele is sufficient to cooperate strongly with BCR-ABL in B-cell leukemogenesis (Virely et al, 2010). Similar IKZFl deletions are also frequently found in BCR-ABL-negative childhood B-ALL cases at high risk of treatment failure and in poor prognosis B-ALL associated with activating mutations in the JAK2 tyrosine kinase gene (Mullighan et al. 2009). Inactivating IKZFl mutations are also found, but at lower frequency, in standard B-ALL (about 10% of the cases) and in acute T cell lymphoblastic leukemia (less than 5% of cases).
  • IKZFl deletion is a predictor of poor therapeutic response (Mullighan et al. 2009).
  • BCR-ABL-targeted therapies are inefficient to induce long term remission in BCR-ABL-induced B-ALL and lymphoid blast crisis CML. Consequently, there is a strong need to improve therapeutic protocols to treat leukemia associated with IKZFl deletion, and in particular IKAROS-deficient BCR-ABL-induced B-ALL and lymphoid blast crisis CML.
  • the object of the present invention is to provide new therapeutic approaches for treating leukemia, in particular leukemia with impaired IKAROS function which is a predictor of poor therapeutic response.
  • IKZFl loss of function in leukemic cells in particular in BCR-ABL + cells, is associated with an increased expression of several gene products, in particular of two receptor-type protein tyrosine kinases, the IGFl receptor (IGFIR) and the vascular endothelial growth factor receptor 1 (FLT1/VEGFR1), which are known to play a facilitating role in a number of cancers.
  • IGFIR IGFl receptor
  • FLT1/VEGFR1 vascular endothelial growth factor receptor 1
  • the present invention concerns an inhibitor of the function of a gene which is upregulated in leukemic cells due to impaired IKAROS function, preferably a gene selected from the group consisting of IGFIR, FLTl, IGF2BP3, EMILINl, DOCKl and LAPTM4B genes, for use in the treatment of a leukemia associated with impaired IKAROS function.
  • the present invention concerns an inhibitor of the IGF1 receptor (IGFIR) or the receptor FLTl, for use in the treatment of a leukemia associated with impaired IKAROS function.
  • IGFIR IGF1 receptor
  • FLTl the receptor FLTl
  • the present invention concerns an inhibitor of the function of a gene selected from the group consisting of IGFIR and FLTl, preferably an inhibitor of the function of the IGFIR gene, for use in the treatment of a leukemia in a subject selected with the method of the invention as described below.
  • the inhibitor may be selected from the group consisting of a neutralizing antibody directed against IGFIR or FLTl, a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR or FLTl, an antibody directed against a IGFIR or FLTl ligand, a nucleic acid molecule interfering specifically with IGFIR or FLTl expression, a soluble decoy IGFIR or FLTl receptor, a dominant negative form of IGFIR or FLTl presenting a kinase dead domain, a peptide antagonist of IGFIR or FLTl and a molecule that reduces the level of at least one IGFIR or FLTl ligand.
  • the inhibitor is selected from the group consisting of a neutralizing antibody directed against IGFIR or FLTl and a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR or FLTl .
  • the inhibitor is an inhibitor of IGFIR.
  • the inhibitor is an inhibitor of FLTl .
  • the inhibitor may be an inhibitor of the function of the IGFIR gene selected from the group consisting of a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR and a neutralizing antibody directed against IGFIR.
  • the inhibitor is a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, more preferably selected from the group consisting of NVP- AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS-536924, BMS-554417, BMS-754807, BVP-51004 and ANT-429.
  • the leukemia to be treated associated with impaired IKAROS function may be a leukemia selected from the group consisting of an acute lymphoblastic leukemia and blast crisis chronic myelogenous leukemia. More particularly, the leukemia may be selected from the group consisting of BCR-ABL induced acute B cell lymphoblastic leukemia, lymphoid blast crisis chronic myelogenous leukemia, acute B cell lymphoblastic leukemia associated with activating mutation in JAK2 tyrosine kinase gene, acute B cell lymphoblastic leukemia and acute T cell lymphoblastic leukemia.
  • the leukemia associated with impaired IKAROS function is a BCR-ABL induced leukemia, preferably selected from the group consisting of BCR-ABL induced acute B cell lymphoblastic leukemia and lymphoid blast crisis chronic myelogenous leukemia.
  • the inhibitor of the invention may be used in combination with a BCR-ABL-targeted therapy, preferably with a BCR-ABL tyrosine kinase inhibitor, more preferably with a BCR-ABL tyrosine kinase inhibitor selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, and a combination thereof.
  • the BCR-ABL tyrosine kinase inhibitor is nilotinib.
  • the BCR-ABL tyrosine kinase inhibitor preferably nilotinib
  • a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS-536924, BMS-554417, BMS-754807, BVP-51004 and ANT-429, more preferably, being AEW541
  • the present invention also concerns a method, preferably an in vitro method, for (a) selecting a subject affected with a leukemia, preferably an acute lymphoblastic leukemia or blast crisis CML, for a therapy with an inhibitor of the invention, preferably an inhibitor of the function of the IGFIR gene, or (b) determining whether a subject affected with a leukemia, preferably an acute lymphoblastic leukemia or blast crisis CML, is susceptible to benefit from a therapy with an inhibitor of the invention, preferably an inhibitor of the function of the IGFIR gene, wherein the method comprises the step of determining the functional state of IKAROS in a sample of leukemic cells of the subject, an impaired IKAROS function indicating that a therapy with said inhibitor is suitable.
  • the step of determining the functional state of IKAROS may comprise detecting a deletion or a mutation in at least one coding exon of the IKZF1 gene.
  • the step of determining the functional state of IKAROS may comprise detecting a reduction of IKAROS protein expression.
  • the impaired IKAROS function is due to haploinsufficiency, expression of a dominant negative IKAROS isoform or the complete loss of IKAROS expression.
  • the present invention further concerns a method, preferably an in vitro method, for screening or identifying a molecule suitable for treating a leukemia associated with impaired IKAROS function, wherein the method comprises the steps of (i) contacting candidate molecules with IGFIR or FLTl receptor, and (ii) selecting molecules having the ability to bind to IGFIR or FLTl and/or to compete with and/or for a ligand of IGFIR or FLTl and/or to decrease the kinase activity of IGFIR or FLTl .
  • the method may further comprise the steps of (iii) administering a molecule selected in step (ii) in a non human animal model of leukemia with impaired IKAROS function, preferably a BCR-ABL +/0 ; IK L/+ mouse, and (iv) analyzing the effect on the disease progression.
  • a non human animal model of leukemia with impaired IKAROS function preferably a BCR-ABL +/0 ; IK L/+ mouse
  • the present invention concerns a method, preferably an in vitro method, for screening or identifying a molecule suitable for treating a leukemia in a subject selected with the method of the invention as described above, wherein the method comprises the steps of (i) contacting candidate molecules with IGFIR or FLTl receptor, preferably IGFIR, and (ii) selecting molecules having the ability to bind to IGFIR or FLTl, preferably IGFIR, and/or to compete with and/or for a ligand of IGFIR or FLTl, preferably IGFIR, and/or to decrease the kinase activity of IGFIR or FLTl, preferably IGFIR.
  • the method may further comprise the steps of (iii) administering a molecule selected in step (ii) in a non human animal model of leukemia with impaired IKAROS function, preferably a BCR-ABL 70 ; IK L/+ mouse, and (iv) analyzing the effect on the disease progression
  • the present invention concerns a combined preparation, product or kit containing (a) an inhibitor of the invention, preferably an inhibitor of the function of the IGFIR gene, and (b) another chemotherapeutic agent or an immunotherapy agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of a leukemia associated with impaired IKAROS, preferably a leukemia in a subject selected with the method of the invention as described above.
  • the other chemotherapeutic agent is an agent used to treat BCR-ABL induced leukemia, preferably BCR-ABL+ acute lymphocytic leukemia or blast crisis CML.
  • the other chemotherapeutic agent is a BCR-ABL tyrosine-kinase inhibitor, preferably selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, and a combination thereof, more preferably, being nilotinib.
  • the combined preparation, product or kit may contain (a) a low molecular weight inhibitor of the tyrosine kinase activity of IGF1R, preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS-536924, BMS-554417, BMS-754807, BVP-51004 and ANT-429, more preferably, being AEW541, and (b) a BCR-ABL tyrosine-kinase inhibitor, preferably nilotinib.
  • a low molecular weight inhibitor of the tyrosine kinase activity of IGF1R preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS-536924, BMS-554417, BMS-754807, BVP-51004 and
  • the present invention further concerns the use of a kit comprising
  • kits for (a) selecting a subject affected with a leukemia, preferably an acute lymphoblastic leukemia or blast crisis CML, for a therapy with an inhibitor of the invention, preferably an inhibitor of the function of the IGF1R gene ; and/or (b) determining whether a subject affected with a leukemia, preferably an acute lymphoblastic leukemia or blast crisis CML, is susceptible to benefit from a therapy with an inhibitor of the invention, preferably an inhibitor of the function of the IGF1R gene.
  • the kit may be used to determine the functional state of IKAROS in a sample of leukemic cells of the subject, an impaired IKAROS function indicating that a therapy with said inhibitor is suitable.
  • the present invention also concerns the use of a kit comprising (i) at least one probe specific to the genomic DNA, mRNA or cDNA of a gene selected from the group consisting of IGF1R, FLT1, IGF2BP3, EMILIN-1, DOCK1, LAPTM4B and H19, and/or
  • Figure 1 Identification of the transcriptional signature linked to the loss of function of Ikaros in BCR-ABL +/0 ; IK L/+ and BCR-ABL +/0 ; IK +/+ tumors.
  • the transcriptome of 5 BCR-ABL +/0 ; IK +/+ was compared to that of 4 BCR-ABL +/0 ; IK L/+ .
  • 240 genes are deregulated between BCR-ABL +/0 ; IK L/+ and BCR-ABL +/0 ; IK +/+ by hierarchical clustering analysis.
  • Figure2 Western blot analysis of IGF-1R and ⁇ -actin expression in sorted leukemic blasts (large B220 + IgM " cells) obtained from the bone marrow of BCR- ABL +/0 ; IK L/+ and BCR-ABL +/0 ; IK +/+ mice. Western blot were normalized using an antibody specific to ⁇ -actin.
  • Figure 3 Western blot analysis of IGFIR expression in cell lines obtained from the bone marrow of leukemic BCR-ABL +/0 ; CDK 2A " " ; IK +/+ and BCR-ABL +/0 ; CDK 2A " " ; IK L/+ mice, as indicated.
  • Figure 4 Flow cytometry analysis of the cell surface expression of IGFIR on cell lines derived from human BCR-ABL + B-ALL (SUP-B15) and CML in lymphoid blast crisis (BV-173), using a mAb to hIGFIR (clone IR3; Calbiochem/Merck) (top). Staining with an isotope control Ig is shown as control (bottom panel).
  • FIG. 5 Functional activation of IGFIR in human BCR-ABL + leukemic cells.
  • the indicated leukemic cell lines were deprived of fetal calf serum for 24 hours and either left untreated (-), or treated IGFl (+) for either 5 or 30 minutes as indicated.
  • Phosphorylation of IGFIR is detected upon IGFl stimulation (lOOng/ml recombinant IGFl) in human leukemic BV-173 cell line (A) and in the SUP-B15 human leukemic cell line (C).
  • Cells were also pre-treated for 60 minutes with 2 ⁇ NVP-AEW541 before IGFl stimulation.
  • the same extracts were analyzed for IGFIR expression using a specific antibody against IGFIR (B, D).
  • NVP-AEW541 treatment induces death of human leukemic cells.
  • Human leukemic cell lines BV-173 and SUP-B15 were treated for 24 hours with NVP- AEW541 at the indicated concentrations and compared to non-treated (DMSO) cells for their survival.
  • FSC LO cells are stained by propidium iodite (PI ) and thus represent dead cells.
  • FSC HI cells are not stained by propidium iodite (PT) and thus represent live cells (right panel).
  • B Treatment of BV-173 cells with the indicated concentration of NVP-AEW541; the percentages of live cells are recorded as relative % to wild-type.
  • FIG. 7 SUP-B15 (Fig. 7A) and BV-173 (Fig. 7B) cells were seeded at 10 6 cells/ml and treated with the indicated amounts of either NVP-AEW541 (1, 1.5 and 2 ⁇ ), or Nilotinib (10, 20, 40 and 50nM) and live cells at day 2 counted by trypan blue exclusion staining.
  • Figure 8 BV-173 cells were seeded at 10 6 cells/ml and treated with the indicated amounts of either NVP-AEW541 (0.5 and ⁇ ), or Nilotinib (5nM) or the indicated combination of Nilotinib and AEW (5nM Nilotinib and 0.5 or ⁇ NVP-AEW541 (0.5 and ⁇ ), or Nilotinib (5nM) or the indicated combination of Nilotinib and AEW (5nM Nilotinib and 0.5 or ⁇ NVP-
  • AEW541) and live cells at day 3 were counted by trypan blue exclusion staining.
  • BV-173 cells were seeded at 10 6 cells/ml and treated for 3 days with the indicated amounts of either NVP-AEW541 (0.5 and ⁇ ), or Nilotinib (5nM) or the combination of Nilotinib and AEW (5nM Nilotinib and 0.5 or ⁇ NVP-AEW541) and stained with either Propidium Iodide (PI) or 7-AAD. % Propidium iodide-positive cells
  • % cells with sub-Gl DNA content B
  • % cells staining positive for cleaved caspase C
  • Figure 10 BV-173 cells were seeded at 10 6 cells/ml and treated for 3 days with the indicated amounts of either NVP-AEW541 ( ⁇ ), or Nilotinib (5nM) or the combination of Nilotinib and AEW (5nM Nilotinib and ⁇ NVP-AEW541), pulse labeled with BrdU and stained with 7-AAD. % cells in S phase of the cell cycle were determined by flow cytometry.
  • FIG. 11 SUP-B15 cells were seeded at 10 6 cells/ml and treated with the indicated amounts of either NVP-AEW541 ( ⁇ ), or Nilotinib (5nM) or the indicated combination of nilotinib and AEW (5nM Nilotinib and ⁇ NVP-AEW541) and live cells were counted at day 3 by trypan blue exclusion staining. * -values were calculated as described in Materials and Methods.
  • Figure 12 SUP-B15 cells were seeded at 10 6 cells/ml and treated for 3 days with the indicated amounts of either NVP-AEW541 ( ⁇ ), or Nilotinib (5nM) or the indicated combination of Nilotinib and AEW (5nM Nilotinib and ⁇ NVP-AEW541) and stained with PI or an antibody to cleaved casapse 3.
  • A % Pi-positive cells; B: % caspase 3-positive cells. * -values were calculated as described in Materials and
  • Figure 13 BCR-ABL + ; CDK 2A " " ; IK L/+ B-ALL mouse leukemic cells were seeded at 0.4x10 6 cells/ml and treated with the indicated amounts of either NVP- AEW541 (1 and 2 ⁇ ), or Nilotinib ( ⁇ ), or the combination of AEW and Nilotinib ( ⁇ Nilotinib and 1 or 2 ⁇ NVP-AEW541) and analyzed 2 days later for cell death by Propidium Iodide (PI) staining and flow cytometry.
  • PI Propidium Iodide
  • Figure 14 BCR-ABL + ; CDKN2A " " ; IK L/+ B-ALL mouse leukemic cells were seeded at 0.4x10 6 cells/ml and treated with the indicated amounts of either NVP- AEW541 (1 and 2 ⁇ ), or Nilotinib ( ⁇ ), or the combination of AEW and Nilotinib ( ⁇ Nilotinib and 1 or 2 ⁇ NVP-AEW541) and counted for live cells by trypan blue exclusion at day 2.
  • * -values were calculated as described in Materials and Methods.
  • FIG. 15 BCR-ABL + ; CDKN2A " " ; IK L/+ B-ALL mouse leukemic cells were seeded at 0.4x10 6 cells/ml and treated with the indicated amounts of either NVP- AEW541 (2 ⁇ ), or Nilotinib ( ⁇ ), or the combination of AEW and Nilotinib ( ⁇ Nilotinib and 2 ⁇ NVP-AEW541) and counted for live cells by trypan blue exclusion at day 2.
  • * -values were calculated as described in Materials and Methods.
  • Figure 16 BCR-ABL + ; CDKN2A " " ; IK L/+ B-ALL mouse leukemic cells were seeded at 0.4x10 6 cells/ml and treated with the indicated amounts of either NVP- AEW541 (0.5 ⁇ ), or Nilotinib ( ⁇ ), or the combination of AEW and Nilotinib ( ⁇ Nilotinib and 0.5 ⁇ NVP-AEW541), pulse-labelled for 30 min with BrdU, stained with 7-AAD and analyzed by flow cytometry for AAD and BrdU staining.
  • IKZF1 loss of function in leukemic cells is associated with an increased expression of several components of signalling pathways known to play facilitating roles in a number of cancers.
  • IKZF1 haploinsufficiency in cells of BCR-ABL-induced B- cell acute lymphoblastic leukemia induces the up-regulation of
  • IGF1R IGF1 receptor
  • IGF2BP3 vascular endothelial growth factor receptor 3
  • FLT1 or VEGFR1 vascular endothelial growth factor receptor 1
  • IGF1R (Gene ID: 3480) is known to be expressed in a number of solid tumors and population studies have provided substantial evidence that cancer risk and prognosis are influenced by IGF1 and insulin levels.
  • Pre-clinical studies in mice xenografted with human solid tumors have shown that targeting IGF1R ligands by specific antibodies (Goya et al, 2004), targeting the IGF1R by specific neutralizing antibodies, or using low molecular weight inhibitors of the tyrosine kinase activity of the IGF1R, have clear anti-neoplastic activity (Pollak et al. 2008; Maki et al. 2010).
  • IGF2BP3 (Gene ID: 10643) encodes a positive regulator of IGF2 mRNA translation.
  • the expression of IGF2BP3 has been associated with an unfavorable outcome in renal clear cell carcinoma (Jiang et al, 2006), in ovarian clear cell carcinoma (Kobel et al., 2009) and in pancreatic ductal adenocarcinoma (Schaeffer et al., 2010).
  • FLT1 also named VEGFR1; Gene ID: 2321
  • VEGFR1 vascular endothelial growth factor B
  • P1GF placental derived growth factor
  • FLT1 and its two ligands, VEGFB and P1GF are upregulated in various solid tumors and multiple myeloma (Fischer et al. 2008).
  • FLT1 signalling favors an invasive phenotype (Fragoso et al. 2006).
  • FLT1/VEGFR1 Besides its direct effect on tumor cells, P1GF signals through FLT1/VEGFR1 in endothelial cells and by recruiting other cell types that upregulate VEGFA and other pro-angiogenic factors including FGF, PDFG, CXCL12, IL-8.
  • the pro-oncogenic function of FLT1/VEGFR1 signalling thus entails several aspects of oncogenesis.
  • Several inhibitors of FLT1 signalling have been developed.
  • a neutralizing antibody to mouse P1GF which inhibits binding to FLT1/VEGFR1 and its co-receptor Neuropilin 1
  • a humanized mAb to human FLT1/VEGFR1 ImClone; mAb IMC-18F1 has also shown anti-tumor effects towards human cells xenografted in mice (Wu et al. 2006).
  • EMILIN-1 (Gene ID: 11117) is a gene encoding an extracellular matrix glycoprotein which associates with elastic fibers at the interface between elastin and microfibrils and may play a role in the development of elastic tissues including large blood vessels, dermis, heart and lung. This protein is characterized by an N-terminal microfibril interface domain, an a-helical domain with high probability for coiled-coil structure formation in the central part, and a region homologous to the globular domain of Clq (gClq domain) at the carboxyl-terminal end. EMILIN-1 was shown to be able to promote tumor cell migration (Spessotto et al, 2003).
  • DOCK1 also named DOCK180; Gene ID: 1793 encodes a protein which is the main target of signal adaptor protein Crk and CrkL, that acts as a guanine-nucleotide exchange factor for small GTPase Racl .
  • signal transfer of Crk/Dockl80/Racl is implicated in actin cytoskeleton reorganization and thus in the cell proliferation, motility, invasion of human ovarian cancer cell line SKOV3 (Wang et al., 2010).
  • LAPTM4B (Gene ID: 55353) encodes a lysosomal/endosomal protein that is overexpressed in about 70% of solid tumors which is associated with metastatic potential. This protein was found to induce multidrug resistance of cancer cells by promoting drug efflux possibly through colocalization and interaction with P-gp, and anti-apoptosis by activating PI3K/AKT signalling (Li et al, 2010a; Li et al 2010b).
  • the present invention concerns an inhibitor of the function of a gene which is upregulated in leukemic cells due to impaired IKAROS function, preferably a gene selected from the group consisting of IGF1R, IGF2BP3, FLTl, EMILINl, DOCKl and LAPTM4B genes, for use in the treatment of leukemia associated with impaired IKAROS function.
  • the term "impaired IKAROS function” refers to the impaired function of the IKAROS protein encoded by the gene IKZF1 (IKAROS family zinc finger 1 ; Gene ID for the human gene IKZF1: 10320; NCBI Reference Sequence: NM 006060.3).
  • this term refers to a complete loss of function of the IKAROS protein.
  • it also refers to a partial loss of function, preferably of at least 50, 60, 70, 80 or 90%.
  • the impaired function may be due to deletion or mutation of IKZF1.
  • the mutation may result in the creation of stop codons, frameshift mutations, amino acid substitutions, particular RNA splicing, processing or translation efficiency, product instability, truncated polypeptide production, etc.
  • the mutation may result in the production of a polypeptide with altered function, stability, targeting or structure. It may also cause a reduction in protein expression that may be assessed for example by immunohistochemistry, semi-quantitative Western-blot or by protein or antibody arrays.
  • the impaired function of IKAROS is due to deletion of IKZF1.
  • Deletions may encompass any region of one, two or more residues in a coding or non- coding portion of the gene locus, such as from two residues up to the entire gene or locus.
  • the human gene IKZF1 encompasses seven exons (Mullighan et al, 2008).
  • the deletion encompasses at least one coding exon, more preferably 2, 3, 4, 5, 6 or 7 exons.
  • the deletion encompasses at least exons 3 to 5 encoding the DNA binding domain.
  • IKZF1 deletion may result in haploinsufficiency, expression of a dominant negative IKAROS isoform (for example the isoform encoded by IKZF1 lacking exons 3 to 6), or the complete loss of IKAROS expression.
  • the IKZF1 deletion may be assessed for example by fluorescence in situ hybridization (FISH), genomic quantitative polymerase chain reaction or by sequencing IKZF1 coding exons and/or regulatory sequences.
  • leukemia associated with impaired IKAROS function or "IKAROS deficient leukemia” refers to leukemia in which leukemic cells have impaired IKAROS function, i.e. have a deletion or mutation of IKZF1 resulting in an impaired function, preferably in a loss of function.
  • Leukemias associated with impaired IKAROS function may be found in several categories of leukemias such as BCR-ABL induced acute B cell lymphoblastic leukemias, lymphoid blast crisis chronic myelogenous leukemias, acute B cell lymphoblastic leukemias associated with an activating mutation in JAK2 tyrosine kinase gene, but also in standard acute B cell or T cell lymphoblastic leukemias.
  • the leukemia associated with impaired IKAROS function belongs to the category selected from the group consisting of BCR-ABL induced acute B cell lymphoblastic leukemia (BCR-ABL+ B-ALL), lymphoid blast crisis chronic myelogenous leukemia (blast crisis CML), acute B cell lymphoblastic leukemia (B-ALL) associated with an activating mutation in JAK2 tyrosine kinase gene, BCR-ABL-negative acute B cell lymphoblastic leukemia and BCR-ABL-negative T cell lymphoblastic leukemia.
  • BCR-ABL+ B-ALL BCR-ABL induced acute B cell lymphoblastic leukemia
  • blast crisis CML lymphoid blast crisis chronic myelogenous leukemia
  • B-ALL acute B cell lymphoblastic leukemia associated with an activating mutation in JAK2 tyrosine kinase gene
  • BCR-ABL-negative acute B cell lymphoblastic leukemia BCR-ABL-
  • the leukemia associated with impaired IKAROS function belongs to the category selected from the group consisting of acute B cell lymphoblastic leukemia (B-ALL) associated with an activating mutation in JAK2 tyrosine kinase gene, BCR-ABL-negative acute B cell lymphoblastic leukemia and BCR-ABL-negative T cell lymphoblastic leukemia, preferably selected from the group consisting of BCR-ABL-negative acute B cell lymphoblastic leukemia and BCR-ABL- negative T cell lymphoblastic leukemia.
  • B-ALL acute B cell lymphoblastic leukemia
  • the leukemia associated with impaired IKAROS function belongs to the category selected from the group consisting of BCR- ABL induced B-ALL, blast crisis CML, and B-ALL associated with an activating mutation in JAK2 tyrosine kinase gene.
  • the leukemia is characterized not only by impaired IKAROS function but also by the presence of the Philadelphia chromosome arising from the t(9;22)(q34;ql 1.2) translocation, which encodes the constitutively activated BCR-ABL1 (also named BCR-ABL) tyrosine kinase.
  • BCR-ABL1 also named BCR-ABL
  • the leukemia associated with impaired IKAROS function belongs to the category selected from the group consisting of BCR-ABL induced B-ALL and blast crisis CML.
  • a gene which is upregulated in leukemic cells due to the impaired IKAROS function refers to a gene for which the expression is increased in leukemic cells with impaired IKAROS function by comparison with the expression of said gene in leukemic cells having normal IKAROS function.
  • the expression level of a gene can be determined from a sample of leukemic cells by measuring the quantity of mRNA or the protein encoded by this gene using any technique known by the skilled person.
  • Leukemic cells may be obtained from bone marrow or peripheral blood sample by any method known by the skilled person such as Fluorescence-Activated Cell Sorting (FACS) using leukemic cell markers (e.g. CD45, CD 19 or CD90.2).
  • FACS Fluorescence-Activated Cell Sorting
  • the quantity of a specific mRNA may be measured, for instance, by quantitative or semi-quantitative RT-PCR, by real-time quantitative or semi-quantitative RT-PCR or by transcriptomic approaches.
  • the quantity of a protein may be measured, for instance, by immunohistochemistry or semi-quantitative western-blot.
  • Expression levels obtained from samples may be normalized by using expression levels of proteins which are known to have stable expression such as RPLPO (acidic ribosomal phosphoprotein PO) (de Cremoux et al, 2004), TBP (TATA box binding protein), GAPDH (glyceraldehyde 3 -phosphate dehydrogenase) or ⁇ -actin.
  • RPLPO acidic ribosomal phosphoprotein PO
  • TBP TATA box binding protein
  • GAPDH glycose dehydrogenase
  • a gene is upregulated in leukemic cells due to the impaired IKAROS function if, after normalization, the expression level of said gene in leukemic cells with impaired IKAROS function, is at least 2-fold higher, preferably 3, 4, 5, 6, 8 or 10-fold higher, than the expression level of said gene in leukemic cells with normal IKAROS function.
  • the upregulated gene is selected from the group consisting of IGF1R, IGF2BP3, FLTl, EMILIN-1, DOCKl and LAPTM4B. More preferably, the upregulated gene is selected from the group consisting of IGF1R, IGF2BP3 and FLTl. Even more preferably, the upregulated gene is IGF1R.
  • the term "inhibitor” refers to a biochemical or chemical compound which reduces or preferably inhibits the activity of gene product.
  • the inhibitor may act by reducing, preferably suppressing, the expression of the targeted gene or by reducing, preferably inhibiting, the activity of the gene product.
  • this inhibitor is specific to its target.
  • This inhibitor can be, for instance, an antibody, nucleic acid molecule interfering specifically with the expression of the targeted gene such as antisense oligonucleotide, RNAi or ribozyme, an aptamer, a binding peptide or protein, a low molecular weight inhibitor or any other class of inhibitors described herein.
  • the choice of the inhibitor essentially depends on the function of the gene product.
  • antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE, and human, humanized or chimeric antibody. In certain embodiments, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and they are most easily manufactured.
  • antibody is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab') 2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art.
  • a “humanized” antibody is an antibody in which the constant and variable framework region of one or more human immunoglobulins is fused with the binding region, e.g. the CDR, of an animal immunoglobulin.
  • “Humanized” antibodies contemplated in the present invention are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof. Such humanized antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody.
  • a "chimeric" antibody is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • RNAi or "interfering RNA” means any RNA which is capable of down-regulating the expression of the targeted gene. It encompasses small interfering RNA (siRNA), double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), micro- RNA (miRNA), and short hairpin RNA (shRNA) molecules.
  • RNAi can comprise naturally occurring RNA, synthetic RNA, or recombinant ly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • RNAi may be administered in free (naked) form or by the use of delivery systems that enhance stability and/or targeting, e.g., liposomes, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (WO 00/53722), or in combination with a cationic peptide (US 2007275923). They may also be administered in the form of their precursors or encoding DNAs.
  • the RNAi molecule is a siRNA of at least about 15-50 nucleotides in length, more preferably about 20-25 nucleotides in length.
  • siRNA are usually designed against a region 50-100 nucleotides downstream the translation initiator codon, whereas 5'UTR (untranslated region) and 3'UTR are usually avoided.
  • the chosen siRNA target sequence should be subjected to a BLAST search against EST database to ensure that the only desired gene is targeted.
  • Various products are commercially available to aid in the preparation and use of siRNA.
  • Antisense nucleic acid can be complementary to all or part of a sense nucleic acid encoding the target gene product e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence, and it thought to interfere with the translation of the target mRNA.
  • An antisense nucleic acid can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Particularly, antisense RNA molecules are usually 18-50 nucleotides in length.
  • An antisense nucleic acid for use in the method of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • antisense RNA can be chemically synthesized, produced by in vitro transcription from linear (e.g. PCR products) or circular templates (e.g., viral or non- viral vectors), or produced by in vivo transcription from viral or non- viral vectors.
  • Antisense nucleic acid may be modified to have enhanced stability, nuclease resistance, target specificity and improved pharmacological properties.
  • antisense nucleic acid may include modified nucleotides designed to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides.
  • the antisense nucleic acid is a RNA molecule complementary to a target mRNA encoding the targeted gene product.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes can be used to catalytically cleave mR A transcripts to thereby inhibit translation of the protein encoded by the mR A. Ribozyme molecules specific for a gene product can be designed, produced, and administered by methods commonly known to the art (see e.g., Fanning and Symonds, 2006, reviewing therapeutic use of hammerhead ribozymes and small hairpin RNA).
  • the term "aptamer” means a molecule of nucleic acid or a peptide which bind with high affinity to a specific target protein.
  • the aptamers are nucleic acids, preferably RNA, generally comprising between 5 and 120 nucleotides (Osborne et al, 1997). They can be selected in vitro according to a process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) (Tuerk and Gold, 1990).
  • the inhibitor is an inhibitor of EMILIN-1.
  • the inhibitor is a function-blocking monoclonal antibody against EMILIN-1 or a nucleic acid molecule interfering specifically with the expression of the EMILIN-1 gene such as a EMILIN-1 -specific RNAi. Examples of EMILIN-1 inhibition resulting in impaired adhesion and invasion of human cells using a function-blocking monoclonal antibody or a specific interfering RNA are described in Spessotto et al, 2003 and Spessotto et al, 2006.
  • the inhibitor is an inhibitor of DOCKl .
  • the inhibitor is a function-blocking monoclonal antibody against DOCKl or a nucleic acid molecule interfering specifically with the expression of the DOCKl gene, such as a DOCKl -specific RNAi. Examples of DOCKl inhibition using a specific interfering RNA that results in inhibition of human ovarian cancer cells is described in Wang et al, 2010.
  • the inhibitor is an inhibitor of LAPTM4B.
  • the inhibitor is a function-blocking monoclonal antibody against LAPTM4B or a nucleic acid molecule interfering specifically with the expression of the LAPTM4B gene, such as a LAPTM4B-spQcific RNAi.
  • LAPTM4B-spQcific RNAi examples of LATPM4B inhibitors, in particular LAPTM4B-spQcific RNAi, may be found in the specification of the European patent EP 1 732 649.
  • the inhibitor is an inhibitor of the function of the gene encoding the receptor IGFIR or FLTl .
  • the inhibitor induces the suppression or the reduction of the transmission of extracellular signals into the cell through IGFIR or FLTl .
  • the inhibitor may directly target the receptor or may target one or several of their ligands.
  • the inhibitor of IGFIR or FLTl may be a neutralizing antibody directed against the receptor IGFIR or FLTl, a low molecular weight inhibitor of the tyrosine kinase activity of the receptor IGFIR or FLTl, an antibody directed against a IGFIR or FLTl ligand, a nucleic acid molecule interfering specifically with IGFIR or FLTl expression, a soluble decoy IGFIR or FLTl receptor, a dominant negative form of IGFIR or FLTl presenting a kinase dead domain, a peptide antagonist of IGFIR or FLTl, or a molecule that reduces the extracellular level of at least one IGFIR or FLTl ligand.
  • the inhibitor of IGFIR or FLTl is a neutralizing antibody directed against the receptor IGFIR or FLTl or a low molecular weight inhibitor of the tyrosine kinase activity of the receptor IGF 1 R or FLT 1.
  • neutralizing antibody directed against IGFIR or FLTl designates an antibody as described above which is able to bind to the extracellular domain of IGFIR or FLTl and to block or reduce its activity. This inhibition can be due to steric hindrance or modification which prevents ligand binding.
  • antibody directed against a IGFIR or FLTl ligand designates an antibody as described above which is able to bind to one or several ligands of IGFIR or FLTl . Such antibodies are thus able to inhibit the activity of the receptor by preventing the binding of the ligand to the receptor.
  • low molecular weight inhibitor of the tyrosine kinase activity of the receptor IGFIR or FLTl refers to a molecule with the ability to inhibit or reduce the activity of the IGFIR or FLTl tyrosine kinase and which exhibits a molecular weight of less than 5000 Da, preferably less than 2000 Da, more preferably less than 1000 Da, most preferably less than 500 Da.
  • This molecule can be an organic or inorganic compound and can be derived from any known organism (including, but not limited to, animals, plants, bacteria, fungi and viruses) or from a library of synthetic molecules using high-throughput procedures.
  • nucleic acid molecule interfering specifically with IGFIR or FLTl expression refers to a nucleic acid molecule which is able to reduce or to suppress the expression of the IGFIR or FLTl gene, preferably in a specific way.
  • nucleic acid molecules include, but are not limited to, RNAi, antisense and ribozyme molecules, as described above.
  • soluble decoy IGFIR or FLTl receptor refers to an extracellular molecule which is able to bind to a IGFIR or FLTl ligand and thus to induce reduction or suppression of the activity of the transmembrane receptor by competition for its ligands or by heterodimerization with the wild type endogenous receptor.
  • This soluble decoy may consist essentially of the extra-cellular domain of the receptor or any peptide which has the ability to bind a IGFIR or FLTl ligand.
  • a "dominant negative form of IGFIR or FLTl presenting a kinase dead domain" is a receptor which is able to bind to its ligand but is defective for the transmission of the signal. Consequently, the over-expression of a dominant- negative receptor affects receptor signalling by blocking signal transduction. The presence of such dominant negative receptor at the cell surface induces a competition for ligand, decreasing the amount of available ligand for the active receptor and thus preventing the activation of this receptor.
  • the dominant negative form of IGFIR or FLTl presents a functional extracellular domain which binds a ligand of said receptor and a non- functional kinase domain which is unable to transmit the signal inside the cell via phosphorylation of intracellular substrates.
  • peptide antagonist of IGFIR or FLTl refers to a peptide of between 5 and 50 amino acids, preferably between 5 and 30 amino acids, more preferably between 5 and 20 amino acids, that is capable of inhibiting or reducing the function of IGFIR or FLTl .
  • peptide is intended to encompass peptide analogues (i.e. comprising one or more non-naturally occurring amino acid), peptide derivatives (i.e. comprising additional chemical or biochemical moieties not normally a part of a naturally occurring peptide) and peptidomimetics (i.e. compounds that are structurally similar to a peptide and contains chemical moieties that mimic the function of the peptide).
  • the term "molecule that reduces the level of at least one IGFIR or FLTl ligand” refers to a molecule which is able to inhibit the expression of a gene encoding a ligand of IGFIR or FLTl, and/or to reduce the level of such a ligand in the serum.
  • the inhibitor is an inhibitor of IGFIR, preferably an inhibitor that is specific to the function of the IGFIR gene.
  • the inhibitor of IGFIR may be a neutralizing antibody directed against IGFIR, a low molecular weight inhibitor of IGFIR, in particular of the tyrosine kinase activity of IGFIR, an antibody directed against a IGFIR ligand, a nucleic acid molecule interfering specifically with IGFIR expression, a soluble decoy IGFIR receptor, a dominant negative form of IGFIR presenting a kinase dead domain, a peptide antagonist of IGFIR, or a molecule that reduces the level of at least one IGFIR ligand.
  • the inhibitor of IGFIR may be a neutralizing antibody directed against IGFIR.
  • examples of such antibody include, but are not limited to, CP-751871 (Pfizer), IMC- A12 (Imclone), R1507 (Roche), AMG-479 (Amgen), SCH-717454 (Schering-Plough), AVE- 1642 (Sanofi-Aventis), MK-0646 (Merck) and BIIB022 (Biogen pou).
  • the inhibitor of IGFIR may also be a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR.
  • Examples of such molecule include, but are not limited to, INSM-18 (Insmed/UCSF), OSI-906 (OSI Pharmaceuticals), XL-228 (Exelixis), NVP-ADW742 (Novartis), NVP-AEW541 (Novartis), AG- 1024 (Merk), BMS-536924 (Bristol-Myers Squibb), BMS-554417 (Bristol-Myers Squibb), BMS- 754807 (Bristol-Myers Squibb), BVP-51004 (Biovitrum) and ANT-429 (Antyra).
  • the inhibitor of IGFIR may be an antibody directed against a IGFIR ligand, preferably against IGFl and/or IGF2.
  • IGFIR ligand preferably against IGFl and/or IGF2.
  • IGFl and/or IGF2 examples include, but are not limited to, KM 1468 (an antibody directed against human IGF-1 and IGF-2; Goya et al., 2004) and the human monoclonal antibody IgGl m610 (as described in Feng et al., 2006).
  • the inhibitor of IGFIR may also be a nucleic acid molecule interfering specifically with IGFIR expression.
  • examples of such molecule include, but are not limited to, the antisense inhibitor ATL-1101 (Antisense Therapeutics) which induced decreased proliferation and increased apoptosis in human prostate cancer cell lines and suppressed their growth as xenografts in mice (Furukawa J. et al. 2010).
  • the inhibitor of IGFIR may be a soluble decoy IGFIR receptor. Examples of such molecule include, but are not limited to, the soluble truncated form of the IGFIR 486/STOP or 482/STOP (Reiss et al, 1998).
  • the inhibitor of IGFIR may be a dominant negative IGFIR receptor presenting a kinase dead domain.
  • IGF1 receptor comprising a point mutation of lysine residue K1003, and/or tyrosine residue Y1131, Y1135, Y1136, Y1250 and/or Y1251 (Burgaud et al, 1995), and a truncated form of the receptor 950/STOP (Sachdev et al, 2004).
  • the inhibitor of IGF1R may be an antagonist peptide.
  • Examples of such molecule include, but are not limited to, the peptides described in the US patent 7,071,300 that antagonize the interaction of IGF 1 with its receptor.
  • the inhibitor of IGF1R may be a molecule that reduces the level of at least one IGF1R ligand, preferably IGF1 or IGF2, more preferably that reduces the level of IGF 1 and IGF2.
  • IGF1R ligand preferably IGF1 or IGF2, more preferably that reduces the level of IGF 1 and IGF2.
  • growth hormone releasing hormone antagonists such as JV-1-38 (Rekasi et al, 2005; Elixir)
  • growth hormone receptor antagonists such as pegvisomant (Pfizer)
  • nucleic acid molecules interfering specifically with IGF1 or IGF2 expression or neutralizing antibodies to human IGF1 or IGF2 as described by Miyamoto et al ((Miyamoto et al. 2005) or Goya et al. (Goya et al. 2004) and that induced tumor growth inhibition in a xenograft model of colorectal cancer.
  • the inhibitor of the function of the IGF1R gene is selected from the group consisting of a low molecular weight inhibitor of the tyrosine kinase activity of IGF1R and a neutralizing antibody directed against IGF1R. More preferably, the inhibitor is a low molecular weight inhibitor of the tyrosine kinase activity of IGF1R.
  • the inhibitor is a low molecular weight inhibitor of the tyrosine kinase activity of IGF1R selected from the group consisting of INSM-18 (Insmed/UCSF), OSI-906 (OSI Pharmaceuticals), XL-228 (Exelixis), NVP-ADW742 (Novartis), NVP-AEW541 (Novartis), AG- 1024 (Merk), BMS-536924 (Bristol-Myers Squibb), BMS-554417 (Bristol-Myers Squibb), BMS-754807 (Bristol-Myers Squibb), BVP-51004 (Biovitrum) and ANT-429 (Antyra).
  • the inhibitor of the function of the IGF1R gene is NVP-AEW541, a specific IGF1R inhibitor.
  • the inhibitor is an inhibitor of FLT1.
  • the inhibitor of FLT1 may be a neutralizing antibody directed against FLT1.
  • Examples of such antibody include, but are not limited to, IMC-18F1 (ImClone_Systems Incorporated) that induced growth inhibition in several mouse xenograft models of breast cancer (Wu et al. 2006).
  • the inhibitor of FLTl may also be a low molecular weight inhibitor of the tyrosine kinase activity of FLTl .
  • Examples of such molecule include, but are not limited to, Sunitinib (Pfizer), Vandetanib (AstraZeneca), Cediranib (AstraZeneca), Pazopanib (GlaxoSmithKline), Axitinib (Pfizer) and AMG706 (Amgen).
  • the inhibitor of FLTl may be an antibody directed against a FLTl ligand, preferably against VEGF-B and/or PIGF.
  • a FLTl ligand preferably against VEGF-B and/or PIGF.
  • examples of such molecule include, but are not limited to, the alphaPlGF antibody which is an antibody against placental growth factor (Fischer et al., 2007).
  • the inhibitor of FLTl may also be a nucleic acid molecule interfering specifically with FLTl expression.
  • examples of such molecule include, but are not limited to, the anti-FLTl ribozyme, as described by Pavco et al. (Pavco et al., 2000), that inhibited primary tumor growth and metastasis in a mouse xenograft model of human colorectal cancer.
  • the inhibitor of FLTl may be a soluble decoy FLTl receptor that prevents FLTl ligands from binding to the transmembrane FLTl receptor.
  • Examples of such molecule include, but are not limited to the decoy-soluble FLTl receptor described by Holash et al. (Holash et al. 2002), that suppressed growth of a variety of human tumors xenotransplanted to mice.
  • the inhibitor of FLTl may be a dominant negative FLTl receptor presenting a kinase dead domain.
  • Examples of such molecule include, but are not limited to the dominant negative FLTl receptor described by Ito et al. (Ito et al, 2001).
  • the inhibitor of FLTl may be a peptide antagonist.
  • examples of such molecule include, but are not limited to the peptide described by Taylor et al. that inhibited motility and invasion of breast cancer cell lines in vitro and their metastasis in vivo in a xenograft model (Taylor et al. 2007 ; Bae et al. 2005).
  • the inhibitor of FLTl may be a molecule that reduces the level of at least one FLTl ligand, preferably VEGF-B or PIGF, more preferably that reduces the level of VEGF-B and PIGF.
  • FLTl ligand preferably VEGF-B or PIGF
  • Examples of such molecule include, but are not limited to, a nucleic acid molecule interfering specifically with VEGF-B or PIGF as described by Akrami et al . (Akrami et al, 2010) or Xu et al. (Xu et al, 2011).
  • the inhibitor according to the invention may be used in combination with other therapy such as other chemotherapy, immunotherapy, radiotherapy, bone marrow or peripheral blood stem cell transplant, or surgery.
  • the inhibitor may also be used in combination with supportive therapy to manage complications from leukemia treatment such as administration of antibiotics or antifungals to fight infections, administration of blood products as replacement therapy when blood cell counts are low or leukapheresis to remove large numbers of white blood cells from the blood.
  • the term "immunotherapy” refers to a cancer therapeutic treatment using the immune system to reject cancer.
  • the therapeutic treatment stimulates the patient's immune system to attack the malignant tumor cells. It includes immunization of the patient with tumoral antigens (eg. by administering a cancer vaccine), in which case the patient's own immune system is trained to recognize tumor cells as targets to be destroyed, or administration of molecules stimulating the immune system such as cytokines, or administration of therapeutic antibodies as drugs, in which case the patient's immune system is recruited to destroy tumor cells by the therapeutic antibodies.
  • radiation therapy is a term commonly used in the art to refer to multiple types of radiation therapy including internal and external radiation therapy, radio immunotherapy, and the use of various types of radiation including X-rays, gamma rays, alpha particles, beta particles, photons, electrons, neutrons, radioisotopes, and other forms of ionizing radiation.
  • radiation therapy can be used to treat disease that may have spread outside the bone marrow, to relieve bone pain or for total body irradiation before a stem cell transplant.
  • the chemotherapy used in combination with the inhibitor of the invention may depend on the type of leukemia to be treated.
  • the inhibitor of the invention may be used, for example, in combination with vincristine, daunorubicin, doxorubicin, idarubicin, mitoxantrone, cytarabine, asparaginase, etoposide, teniposide, mercaptopurine, methotrexate, cyclophosphamide, prednisone, dexamethasone, busalfan, hydroxyurea or interferon alpha.
  • the leukemia to be treated is BCR-ABL induced, preferably BCR-ABL + acute lymphoblastic leukemia or blast crisis CML.
  • the inhibitor of the invention may be used in combination with one or several BCR-ABL-targeted therapies.
  • BCR-ABL-targeted therapies include, but are not limited to BCR-ABL tyrosine kinase inhibitors, farnesyl transferase inhibitors, MAP/ER kinase inhibitors, phosphatidylinosito 1-3 '-kinase inhibitors, PDK1 inhibitors and mTOR inhibitors.
  • BCR-ABL tyrosine kinase inhibitors include, but are not limited to, imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-354825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), ON012380, the pyrazolopyrimidine PP1 (Tatton et al., 2003), CGP76030 and PD166326.
  • Farnesyl transferase inhibitors include, but are not limited to, tipifarnib (Rl 15777), lonafarnib (SCH66336) and BMS-214662.
  • MAP/ERK kinase inhibitors include, but are not limited to, U0126 and CI- 1040 (PD 184352).
  • Phosphatidylinosito 1-3 '-kinase inhibitors include, but are not limited to, LY294002, wortmannin and PX-866.
  • PDK1 inhibitors include, but are not limited to, OSU-03012.
  • mTOR inhibitors include, but are not limited to, sirolimus (rapamycin), temsirolimus (CCI-779), everolimus (RAD001) and deforolimus (AP23573).
  • the leukemia to be treated is BCR-ABL induced and the inhibitor of the invention is used in combination with a BCR-ABL tyrosine-kinase inhibitor.
  • the BCR-ABL tyrosine kinase inhibitor is selected from the group consisting of imatinib, nilotinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, the pyrazolopyrimidine PP1, CGP76030 and PD 166326, and a combination thereof.
  • the BCR-ABL tyrosine kinase inhibitor is selected from the group consisting of imatinib, nilotinib, dasatinib, bosutinib, ponatinib, bafetinib and ON012380, and a combination thereof. Even more preferably, the BCR-ABL tyrosine kinase inhibitor is nilotinib.
  • an inhibitor of the function of the IGFIR gene in particular a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, in combination with a BCR-ABL tyrosine-kinase inhibitor, induces a synergistic effect (see for example Figure 16).
  • an IGFIR inhibitor namely NVP- AEW541
  • a BCR-ABL tyrosine-kinase inhibitor namely Nilotinib
  • the administered amounts of drugs in particular the amounts of IGFIR inhibitor and/or BCR-ABL tyrosine-kinase inhibitor, may be decreased and adverse effects may be prevented or reduced.
  • this potentiating effect may allow to reduce the administration frequency or the treatment period.
  • the leukemia to be treated is BCR-ABL induced and a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, preferably selected from the group consisting of INSM-18, OSI-906, XL-228, NVP- ADW742, NVP-AEW541, AG-1024, BMS-536924, BMS-554417, BMS-754807, BVP- 51004 and ANT-429, more preferably being NVP-AEW541, is used in combination with a BCR-ABL tyrosine-kinase inhibitor, preferably selected from the group consisting of imatinib, nilotinib, dasatinib, bosutinib, ponatinib, bafetinib and ON012380, more preferably being nilotinib.
  • a BCR-ABL tyrosine-kinase inhibitor preferably selected from the group consisting of imatinib,
  • the leukemia to be treated is BCR-ABL induced, preferably BCR-ABL + acute B cell lymphoblastic leukemia or blast crisis CML, and the IGF1R inhibitor NVP-AEW541 is used in combination with the BCR- ABL tyrosine-kinase inhibitor nilotinib.
  • the leukemia to be treated is associated with an activating mutation in JAK2 tyrosine kinase gene, preferably is B-ALL associated with an activating mutation in JAK2 tyrosine kinase gene.
  • the inhibitor of the invention may be used in combination with one or several JAK2 tyrosine kinase inhibitors such as AG490 or lestaurtinib (CEP-701 ) .
  • the leukemia to be treated is BCR-ABL induced and associated with activating mutation in JAK2 tyrosine kinase gene.
  • the inhibitor of the invention may be used in combination with one or several JAK2 tyrosine kinase inhibitors and a BCR-ABL-targeted therapy.
  • the pharmaceutical composition comprising the inhibitor of the invention is formulated in accordance with standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art.
  • standard pharmaceutical practice see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York
  • Possible pharmaceutical compositions include those suitable for oral, rectal, topical (including transdermal, buccal and sublingual), or parenteral (including subcutaneous, intramuscular, intraspinal, intravenous and intradermal) administration.
  • parenteral including subcutaneous, intramuscular, intraspinal, intravenous and intradermal.
  • conventional excipient can be used according to techniques well known by those skilled in the art.
  • the pharmaceutical composition is suitable for parenteral administration.
  • compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions which can optionally be prepared immediately before use from solid or lyophilized form.
  • Adjuvants such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle and a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the active ingredient.
  • the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules and liquid preparations such as syrups, elixirs, and concentrated drops.
  • Non toxic solid carriers or diluents may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like.
  • binders which are agents which impart cohesive qualities to powdered materials, are also necessary.
  • starch, gelatine, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders.
  • Disintegrants are also necessary in the tablets to facilitate break-up of the tablet.
  • Disintegrants include starches, clays, celluloses, algins, gums and crosslmked polymers.
  • lubricants and glidants are also included in the tablets to prevent adhesion to the tablet material to surfaces in the manufacturing process and to improve the flow characteristics of the powder material during manufacture.
  • Colloidal silicon dioxide is most commonly used as a glidant and compounds such as talc or stearic acids are most commonly used as lubricants.
  • composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethy lformamide .
  • nasal sprays for transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used.
  • the active compound can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate.
  • compositions according to the invention may be formulated to release the active drug substantially immediately upon administration or at any predetermined time or time period after administration.
  • compositions may comprise one or several inhibitors of the invention associated with pharmaceutically acceptable excipients and/or carriers. These excipients and/or carriers are chosen according to the form of administration as described above.
  • the pharmaceutical compositions may also comprise at least one another active compound, in particular another antitumoral drug.
  • the amount of inhibitor of the invention to be administered has to be determined by standard procedure well known by those of ordinary skill in the art. Physiological data of the patient (e.g. age, size, and weight), the routes of administration and the disease to be treated have to be taken into account to determine the appropriate dosage. The appropriate dosage of each inhibitor may also vary if it is used alone or in combination.
  • each unit dosage may contain, for example, from 200 to 1000 mg/kg of body weight, particularly from 500 to 800 mg/kg of body weight. If the inhibitor is an antibody as defined above, each unit dosage may contain, for example, from 0.1 to 20 mg/kg of body weight, particularly from 4 to 10 mg/kg of body weight. If the inhibitor is an interfering nucleic acid molecule as defined above, each unit dosage may contain, for example, from 2 to 50 mg/kg of body weight, particularly from 5 to 20 mg/kg of body weight. If the inhibitor is a dominant negative form or a soluble bait as defined above, each unit dosage may contain, for example, from 5 to 100 mg/kg of body weight, particularly from 15 to 70 mg/kg of body weight.
  • the present invention also relates to
  • composition comprising an inhibitor of the invention, and optionally a pharmaceutically acceptable carrier, for use in the treatment of a leukemia associated with impaired IKAROS function, optionally in combination with radiotherapy, an immunotherapy agent or another chemotherapeutic agent;
  • an inhibitor of the invention and optionally a pharmaceutically acceptable carrier, for use in the treatment of a leukemia associated with impaired IKAROS function, optionally in combination with radiotherapy, an immunotherapy agent or another chemotherapeutic agent;
  • an inhibitor of the invention for the manufacture of a medicament for the treatment of a leukemia associated with impaired IKAROS function, optionally in combination with radiotherapy, an immunotherapy agent or another chemotherapeutic agent;
  • a method for treating a leukemia associated with impaired IKAROS function in a subject in need thereof comprising administering a therapeutically efficient amount of a pharmaceutical composition comprising an inhibitor of the invention and optionally a pharmaceutically acceptable carrier;
  • a combined preparation, product or kit containing (a) an inhibitor of the invention and (b) an immunotherapy agent or another chemotherapeutic agent as a combined preparation for simultaneous, separate or sequential use, in particular in the treatment of a leukemia associated with impaired IKAROS;
  • a method for treating a leukemia associated with impaired IKAROS in a subject in need thereof comprising administering an effective amount of a pharmaceutical composition comprising an inhibitor of the invention, and a therapeutically efficient amount of a pharmaceutical composition comprising, an immunotherapy agent or another chemotherapeutic agent; and,
  • a method for treating a leukemia associated with impaired IKAROS in a subject in need thereof comprising administering a therapeutically efficient amount of a pharmaceutical composition comprising an inhibitor of the invention in combination with radiotherapy.
  • the inhibitor of the invention is an inhibitor of the function of the
  • IGF1R gene in particular a low molecular weight inhibitor of the tyrosine kinase activity of IGF1R.
  • the inhibitor is selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS- 536924, BMS-554417, BMS-754807, BVP-51004 and ANT-429. Even more preferably, the inhibitor is NVP-AE W541.
  • the other chemotherapeutic agent is an agent used to treat BCR-ABL induced leukemia, in particular BCR-ABL + acute lymphocytic leukemia or blast crisis CML.
  • the other chemotherapeutic agent is a BCR-ABL tyrosine- kinase inhibitor, as described herein and preferably is selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, and a combination thereof. Even more preferably, the other chemotherapeutic agent is nilotinib.
  • the present invention relates to - a pharmaceutical composition
  • a pharmaceutical composition comprising a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS-536924, BMS-554417, BMS-754807, BVP-51004 and ANT-429, more preferably being NVP- AEW541, and optionally a pharmaceutically acceptable carrier, for use in the treatment of a leukemia associated with impaired IKAROS function, in combination with a BCR- ABL tyrosine-kinase inhibitor, preferably selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, more preferably being nilotinib;
  • composition comprising (a) a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG-1024, BMS- 536924, BMS-554417, BMS-754807, BVP-51004 and ANT-429, more preferably being NVP-AEW541, (b) a BCR-ABL tyrosine-kinase inhibitor, preferably selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, more preferably being nilotinib, and optionally a pharmaceutically acceptable carrier, for use in the treatment of a leukemia associated with impaired IKAROS function;
  • a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR preferably selected from the group consisting of NVP-AEW541 , INSM-18, OSI-906,
  • NVP-ADW742 AG-1024, BMS-536924, BMS-554417, BMS-754807, BVP- 51004 and ANT-429, more preferably being NVP-AEW541, and optionally a pharmaceutically acceptable carrier, for use in the treatment of a leukemia associated with impaired IKAROS function, in combination with a BCR-ABL tyrosine-kinase inhibitor, preferably selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, more preferably being nilotinib;
  • a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR preferably selected from the group consisting of NVP-AEW541 , INSM-18, OSI-906, XL-228, NVP-ADW742, AG-1024, BMS-536924, BMS-554417, BMS-754807, BVP- 51004 and ANT-429, more preferably being NVP-AEW541,
  • a BCR-ABL tyrosine- kinase inhibitor preferably selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, more preferably being nilotinib and optionally a pharmaceutically acceptable carrier, for use in the treatment of a leukemia associated with impaired IKAROS function;
  • a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS-536924, BMS-554417, BMS- 754807, BVP-51004 and ANT-429, more preferably being NVP-AEW541, for the manufacture of a medicament for the treatment of a leukemia associated with impaired IKAROS function, in combination with a BCR-ABL tyrosine-kinase inhibitor, preferably selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, more preferably being nilotinib;
  • a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS-536924, BMS-554417, BMS- 754807, BVP-51004 and ANT-429, more preferably being NVP-AEW541,
  • a BCR- ABL tyrosine-kinase inhibitor preferably selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, more preferably being nilotinib, for the manufacture of a medicament for the treatment of a leukemia associated with impaired IKAROS function;
  • a method for treating a leukemia associated with impaired IKAROS function in a subject in need thereof comprising administering a therapeutically efficient amount of a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS-536924, BMS-554417, BMS-754807, BVP-51004 and ANT-429, more preferably being NVP- AEW541, (b) a BCR-ABL tyrosine-kinase inhibitor, preferably selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, more preferably being nilotinib, and optional
  • a combined preparation, product or kit containing (a) a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG-1024, BMS-536924, BMS-554417, BMS-754807, BVP-51004 and ANT-429, more preferably being NVP-AEW541 and (b) a BCR-ABL tyrosine-kinase inhibitor, preferably selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, more preferably being nilotinib, as a combined preparation for simultaneous, separate or sequential use, in particular in the treatment of a leukemia associated with impaired IKAROS;
  • a method for treating a leukemia associated with impaired IKAROS in a subject in need thereof comprising administering a therapeutically efficient amount of a pharmaceutical composition comprising a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS-536924, BMS-554417, BMS-754807, BVP-51004 and ANT-429, more preferably being NVP- AEW541, and a therapeutically efficient amount of a pharmaceutical composition comprising a BCR-ABL tyrosine-kinase inhibitor, preferably selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, more preferably being nilotinib
  • a method for treating a leukemia associated with impaired IKAROS in a subject in need thereof comprising administering a therapeutically efficient amount of a pharmaceutical composition comprising (a) a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS-536924, BMS-554417, BMS-754807, BVP-51004 and ANT-429, more preferably being NVP- AEW541, and (b) a BCR-ABL tyrosine-kinase inhibitor, preferably selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, more preferably being nilotinib; and,
  • a method for treating a leukemia associated with impaired IKAROS in a subject in need thereof comprising administering a therapeutically efficient amount of a pharmaceutical composition comprising a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS-536924, BMS-554417, BMS-754807, BVP-51004 and ANT-429, more preferably being NVP- AEW541, in combination with radiotherapy.
  • a pharmaceutical composition comprising a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG- 1024, BMS-536924, BMS-554417, BMS-7548
  • the leukemia to be treated is a leukemia in a subject selected with the method of the invention as described below, i.e. the method for selecting a subject affected with a leukemia for a therapy with an inhibitor of the invention.
  • subject refers to an animal, preferably to a mammal, even more preferably to a human, including adult, child and human at the prenatal stage.
  • treatment refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease.
  • such term refers to the amelioration or eradication of a disease or symptoms associated with a disease.
  • this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.
  • a “therapeutically efficient amount” is intended an amount of therapeutic agent, an inhibitor of the invention, administered to a subject that is sufficient to constitute a treatment of a leukemia associated with impaired IKAROS as defined above.
  • IKAROS- deficient leukemic cells with the combination of a IGFIR inhibitor, namely NVP- AEW541, and a BCR-ABL tyrosine-kinase inhibitor, namely nilotinib, induced a massive cell death, greater than their additive effects when used individually (see for example Figures 8 and 15).
  • a IGFIR inhibitor namely NVP- AEW541
  • a BCR-ABL tyrosine-kinase inhibitor namely nilotinib
  • leukemic cells a human cell line established from IKAROS-deficient BCR-ABL- induced acute B-cell lymphoblastic leukemia (SUP-B15), a human cell line established from IKAROS-deficient CML blast crisis (BV-173), and BCR-ABL +/0 ; IK L/+ mouse leukemic cells.
  • the present invention concerns
  • a pharmaceutical composition comprising (a) an inhibitor of the function of the IGFIR gene and (b) a BCR-ABL tyrosine-kinase inhibitor and optionally a pharmaceutically acceptable carrier, for use in the treatment of a leukemia associated with impaired IKAROS function, wherein the relative amounts of the IGFIR inhibitor and the BCR-ABL tyrosine-kinase inhibitor are such that they exhibit a synergistic therapeutic effect upon administration to a subject;
  • a pharmaceutical composition comprising an inhibitor of the function of the IGFIR gene, and optionally a pharmaceutically acceptable carrier, for use in the treatment of a leukemia associated with impaired IKAROS function, in combination with a BCR-ABL tyrosine-kinase inhibitor, wherein the relative amounts of the IGFIR inhibitor and the BCR-ABL tyrosine-kinase inhibitor are such that they exhibit a synergistic therapeutic effect upon administration to a subject
  • an inhibitor of the function of the IGFIR gene and optionally a pharmaceutically acceptable carrier, for use in the treatment of a leukemia associated with impaired IKAROS function, in combination with a BCR-ABL tyrosine-kinase inhibitor, wherein the relative amounts of the IGFIR inhibitor and the BCR-ABL tyrosine-kinase inhibitor to be administered are such that they exhibit a synergistic therapeutic effect;
  • an inhibitor of the function of the IGFIR gene and a BCR-ABL tyrosine-kinase inhibitor an inhibitor of the function of the IGFIR gene and a BCR-ABL tyrosine-kinase inhibitor, and optionally a pharmaceutically acceptable carrier, for use in the treatment of a leukemia associated with impaired IKAROS function, wherein the relative amounts of the IGFIR inhibitor and the BCR-ABL tyrosine-kinase inhibitor to be administered are such that they exhibit a synergistic therapeutic effect;
  • an inhibitor of the function of the IGFIR gene for the manufacture of a medicament for the treatment of a leukemia associated with impaired IKAROS function, in combination with a BCR-ABL tyrosine-kinase inhibitor, wherein the relative amounts of the IGFIR inhibitor and the BCR-ABL tyrosine-kinase inhibitor are such that they exhibit a synergistic therapeutic effect upon administration to a subject;
  • a method for treating a leukemia associated with impaired IKAROS function in a subject in need thereof comprising administering a pharmaceutical composition comprising of an inhibitor of the function of the IGFIR gene and a BCR-ABL tyrosine- kinase inhibitor, and optionally a pharmaceutically acceptable carrier, wherein the relative amounts of the IGFIR inhibitor and the BCR-ABL tyrosine-kinase inhibitor to be administered are such that they exhibit a synergistic therapeutic effect upon administration to the subject;
  • a method for treating a leukemia associated with impaired IKAROS in a subject in need thereof comprising administering a pharmaceutical composition comprising an inhibitor of the function of the IGF1R gene and a pharmaceutical composition comprising a BCR-ABL tyrosine-kinase inhibitor, wherein the relative amounts of the IGF1R inhibitor and the BCR-ABL tyrosine-kinase inhibitor to be administered are such that they exhibit a synergistic therapeutic effect upon administration to the subject;
  • a combined preparation, product or kit containing an inhibitor of the function of the IGF1R gene and a BCR-ABL tyrosine-kinase inhibitor as a combined preparation for simultaneous, separate or sequential use, in particular in the treatment of a leukemia associated with impaired IKAROS, wherein the relative amounts of the IGF1R inhibitor and a BCR-ABL tyrosine-kinase inhibitor are such that they exhibit a synergistic therapeutic effect upon administration to a subject.
  • the term "synergistic therapeutic effect” means that the therapeutic effect obtained with the combination of the IGF1R inhibitor and the BCR- ABL tyrosine-kinase inhibitor is greater than the addition of the therapeutic effect of each compound used alone. In other words, the inhibition of the leukemic cell proliferation or the induction of leukemic cell death of the composition or the combination is greater than the sum of the effect of each compound alone.
  • the potentiating effect allows to use decreased amounts of IGF1R inhibitor and/or BCR-ABL tyrosine-kinase inhibitor.
  • the two inhibitors are present in the synergistic composition or combination, or are administered to the subject in need thereof at subtherapeutic doses.
  • the term "subtherapeutic dose” refers to an amount or dose of a therapeutic agent lower than the conventional dose administered to a subject for the same indication and the same administration route when it is used alone. In particular, it refers to an amount or dose of a therapeutic agent which has no or only slight effect on leukemic cell proliferation or death when used alone.
  • the IGFIR inhibitor is present in the synergistic composition or combination, or is administered to the subject in need thereof at subtherapeutic dose and the BCR-ABL tyrosine kinase inhibitor is present in the synergistic composition or combination, or is administered to the subject in need thereof at therapeutic effective dose, i.e. at dose that is sufficient to induce leukemic cell death or inhibition of leukemic cell proliferation when used alone.
  • the IGFIR inhibitor is present in the synergistic composition or combination, or is administered to the subject in need thereof at therapeutic effective dose and the BCR-ABL tyrosine kinase inhibitor is present in the synergistic composition or combination, or is administered to the subject in need thereof at subtherapeutic dose.
  • the IGFIR inhibitor and the BCR-ABL tyrosine kinase inhibitor are present in the synergistic composition or combination, or is administered to the subject in need thereof at therapeutic effective doses.
  • the administration frequency or the treatment period may be reduced.
  • the inhibitor of the function of the IGFIR gene is a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, more preferably selected from the group consisting of NVP-AEW541, INSM-18, OSI-906, XL-228, NVP-ADW742, AG-1024, BMS-536924, BMS-554417, BMS-754807, BVP-51004 and ANT-429.
  • the low molecular weight inhibitor of the tyrosine kinase activity of IGFIR is NVP-AEW541.
  • the BCR-ABL tyrosine-kinase inhibitor is selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, and a combination thereof. More preferably, the BCR-ABL tyrosine-kinase inhibitor is nilotinib.
  • the IGFIR inhibitor is NVP-AEW541 and the BCR-ABL tyrosine-kinase inhibitor is nilotinib.
  • the leukemia to be treated is a leukemia in a subject selected with the method of the invention as described below, i.e. the method for selecting a subject affected with a leukemia for a therapy with an inhibitor of the invention, in particular with an inhibitor of the function of the IGFIR gene.
  • the present invention also concerns a method, preferably an in vitro method, for (a) selecting a subject affected with a leukemia, preferably an acute lymphoblastic leukemia or blast crisis CML, for a therapy with an inhibitor of the invention, preferably an inhibitor of the function of the IGF1R gene, or (b) determining whether a subject affected with a leukemia, preferably an acute lymphoblastic leukemia or blast crisis, CML is susceptible to benefit from a therapy with an inhibitor of the invention, preferably an inhibitor of the function of the IGF1R gene, wherein the method comprises the step of determining the functional state of IKAROS in a sample of leukemic cells of the subject, an impaired IKAROS function, preferably a loss of function, indicating that a therapy with an inhibitor of the invention is suitable.
  • the leukemia is selected from the group consisting of an acute lymphoblastic leukemia and blast crisis chronic myelogenous leukemia. More preferably, the leukemia is selected from the group consisting of BCR-ABL induced acute B cell lymphoblastic leukemia, lymphoid blast crisis chronic myelogenous leukemia, acute B cell lymphoblastic leukemia associated with activating mutation in JAK2 tyrosine kinase gene, acute B cell lymphoblastic leukemia and acute T cell lymphoblastic leukemia.
  • the leukemia may also be an acute lymphoblastic leukemia selected from the group consisting of BCR-ABL induced acute B cell lymphoblastic leukemia, acute B cell lymphoblastic leukemia associated with activating mutation in JAK2 tyrosine kinase gene, acute B cell lymphoblastic leukemia and acute T cell lymphoblastic leukemia. More preferably, the acute lymphoblastic leukemia is selected from the group consisting of BCR-ABL induced acute B cell lymphoblastic leukemia, acute B cell lymphoblastic leukemia associated with activating mutation in JAK2 tyrosine kinase gene.
  • the leukemia is a BCR-ABL induced leukemia, preferably a BCR-ABL induced acute lymphoblastic leukemia or lymphoid blast crisis chronic myelogenous leukemia.
  • the leukemia is selected from the group consisting of BCR-ABL induced acute B cell lymphoblastic leukemia and lymphoid blast crisis chronic myelogenous leukemia. More preferably, the leukemia is BCR-ABL induced acute B cell lymphoblastic leukemia.
  • determining the functional state of IKAROS means determining if the IKAROS function in leukemic cells is normal, impaired or completely lost.
  • the functional state of IKAROS may be assessed by determining the level of expression of the IKAROS protein, for example by immunohistochemistry, semi-quantitative Western-blot or by protein or antibody arrays.
  • the functional state of IKAROS is assessed by determining if the IKZFl gene is mutated or deleted, for example by fluorescence in situ hybridization (FISH), genomic quantitative polymerase chain reaction, or sequencing IKZFl exons and/or regulatory sequences, as described above.
  • the subject selected with the method of the invention may be treated using the inhibitor of the invention, in particular using an inhibitor of the function of the IGF1R gene. All the embodiments of the inhibitor of the invention are also contemplated in this method.
  • the present invention further concerns a method for screening or identifying a molecule suitable for treating a leukemia associated with impaired IKAROS function.
  • the method may be in vivo, ex vivo or in vitro method, preferably in vitro method.
  • the leukemia to be treated is in a subject selected with the method of the invention.
  • the leukemia associated with impaired IKAROS function is selected from the group consisting of BCR-ABL induced acute B cell lymphoblastic leukemia (BCR-ABL induced B-ALL), blast crisis CML, acute B cell lymphoblastic leukemia associated with activating mutation in JAK2 tyrosine kinase gene, acute B cell lymphoblastic leukemia and T cell lymphoblastic leukemia.
  • BCR-ABL induced B-ALL blast crisis CML
  • B-ALL associated with activating mutation in JAK2 tyrosine kinase gene
  • the leukemia is selected from the group consisting of BCR-ABL induced B- ALL and lymphoid blast crisis CML.
  • the method comprises the steps of (i) contacting candidate molecules with leukemic cells having impaired IKAROS function and (ii) selecting molecules having the ability to reduce or inhibit the expression of at least one gene selected from the group consisting of IGF1R, IGF2BP3, FLT1, EMILIN-1, DOCK1, LAPTM4B and H19, more preferably from the group consisting of IGF1R, IGF2BP3, FLT1, EMILIN-1, DOCK1 and LAPTM4B, even more preferably from the group consisting of IGF1R, IGF2BP3 and FLT1.
  • the method comprises the steps of (i) contacting candidate molecules with leukemic cells with impaired IKAROS function and (ii) selecting molecules having the ability to inhibit or to reduce the activity of at least one protein encoded by a gene selected from the group consisting of IGFIR, IGF2BP3, FLTl, EMILIN-1, DOCK1, LAPTM4B and H19, more preferably from the group consisting of IGFIR, IGF2BP3, FLTl, EMILIN-1, DOCK1 and LAPTM4B, even more preferably from the group consisting of IGFIR and FLTl.
  • the method comprises the steps of (i) contacting candidate molecules with IGFIR or FLTl receptor, and (ii) selecting molecules having the ability to bind to IGFIR or FLTl and/or to compete with and/or for a ligand of IGFIR or FLTl and/or to decrease the kinase activity of IGFIR or FLTl .
  • the method comprises the steps of (i) contacting candidate molecules with IGFIR, and (ii) selecting molecules having the ability to bind to IGFIR and/or to compete with and/or for a ligand of IGFIR and/or to decrease the kinase activity of IGF 1 R.
  • the binding of a molecule to IGFIR or FLTl can be measured by well-known techniques such as surface plasmon resonance, calorimetry or Biacore technology.
  • the ability of a molecule to compete with or for a ligand of IGFIR or FLTl can be evaluated, for example, by competition experiments with labelled ligand, in particular radio-labelled ligand, Biacore or spectroscopic observations.
  • the IGFIR or FLTl gene expression can be evaluated with different well known techniques, such as quantitative RT-PCR, Northern-blot, ELISA or Western-blot.
  • the IGFIR or FLTl phosphorylation level can be assessed by western-blot using an anti-phosphotyrosine antibody, radioactive FlashPlate assay, fluorescent resonance energy transfer (FRET) assay or dissociation-enhance lanthanide fluorescence immunoassay (DELFIA). All these techniques are well known by the skilled person.
  • the method as described above can further comprise a subsequent step consisting of administering a molecule previously selected by the in vitro method of the invention as disclosed above, in a non human animal model of leukemia with impaired IKAROS function, and analyzing the effect on the disease progression.
  • the non human animal model may be a BCR-ABL +/0 ; IK L/+ mouse as described in the article of Virely et al, 2010, or a NOD/SCID mouse xenotranplanted with IKAROS -mutated primary human BCR-ABL + B-ALL cells (as described for example in the article of Notta et al, 2011) or with cell lines derived from such tumors (as described for example in the article of Janes et al, 2010).
  • the non human animal model is a BCR-ABL +/0 ; IK L/+ mouse as described in the article of Virely et al., 2010.
  • the efficiency of the molecule can be evaluated, for instance, by analyzing the life span of animals, the spread of leukemia outside the bone marrow, e.g. to the central nervous system, testicles or skin. All these characteristics have to be compared with those of controls consisting of the same non human animal models with no treatment.
  • the present invention concerns the use of a kit comprising (i) at least one probe specific to the IKZF1 genomic DNA, mR A or cDNA, and/or (ii) at least one nucleic acid primer pair specific to the IKZF1 genomic DNA, mRNA or cDNA, and optionally, a leaflet providing guidelines to use such a kit, for (a) selecting a subject affected with a leukemia, preferably an acute lymphoblastic leukemia or blast crisis CML, for a therapy with an inhibitor of the invention, preferably an inhibitor of the function of the IGFIR gene; and/or (b) determining whether a subject affected with a leukemia, preferably an acute lymphoblastic leukemia or blast crisis CML, is susceptible to benefit from a therapy with an inhibitor of the invention, preferably an inhibitor of the function of the IGFIR gene. All the embodiment of the inhibitor of the invention are also contemplated in this aspect.
  • the present invention also concerns the use of a kit comprising (i) at least one probe specific to the genomic DNA, mRNA or cDNA of a gene selected from the group consisting of IGFIR, IGF2BP3, FLTI, EMILIN-1, DOCK1, LAPTM4B and HI 9, and/or (ii) at least one nucleic acid primer pair specific to the genomic DNA, mRNA or cDNA of a gene selected from the group consisting of IGFIR, IGF2BP3, FLTI, EMILIN-1, DOCK1, LAPTM4B and H19, and optionally, a leaflet providing guidelines to use such a kit, for screening or identifying a molecule suitable for treating a leukemia associated with impaired IKAROS function.
  • BCR-ABL transgenic mice that exclusively develop B-ALL to mice heterozygous for an hypomorphic allele of IKZF1 (Ik L/+ mice; Kirstetter et al., 2002) to generate BCR- ABL ⁇ 0 mice carrying either (i) 2 copies of wild-type IKZF1 (control group; BCR- ABL +/0 IK +/+ ) or, (ii) 1 wild type and 1 IK L allele (test group; BCR-ABL +/0 ; IK L/+ ) (Virely et al, 2010).
  • IK L + and Ik + + littermates generated in parallel were used as controls. Comparison of tumor onset in the BCR-ABL +/0 ; IK + + and BCR-ABL +/0 ; IK L + cohorts showed that loss of 1 wild type IKZF1 allele and its replacement by the IK L allele considerably accelerated B-ALL onset, providing the first experimental evidence and mouse model of a tumor suppressive function of IKZF1 in BCR-ABL- induced leukemogenesis (Virely et al, 2010). They also showed that deletion of a single wild-type IKZF1 allele is sufficient to strongly cooperate with BCR-ABL in lymphoid leukemogenesis.
  • Transgenic BCR-ABL mice carried an expression cassette in which the BCR-ABL cDNA encoding the pl90 BCR"ABL variant was placed under control of the metallothionein promoter (Heisterkamp et al, 1990). These mice were obtained from the Jackson laboratory on a mixed genetic background. They were backcrossed for more than 10 generations on C57B/6 mice before use.
  • the second model involved retrovirally-mediated transduction by spinoculation of bone marrow cells obtained from unconditionned C57B/6 mice deleted for the CDK 2A locus (eliminating the pl6INK4A and ARF tumor suppressor genes) and heterozygous for the IK L allele (IK L/+ ) with a pMIG-based retroviral vector encoding pl90 BCR ABL and EGFP.
  • transduced cells were inoculated by intravenous injection into non conditionned, wild type C57B/6 mice. These mice developed B-ALL within 2-3 weeks after transduction/reinoculation.
  • mice carrying the hypomorphic IKZF1 allele (IK L ) (Kirstetter et al., 2002) were maintained on a pure C57B/6 background by backcrossing to C57B/6 mice (Charles River Laboratories).
  • the pMIG retroviral vector encoding pl90 BCR ABL was previously described (Kharas et al. 2004).
  • the corresponding viral stocks were generated following calcium phosphate co -precipitation of 20 ⁇ g retroviral DNA in the ecotropic Plat-E encapsidation cell line.
  • the culture medium (DMEM supplemented with 10% heat- inactivated fetal calf serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2 mM glutamine, all from Life Technologies) was replaced after 18 hours by fresh medium and supernatants collected 24 and 48 hours later, aliquoted and stored frozen at -80°C.
  • BCPv-ABL + ; IK L/+ ; CDK 2A " cell lines were established by co-cultivating leukemic cells obtained from the bone marrow of leukemic mice with cells of the MS5 stromal cell line (2.10 6 leukemic cells/ml/well on 12 wells plates containing a confluent layer of MS5 cells) in RPMI supplemented with 15 % heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2 mM glutamine (all from Life Technologies).
  • BV-173 cell line Human CML blast crisis BCR-ABL positive Leukemia Cell Line; Pegoraro et al, 1983
  • RPMI 1640 medium supplemented with 20% FBS
  • SUP-B15 cell line Human pre-B cell Acute Lymphoblastic Leukemia; Naumovski et al, 1988
  • McCoy medium supplemented with 20%>FBS. All the cell lines were maintained at 37°C with 5% C0 2 ; 10 6 cells/ml and split every two days.
  • Nitrocellulose membranes were then incubated in low-fat, skimmed milk (w/v) 5%; PBS IX; 0,2%> (v/v) Tween20 (Sigma) at room temperature for 60 minutes. Membranes were then incubated with different primary antibodies overnight (dilution 1/1000) in a solution of skimmed milk 5% (w/v); PBS IX; 0,2%> (v/v) Tween20 (Sigma).
  • Primaries antibodies used were: an IKAROS-specific antibody (E20; sc-9861; Santa Cruz Biotechnology), a polyclonal rabbit antibody to IGF1R (N-20; Sc712; Santa Cruz Biotechnology), an ABL-specific antibody (Ab-3, 24-21) from Calbiochem and a rabbit antibody to phospho-IGFIR ⁇ chain (Yl 135 and Yl 136) / Insulin Receptor ⁇ chain (Yl 150/Y1151) (Cell Signaling).
  • IKAROS-specific antibody E20; sc-9861; Santa Cruz Biotechnology
  • N-20 Sc712; Santa Cruz Biotechnology
  • ABL-specific antibody Ab-3, 24-21
  • a rabbit antibody to phospho-IGFIR ⁇ chain Yl 135 and Yl 136) / Insulin Receptor ⁇ chain (Yl 150/Y1151) (Cell Signaling).
  • Bone marrow cells were obtained from wild-type and leukemic C57B/6 mice by flushing the long bones in PBS IX and filtration on cell strainer 40 mm (BD Biosciences). Cells (5xl0 6 ) were incubated with antibodies to B220 (RA3-6B2; BD Biosciences; 1/500), Thy 1.2 (53-2.1; BD Biosciences; 1/500), CD43 (S7; BD Biosciences, 1/200), CD19 (1D3; BD Biosciences, 1/200); slgM (R6-60.2 ; BD Biosciences, 1/200).
  • Sorting of leukemic cells was performed on a FACS Aria II (BD Bioscienes), gating the FSC large, B220+, IgM- population and analyses performed on gated FSC large B220+ leukemic cells on FACSCalibur (BD Biosciences).
  • cRNA synthesis, labeling and hybridization to the arrays were performed according to the manufacturer's instructions (Affymetrix). Arrays washing, and scanning were performed according to the protocols recommended by Affymetrix in their GeneChip Expression Analysis Technical Manual.
  • Raw feature data were normalized and log2 intensity expression summary values for each probe set were calculated using Robust Multi-array (RMA, R package affy VI .4.32). The coefficient of variation was used as a pre-processing step for getting the 10570 most variable probe sets and the Student t-test was used for selecting the significant genes. Only probe sets with a p-value less than or equal to 0,008 were considered.
  • NVP-AEW-541 Novartis
  • Nilotinib Novartis
  • Apoptosis assays were carried out using a FITC-conjugated Rabbit antibody to cleaved caspase 3, using the Active Caspase-3 Apoptosis Kit (BD Biosciences) according to the manufacturer instructions.
  • leukemic cells were pulse-labeled 30 min with 10 ⁇ Bromodeoxyuridine (BrdU), fixed and stained with APC-conjugated anti-BrdU antibody (1/50 dilution; APC BrdU Flow Kit, BD Biosciences), washed and stained by 7-AAD ( ⁇ g/ml; BD Biosciences).
  • the inventors have compared the transcriptome of BCR-ABL +/0 ; Ik L/+ and BCR- ABL +/0 ; IK +/+ leukemic cells.
  • Leukemic cells were obtained from mice at the terminal stage of the disease, purified by FACS-sorting (high forward scatter, B220+; IgM-) and their total RNA extracted. Probes were synthesized from each samples and hybridized to Affymetrix pangenomic DNA oligonucleotide chips. About 240 genes were found differentially expressed between BCR-ABL +/0 ; IK L/+ and BCR-ABL +/0 ; IK +/+ leukemic cells ("Ikaros signature", Fig. 1).
  • IK L/+ leukemic cells were involved in cell adhesion, cellular signaling events or integrin signaling such as Dockl, Emilinl or VEGFR1. Upregulation of these genes may thus contribute to the aggressive phenotype of Ik +/L BCR-ABL + leukemic cells by changing their interactions with the leukemic niche and/or their trafficking dynamics and systemic dissemination.
  • IK L/+ are hematopoietic stem cell specific, such as VEGFR1, Laptm4b, Emilinl or IGF1R. Ikzfl haploinsufficiency may thus stabilize a stem cell-related program that might be critical for leukemic cell self- renewal.
  • Laptm4b upregulated to more than 10 times
  • Laptm4b may contribute to the dissemination and drug resistance of Ikaros-deficient leukemias
  • the table 1 below shows the fold expression ratio between BCR-ABL +/0 IK L/+ and IK +/+ leukemias for these seven genes.
  • Table I A selection of genes differentially expressed between BCR-ABL ; IK and BCR-ABL 10 ; IK /+ leukemic cells.
  • the fold expression ratio between BCR-ABL 70 IK L/+ and IK /+ leukemias (Ratio L+/++) is expressed in log 2 .
  • the coefficient of variation (CV) and t-test were carried out as described in Materials and Methods.
  • IK L/+ included two receptor-type protein tyrosine kinases, the receptor for IGF1 (Insulin growth factor 1) (IGFIR) and the receptor FLT1 (VEGFR1) for VEGF-B and the Placental growth factor.
  • IGFIR Insulin growth factor 1
  • VEGFR1 receptor FLT1
  • IGFIR was about 10-fold- and FLT1/VEGFR1 about 2-fold upregulated in
  • BCR-ABL 70 IK L/+ leukemic cells. Upregulation of the expression of both IGFIR and FLT1/VEGFR1 were validated in independent BCR-ABL +/0 ; IK L/+ leukemias as compared to BCR-ABL 70 ; IK /+ tumor cells by RT-PCR analyses (data not shown) and western blot analyses (Figure 2). Strikingly, IGF2BP3, a positive regulator of IGF2 mRNA translation, one of the ligands of IGFIR is also more than 8 times upregulated in BCR-ABL +/0 ; IK L/+ as compared to BCR-ABL +/0 ; IK +/+ .
  • BCR-ABL Similar to what was observed in the transgenic BCR-ABL model, BCR-ABL; CDK 2A-/-; IK L/+ leukemias expressed IGFIR at higher levels as compared to BCR-ABL; CDK 2A-/-; IK +/+ leukemic cells (Figure 3).
  • These two mouse models thus concur to show that impaired Ikaros function in BCR-ABL leukemias is linked to upregulation of IGFIR and IGFIR ligands and thus persistent IGFR signaling.
  • IGFIR expression was detected at the cell surface of two cell lines derived from BCR-ABL+ human B-ALL, BV-173 and SUP-B15 cell lines, both carrying an IKZF1 mutation leading to expression of a dominant-negative IKAROS protein (Figure 4).
  • FBS Fetal Bovine Serum
  • cells were pre-treated with an IGFIR inhibitor (NVP-AEW541; ⁇ ; Novartis Pharmaceuticals Corporation, also named herein AEW) for 60 minutes and subsequently incubated with a human IGF1 at (lOOng/ml) for 5 and 30 minutes, respectively.
  • IGFIR inhibitor NBP-AEW541; ⁇ ; Novartis Pharmaceuticals Corporation, also named herein AEW
  • a human IGF1 at (lOOng/ml) for 5 and 30 minutes, respectively.
  • Cellular extracts were prepared, protein were separated by SDS/PAGE, followed by nitrocellulose transfer and western blot analysis.
  • Membranes were incubated with either an antibody specific to phosphorylated tyrosine residues Y1135 and Y1136 of human IGFIR, or a monoclonal antibody to human IGFIR.
  • NVP-AEW541 (2,5 ⁇ ; 5 ⁇ ; 7,5 ⁇ ; ⁇ ) cells were collected and analyzed for the proportion of FSC hl /PT, live cells.
  • BCR-ABL + ; CDKN2A " " ; IK L/+ B-ALL mouse leukemic cells express high levels IGFIR. It was thus analyzed whether treatment of mouse BCR-ABL-induced B-ALL cells with NVP-AEW541 affected cell survival. Cells were treated with increasing concentrations of NVP-AEW-541 ( ⁇ and 2 ⁇ ), and survival was analyzed by following the proportion of FSC hl /PT, live cells by flow cytometry. The results showed significant induction of cell death at concentrations equal or superior to 2 ⁇ AEW ( Figure 13). This translated in a reduction on the number of live cells as measured by Trypan blue exclusion staining ( Figure 14). Thus IGF1R/IR signalling contributes to the survival of BCR-ABL + ; CDKN2A " " ; IK L/+ B- ALL mouse leukemic cells.
  • Nilotinib concentrations higher than 20nM were found to induce massive cell death while Nilotinib ⁇ and 20nM concentrations only induce a slight effect on cell survival (data not shown). It was then analyzed whether treatment of BCR-ABL + ; CDK 2A " " ; IK L/+ B- ALL mouse leukemic cells with amounts of AEW which by themselves did not or only marginally affected cell expansion were able to cooperate with Nilotinib to inhibit cell survival and proliferation.
  • BCR-ABL + /CDKN2A _/ 7Ik L/+ leukemic cells were then co-treated with low concentrations of Nilotinib (10 nM) either alone or with increasing amounts of NVP-AEW541 (0,5 ⁇ ; ⁇ and 2 ⁇ ). Cells treated with NVP-AEW541 alone were also analyzed. The results showed that AEW treatment clearly sensitized these leukemic cells to the cytotoxic effects induced by Nilotinib ( Figures 13-15).
  • BCR-ABL + /CDKN2A _/ 7Ik L/+ leukemic cells were either left untreated, or treated with ⁇ Nilotinib, or 0.5 ⁇ AEW, or ⁇ Nilotinib and 0.5 ⁇ AEW. After 2 days, cells were pulse-labelled with BrdU, stained with 7-AAD (to measure DNA content) and examined by flow cytometry to quantify cell death (% of cells with sub-Gl, fragmented DNA content) and cell proliferation (% BrdU-positive cells).
  • An inhibitor of the function of a gene which is upregulated in leukemic cells due to impaired IKAROS function wherein said gene is selected from the group consisting of IGF1R and FLT1, for use in the treatment of a leukemia associated with impaired IKAROS function.
  • the inhibitor is selected from the group consisting of a low molecular weight inhibitor of the tyrosine kinase activity of IGF1R or FLT1, a neutralizing antibody directed against IGF1R or FLT1, an antibody directed against a IGF1R or FLT1 ligand, a nucleic acid molecule interfering specifically with IGF1R or FLT1 expression, a soluble decoy IGF1R or FLT1 receptor, a dominant negative form of IGFIR or FLTl presenting a kinase dead domain, a peptide antagonist of IGFIR or FLTl and a molecule that reduces the level of at least one IGFIR or FLTl ligand.
  • inhibitor according to aspect 1 or 2 wherein the inhibitor is selected from the group consisting of a neutralizing antibody directed against IGFIR or FLTl and a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR or FLTl .
  • the leukemia associated with impaired IKAROS function is a leukemia selected from the group consisting of BCR-ABL induced acute B cell lymphoblastic leukemia, lymphoid blast crisis chronic myelogenous leukemia, acute B cell lymphoblastic leukemia associated with activating mutation in JAK2 tyrosine kinase gene, acute B cell lymphoblastic leukemia and acute T cell lymphoblastic leukemia.
  • the leukemia associated with impaired IKAROS function is a BCR-ABL induced leukemia, preferably selected from the group consisting of BCR-ABL induced acute B cell lymphoblastic leukemia and lymphoid blast crisis chronic myelogenous leukemia.
  • the inhibitor is used in combination with a BCR-ABL-targeted therapy, preferably with a BCR-ABL tyrosine kinase inhibitor, more preferably selected from the group consisting of imatinib, nilotinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, and a combination thereof.
  • a BCR-ABL tyrosine kinase inhibitor more preferably selected from the group consisting of imatinib, nilotinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, and a combination thereof.
  • An in vitro method for (a) selecting a subject affected with an acute lymphoblastic leukemia or blast crisis CML for a therapy with an inhibitor of any of aspects 1 to 8 or (b) determining whether a subject affected with an acute lymphoblastic leukemia or blast crisis CML is susceptible to benefit from a therapy with an inhibitor of any of aspects 1 to 8, wherein the method comprises the step of determining the functional state of IKAROS in a sample of leukemic cells of the subject, an impaired IKAROS function indicating that a therapy with said inhibitor is suitable. 10.
  • An in vitro method for screening or identifying a molecule suitable for treating a leukemia associated with impaired IKAROS function comprises the steps of (i) contacting candidate molecules with IGFIR or FLT1 receptor, and (ii) selecting molecules having the ability to bind to IGFIR or FLT1 and/or to compete with and/or for a ligand of IGFIR or FLT1 and/or to decrease the kinase activity o f IGF 1 R or FLT 1.
  • step (iii) administering a molecule selected in step (ii) in a non human animal model of leukemia with impaired IKAROS function, preferably a BCR-ABL 70 ; IK L/+ mouse, and (iv) analyzing the effect on the disease progression.
  • a non human animal model of leukemia with impaired IKAROS function preferably a BCR-ABL 70 ; IK L/+ mouse
  • a combined preparation, product or kit containing (a) an inhibitor according to any of aspects 1 to 8 and (b) another chemotherapeutic agent or an immunotherapy agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of a leukemia associated with impaired IKAROS.
  • the other chemotherapeutic agent is an agent used to treat BCR-ABL induced leukemia, preferably BCR-ABL+ acute lymphocytic leukemia or blast crisis CML.
  • kits comprising (i) at least one probe specific to the IKZF1 genomic
  • DNA, mRNA or cDNA and/or (ii) at least one nucleic acid primer pair specific to the IKZF1 genomic DNA, mRNA or cDNA, and optionally, a leaflet providing guidelines to use such a kit, for (a) selecting a subject affected with an acute lymphoblastic leukemia or blast crisis CML for a therapy with an inhibitor of any of aspects 1 to 8; and/or (b) determining whether a subject affected with an acute lymphoblastic leukemia or blast crisis CML is susceptible to benefit from a therapy with an inhibitor of any of aspects 1 to 8.
  • An in vitro method for (a) selecting a subject affected with a leukemia for a therapy with an inhibitor of the function of the IGFIR gene, or (b) determining whether a subject affected with a leukemia is susceptible to benefit from a therapy with an inhibitor of the function of the IGFIR gene, wherein the method comprises the step of determining the functional state of IKAROS in a sample of leukemic cells of the subject, an impaired IKAROS function indicating that a therapy with said inhibitor is suitable.
  • step of determining the functional state of IKAROS comprises detecting a deletion or a mutation in at least one coding exon of the IKZF1 gene, or a reduction of IKAROS protein expression.
  • An inhibitor of the function of the IGF1R gene for use in the treatment of a leukemia in a subject selected with the method of any of the preceding aspects.
  • the inhibitor is selected from the group consisting of a low molecular weight inhibitor of the tyrosine kinase activity of IGF1R, a neutralizing antibody directed against IGF1R, an antibody directed against a IGF1R ligand, a nucleic acid molecule interfering specifically with IGF1R expression, a soluble decoy I
  • the leukemia is selected from the group consisting of an acute lymphoblastic leukemia and blast crisis chronic myelogenous leukemia.
  • the leukemia is selected from the group consisting of BCR- ABL induced acute B cell lymphoblastic leukemia, lymphoid blast crisis chronic myelogenous leukemia, acute B cell lymphoblastic leukemia associated with activating mutation in JAK2 tyrosine kinase gene, acute B cell lymphoblastic leukemia and acute T cell lymphoblastic leukemia.
  • BCR- ABL tyrosine kinase inhibitor is selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, and a combination thereof.
  • An in vitro method for screening or identifying a molecule suitable for treating a leukemia in a subject selected with the method according to any of aspects 1 to 3, wherein the method comprises the steps of (i) contacting candidate molecules with IGFIR, and (ii) selecting molecules having the ability to bind to IGFIR and/or to compete with and/or for a ligand of IGFIR and/or to decrease the kinase activity of IGFIR. 20.
  • the method of aspect 19, further comprising the steps of (iii) administering a molecule selected in step (ii) in a non human animal model of leukemia with impaired IKAROS function, preferably a BCR-ABL +/0 ; IK L/+ mouse, and (iv) analyzing the effect on the disease progression.
  • a non human animal model of leukemia with impaired IKAROS function preferably a BCR-ABL +/0 ; IK L/+ mouse
  • a combined preparation, product or kit containing (a) an inhibitor of the function of the IGFIR gene and (b) another chemotherapeutic agent or an immunotherapy agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of a leukemia in a subject selected with the method of any of aspects 1 to 3.
  • the inhibitor of the function of the IGFIR gene is selected from the group consisting of a low molecular weight inhibitor of the tyrosine kinase activity of IGFIR, a neutralizing antibody directed against IGFIR, an antibody directed against a IGFIR ligand, a nucleic acid molecule interfering specifically with IGFIR expression, a soluble decoy IGFIR, a dominant negative form of IGFIR presenting a kinase dead domain, a peptide antagonist of IGFIR and a molecule that reduces the level of at least one IGFIR ligand.
  • BCR- ABL tyrosine kinase inhibitor is selected from the group consisting of nilotinib, imatinib, dasatinib, bosutinib, ponatinib, bafetinib, ON012380, and a combination thereof.
  • BCR-ABL tyrosine kinase inhibitor is nilotinib.
  • kits comprising (i) at least one probe specific to the IKZF1 genomic DNA, mRNA or cDNA, and/or (ii) at least one nucleic acid primer pair specific to the IKZF1 genomic DNA, mRNA or cDNA, and optionally, a leaflet providing guidelines to use such a kit, for (a) selecting a subject affected with a leukemia for a therapy with an inhibitor of the function of the IGFIR gene; and/or (b) determining whether a subject affected with a leukemia is susceptible to benefit from a therapy with an inhibitor of the function of the IGFIR gene, using the method of any of aspects 1 to 3 and 5 to 18.

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Abstract

La présente invention concerne un procédé de sélection d'un sujet atteint de leucémie pour un traitement avec un inhibiteur de la fonction du gène IGF1R. La présente invention concerne notamment l'utilisation d'inhibiteurs d'IGFR1 pour le traitement d'une leucémie avec déficience en IKAROS, de préférence de leucémies lymphoblastiques à cellules B induites par BCR-ABL avec déficience en IKAROS ainsi que de leucémies myéloïdes chroniques avec crise blastique lymphoïde.
PCT/EP2012/061675 2011-06-20 2012-06-19 Compositions et procédés destinés au traitement de la leucémie WO2012175481A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11407732B1 (en) 2019-04-12 2022-08-09 C4 Therapeutics, Inc. Tricyclic degraders of Ikaros and Aiolos
WO2022032132A1 (fr) * 2020-08-07 2022-02-10 C4 Therapeutics, Inc. Thérapies avantageuses pour des troubles médiés par ikaros ou aiolos

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