WO2016014992A2 - Compositions pour le traitement, le diagnostic et le pronostic de maladies - Google Patents

Compositions pour le traitement, le diagnostic et le pronostic de maladies Download PDF

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WO2016014992A2
WO2016014992A2 PCT/US2015/042084 US2015042084W WO2016014992A2 WO 2016014992 A2 WO2016014992 A2 WO 2016014992A2 US 2015042084 W US2015042084 W US 2015042084W WO 2016014992 A2 WO2016014992 A2 WO 2016014992A2
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pip5k1a
subject
disease
genes
inhibitor
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PCT/US2015/042084
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WO2016014992A3 (fr
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Jenny L. PERSSON
Heather H. JOHNSON
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Everest Biosciences, Inc.
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01068—1-Phosphatidylinositol-4-phosphate 5-kinase (2.7.1.68)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to novel pharmaceutically-useful compositions, which compositions may be useful in the treatment of diseases such as cancer.
  • the invention also relates to use of the compositions for disease diagnosis and prognosis, treatment selection, and disease progression monitoring.
  • Phosphoinositides has long beene known to present in cellular membranes.
  • the phosphoinositide family consists of seven derivatives of phosphatidylmositol (Ptdlns) that are formed through the phosphorylation of the 3-, 4- and 5-positions of the inositol ring.
  • Ptdlns phosphatidylmositol
  • phosphoinositides are important regulators of a large variety of cellular processes.
  • the production of the different phosphoinositide species is spatially and temporally regulated through the actions of kinases, phosphatases and phospholipases, some of which can be localized in different subcellular compartments.
  • PIP5Ks Phosphatidylmositol 4-phosphate 5-kinases
  • PIP5K1A PIP5K
  • ⁇ 5 ⁇ also known as PIP5K1B
  • PIP5Ky PIP5K1C
  • PIP5Ks has been linked to diseases in humans.
  • a report has linked the mutation of human ⁇ 5 ⁇ to a lethal congenital contractural syndrome type 3 (LCCS3) characterized by multiple joint contractures, micrognathia and anterior-horn atrophy in the spinal cord.
  • LCCS3 lethal congenital contractural syndrome type 3
  • the origin of this disease was traced back to a mutation (G757A) in the kinase domain of ⁇ 5 ⁇ that renders the protein unable to phosphorylate PtdIns(4)/ > .
  • ⁇ 5 ⁇ is highly expressed in the brain and the symptoms are linked to major neurological defects.
  • PtdIns(4,5)P2 is important for different processes, including synaptic-vesicle endocytosis and neurite outgrowth.
  • PtdIns(4,5)/ > 2 might impinge on these processes, resulting in the neurological defects found in LCCS3.
  • the activity of phosphatidylmositol 4-kinase (PI4K) and of PIP5K is increased in different hepatoma cell lines as compared with that in normal liver cells.
  • PtdIns(3,4,5)P3 levels are altered in many cancers because of mutations of the phosphatase and tensin homolog (PTEN) (which negatively regulates the levels of PtdIns(3,4,5)P3), it was suggested increased PtdIns(4,5)P2 levels (through changes in PIP5K activity) might also be important to sustain increased PtdIns(3,4,5).P3 production during cancer progression.
  • PTEN phosphatase and tensin homolog
  • Cancer is a class of diseases that affects people world-wide. Generally, cells in a benign tumor retain their differentiated features and do not divide in a completely uncontrolled manner. A benign tumor is usually localized and non-metastatic.
  • Malignant tumors In a malignant tumor, cells become undifferentiated, do not respond to the body's growth control signals, and multiply in an uncontrolled manner.
  • Malignant tumors are generally divided into two categories: primary and secondary. Primary tumors arise directly from the tissue in which they are found. Secondary tumors may be originated from the primary tumors or may be originated elsewhere in the body, and are capable of spreading to distant sites (metastasizing) or metastasis. The common routes for metastasis are direct growth into adjacent structures, spread through the vascular or lymphatic systems or blood streams.
  • the present invention provides compounds and formulation thereof for treatment of diseases, such as cancer, spinal cord injur, and AIDS.
  • the present invention provides a method for treatment of a disease in a subject in which inhibition of PIP5K1A is desired and/or required, comprising: administering a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.
  • the present invention provides a method for treatment of a disease in a subject characterized by over activation of PIP5K1A and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.
  • the inhibitor of PIP5K1A is capable of reducing PIP5K1A activity by between about 5% and 100% as determined by an in vitro or in vivo assay. In some embodiments, the the inhibitor of PIP5K1A is capable of reducing PIP5K1A activity by between about 40% and 80% as determined by an in vitro or in vivo assay.
  • the present invention provides a method for treatment of a disease in a subject characterized by over activation of Akt and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.
  • the inhibitor of PIP5K1A is capable of reducing Akt activity to between about 50 % and 150% of normal Akt activity as determined by an in vitro or in vivo assay. In some embodiments, the inhibitor of PIP5K1A is capable of reducing Akt activity to between about 80 % and 120% of normal Akt activity as determined by an in vitro or in vivo assay.
  • the present invention provides a method for treatment of a disease in a subject characterized by over activation of PI3 Kinase and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.
  • the inhibitor of PIP5K1A is capable of reducing production level of phosphatidylinositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) to between about 50 %> and 150%o of normal production level of phosphatidylinositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay.
  • the inhibitor of PIP5K1A is capable of reducing production level of phosphatidylinositol 3,4,5- bisphosphate (PtdIns(3,4,5)P2) to between about 80 %> and 120%o of normal production level of phosphatidylinositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal , as determined by an in vitro or in vivo assay.
  • the present invention provides a method for treatment of a disease in a subject characterized by over activation of phospholipase C (PLC) and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.
  • PLC phospholipase C
  • the inhibitor of PIP5K1A is capable of reducing PLC activity to between about 50 % and 150% of normal PLC activity as determined by an in vitro or in vivo assay. In some embodiments, the inhibitor of PIP5K1A is capable of reducing PLC activity to between about 80 % and 120% of normal PLC activity as determined by an in vitro or in vivo assay.
  • the present invention provides method for treatment of a disease in a subject characterized by over activation of Akt and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of modulator of PDK/Akt pathway, wherein the modulator is capable of reducing Akt activity to between about 50 % and 150% of normal Akt activity as determined by an in vitro or in vivo assay.
  • the modulator of PI3K/Akt pathway is capable of reducing Akt activity to between about 80 % and 120% of normal Akt activity as determined by an in vitro or in vivo assay.
  • the disease is cancer, spinal cord injury, or AIDS.
  • the cancer is selected from the group consisting of: hemangioma, hepatocellular adenoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas, pyogenic granulomas, leukemia, myelodysplastic syndromes (MDS), prostate cancer, breast cancer, skin cancer, bone cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, bladder, gall bladder, ovary, cervix, pancreas, rectum, parathyroid, thyroid, esophagus, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, s
  • MDS
  • the inhibitor of PIP5K1A, or modulator of PI3K/Akt pathway comprises a small molecule, an RNA, a peptide, or an antibody.
  • the present invention provides a method of diagnosing a subject suspect of having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to diagnosis of the disease, thereby diagnosing the subject with regard to the disease.
  • the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient diagnosed as having the disease.
  • the present invention provides a method for determining disease stage of a subject diagnosed as having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to disease stage of the disease, thereby determining disease stage of the subject with regard to the disease.
  • the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient has been diagnosed as having the disease of different disease stages.
  • the present invention provides a method for monitoring disease progression of a subject diagnosed as having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to disease progression of the disease, thereby determining disease progression of the subject with regard to the disease.
  • the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient has been diagnosed as having the disease of different disease progression.
  • the present invention provides a method for determining a prognosis for a subject diagnosed as having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to prognosis of the disease, thereby determining prognosis of the subject with regard to the disease.
  • the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient determined as having the disease of different prognosis outcome.
  • the present invention provides a method of determining effect of treatment of a subject having a disease condition with a pharmaceutical composition, comprising: (a) providing a biological sample from a subject that has been subject to a treatment of a disease; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to treatment effect of the disease, thereby determining the treatment effect of the pharmaceutical composition.
  • the present invention provides a method of determining whether a subject having a disease condition should be treated with a pharmaceutical composition comprising an inhibitor of PIP5K1A, and/or an inhibitor of PI3K/Akt pathway, and/or an inhibitor of PLC pathway, comprising: (a) providing a biological sample from a subject t having a disease condition; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to whether a subject having a disease condition should be treated with a pharmaceutical composition comprising an inhibitor of PIPK1 A, and/or an inhibitor of PDK/Akt pathway, and/or an inhibitor of PLC pathway, thereby determining whether the subject should be treated with the pharmaceutical composition.
  • the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient that has been subjected to the treatment and achieved treatment effect.
  • the one or more genes comprises a gene selected from the group consisting of: Akt, pAkt (phosphorylated Akt), Alpha-fetoprotein (AFP), Beta-2-microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), BCR-ABL fusion gene, BRAF mutation V600E, CA15-3/CA27.29, CA19-9, CA-125, Carcinoembryonic antigen (CEA), CD20, Chromogranin A (CgA), Chromosomes 3, 7, 17, and 9p21 , Cytokeratin fragments 21 -1, EGFR mutations, Estrogen receptor (ER)/progesterone receptor (PR), Androgen receptor (AR), Fibrin/fibrinogen, HER4, HER2/neu, Immunoglobulins, KIT, KRAS , mutation analysis, Lactate dehydrogenase, Nuclear matrix protein 22, Prostate-specific antigen (PSA), Urokina
  • PSA Prostate-specific
  • the expression levels are mRNA expression level or protein expression level, or both.
  • the present invention provides a method for reducing over activation of PI3K/Akt pathway in a subject, comprising administering the subject with an inhibitor of PIP5K1A, thereby reducing over activation of the PDK/Akt pathway in the subject.
  • the present invention provides a method for reducing over activation of PI3K/Akt pathway in a cell, comprising contacting the cell with an inhibitor of PIP5K1A, thereby reducing over activation of the PI3K/Akt pathway in the cell.
  • the present invention provides a method for reducing over activation of PLC pathway in a subject, comprising administering the subject with an inhibitor of PIP5K1A, thereby reducing over activation of the PLC pathway in the subject.
  • the present invention provides a method for reducing over activation of PLC pathway in a cell, comprising contacting the cell with an inhibitor of PIP5K1A, thereby reducing over activation of the PLC pathway in the cell.
  • Figure 1 depicts depletion of PIP5K1A by RNAi in prostate cancer PC-3 cells. PIP5K1A expression was completely blocked by RNAi treatment at 48 and 72 hrs.
  • Figure 2A-B Figure 2 A and Figure 2B depict inhibition of Akt phosphorylation by PIP5K1A RNAi treatment in prostate cancer PC-3 cells. Akt phosphorylation and activation were significantly inhibited by blocking PIP5K1 A expression with RNAi treatment.
  • Figure 3A-B Figure 3A and Figure 3B depict inhibition of expression of Akt downstream effector cyclin Dl by PIP5K1A RNAi treatment in prostate cancer PC-3 cells. Cyclin Dl expression was significantly inhibited by blocking PIP5K1 A expression with RNAi treatment.
  • Figure 4 depicts increased expression of Akt downstream negative effector p27 by PIP5K1A RNAi treatment in prostate cancer PC-3 cells. P27 expression was significantly increased by blocking PIP5K1A expression with RNAi treatment.
  • FIG. 5 depicts expression of PIP5K1A in prostate cancer patient tumor samples.
  • PIP5K1A is over-expressed in prostate cancer patient tissues and its expression level correlates with cancer grade: very low in benign prostatic hyperplasia (BPH) patients, low in low grade prostate cancer patients and at the highest level in high grade metastatic prostate cancer patients.
  • BPH benign prostatic hyperplasia
  • Figure 6 depicts statistical analysis of PIP5K1A expression level in benign patients and malignant prostate cancer patients. There is a statistically significant difference between PIP5K1A levels in benign and malignant prostate cancer patients (P ⁇ 0.001).
  • FIG. 7C depicts correlation (FIG. 7C) of PIP5K1A expression (FIG. 7B) and Androgen Receptor (AR) expression (FIG. 7A) in prostate cancer patients. Both AR and PIP5K1A are over-expressed in malignant prostate cancer patients. There is a strong and statistically significant correlation between AR and PIP5K1A expression with correlation coefficient at 0.633 (P ⁇ 0.01).
  • Figure 8 depicts expression of PIP5K1A in healthy and lung cancer patient samples.
  • PIP5K1A is over-expressed in lung cancer patient tissues and its expression level correlates with cancer grade: very low in healthy subjects, low in non-invasive Lung Squamous Cell Carcinoma and very high in invasive Non-Small Cell Lung Cancer patients.
  • Figure 9A-G Characterization of ⁇ 5 ⁇ 1 ⁇ as a specific target for a novel anticancer compound ISA-201 1B.
  • Figure 9A The chemical structure of ISA-201 1B is presented.
  • Bound kinase levels in test compound and control wells are compared. The experiments were performed in triplicates.
  • Figure 9E The binding affinity of ISA-201 IB with ⁇ 5 ⁇ 1 ⁇ and a group of kinases was obtained from the assay indicated in Figure 9D is shown.
  • Figure 9F PC-3 cells were treated with DMSO as control, docetaxel (DTX) at 50 nM, ISA-201 IB and its analog 2011 A at 20 ⁇ for 48 hours. Protein lysates from each treatment were subjected to immunoblot analysis. Antibody against ⁇ 5 ⁇ 1 ⁇ was used as a probe. Data are the means of three independent experiments. *P ⁇ 0.05.
  • Figure 9G The binding affinity of ISA-201 IB with ⁇ 5 ⁇ 1 ⁇ and a group of kinases was obtained from the assay indicated in Figure 9D is shown.
  • Figure 9F PC-3 cells were treated with DMSO as control, docetaxel (DTX) at 50 nM, ISA-
  • FIG. 10A-B The effect of ISA-201 1B on growth of PC-3 tumor xenografts in vivo.
  • Figure 10A Growth of tumor xenografts treated with vehicle (Control), docetaxel (10 mg/kg), ISA- 201 IB (40 mg/kg), and docetaxel (10 mg/kg) in combination with ISA-2011B (40 mg/kg) every second day.
  • Figure 11A-D Evaluation of the clinical importance of ⁇ 5 ⁇ 1 ⁇ and its link with PIP2 and AR in PCa patients.
  • Figure 1 1A Immunohistochemical analysis of a TMA containing BPH and paired PCa specimens from 48 PCa patients. Representative microphotographs are shown. Scale bars are indicated and are applied to all images in panel a.
  • Figure 11B Box-plot quantitative comparison between BPH and paired cancer specimens from 48 PCa patients are shown. The paired Wilcoxon's rank sum test analyses are shown for ⁇ 5 ⁇ 1 ⁇ , PIP2, and AR. **P ⁇ 0.01.
  • Figure 11C Box-plots presenting expression of genes encoding ⁇ 5 ⁇ 1 ⁇ , AKT2 and AR.
  • Figure 12A-K ⁇ 5 ⁇ 1 ⁇ overexpression promotes malignant phenotype in non-malignant epithelial PNTIA cells via AKT/AR/CDKl pathways.
  • Figure 12B The effect of ⁇ 5 ⁇ 1 ⁇ overexpression on the morphology of PNTIA cells.
  • Representative immune-fluorescent images show the overexpression of ⁇ 5 ⁇ 1 ⁇ and its co -localization with ⁇ -Tubulin. DAPI was used to stain the nucleus of cells.
  • FIG. 12C Representative images of the immunofluorescent cells overexpressing ⁇ 5 ⁇ 1 ⁇ or control vector, stained with PIP2 antibody.
  • Figure 12D Immunoblots show the effect of ⁇ 5 ⁇ 1 ⁇ overexpression on PNTIA cells.
  • Immunoblots show the effect of overexpression of ⁇ 5 ⁇ 1 ⁇ on cyclin Dl , cyclin El , and ( Figure 12F) cyclin A2, CDKl , and cyclin Bl or ( Figure 12G, H) phosphorylated FAK, Twist, VEGF and MMP9 in PNT1A cells.
  • Figure 12J Immunoblots show the expression of AR in PNT1A cells overexpressing ⁇ 5 ⁇ 1 ⁇ or control vector.
  • Figure 12K The expression of AR in PNT1A cells overexpressing ⁇ 5 ⁇ 1 ⁇ or control vector.
  • Cytoplasmic (Cyt) and nuclear (Nuc) fractions were separated from PNT1A cells overexpressing ⁇ 5 ⁇ 1 ⁇ .
  • Cells were subjected to immunoprecipitation (IP) assay as shown in the right panel.
  • Antibody to CDKl was used to pull down the immunocomplexes, and antibody to IgG was used as a negative control.
  • Antibodies against AR or CDKl were used for immunoblot analysis (IB).
  • the cell lysates from cytoplasmic and nuclear fractions were used as "Input" controls as indicated in the left panel. Blotting of actin served as loading control, and antibody against lamin B was used as a control for the nuclear fraction.
  • Figure 13A-H The effect of ISA-201 1B on ⁇ 5 ⁇ 1 ⁇ and its downstream regulators in androgen-sensitive LNCaP cells.
  • Figure 13 A LNCaP cells were treated with docetaxel at 50 nM and ISA-201 1B (20 ⁇ ) and the lysates were subjected to immunoblot analysis. Antibodies against ⁇ 5 ⁇ 1 ⁇ and ⁇ -Actin were used.
  • Figure 13B LNCaP cells were treated with DMSO as vehicle control (Control), etoposide (20 ⁇ ), ISA-201 1A (20 ⁇ ), ISA-201 1B (20 ⁇ ), tadalafil (20 ⁇ ), and docetaxel (DTX) at 50 nM.
  • FIG. 13C Representative immunofluorescent images show the expression and subcellular localization of phosphorylated AKT in LCaP cells treated with vehicle control or ISA-201 1B.
  • Figure 13D Immunoblots show the expression of phosphorylated CREB in LCaP cells treated with different agents as indicated.
  • Figure 13E Representative immunofluorescent images show the expression and subcellular localization of CDKl in LCaP cells treated with vehicle control or ISA-201 IB.
  • Figure 13F Representative immunofluorescent images show the expression and subcellular localization of CDKl in LCaP cells treated with vehicle control or ISA-201 IB.
  • LNCaP cells were treated with docetaxel (50 nM), ISA-2009, ISA- 2011 A, and ISA-201 IB (20 ⁇ ).
  • the lysates were subjected to immunoblot analysis using antibodies against P27 and SKP2 as probes.
  • Figure 13G Representative immunofluorescent images show the expression and subcellular localization of AR and PSA in LNCaP cells treated with vehicle control or ISA-201 IB.
  • Figure 13H Immunoblots show the expression of AR and PSA in LNCaP cells that were treated with docetaxel (50 nM), ISA-2009, ISA-201 1A, and ISA-201 IB (20 ⁇ ).
  • Figure 14A-J ISA-201 1 inhibits tumor invasion through inhibition PIP5K la-mediated cancer cell pathways. Holomonitor M3 imaging system was applied to capture living PC-3 cells in culture during treatment.
  • Figure 14A Representative microphotographs of PC-3 cells treated with vehicle control, etoposide (50 ⁇ ), docetaxel (100 nM), and ISA-201 IB (50 ⁇ ) for 48 hours are shown. The scale bar is indicated.
  • Figure 14B Representative immunofluorescent images show the expression and subcellular localization of PIP3 in PC-3 cells treated with vehicle control or ISA- 201 IB. The scale bar is indicated.
  • Figure 14C Representative immunofluorescent images show the expression and subcellular localization of PIP3 in PC-3 cells treated with vehicle control or ISA- 201 IB. The scale bar is indicated.
  • Figure 14C Representative immunofluorescent images show the expression and subcellular localization of PIP3 in PC-3 cells treated with vehicle control or ISA- 201 IB. The scale bar is indicated.
  • PC-3 cells were treated with vehicle control (DMSO), 50 ⁇ of etoposide, ISA-2011 A, ISA-201 IB, tadalafil, and docetaxel (DTX) at 100 nM for 48 hours.
  • the protein lysates were subjected to immunoblots. Antibodies to phosphorylated AKT and total AKT were used as probes. Data is representative of three independent experiments. *P ⁇ 0.05.
  • Figure 14D and E Immunoblots shows the expression of the key cell cycle proteins in PC-3 cells treated with different agents as indicated.
  • Figure 14F Cell cycle distribution of PC-3 cells treated with vehicle control or ISA-2011B.
  • Figure 14G Cell cycle distribution of PC-3 cells treated with vehicle control or ISA-2011B.
  • FIG. 14H Representative FACS plots show the apoptosis status of cells treated with DMSO as vehicle control, docetaxel (DTX) or ISA-2011 (left panel). Data is representative of three independent experiments. **P ⁇ 0.01 (right panel).
  • Figure 14H Adhesion assay of PC-3 cells treated with DMSO as vehicle control, etoposide, and ISA-201 1B. Plates were coated with fibronectin prior to seeding the cells.
  • Figure 15A-I The effect of ⁇ 5 ⁇ 1 ⁇ inhibition on AKT pathways in PC-3 cells.
  • Figure 15A and B ⁇ 5 ⁇ 1 ⁇ was depleted by transfecting PC-3 cells with ⁇ 5 ⁇ 1 ⁇ siRNA (KD) or scramble control (Ct). Immunoblots for ⁇ 5 ⁇ 1 ⁇ and phosphorylated AKT in PC-3 cells that were transfected with ⁇ 5 ⁇ 1 ⁇ siRNA (KD) or scramble control (Ct) are shown. Data is presented as average of three independent experiments ( ⁇ SD). **P ⁇ 0.01.
  • Figure 15C and D The effect of ⁇ 5 ⁇ 1 ⁇ inhibition on AKT pathways in PC-3 cells.
  • Figure 15A and B ⁇ 5 ⁇ 1 ⁇ was depleted by transfecting PC-3 cells with ⁇ 5 ⁇ 1 ⁇ siRNA (KD) or scramble control (Ct). Immunoblots for ⁇ 5 ⁇ 1 ⁇ and phosphorylated AKT in PC-3 cells that were transfected with ⁇ 5 ⁇ 1 ⁇ siRNA (KD
  • FIG. 15E Immunoblots showing the effect of ⁇ 5 ⁇ 1 ⁇ knockdown on cyclin Dl, cyclin A2, CDK1, and cyclin B l in PC-3 cells.
  • Figure 15E Morphology of PC-3 cells expressing siRNA to ⁇ 5 ⁇ 1 ⁇ and PNT1A cells overexpressing ⁇ 5 ⁇ 1 ⁇ is shown. Antibody to alpha-tubulin was used for staining. The scale bar is indicated.
  • Figure 15F Immunoblots of phosphorylated FAK in PC-3 cells transfected with ⁇ 5 ⁇ 1 ⁇ siRNA or scramble control are shown. Data is presented as average of three independent experiments ( ⁇ SD).
  • FIG. 15G Immunoblots of P27, Twist, and MMP9 in PC-3 cells transfected with ⁇ 5 ⁇ 1 ⁇ siRNA or scramble control are shown.
  • Figure 15H Immunoblots of c-PARP in PC-3 cells treated with vehicle control (DMSO) or ISA-201 IB (20 ⁇ ' and 50 ⁇ ⁇ ), with or without ⁇ 5 ⁇ 1 ⁇ or scramble siRNA co-transfection are shown.
  • Figure 151 Immunoblots of c-PARP in PC-3 cells treated with vehicle control (DMSO) or ISA-201 IB (20 ⁇ ' and 50 ⁇ ⁇ ), with or without ⁇ 5 ⁇ 1 ⁇ or scramble siRNA co-transfection are shown.
  • PIP2 serves as a precursor molecule required for PI3K activity and generation of PIP3.
  • PIP3 is involved in the downstream activation of AKT, and AKT -related signaling pathways.
  • ISA-2011B is able to enter into the cell, and inhibit ⁇ 5 ⁇ 1 ⁇ . This leads to the reduced production of PIP2 and PIP3 and a subsequent inhibition of PI3K/AKT, but sustained P27 and down-regulation of CDK1 and other cell cycle regulators. Inhibition of AR signaling pathways is mediated through CDK1 in part via feedback loop.
  • FIG. 17A-D Effect of ISA-2011B in various aggressive cancer cell lines.
  • HTC-1 16 Figure 17A
  • CaCo2 Figure 17B
  • colon cancer cells MDA-MB-231 breast cancer cells
  • Figure 17D U937 leukemic cells
  • the cells were treated with vehicle control (DMSO) or ISA-2011B dissolved in 100% DMSO.
  • the treatments were carried out for 48 h, at 10 ⁇ , 50 ⁇ , and 100 ⁇ of ISA-201 IB.
  • the figure shows the viable cell count for every treatment group.
  • FIG. 19 Correlation between level of PTEN mRNA expression and disease-free survival in PCa patients.
  • Kaplan-Meier survival analysis based on biochemical recurrence-free (BCR-free) in PCa patients with high or low PTEN expression.
  • the follow-up time is 160 months, as indicated. Differences in BCR-free survivals between two groups were calculated using the log-rank test, P ⁇ 0.001.
  • Figure 20 Effect of ⁇ 5 ⁇ 1 ⁇ overexpression on AR in PNT1A cells.
  • Representative immunofluorescent images show the expression and localization of AR in PNT1A cells overexpressing ⁇ 5 ⁇ 1 ⁇ or control vector. The cells were stained with antibody to AR (green) and DAPI (blue). The merge of these two images is shown.
  • FIG. 21 Interaction between CDK1 and AR in PNT1A cells.
  • Cytoplasmic (Cyt) and nuclear (Nuc) fractions were separated from PNT1A cells expressing pPLPS Control vector and were subjected to immunoprecipitation (IP) assay as shown (Right).
  • IP immunoprecipitation
  • Antibody to CDK1 was used to pull down the immunocomplexes, and antibody to IgG was used as a negative control.
  • Antibodies against AR or CDK1 were used for immunoblot analysis (IB) to detect AR or CDK1 in cytoplasmic or nuclear compartment.
  • the cell lysates from cytoplasmic and nuclear fractions were used as "Input" controls, as indicated (Left). Blotting of actin served as loading control, and antibody against lamin B was used as a control for the nuclear fraction.
  • FIG. 22 Effect of ISA-2011B in the androgen-dependent LNCaP cell line.
  • LNCaP prostate cancer cells were purchased from American Type Culture Collection. The cells were treated with vehicle control (DMSO) or ISA-2011B dissolved in 100% DMSO. The treatments were carried out for 48 h, at 10 ⁇ , 50 ⁇ , and 100 ⁇ of ISA-201 IB. The figure shows the viable cell count for every treatment group.
  • DMSO vehicle control
  • ISA-2011B dissolved in 100% DMSO.
  • the figures shows the viable cell count for every treatment group.
  • the present invention provides compositions and methods related to using inhibitors of PIP5K1A for treatment of diseases.
  • PIP5K1A catalyses the phosphorylation of phosphatidylmositol 4- phosphate (PtdIns4P) to form phosphatidylmositol 4,5-bisphosphate (PtdIns(4,5)P2).
  • PtdIns(4,5)P2 is involved in a variety of cellular processes and is the substrate to form phosphatidylmositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), another second messenger.
  • PtdIns(4,5)P2 is thought to be produced via type I phosphatidylmositol 4-phosphate 5-kinases (PIP5Ks) given the abundance of PtdIns4P.
  • PIP5K1A can catalyse the phosphorylation of phosphatidylmositol 3,4- bisphosphate (PtdIns(3,4)P2) to form phosphatidylmositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3).
  • PIP5K1A has been known as one of the PIP5Ks that participates in a variety of cellular processes such as actin cytoskeleton organization, cell adhesion, migration and phagocytosis.
  • PIP5K1A is required for membrane ruffling formation, actin organization and focal adhesion formation during directional cell migration by controlling integrin-induced translocation of RAC1 to the plasma membrane.
  • PIP5K1A is required for phagocytosis, but they regulate different types of actin remodeling at sequential steps.
  • PIP5K1A promotes particle ingestion by activating WAS that induces Arp2/3 dependent actin polymerization at the nascent phagocytic cup.
  • PIP5K1A, together with PIP5K1B, is required after stimulation of G-protein coupled receptors for stable platelet adhesion.
  • PIP5K1A plays a role during calcium-induced keratinocyte differentiation.
  • PIP5K1A is recruited to the plasma membrane by the E-cadherin/beta-catenin complex where it provides the substrate PtdIns(4,5)P2 for the production of PtdIns(3,4,5)P3, diacylglycerol and inositol 1,4,5-trisphosphate that mobilize internal calcium and drive keratinocyte differentiation.
  • PIP5K1A, together with PIP5K1C, also have a role during embryogenesis.
  • PIP5K1A is highly expressed in heart, placenta, skeletal muscle, kidney and pancreas.
  • PIP5K1A is detected at lower levels in brain, lung and liver.
  • Four isoforms of the human protein PIP5K1 A are produced by alternative splicing.
  • the present invention provides a method for treatment of a disease in a subject in which inhibition of PIP5K1A is desired and/or required.
  • the method comprises administering a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5KlA.
  • PIP5K1A is a regulator of multiple signal transduction pathways that involved in various diseases, such as cancer. Exemplary paths are the PDK/Akt pathway and the phospholipase C (PLC) pathway.
  • PLC phospholipase C
  • the expression level and/or activity in the cells of disease tissues may be normal or more likely, higher than normal (over-expressed). Nevertheless, as over activation of many signal transduction pathways, such as the PDK/Akt pathway and the PLC pathway, are involved in various diseases conditions, such as cancer, downregulation of PIP5K1A activity reduces the activity of these pathways. Downregulation of PI3K/Akt and/or PLC pathway activity in turn results in reversion or curing of disease conditions.
  • PIP5K1A is over-expressed.
  • PIP5K1A is over-expressed in prostate cancer patient tissues.
  • its expression level correlates with cancer grade: very low in benign prostatic hyperplasia (BPH) patients, low in low grade prostate cancer patients and at the highest level in high grade metastatic prostate cancer patients. See Figure 5.
  • the present invention provides various inhibitors of PIP5K1 A that can be used to reduce PIPKIA activity.
  • Reduction of PIP5K1A activity can be measured either as relative to the level of over- expression or relative to the level of normal expression, as determined by an in vitro or in vivo assay.
  • PIP5K1A activity is reduced by about 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% in comparison to the over-expression level.
  • the inhibitor of PIP5K1A is capable of reducing PIP5K1A activity by between about 5% and 100% as determined by an in vitro or in vivo assay.
  • the inhibitor of PIP5K1A is capable of reducing PIP5K1A activity by between about 40% and 80% as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing PIP5K1A activity to 1 , 2, 3, 4, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,1 15, 120, 125, 130, 135, 140, 145, 150, 155, or 160 % of normal PIP5K1A activity as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing PIP5K1A activity to about 1 % and 150% of normal PIP5K1A activity of as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing PIP5K1A activity to between about 50 % and 150% of normal PIP5K1A activity of as determined by an in vitro or in vivo assay.
  • PIP5K1A activity herein is meant the mRNA expression level, protein expression level, or kinase activity of PIPKIA. Measurement of mRNA expression level, protein expression level, or kinase level is carried out using methods known in the art and/or disclosed herein.
  • the present invention provides various PIP5K1A modulators obtained using the methods provided herein.
  • PIP5K1A phosphatidylinositol-4-phosphate 5-kinase, type I, alpha.
  • the DNA and protein sequences of PIPKIA from various species are known in the art and can be obtained from various public domain sources, such as GeneBank.
  • the PIP5K1A used here is of a mammal, preferably of human, rat, or mice.
  • PIP5K1A nucleic acids e.g. cDNA or genomic DNA
  • PIP5K1A nucleic acids is synthesized or cloned from various sources and used to generate transgenic cells or animals, or to generate PIP5K1 A polypeptide or a fragment thereof to be used in the assays provided herein.
  • PIP5K1A modulator herein is meant a molecule that can affect the expression (mRNA level or protein level) and/or the activity (e.g., kinase activity) of PIP5K1A, as determined by an in vivo or in vitro assay.
  • a modulator can either decrease (inhibitor) or enhance (activator) the expression and/or the activity of PIP5K1 A.
  • the modulator is an inhibitor.
  • the methods of the invention utilize PIP5K1A polypeptides, antibodies or nucleic acids which encode PIP5K1A polypeptides for identifying candidate bioactive agents which bind to PIP5K1A, which modulate the activity of PIP5K1A, or which alter the expression of PIP5K1A within cells.
  • candidate bioactive agent any molecule which binds to PIP5K1A, modulates the kinase activity of PIP5K1A, and/or alters the expression of PIP5K1A within cells.
  • a molecule, as described herein can be an oligopeptide, small organic molecule, polysaccharide, antibody or polynucleotide, etc.
  • a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons (D).
  • Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
  • the candidate bioactive agents are proteins.
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo- phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
  • Amino acid also includes amino acid residues such as proline and hydroxyproline.
  • the side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
  • the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins.
  • cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts may be used.
  • libraries of multicellular eukaryotic proteins may be made for screening in the methods of the invention.
  • Particularly preferred in this embodiment are libraries of multicellular eukaryotic proteins, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
  • the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred.
  • the peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased” random peptides.
  • randomized or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position.
  • the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
  • the library is fully randomized, with no sequence preferences or constants at any position.
  • the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
  • the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
  • the candidate bioactive agents are nucleic acids.
  • nucleic acid candidate bioactive agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids.
  • digests of prokaryotic or eukaryotic genomes may be used as is outlined above for proteins.
  • the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.
  • RNAi short RNA
  • anti-sense RNAs anti-sense RNAs
  • microRNMs microRNMs
  • DNAs can be used as therapeutic agents for blocking the expression of certain PIP5K1A genes in vivo.
  • short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane (Zamecnik et al., (1986), Proc. Natl. Acad. Sci. USA 83:4143-4146).
  • the anti- sense oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
  • PIP5K1A anti-sense RNAs and DNAs can be used to prevent PIP5K1A gene transcription into mRNAs, to inhibit translation of PIP5K1A mRNAs into proteins, and to block activities of pre-existing PIP5K1A proteins.
  • a multivalent cation indicator is a molecule that is readily permeable to a cell membrane or otherwise amenable to transport into a cell e.g., via liposomes, etc., and upon entering a cell, exhibits a fluorescence that is either enhanced or quenched upon contact with a multivalent cation.
  • Examples of multivalent cation indicators useful in the invention are set out in Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals. 1 1th ed. Molecular Probes, Inc. Eugene, OR (2013); incorporated herein by reference in its entirety.
  • either PIP5K1A or the candidate bioactive agent is labelled with, for example, a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide means of detecting the binding of the candidate agent to PIP5K1A.
  • the label also can be an enzyme, such as, alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that can be detected.
  • the label can be a labelled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalysed or altered by the enzyme.
  • the label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin.
  • the streptavidin is labelled as described above, thereby, providing a detectable signal for the bound PIP5K1A.
  • unbound labelled streptavidin is removed prior to analysis.
  • PIP5K1A can be immobilized or covalently attached to a surface and contacted with a labelled candidate bioactive agent.
  • a library of candidate bioactive agents can be immobilized or covalently attached to a biochip and contacted with a labelled PIP5K1A. Procedures which employ biochips are well known in the art.
  • the kinase activity PIP5K1A is measured in intact cells, preferably PC-3 cells, which have high PIP5K1A activity.
  • candidate agents can be used which wholly suppress the expression of PIP5K1A within cells, thereby altering the cellular phenotype.
  • candidate agents can be used which reduce the expression of PIP5K1A within cells, thereby altering the cellular phenotype.
  • these candidate agents include antisense cDNAs and DNAs, regulatory binding proteins and/or nucleic acids, as well as any of the other candidate bioactive agents herein described which modulate transcription or translation of nucleic acids encoding PIP5K1A.
  • the invention provides antibodies which specifically bind to unique epitopes on the human PIP5K1 A polypeptide, e.g. unique epitopes of the protein.
  • the invention provides antibodies which specifically bind to unique epitopes on the mouse PIP5K1A polypeptide, e.g. unique epitopes of the protein.
  • the anti-PIP5Kl A polypeptide antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include the PIP5K1A polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the immunization protocol may be selected by one skilled in the art without undue experimentation.
  • the anti-PIP5KlA polypeptide antibodies may further comprise monoclonal antibodies.
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include the PIP5K1A polypeptide or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Rockville, Md. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against a PIP5K1A polypeptide.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme- linked immunosorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S. Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • a non- immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the anti-PIP5KlA polypeptide antibodies may further comprise monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
  • the anti-PIP5KlA polypeptide antibodies may further comprise humanized antibodies or human antibodies.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fabi, F(abi) 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non- human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain.
  • Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)].
  • human antibodies can be made by the introducing of human immunoglobulin loci into transgenic animals, e.g. mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • the anti-PIP5KlA polypeptide antibodies may further comprise heteroconjugate antibodies.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Pat. No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089].
  • the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.
  • anti-PIP5KlA polypeptide antibodies may have various utilities.
  • anti-PIP5KlA polypeptide antibodies may be used in diagnostic assays for PIP5K1A polypeptides, e.g., detecting its expression in specific cells, tissues, or serum.
  • diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158].
  • the antibodies used in the diagnostic assays can be labelled with a detectable moiety.
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as .sup.3H, .sup. l4C, .sup.32P, .sup.35S, or .sup.1251, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta- galactosidase or horseradish peroxidase.
  • any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13 : 1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).
  • the PIP5K1A inhibitor comprises a small interference RNA (siRNA) and modulates kinase expression through RNA interference (RNAi).
  • siRNA small interference RNA
  • RNAi RNA interference
  • Figure 1 depicts depletion of PIP5K1A by RNAi in prostate cancer PC-3 cells. PIP5K1A expression was completely blocked by RNAi treatment at 48 and 72 hrs.
  • FIGS. 2A and 2B depict inhibition of Akt phosphorylation by PIP5K1A RNAi treatment in prostate cancer PC-3 cells. Akt phosphorylation and activation were significantly inhibited by blocking PIP5K1A expression with RNAi treatment.
  • Figure 3 A and 3B depict inhibition of expression of Akt downstream effector cyclin Dl by PIP5K1A RNAi treatment in prostate cancer PC-3 cells. Cyclin Dl expression was significantly inhibited by blocking PIP5K1 A expression with RNAi treatment.
  • Figure 4 depicts increased expression of Akt downstream negative effector p27 by PIP5K1A RNAi treatment in prostate cancer PC-3 cells. P27 expression was significantly increased by blocking PIP5K1A expression with RNAi treatment. 3. PBK/Akt Pathway
  • the present invention provides a method for treatment of a disease in a subject characterized by over activation of Akt and is in need of such treatment, comprising: administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.
  • the PI3K/AKT/mTOR pathway is an intracellular signalling pathway important in cell proliferation, apoptosis and angiogenesis, and hence in cancers, e.g. breast cancer and non-small-cell lung cancer.
  • PI3K activation activates AKT which activates mTOR.
  • this pathway is overactive thus increasing cancer cell proliferation, reducing cancer cell apoptosis, and promoting tumor angiogenesis and metastasis.
  • some experimental cancer drugs aim to inhibit the signalling pathway of PDK/AKT/mTOR.
  • the PI3K pathway may be overactive because PTEN is faulty or deficient.
  • Akt also known as Protein Kinase B (PKB)
  • PPKB Protein Kinase B
  • Akt also known as Protein Kinase B (PKB)
  • PKA Protein Kinase B
  • Akt has three family members, Aktl, Akt2 and Akt3.
  • Akt is associated with tumor cell survival, proliferation, and invasiveness. The activation of Akt is also one of the most frequent alterations observed in human cancer and tumor cells. Tumor cells that have constantly active Akt may depend on Akt for survival.
  • a mosaic activating mutation (c. 49G ⁇ A, p.Glul7Lys) in AKT1 is associated with the Proteus Syndrome, which causes overgrowth of skin, connective tissue, brain and other tissues.
  • Akt inhibitors may treat cancers such as neuroblastoma.
  • miltefosine is approved for leishmaniasis and under investigation for other indications including HIV.
  • AKT activation is associated with many malignancies, however, and it has been observed a converse role for AKT and one of its downstream effector FOXOs in acute myeloid leukemia (AML). It was claimed that low levels of AKT activity associated with elevated levels of FOXOs are required to maintain the function and immature state of leukemia-initiating cells (LICs). FOXOs are active, implying reduced Akt activity, in about 40% of AML patient samples regardless of genetic subtype; and either activation of Akt or compound deletion of FOXO 1/3/4 reduced leukemic cell growth in a mouse model.
  • Akt activity herein is meant the mRNA expression level, protein expression level, or kinase activity of Akt. Measurement of mRNA expression level, protein expression level, or kinase level is carried out using methods known in the art and/or disclosed herein. [0131] Reduction of Akt activity can be measured either as relative to the level of over-expression or relative to the level of normal expression, as determined by an in vitro or in vivo assay.
  • Akt activity is reduced by about 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% in comparison to the over-expression level.
  • inhibitor of PIP5K1A is capable of reducing Akt activity by between about 5% and 100% as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing Akt activity by between about 40% and 80% as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing Akt activity to 1 , 2, 3, 4, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10,1 15, 120, 125, 130, 135, 140, 145, 150, 155, or 160 % of normal Akt activity as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing Akt activity to about 1 % and 150% of normal Akt activity of as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing Akt activity to between about 50 % and 150% of normal Akt activity of as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing Akt activity to between about 80 % and 120% of normal Akt activity of as determined by an in vitro or in vivo assay.
  • the present invention provides method for treatment of a disease in a subject characterized by over activation of Akt and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of modulator of PDK/Akt pathway, wherein the modulator is capable of reducing Akt activity to between about 50 % and 150% of normal Akt activity as determined by an in vitro or in vivo assay.
  • the inhibitor of PIP5K1A is capable of reducing Akt activity to between about 80 % and 120% of normal Akt activity of as determined by an in vitro or in vivo assay.
  • the modulator of PDK/Akt pathway comprises a small molecule, an RNA, a peptide, or an antibody.
  • the modulator of PDK/Akt pathway comprises a PIP5K1A inhibitor as provided herein.
  • the modulator of PDK/Akt is obtained using methods similar to the methods provided herein for obtaining PIP5klA modulator, as a person skilled in the art will appreciate.
  • the present invention provides a method for treatment of a disease in a subject characterized by over activation of PI3 Kinase and is in need of such treatment, comprising: administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.
  • Phosphatidylinositide 3-kinases are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer.
  • PBKs are a family of related intracellular signal transducer enzymes capable of phosphorylating the 3 position hydroxyl group of the inositol ring of phosphatidylmositol (Ptdlns). They are also known as phosphatidylinositol-3- kinases.
  • PI3K catalyses the phosphorylation of phosphatidylmositol 4,5-bisphosphate (PtdIns(4,5)P2) to form phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2).
  • Phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) then activates Akt and a myriad of downstream effectors.
  • This pathway with oncogene PIK3CA and tumor suppressor PTEN (gene), is implicated in tumorigenesis of many cancers, and insensitivity of cancer tumors to insulin and IGF1 in calorie restriction.
  • Such reduction of PI3K activity can be measured by level of its enzymatic products, such as phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2), either as relative to the level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K is over-expressed or relative to the level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay.
  • PtdIns(3,4,5)P2 phosphatidylmositol 3,4,5-bisphosphate
  • PIP5K1A inhibitor may not directly inhibit PI3K activity, but rather inhibits production of PI3K enzymatic substrate phosphatidylmositol 4,5-bisphosphate (PtdIns(4,5)P2, so it cannot catalyse production of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2).
  • reduction of PI3K activity generally is measured by level of its enzymatic products, such as phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2), either as relative to the level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K is over-expressed or relative to the level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay.
  • PtdIns(3,4,5)P2 phosphatidylmositol 3,4,5-bisphosphate
  • production level of phosphatidylmositol 3,4,5-bisphosphate is reduced by about 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% in comparison to production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K is over-expressed, as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) to between about 1 , 2, 3, 4, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10,115, 120, 125, 130, 135, 140, 145, 150, 155, or 160 % of normal production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay.
  • PtdIns(3,4,5)P2 phosphatidylmositol 3,4,5-bisphosphate
  • inhibitor of PIP5K1A is capable of reducing production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) to between about 1 % and 150% of normal production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay.
  • PtdIns(3,4,5)P2 phosphatidylmositol 3,4,5-bisphosphate
  • inhibitor of PIP5K1A is capable of reducing production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) to between about 50 %> and 150%o of normal production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay.
  • the present invention provides method for treatment of a disease in a subject characterized by over activation of phospholipase C (PLC) and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.
  • PLC phospholipase C
  • Phospholipase C is a class of enzymes that cleave phospholipids just before the phosphate group. It is most commonly taken to be synonymous with the human forms of this enzyme, which play an important role in eukaryotic cell physiology, in particular signal transduction pathways. Thirteen types of mammalian phospholipase C are classified into six isotypes ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) according to structure.
  • Reduction of PLC activity can be measured either as relative to the level of over-expression or relative to the level of normal expression, as determined by an in vitro or in vivo assay.
  • PLC activity is reduced by about 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%o in comparison to the over-expression level.
  • inhibitor of PIP5K1A is capable of reducing PLC activity by between about 5% and 100% as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing PLC activity by between about 40% and 80% as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing PLC activity to 1 , 2, 3, 4, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10,115, 120, 125, 130, 135, 140, 145, 150, 155, or 160 % of normal PLC activity of as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing PLC activity to about 1 % and 150% of normal PLC activity of as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing PLC activity to between about 50 % and 150% of normal PLC activity of as determined by an in vitro or in vivo assay.
  • PLC activity herein is meant the mRNA expression level, protein expression level, or lipase activity of PLC. Measurement of mRNA expression level, protein expression level, or lipase level is carried out using methods known in the art and disclosed herein.
  • inhibitor of PIP5K1A is capable of reducing PLC activity to between about 50 % and 150% of normal PLC activity of as determined by an in vitro or in vivo assay.
  • inhibitor of PIP5K1A is capable of reducing PLC activity to between about 80 % and 120% of normal PLC activity of as determined by an in vitro or in vivo assay.
  • the present invention provides compositions and methods related to using PIP5K1A gene as a biomarker for diagnosis, prognosis, or monitoring of various diseases (preferably cancer) and selection of patients for treatment with certain diseases.
  • the present invention provides a method of diagnosing a subject suspect of having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of one or more genes in the biological sample, wherein at least one of said one or more genes is PIP5K1A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to diagnosis of the disease, thereby diagnosing the subject with regard to the disease.
  • biological sample herein is meant any biological sample suspected of containing a target gene and/or gene product to be detected (e.g. PIP5K1A), such as polynucleotides or polypeptides or fragments thereof, and may comprise a cell, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern analysis), RNA (in solution or bound to a solid support such as for northern analysis), cDNA (in solution or bound to a solid support), protein (in solution or bound to a solid support such as for western blot analysis), peptide (in solution or bound to a solid support such as for western blot analysis), an extract from cells, blood, urine, marrow, body fluid, or a tissue, and the like.
  • a target gene and/or gene product to be detected e.g. PIP5K1A
  • genomic DNA in solution or bound to a solid support such as for Southern analysis
  • Biological samples useful in the practice of the methods of the invention may be obtained from any mammal in which a diseases condition is suspected, confirmed, or developing.
  • the mammal is a human, and the human may be a candidate for a therapeutic for the treatment of a cancer, e.g. prostate cancer.
  • the human candidate may be a patient currently being treated with, or considered for treatment with, a PIP5K1 A inhibitor, such as those provided herein.
  • the mammal is large animal, such as a horse or cow, while in other embodiments, the mammal is a small animal, such as a dog or cat, all of which are known to develop cancers, including lung carcinomas.
  • any biological sample comprising cells (or extracts of cells) from a mammalian cancer is suitable for use in the methods of the invention. Circulating tumor cells may also be obtained from serum using tumor markers, cytokeratin protein markers or other methods of negative selection as described (see Ma et al., Anticancer Res. 23(1A): 49-62 (2003)). Serum and bone marrow samples may be particularly preferred for patients with leukemia.
  • the biological sample may comprise cells obtained from a tumor biopsy, which maybe be obtained according to standard clinical techniques.
  • Circulating tumor cells may be purified, for example, using the kits and reagents sold under the trademarks VITA-ASSAYSTM, VITA-CAPTM, and CELLSEARCH® (commercially available from Vitatex, LLC (a Johnson and Johnson corporation). Other methods for isolating CTCs are described (see, for example, PCT Publication No. WO/2002/020825, CristofaniUi et al., New Engl. J. of Med. 351 (8):781-791 (2004), and Adams et al., J. Amer. Chem. Soc. 130(27): 8633-8641 (July 2008)).
  • a CTC may be isolated and identified as having originated from the lung.
  • the PIP5K1A Polypeptide is detected by an immunoassay.
  • An PIP5K1A protein or peptide is generated to produce antibodies (monoclonal or polyclonal) specific for PIP5K1A proteins. Such antibodies are then used in an assay to detect the presence of PIP5K1A.
  • PIP5K1A is generally detected using a PIP5K1A polypeptide-specific reagent.
  • PIP5K1A polypeptide-specific reagent herien is meant any reagent, biological or chemical, capable of specifically binding to, detecting and/or quantifying the presence/level of expressed PIP5K1A polypeptide in a biological sample.
  • the term includes, but is not limited to, the preferred antibody and reagents discussed below, and equivalent reagents are within the scope of the present invention.
  • Reagents suitable for use in practice of the methods of the invention include an PIP5K1A polypeptide-specific antibody.
  • Human PIP5K1A polypeptide-specific antibodies may also bind to highly homologous and equivalent epitopic peptide sequences in other mammalian species, for example murine or rabbit, and vice versa.
  • Antibodies useful in practicing the methods of the invention include (a) monoclonal antibodies, (b) purified polyclonal antibodies that specifically bind to the target polypeptide (e.g. the PIP5K1A polypeptide), (c) antibodies as described in (a)-(b) above that bind equivalent and highly homologous epitopes or phosphorylation sites in other non-human species (e.g. mouse, rat), and (d) fragments of (a)-(c) above that bind to the antigen (or more preferably the epitope) bound by the exemplary antibodies disclosed herein
  • antibody or “antibodies” herein is meant all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE.
  • the antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26: 403-1 1 (1989); Morrision et al., Proc. Nat'l. Acad. Sci. 81 : 6851 (1984); Neuberger et al., Nature 312: 604 (1984)).
  • the antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 (Reading) or U.S. Pat. No. 4,816,567 (Cabilly et al.).
  • the antibodies may also be chemically constructed specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980 (Segel et al.)
  • the invention is not limited to use of antibodies, but includes equivalent molecules, such as protein binding domains or nucleic acid aptamers, which bind, in a fusion-protein or truncated-protein specific manner, to essentially the same epitope to which an PIP5K1A polypeptide-specific antibody in the methods of the invention binds. See, e.g., Neuberger et al., Nature 312: 604 (1984). Such equivalent non-antibody reagents may be suitably employed in the methods of the invention further described below.
  • Polyclonal antibodies useful in practicing the methods of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen encompassing a desired fusion-protein specific epitope (e.g. the fusion junction of an Rspo fusion protein described herein), collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, and purifying polyclonal antibodies having the desired specificity, in accordance with known procedures.
  • the antigen may be a synthetic peptide antigen comprising the desired epitopic sequence, selected and constructed in accordance with well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 5, p.
  • U.S. Pat. No. 5,194,392 Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) that is a topological equivalent of the epitope (i.e., a "mimotope") that is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, this method involves detecting or determining a sequence of monomers that is a topographical equivalent of a ligand that is complementary to the ligand binding site of a particular receptor of interest.
  • U.S. Pat. No. 5,480,971 Houghten et al.
  • Antibodies employed in the methods of the invention may be further characterized by, and validated for, use in a particular assay format, for example flow cytometry (FC), immunohistochemistry (IHC), and/or Immunocytochemistry (ICC).
  • Antibodies may also be advantageously conjugated to fluorescent dyes (e.g. Alexa488, PE), or labels such as quantum dots, for use in multi-parametric analyses along with other signal transduction (phospho-AKT, phospho- Erk 1/2) and/or cell marker (cytokeratin) antibodies.
  • PIP5K1A -specific reagents provided by the invention also include nucleic acid probes and primers suitable for detection of a PIP5K1A polynucleotide.
  • the PIP5K1A gene is detected by PCR, such as regular PCR, Reverse Transcription PCR, Real-time PCR (Q-PCR) or digital PCR.
  • PCR such as regular PCR, Reverse Transcription PCR, Real-time PCR (Q-PCR) or digital PCR.
  • a pair of primers is used to amplify the PIP5K1A gene.
  • the primers are designed based on the target gene sequence to be amplified.
  • the PIP5K1A gene is detected by other hybridization-based methods, such as microarray, branched DNA (QUANTIGENE ® ), VIEWRNA ® or RNASCOPE ® .
  • the present invention provides compositions and methods for detection of PIP5K1A overexpression.
  • Overexpression of PIP5K1A may or may not co-exist with overexpression or activation of PI3K, Akt, or PLC.
  • PIP5K1A overexpression can be overexpression of either PIP5K1A mRNA or polypeptide, or both.
  • the PIP5K1 A can be either wild-type or a variant of PIP5K1 A.
  • PIP5K1A overexpression is determined relevant to a baseline expression level, which is obtained by measuring expression level of PIP5K1A (mRNA or polypeptide) in normal cells or a normal subject population (e.g., normal human population).
  • the expression level of PIP5K1A mRNA level is measured using methods known in the art, such as Northern blot, RT-PCR, RT-PCT combined with Real-time PCR, digital PCR, DNA array, high throughput sequencing, or in situ hybridization, and the like.
  • the expression level of PIP5K1A is measured using methods known in the art, such as Western blot, protein array, immunohistology staining, and the like.
  • the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient diagnosed as having the disease.
  • the present invention provides a method for monitoring disease progression of a subject diagnosed as having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to disease progression of the disease, thereby determining disease progression of the subject with regard to the disease.
  • the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient has been diagnosed as having the disease of different disease progression.
  • the present invention provides a method for determining disease stage of a subject diagnosed as having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to disease stage of the disease, thereby determining disease stage of the subject with regard to the disease.
  • the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient has been diagnosed as having said disease of different disease stages.
  • PIP5kl A expression level can be used for determining disease stage of (e.g. cancer grade) as the level correlates to disease stage (e.g. low level in BPH, medium level in low grade prostate cancer, high level in high grade of cancer, highest level in metastatic cancer).
  • the present invention provides a method for determining a prognosis for a subject diagnosed as having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of one or more genes, wherein the reference expression levels correlate to prognosis of the disease, thereby determining prognosis of the subject with regard to the disease.
  • the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient diagnosed as having the disease of different progression stages.
  • the present invention provides a method of determining effect of treatment of a subject having a disease condition with a pharmaceutical composition, comprising: (a) providing a biological sample from a subject that has been subject to a treatment of a disease; (b) measuring expression levels for one or more genes and/or product of one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of one or more genes, wherein the reference expression levels correlate to treatment effect of the disease, thereby determining the treatment effect of the pharmaceutical composition.
  • the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient that has been subjected to said treatment and achieved treatment effect.
  • the present invention provides a method of determining whether a subject having a disease condition should be treated with a pharmaceutical composition comprising an inhibitor of PIP5K1A, and/or an inhibitor of PI3K/Akt pathway, and/or an inhibitor of PLC pathway, comprising: (a) providing a biological sample from a subject t having a disease condition; (b) measuring expression levels for one or more genes and/or product of one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of one or more genes, wherein the reference expression levels correlate to whether a subject having a disease condition should be treated with a pharmaceutical composition comprising an inhibitor of PIPK1 A, and/or an inhibitor of PDK/Akt pathway, and/or an inhibitor of PLC pathway, thereby determining whether the subject should be treated with the pharmaceutical composition.
  • the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient that has been subjected to said treatment and achieved treatment effect.
  • one or more genes is combined with PIP5KA as a biomarker, and such gene is selected from the group consisting of: Akt, pAkt (phosphorylated Akt), Alpha- fetoprotein (AFP), Beta-2 -microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), BCR-ABL fusion gene, BRAF mutation V600E, CA15-3/CA27.29, CA19-9, CA-125, Carcinoembryonic antigen (CEA), CD20, Chromogranin A (CgA), Chromosomes 3, 7, 17, and 9p21, Cytokeratin fragments 21-1 , EGFR mutations, Estrogen receptor (ER)/progesterone receptor (PR), Androgen receptor (AR), Fibrin/fibrinogen, HER4, HER2/neu, Immunoglobulins, KIT, KRAS , mutation analysis, Lactate dehydrogenase, Nuclear matrix protein
  • the present invention provides methods for reducing PI3K/Akt over activation using an inhibitor of PIP5KA.
  • the methods can be used in screening for new inhibitor of PIP5K1A.
  • the present invention provides a method for reducing over activation of PI3K/Akt in a subject, comprising administering the subject with an inhibitor of PIP5K1A, thereby reducing over activation of the PI3K/Akt in the subject.
  • the present invention provides a method for reducing over activation of PI3K/Akt in a cell, comprising contacting the cell with an inhibitor of PIP5K1A, thereby reducing over activation of the PDK/Akt in the cell.
  • Compounds disclosed herein include small molecules, biologies (RNA, DNA, RNA/DNA hybrid, antibody, peptide and polymer).
  • Compounds of the invention are indicated as pharmaceuticals.
  • a compound of the invention as hereinbefore (but without any provisos, where applicable), for use as a pharmaceutical.
  • a synthetic form of a compound of the invention (but without any provisos, where applicable), for use as a pharmaceutical.
  • compounds of the invention may possess pharmacological activity as such, certain pharmaceutically-acceptable (e.g. "protected") derivatives of compounds of the invention may exist or be prepared which may not possess such activity, but may be administered, including bot not limited to parenterally or orally and thereafter be metabolized in the body to form compounds of the invention.
  • Such compounds (which may possess some pharmacological activity, provided that such activity is appreciably lower than that of the "active" compounds to which they are metabolized) may therefore be described as "prodrugs" of compounds of the invention.
  • prodrug of a compound of the invention we include compounds that form a compound of the invention, in an experimentally-detectable amount, within a predetermined time (e.g. about 1 hour), following an administration, including but not limited to oral or parenteral administration. All prodrugs of the compounds of the invention are included within the scope of the invention.
  • certain compounds of the invention may possess no or minimal pharmacological activity as such, but may be administered, including but not limited to parenterally or orally, and thereafter be metabolized in the body to form compounds of the invention that possess pharmacological activity as such.
  • Such compounds (which also includes compounds that may possess some pharmacological activity, but that activity is appreciably lower than that of the "active" compounds of the invention to which they are metabolized), may also be described as "prodrugs".
  • the compounds of the invention are useful because they possess pharmacological activity, and/or are metabolized in the body following an administration, including but not limited to oral or parenteral administration, to form compounds which possess pharmacological activity.
  • cancer we mean any disease that arises from an uncontrolled growth of cells (e.g. uncontrolled division), invasion (e.g. direct growth into adjacent tissue) or metastasis.
  • uncontrolled growth we include an increase in the number and/or size of cancer cells (also referred to herein as “proliferation”).
  • metastasis we mean the movement or migration (e.g. invasiveness) of cancer cells from a primary tumor site in the body of a subject to one or more other areas within the subject's body (where the cells can then form secondary tumors).
  • the invention provides compounds and methods for inhibiting, in whole or in part, the formation of secondary tumors in a subject with cancer.
  • the compounds of the invention may be capable of inhibiting the proliferation and/or metastasis of cancer cells selectively.
  • the compounds of the invention may inhibit the proliferation and/or metastasis of cancer cells to a greater extent than it modulates the function (e.g. proliferation) of non-cancer cells.
  • the compounds of the invention inhibit the proliferation and/or metastasis of cancer cells only.
  • Compounds of the invention may be suitable for use in the treatment of various diseases, such as cancer and spinal cord injury.
  • Compounds of the invention may be suitable for use in the treatment of any cancer type, including all tumors (non-solid and solid tumors).
  • the cancer type may include (but is not limited to) benign tumors (such as hemangiomas, hepatocellular adenoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas and pyogenic granulomas) and malignant tumors (such as leukemia, myelodysplastic syndromes (MDS), prostate cancer, breast cancer, skin cancer, bone cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, bladder, gall bladder, ovary, cervix, pancrea
  • cancers of the testis include cancers of the testis, genitourinary tract, glioblastoma, neuroblastoma, keratoacanthoma, epidermoid carcinoma, large cell carcinoma, follicular carcinoma, undifferentiated carcinoma, papilliary carcinoma, seminona, myeloid disorders, lymphoid disorders, hairy cells, buccal cavity and pharynx (oral), lip, tongue, mouth, small intestine, colon-rectum, large intestine, rectum, brain and central nervous system, Hodgkin's and leukaemia; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocitic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopo
  • compounds of the invention may possess potent inhibitory activity on the growth and invasion of hormone-refractory and aggressive human prostate tumors (for example, as may be shown in xenograft mouse models). It is particularly preferred therefore that the compounds of the invention may be useful in the treatment of aggressive cancers such as prostate cancer.
  • Compounds of the invention may reduce the rate of cell proliferation when tested in an assay using a PC-3 cancer cell line (e.g. obtained from ATCC).
  • the compounds may thus possess a beneficial inhibitory effect on the ability of tumors of this type, and of cancers generally, to survive.
  • the PC-3 cancer cell line has several properties that represent the hormone-independent and invasive prostate cancer.
  • PC-3 cells lack functional androgen receptor (AR) signalling, grow rapidly in culture medium and can form large and very aggressive tumors when implanted into nude mice.
  • the biological tests described hereinafter e.g. PC-3 xenograft mouse models
  • the compounds of the invention may target multiple cellular pathways that are associated with tumor growth, apoptosis, angiogenesis and metastasis.
  • the compounds of the invention may also affect other cancer pathways, such as cell cycle regulation and inflammation.
  • the compounds of the invention may therefore be VEGF (vascular endothelial growth factor) inhibitors, e.g. they may inhibit the expression of VEGF and/or VEGF receptors including but not limited to VEGF and/or VEGF receptor 2 (as may be shown in a test described herein). This may occur selectively, or, may be one of a plurality of the mechanisms by which the compounds of the invention act to treat cancer.
  • the VEGF signalling pathway is known to be linked to tumor vascularisation and invasion, and hence compounds of the invention may possess anti-cancer effects by inhibiting angiogenesis (and may therefore be classed as anti-angiogenesis agents).
  • the compounds of the invention may be useful in the treatment of a disease in which the inhibition of angiogenesis (and/or VEGF) is desired and/or required.
  • the term "inhibition” may refer to any measurable reduction and/or prevention, which in the context of angiogenesis refers to the reduction and/or prevention of angiogenesis (e.g. the expression of VEGF receptors including but not limited to VEGF and VEGF receptor 2).
  • the inhibitory activity may be measured by comparing the angiogenesis inhibition in a sample containing a compound of the invention and (a) VEGF receptor(a), such as VEGF and/or VEGF receptor 2, with an equivalent sample of in the absence of a compound of the invention.
  • the measurable change may be objective (e.g.
  • test or marker for example in an in vitro or in vivo assay or test, such as one described hereinafter, or otherwise another suitable assay or test known to those skilled in the art) or subjective (e.g. the subject gives an indication of or feels an effect).
  • a method of treatment of a disease which may be associated with, or affected by, angiogenesis (and/or VEGF; e.g. an inhibition of the expression of VEGF and/or VEGF receptor 2) is desired and/or required, which method comprises administration of a therapeutically effective amount of a compound of the invention but without the proviso(s), as hereinbefore defined, to a patient suffering from, or susceptible to, such a condition.
  • a disease e.g. cancer or another disease as mentioned herein
  • angiogenesis and/or VEGF; e.g. an inhibition of the expression of VEGF and/or VEGF receptor 2
  • VEGF e.g. an inhibition of the expression of VEGF and/or VEGF receptor 2
  • Patients include mammalian (including human) patients.
  • the method of treatment discussed above may include the treatment of a human or animal body.
  • the term "effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient.
  • the effect may be objective (e.g. measurable by some test or marker) or subjective (e.g. the subject gives an indication of or feels an effect).
  • Compounds of the invention may be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, sublingually, by any other parenteral route or via inhalation, in a pharmaceutically acceptable dosage form.
  • Compounds of the invention may be administered alone, but are preferably administered by way of known pharmaceutical formulations, including tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for parenteral or intramuscular administration, and the like.
  • the type of pharmaceutical formulation may be selected with due regard to the intended route of administration and standard pharmaceutical practice.
  • Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use.
  • Such formulations may be prepared in accordance with standard and/or accepted pharmaceutical practice. Otherwise, the preparation of suitable formulations may be achieved non- inventively by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.
  • a pharmaceutical formulation including a compound of the invention, as hereinbefore defined but without the proviso(s), in admixture with a pharmaceutically acceptable adjuvant, diluent and/or carrier.
  • compositions that may be mentioned include those in which the active ingredient is present in at least 1% (or at least 10%, at least 30% or at least 50%) by weight. That is, the ratio of active ingredient to the other components (i.e. the addition of adjuvant, diluent and carrier) of the pharmaceutical composition is at least 1 :99 (or at least 10:90, at least 30:70 or at least 50:50) by weight.
  • the amount of compound of the invention in the formulation will depend on the severity of the condition, and on the patient, to be treated, as well as the compound(s) which is/are employed, but may be determined non-inventively by the skilled person.
  • the invention further provides a process for the preparation of a pharmaceutical formulation, as hereinbefore defined, which process comprises bringing into association a compound of the invention, as hereinbefore defined, or a pharmaceutically acceptable ester, amide, solvate or salt thereof with a pharmaceutically-acceptable adjuvant, diluent or carrier.
  • Compounds of the invention may also be combined with other therapeutic agents that may be useful in the treatment of a cancer and/or a proliferative disease (e.g. another VEGF inhibitor as described herein). Compounds of the invention may also be combined with other therapies.
  • a proliferative disease e.g. another VEGF inhibitor as described herein.
  • Compounds of the invention may also be combined with other therapies.
  • each of components (A) and (B) is formulated in admixture with a pharmaceutically- acceptable adjuvant, diluent or carrier.
  • a pharmaceutically- acceptable adjuvant diluent or carrier.
  • Such combination products provide for the administration of a compound of the invention in conjunction with the other therapeutic agent, and may thus be presented either as separate formulations, wherein at least one of those formulations comprises a compound of the invention, and at least one comprises the other therapeutic agent, or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including a compound of the invention and the other therapeutic agent).
  • a pharmaceutical formulation including a compound of the invention, as hereinbefore defined but without the proviso(s), one or more therapeutic agent(s) that is useful in the treatment of cancer and/or a proliferative disease, and a pharmaceutically-acceptable adjuvant, diluent or carrier; and
  • a pharmaceutical formulation including one or more therapeutic agent(s) that is useful in the treatment of cancer and/or a proliferative disease in admixture with a pharmaceutically- acceptable adjuvant, diluent or carrier,
  • components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other.
  • the invention further provides a process for the preparation of a combination product as hereinbefore defined but without the proviso(s), which process comprises bringing into association a compound of the invention, as hereinbefore defined, or a pharmaceutically acceptable ester, amide, solvate or salt thereof with the other therapeutic agent (or agents) that is (or are) useful in the treatment of cancer and/or a proliferative disease, and at least one pharmaceutically-acceptable adjuvant, diluent or carrier.
  • compounds of the invention may be administered at varying therapeutically effective doses to a patient in need thereof.
  • the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the mammal over a reasonable timeframe.
  • the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.
  • Administration may be continuous or intermittent (e.g. by bolus injection).
  • the dosage may also be determined by the timing and frequency of administration.
  • the dosage can vary from about 0.01 mg to about 10 g (e.g. 1000 mg) per day of a compound of the invention.
  • the dose range may be between 1 mg/kg and 1000 mg/kg (e.g. between 10 mg/kg and 500 mg/kg, preferably between about 20 mg/kg and 200 mg/kg) such as the does ranges employed in the mouse models described hereinafter.
  • Compounds of the invention may have the advantage that they target multiple pathways involving tumor growth, apoptosis, angiogenesis and metastases.
  • the compounds of the invention may also be effective angiogenesis inhibitors (and/or VEGF inhibitors), i.e. they may (for example, selectively, or as one mode of action) inhibit angiogenesis (and/or VEGF; e.g. they may inhibit the expression of VEGF and VEGF receptor 2).
  • Compounds of the invention may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g. higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the above-stated indications or otherwise.
  • a better pharmacokinetic profile e.g. higher oral bioavailability and/or lower clearance
  • compounds of the invention may be well tolerated by the patient, i.e. show less or no side effects (e.g. weight loss or other toxic side effects) for example as compared to other therapeutic agents. This may be the case even at high concentrations/doses of the compounds of the invention.
  • the compounds of the invention may also display good potency (e.g. better potency than other therapeutic agents) at a relatively or comparatively lower dose. Hence the compound of the invention may have a large therapeutic window.
  • the compounds of the invention may have advantages over other known chemotherapeutic agents (e.g. AVASTINTM, docetaxel and/or etoposide), which includes better potency and better safety profile (i.e. reduced side effects). Such comparative advantages may be shown in biological tests such as those described hereinafter.
  • Akt activity is assessed by obtaining cells/tissue ⁇ ood/urine/body fluid sample from a subject with a disease, and to perform an in vitro Akt activity assay, which utilizes an Akt-specific antibody to immunoprecipitate Akt in the lysate obtained from collected cells/tissue/blood/urine/body fluid sample.
  • Akt activity is determined in a kinase reaction using recombinant GSK-3a as substrate.
  • Phosphorylation of the GSK-3a is analyzed by Western blot analysis using the phospho-GSK-3a specific antibody.
  • Akt activity in the subject with a disease is measured by the level of phospho- GSK-3a.
  • the following antibodies were used: polyclonal antibodies against PIP5K1A and phoshorylated Akt and Cyclin Dl (Cell Signalling Technology, Danvers, MA), P27 (Santa Cruz Technology, Santa Cruz) and ⁇ -Actin (MP Biomedicals, Illkirch France). Secondary antibodies were HRP-conjugated anti-mouse IgG and anti-rabbit IgG (GE Healthcare).
  • siRNA experiments 2 xlO 6 human prostate cancer PC-3 cells were transfected with 10 nM siRNA non-targeting (Ctrl) or oligos of siRNA to PIP5K1A (Invitrogen, Grand Island, NY) and cultured for 24, 48 or 72 hours. Transfections were performed with Microporator MP-100 (Digital- Bio Technology, Seoul, Korea) according to the manufacturer's instructions.
  • the siRNAs were designed to target exon 14 of PIP5K1A gene to specifically silence PIP5K1A.
  • the first oligo duplex contains sense and anti-sense strands with the sequences of GCG UUC ACC UUG GUC GUC CUG AUG U (SEQ ID NO:l) and ACA UCA GGA CGA CCA AGG UGA ACG C (SEQ ID NO:2).
  • the second oligo duplex contains sense and anti-sense strands with the sequences of CCU UCC GCU ACU UCC GGG AGC UAU U (SEQ ID NO:3) and AAU AGC UCC CGG AAG UAG CGG AAG G (SEQ ID NO:4).
  • the annealed oligos were produced and mixed by Invitrogen.
  • the cells were harvested and lysed in ice-cold RIPA buffer (120 mM NaCl, 50 mM Tris- HC1 pH 7.6, 50 mM NaF, O.lmM Na3V04, 1% NP40, 1 mM phenylmethylsulfonyl fluoride (PMSF)) (Sigma, St. Louis, MD) and 15 % protease inhibitor cocktail Complete Mini (Roche, Basel, Switzerland). 20 ⁇ g of the protein was separated with 12% SDS-PAGE gels and transferred onto nitrocellulose membranes. Signals were visualized using the Enhanced ChemiLuminescence detection system (Millipore Corp Sweden, Solna, Sweden) and documented with an Alphalmager CCD system. Densitometric quantification of immunoblots was performed by the ImageJ Image Analysis Software (NIH, Baltimore, MD) and represented as fold change relative to control and was normalized with actin staining.
  • RIPA buffer 120 mM NaCl, 50 mM Tris-
  • PIP5K1A expression was silenced by RNAi-mediated knockdown in PC-3 cells. PIP5K1A expression was completely inhibited after 48 and 72 hours of treatment with siRNA to PIP5K1A ( Figure 1). Knockdown of PIP5K1A led to significantly lower level of phosphorylated Akt ( Figure 2A-B). Akt phosphorylation is a critical step in activation of PI3 Kinase/ Akt pathway, so the result demonstrates the role of PIP5K1A in activation of PI3 Kinase/ Akt pathway.
  • Cyclin Dl is a known down-stream effector of the PI3 Kinase/ Akt pathway. Inhibition of PIP5K1 A also led to the decreased level of cyclin Dl expression ( Figure 3A-B).
  • P27 is a known down-stream negative effector of the PI3 Kinase/ Akt pathway. Inhibition of PIP5K1A led to increased expression of P27 ( Figure 4). These results supports the role of PIP5K1 A in regulating PI3 Kinase/ Akt pathway.
  • PIP5K1A pathway protein expression level of PIP5K1A in clinical specimens obtained from patients with prostate hyperplasia tissues (BPH) and malignant prostate cancers was evaluated. Prostate cancer specimens had significantly higher level of PIP5K1A expression than that of the BPH prostate tissues (p ⁇ 0.001) ( Figure 5 and 6). More importantly, PIP5K1A expression level correlates with cancer grade: very low in benign prostatic hyperplasia (BPH) patients, low in low grade prostate cancer patients and at the highest level in high grade metastatic prostate cancer patients ( Figure 5). [0269] Expression of PIP5K1A Correlates with AR Expression in Prostate Hyperplasia Tissues and Prostate Cancer Specimens
  • PIP5K1A was evaluated in normal lung tissues, and non-invasive lung sequamous cell carcinoma and invasve non-small cell lung cancer specimens.
  • PIP5K1A is over- expressed in lung cancer patient tissues as compared to normal lung tissue. More importantly, its expression level correlates with cancer grade and invasiveness: very low in normal lung tissues, low in non-invasive lung sequamous cell carcinoma specimens and very high in invasive non-small cell lung cancer specimens ( Figure 8).
  • Protein kinases are phosphoryl transferases that transfer the ⁇ -phosphate of ATP to conserved serine, threonine, or tyrosine residues on specific substrate proteins.
  • Kinase activity assay involves the quantification of this phosphoryl transfer by detection of the production of the phosphorylated product or the change in the ratio of ATP to ADP.
  • the radioisotope filtration binding assay is used to measure kinase inhibition activity of a putative kinase inhibitor. The reactions are performed using radioisotope labeled ⁇ - ⁇ . The incorporation of this radiolabeled phosphate into the kinase substrate is then assayed after a series of binding and washing steps to remove unincorporated radioisotope.
  • kinase phosphoryl transfer activity which is directly proportional to the amount of phosphorylated substrate.
  • the kinase activity in the presence and absence of the putative kinase inhibitor is measured to determine and quantify the kinase inhibition activity.
  • kinase inhibitors For high-throughput screening of kinase inhibitors, many convenient and automation methods to assess kinase activity can be used with detection methods using radioisotope, fluorescence emission, chemiluminescence emission, production of phosphorylated substrates or the binding of potential chemical inhibitors to the target kinase.
  • the high-throughput screening methods include radiometric based filtration binding assay, radiometric based scintillation proximity assay (SPA), fluorescence Intensity assay (FI), fluorescence polarization assay (FP), fluorescence resonance energy transfer assay (FRET), time-resolved fluorescence assay (TRF), time-resolved fluorescence resonance energy transfer assay (TR-FRET), ELISA-based assay, luminescence detection assay, mobility shift assay and ligand-kinase binding assay.
  • SPA radiometric based scintillation proximity assay
  • FI fluorescence Intensity assay
  • FP fluorescence polarization assay
  • FRET fluorescence resonance energy transfer assay
  • TRF time-resolved fluorescence assay
  • TR-FRET time-resolved fluorescence resonance energy transfer assay
  • a library of chemical compounds that are potential PIP5K1A inhibitors can be tested in a high-throughput PIP5K1A inhibition screening to identify compounds that can inhibit PIP5K1A in the ⁇ range. Further tests on the putative inhibitors can be performed to identify compounds with kinase inhibition activity in nM concentration range. These molecules will also be tested in membrane preparations and only compounds capable of inhibiting PIP5K1A activity in its natural environment as well as in the original high throughput assay will be identified as PIP5K1A inhibitors.
  • kinase inhibition selectivity can be tested to identify specific PIP5K1A inhibitors. The identified inhibitors can be further tested in vivo such as in xenograft animal model studies.
  • Nitrogen-containing heterocyclic compounds are important class of molecules that are commonly used for the synthesis of candidate drugs.
  • Phosphatidylinositol-4- phosphate 5-kinase alpha (PIP5Ka) is a lipid kinase, similar to phosphatidylinositol 3-kinase (PI3K).
  • PI3K phosphatidylinositol 3-kinase
  • ⁇ 5 ⁇ 1 ⁇ in PNT1A non-malignant cells results in an increased AKT activity, and an increased survival as well as invasive malignant phenotype, while siRNA-mediated knockdown of ⁇ 5 ⁇ 1 ⁇ in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice.
  • ISA-201 1B is discovered during our synthetic studies of C-l indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach.
  • ISA-201 1B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting ⁇ 5 ⁇ 1 ⁇ associated PI3K/AKT and the downstream survival, proliferation and invasion pathways. Further, siRNA-mediated knockdown of ⁇ 5 ⁇ 1 ⁇ exerts similar effects on PC3 cells as ISA-201 1B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, ⁇ 5 ⁇ 1 ⁇ has a high potential as a drug target, and compound ISA-201 1B is interesting for further development of targeted cancer therapy. [0281] Prostate cancer is the most common malignancy, and the third leading cancer-related cause of death among men of western world.
  • lipid kinase ⁇ 5 ⁇ 1 ⁇ plays an important role in cancer cell invasion and survival by regulating the PI3K/AKT/AR pathways. Elevated level of ⁇ 5 ⁇ 1 ⁇ contributes to cancer cell proliferation, survival and invasion.
  • ISA-201 IB with promising anticancer effects by inhibiting the ⁇ 5 ⁇ 1 ⁇ associated AKT pathways.
  • ⁇ 5 ⁇ 1 ⁇ may be used as a potential therapeutic target for treatment of advanced prostate cancer.
  • the mainstay of cancer treatment largely consists of nonspecific cytotoxic agents with severe side-effects in treated cancer patients (1). It is thus essential to discover novel drugs and their targets to more selectively eradicate cancer cells.
  • the 1 ,2,3,4-tetrahydroquinoline and 1,2,3,4-tetrahydroisoquinoline ring systems are common structural motifs found in several biologically active compounds (2-6).
  • the discovery and development of a novel class of 1,2,3,4-tetrahydroisoquinoline derivatives as selective anticancer drugs represents new challenges in discovery and development of anticancer drugs.
  • the phosphoinositide family of lipids consists of several derivatives of phosphatidylinositols (Ptdlns) that are formed through series of phosphorylation by enzymes termed phosphatidylinositol-phosphate kinases (PIPKs) (7).
  • PIPKs phosphatidylinositol-phosphate kinases
  • PI3K PI3 -kinases
  • PIP5K PI-4-phosphate 5 kinases
  • the PIP5K family of lipid kinases consists of the three isozymes ⁇ , ⁇ , and ⁇ (11 -13). ⁇ 5 ⁇ 1 ⁇ is located in the chromosomal region lq21.3 (14), the product of which is predominantly responsible for the synthesis of PtdIns-4,5-P 2 (PIP2), a substrate used by PI3K to produce PtdIns-3,4,5-P 3 (PIP3) (15). PIP3 in turn activates the AKT family of serine/threonine kinases (16, 17).
  • AKT kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase and kinapTEN gene in metastatic cancers (18, 19).
  • PCa prostate cancer
  • AR cross-activate androgen receptor
  • AR signaling has also been used as targets for designing drugs to treat lethal metastatic PCa (24-28).
  • ⁇ 5 ⁇ 1 ⁇ produces PIP2, which is required for the activation of PI3K/AKT
  • ⁇ 5 ⁇ 1 ⁇ may play an important role in cancer progression. It is of importance to investigate whether ⁇ 5 ⁇ 1 ⁇ may be used as potential target for developing effective novel anticancer drugs. It is known that ⁇ 5 ⁇ 1 ⁇ is expressed at low level in lipid tissues and is dispensable during organ development, as deletion of ⁇ 5 ⁇ 1 ⁇ does not result in lethal defects in mice but causes impaired spermatogenesis in males (29, 30). A recent reported study shows that ⁇ 5 ⁇ 1 ⁇ is highly expressed in the human MDA-MB-231 breast cancer cell line, suggesting that overexpression of ⁇ 5 ⁇ 1 ⁇ is associated with malignant diseases (31).
  • ISA-201 IB a diketopiperazine fused C-1 indol-3-yl substituted tetrahydroisoquinoline
  • ISA-201 IB a novel anticancer drug that effectively inhibits growth of PCa tumor in vivo, and invasion of PCa cells in vitro.
  • ⁇ 5 ⁇ 1 ⁇ a target for ISA-201 IB
  • PI3K/AKT and AR signaling pathways which are involved in regulation of cell proliferation, survival, and invasion.
  • ISA-201 IB a diketopiperazine fused C-1 indol-3-yl substituted 1 ,2,3,4- tetrahydroisoquinoline derivative
  • ISA-201 IB a diketopiperazine fused C-1 indol-3-yl substituted 1 ,2,3,4- tetrahydroisoquinoline derivative
  • Figure 9A a Pictet-Spengler approach
  • Figure 17 The dose-dependent effect of ISA-201 IB on proliferation of PC-3 cells was determined by MTS assay.
  • TMA tissue microarray
  • PNT1A cells overexpressing ⁇ 5 ⁇ 1 ⁇ may have gained malignant invasive feature by performing invasion assay.
  • overexpression of ⁇ 5 ⁇ 1 ⁇ greatly increased expression of AR as determined by immunoblot analysis ( Figure 12 J) and immunostaining ( Figure 20).
  • Immunoprecipitation assays further revealed that AR formed protein-protein complexes with CDK1 in the nuclear compartment of PNT1A cells expressing control vector or ⁇ 5 ⁇ 1 ⁇ vector ( Figure 21 ; Figure 12K).
  • ⁇ 5 ⁇ 1 ⁇ is able to activate PI3K/AKT pathway and enhance cross-interactions of AKT with AR, probably through CDK1.
  • ISA-2011B inhibits the elevated survival, proliferation and invasion signaling
  • ISA-201 IB treatment inhibited SKP2, a key ubiquitin enzyme that mediates P27 degradation, and ISA-201 IB increased expression of P27, an inhibitor that is required to block cell proliferation by inhibiting cyclins and CDKs (Figure 13F).
  • ISA-201 IB treatment also led to a remarkable inhibition of AR and PSA expression ( Figure 13G, H).
  • docetaxel, ISA-2009 and ISA-201 1 A had no effect on AR and PSA expression (Figure 13H).
  • This data suggest that ISA-201 IB mediated inhibitory effect on PCa proliferation is associated with its ability to inhibit ⁇ 5 ⁇ 1 ⁇ , which transduce the inhibitory effect to its downstream signaling pathways including AKT, AR, and cell cycle.
  • ISA-2011B target PIP5Kla/AKT/AR pathways to inhibit tumor cell growth and induce apoptosis in aggressive PCa cells
  • ISA- 201 IB inhibited invasiveness of PC-3 cells down to 55.96% of control ( Figure 141).
  • Expression of phosphorylated FAK, a factor that promotes invasion was almost diminished in PC-3 cells treated with ISA-201 IB and ISA-201 1 A at dose-dependent fashion ( Figure 13 J).
  • docetaxel did not show pronounced effect on FAK phosphorylation ( Figure 14J).
  • ISA-201 IB inhibits proliferation, survival, and invasion in androgen-insensitive PCa cells. Moreover, this effect may be mediated through ⁇ 5 ⁇ 1 ⁇ and its downstream PI3K/AKT.
  • An apoptotic marker c-PARP is used to detect early apoptotic process.
  • Immunoblot analysis for c-PARP expression was performed using lysates from PC-3 cells transfected with control siRNA or siPIP5Kl a, which were treated with ISA-201 IB or vehicle ( Figure 15H).
  • Two distinct isoforms of c-PARP were detected in PC-3 cells that were either treated with ISA- 201 IB at 50 ⁇ alone, expressed siPIP5Kla alone, or expressed siPIP5Kl a and were treated with ISA-201 IB in a combination. This suggests that downregulation of ⁇ 5 ⁇ 1 ⁇ through treatment or knockdown induced apoptosis (Figure 15H).
  • ISA-201 IB has a unique structure consisting of a diketopiperazine fused, methylenedioxy protected, 1 ,2,3,4- tetrahydroisoquinoline core with an electron rich trans-substituent at position 1.
  • ISA- 201 IB inhibits tumor growth by inhibiting the expression and activity of ⁇ 5 ⁇ 1 ⁇ thereby affecting the downstream PI3K/AKT, AR and cell cycle pathways ( Figure 16).
  • AKT can be activated by phosphorylation through two upstream pathways: through PI3K/PIP3, or through cAMP/PKA (36).
  • ISA-201 IB inhibits AKT activity through ⁇ 5 ⁇ 1 ⁇ / ⁇ 3 ⁇ / ⁇ 3.
  • ISA-201 IB treatment has no effect on PKA receptor and the key protein phosphorylated CREB in cAMP/PKA pathway in LNCaP or PC-3 cells.
  • the effect of ISA-201 IB is significantly stronger on PC-3 cells with PTEN mutation than on 22Rvl cells which contain intact PTEN gene. 22Rvl cells, though less sensitive to ISA-201 IB, also displayed reduced proliferation after ISA-201 IB treatment.
  • CDKl and AR form protein-protein complexes predominantly in the nuclear compartment of cells.
  • the complexes of CDK1-AR are persistent in PNT1A cells overexpressing ⁇ 5 ⁇ 1 ⁇ .
  • ISA-201 IB targets CDKl associated pathways that regulate AR activity.
  • Tissue microarrays containing BPH and PCa tissues from 48 patients were purchased from Pantomics Inc. (Richmond, CA). mRNA expression data of ⁇ 5 ⁇ 1 ⁇ , AKT2, PTEN and AR was extracted from the dataset in the cBioPortal database (35). The study was approved by the Ethics Committee, Lund University and the Helsinki Declaration of Human Rights was strictly observed.
  • pLPS-3'EGFP vector containing full-length human ⁇ 5 ⁇ 1 ⁇ cDNA as "PIP5K1 a-EGFP" or control empty vector were used.
  • Cells overexpressing PIP5Kla- pLPS-3'EGFP or pLPS-3'EGFP vector were selected by culturing cells in medium containing G418 antibiotics (400 g/ml) (Sigma Aldrich, Sweden).
  • KINOMEscan The interaction of ISA-201 IB with 442 kinases covering more than 80% of the human catalytic protein kinome was tested using the KINOMEscan assay.
  • Kd quantitative dissociation constant
  • Escherichia coli or mammalian cell-expressed kinases labeled with DNA tags for quantitative PCR readout are equilibrated together with test compound (ISA-201 IB) or DMSO as control. Briefly, known active site binding ligands are immobilized on each well of the microplates. After equilibration the wells are washed to remove unbound kinases. Screening "hits" are identified by measuring the amount of kinase captured in test vs. control samples by using quantitative RT-PCR. Bound kinase levels in test compound and control wells are compared. In a similar manner, dissociation constants (Kd) for test compound-kinase interactions are determined by measuring the amount of kinase capture.
  • Kd dissociation constants
  • Paraffin- embedded sections containing representative tissue cores of prostate cancer (PCa) and corresponding benign prostate samples from 48 patients were purchased from Pantomics Inc.
  • mRNA expression data of ⁇ 5 ⁇ 1 ⁇ , AKT2, phosphatase and tensin homolog (PTEN), and AR were extracted from the dataset in the cBioPortal database.
  • the study was approved by the Ethics Committee, Lund University, and the Helsinki Declaration of Human Rights was strictly observed.
  • the Mann- Whitney test was performed to compare expression levels in metastatic cancers with those observed in nonmetastatic primary tumor tissues; **P ⁇ 0.01.
  • PC-3, 22Rvl , and PNT1A cells were purchased from American Type Culture Collection. Treatments with ISA-201 1B dissolved in DMSO and various types of drugs, including docetaxel, etoposide (Sigma-Aldrich), and tadalafil, was performed. A 0.5% DMSO concentration was used in in vitro experiments. Cells (0.2 ⁇ 106 cells/mL) were seeded and allowed to attach to the plates by growing in 10% FBS phenol red-free RPMI-1640 medium for 24 h and then were treated with drugs alone or in combination for 24, 48, and 72 h.
  • ISA-201 IB ISA-2009
  • ISA-201 1A ⁇ , 5 ⁇ , 10 ⁇ , 50 ⁇ , and 100 ⁇
  • etoposide tadalafil (20-50 ⁇ )
  • docetaxel 25 nM
  • MTS Proliferation Assay The effects of ISA-201 IB, docetaxel, etoposide, and tadalafil on PCa cell lines and various types of cancer cell lines were determined using the nonradioactive MTS proliferation assay (Promega Biotech) according to the manufacturer's protocol. Cancer cells (5 x 103 cells per well) were incubated for 24 h in a 96-well plastic plate that contained complete growth medium to allow attachment. The medium was then replaced with fresh medium containing ISA- 201 IB or the anticancer drugs as mentioned above at different concentrations and cultured for 24, 48, or 72 h. MTS reagent (20 ⁇ ) was then added, and cells were cultured for another 4 h. Viability was determined by measuring the absorbance at 490-nm wavelength, on an Infinite M200 multimode microplate reader (Tecan Sunrise).
  • Tumor xenografts were treated with vehicle (control), docetaxel (10 mg/kg), ISA- 201 IB (40 mg/kg), and docetaxel (10 mg/kg) in combination with ISA-2011B (40 mg/kg) every second day.
  • Plasmids, Stable Transfection, and siRNA Knockdown Assay Plasmids, Stable Transfection, and siRNA Knockdown Assay.
  • pLPS-3'EGFP vector containing full-length human ⁇ 5 ⁇ 1 ⁇ cDNA as "PIP5K1 a- EGFP" or control empty vector were used.
  • the vectors were purchased from Harvard Medical School.
  • Transient transfection was performed using Lipofectamine 2000 transfection reagent (Life Technologies) according to the manufacturer's instructions.
  • PNT1A cells were transfected with Lipofectamine 2000 transfection reagent. Vectors (2-5 ⁇ g) were used in transfection experiments.
  • Cells overexpressing ⁇ 5 ⁇ 1 ⁇ - pLPS-3'EGFP or pLPS-3'EGFP vector were selected by culturing cells in medium containing G418 antibiotics (400 g/mL) ( Sigma- Aldrich). Cells were maintained in medium with G418 antibiotics for a total of 2 wk.
  • G418 antibiotics 400 g/mL
  • SiRNAs against ⁇ 5 ⁇ 1 ⁇ or siRNA negative control duplex were purchased from Life Technologies.
  • SiRNAs (50 nM) were transfected into 1 x 105 PCa cells using Transfection Reagent TransIT-TKO according to the manufacturer's protocol (Minis Bio, LCC). After introduction of respective siRNA complexes into PCa cells, cells were then collected at 24, 48, and 72 h after transfection.
  • Antibodies Primary antibodies against ⁇ 5 ⁇ 1 ⁇ , ⁇ -actin, pAkt, p27, cyclin Dl , cyclin E, cyclin B, cyclin-dependent kinase 1 (CDK1), pCDKl(Tyrl4-15), and PKA RI- ⁇ / ⁇ at 1 :500 dilutions (Cell Signaling Technology); PKA RIi at 1 : 1 ,000 (BD Biosciences Transduction Laboratories); androgen receptor (AR) at 1 :250; the p-CREB-1 at Ser-133 sites at 1 :500; P27, cyclin A2 (Santa Cruz Biotechnology Inc.), prostate-specific antigen (PSA) at 1 :500 (DAKO); and MMP-9 (Abeam) and anti ⁇ -actin at 1 : 10,000 (MP Biochemicals) were used. Secondary antibodies were HRP-conjugated anti- mouse IgG and anti-rabbit IgG (GE Healthcare).
  • the cells were stained with 50 g/mL propidium iodide (PI) (Sigma-Aldrich), 0.1 % Triton X-100 (Sigma-Aldrich), and 100 g/mL RNase-A (AppliChem) for 40 min at room temperature in the dark, and the ⁇ -elicited fluorescence of individual cells was measured using flow cytometry (FACS Calibur; Becton Dickinson). To measure apoptosis, cells were collected after treatment for 48 h and were subsequently stained with FITC-conjugated Annexin V and 7-AAD according to the manufacturer's protocol (BD Biosciences). The cells were then subsequently subjected to flow cytometry analysis on a FACSCalibur (Becton Dickinson). The data were assessed using FCS Express software (DeNovo Software).
  • Subcellular Fractionation and Immunoprecipitation were prepared as in previously published work (4). Cell pellets were resuspended in ice-cold nuclei isolation buffer [10 mM HEPES (pH 7.9), 1.5 mM MgC12, 10 mM KC1, 0.5 mM DTT, 1 % Triton X- 100, 15% PI Complete Mini, and 1 mM PMSF], incubated on ice for 10 min, and then centrifuged.
  • nuclei isolation buffer 10 mM HEPES (pH 7.9), 1.5 mM MgC12, 10 mM KC1, 0.5 mM DTT, 1 % Triton X- 100, 15% PI Complete Mini, and 1 mM PMSF
  • the supernatant containing the cytoplasmic fraction was collected, and the pellet was resuspended in ice-cold RIPA buffer [120 mM NaCl, 50 mM Tris HCl (pH 7.6), 50 mM NaF, 0.1 mM Na3V04, 1 % Nonidet P-40, and 1 mM PMSF; Sigma] and 15% protease inhibitor mixture Complete Mini (Roche), followed by incubation on ice for 20 min. After centrifugation the supernatant containing the final nuclear fraction was collected. The nuclear and cytoplasmic fractions were subjected to immunoblot analysis. The subcellular fractionation was controlled by detection of ⁇ -tubulin and Lamin B in the cytoplasmic and nuclear fractions, respectively.
  • Cytoplasmic (Cyt) and nuclear (Nuc) fractions were separated from PNT1A cells overexpressing ⁇ 5 ⁇ 1 ⁇ or control vector and were subjected to immunoprecipitation assay.
  • Antibody to CDK1 was used to pull down the immunocomplexes, and antibody to IgG was used as a negative control.
  • CDK1 antibody and antibody to IgG (negative control) (BD Biosciences) were incubated with 500 x,g of freshly prepared protein lysates and 30 of G-Sepharose beads (GE Healthcare) for 3 h at 4 °C. After incubation the samples were washed in RIPA buffer and prepared for immunoblot analysis. The cell lysates from cytoplasmic and nuclear fractions were used as "Input" controls. Blotting of actin served as loading control, and antibody against lamin B was used as a control for the nuclear fraction.
  • PCa cells were grown on the glass coverslips in phenol red-free RPMI-1640 medium containing 10% FBS for 24 h and were then treated with ISA-201 IB or indicated drugs for 24 h. Cells were fixed with 4% paraformaldehyde in PBS. For blocking background staining from nonspecific interactions, Image-iT FX signal enhancer (Molecular Probes) was used. Primary antibodies against ⁇ 5 ⁇ 1 ⁇ , PIP2, Phosphor-473 AKT, ⁇ -tubulin, AR, and PSA were used.
  • the secondary antibodies including rabbit anti-donkey conjugated to Rhodamine (Chemicon/Millipore International Inc.) or anti-goat conjugated to FITC antibodies at 1 :200 and goat anti-rabbit Alexa Fluor 488 at 1 :500 (Invitrogen, Swiss, Sweden), were used.
  • the counterstain 4',6-diamidino-2-phenylindole (SERVA Electrophoresis GmbH) was used to visualize cell nuclei.
  • the slides were detected under an Olympus AX70 fluorescent microscope (Nikon DS-U1).
  • the software ACT2U was used (ACT2U version. 1.5).
  • the images were accessed, and photomicrographs were taken at x lO magnification using the HoloMonitor M3 (Phase Holographic Imaging AB).
  • Invasion Assay For invasion assay using PNT1A cells, cells stably expressing control vector or PIP5Kla vector were cultured in RPMI-1640 medium containing 10% FBS for 5 d and followed by culturing in serum-free medium for an additional 2 d before being subjected to invasion assays. PNT1A cells in RPMI-1640 medium containing 10% FBS were also subjected to invasion assay. The effect of ISA-201 IB (50 ⁇ ) on the invasiveness of PC-3 cells was evaluated with Boyden transwell chambers (Merck KGaA) according to the manufacturer's protocol. In brief, 5 x 104 PC-3 cells in 300 of serum-free medium supplemented with 50 ⁇ ISA-201 IB were seeded into the upper chamber of the system.
  • the lower well was filled with 10% FBS containing Ham's F-12 medium as chemoattractant and the same reagent treatment as the upper chamber. After 48 h of incubation, the noninvading cells in the upper chamber were wiped off, and the invasive cells on the lower membrane were stained for 15 min at room temperature and then dissolved in 10% acetic acid. The absorbance of the stained cells was measured on an ELISA plate reader. The effects of ⁇ 5 ⁇ 1 ⁇ overexpression on the invasion capacity of PNT1A cells were evaluated using QCM High Sensitivity Noncross- linked Collagen invasion assay (Merck Millipore) as per the manufacturer's protocol.
  • control and PIP5Kla-overexpressing PNTA cells were starved in serum- free growth medium for 96 h before seeding.
  • the invasion assay was initiated by seeding 1.25 x 105 cells into respective inserts, in a total of 250 of serum-free growth medium (upper chamber). At the same time, 500 of complete growth medium supplemented with 10% FBS was added into the lower chamber. The cells were cultured for 48 h to allow migration through the collagen layer. After incubation the cells were stained, and the invaded cell number was determined by manual counting.
  • Adhesion Assay was initiated by seeding 1.25 x 105 cells into respective inserts, in a total of 250 of serum-free growth medium (upper chamber). At the same time, 500 of complete growth medium supplemented with 10% FBS was added into the lower chamber. The cells were cultured for 48 h to allow migration through the collagen layer. After incubation the cells were stained, and the invaded cell number was determined by manual counting. Adhesion Assay.
  • a 96-well plate was coated overnight at 4 °C with Fibronectin (AMS Biotechnology) diluted in Dulbecco's PBS at a final concentration 50 g/mL in a volume of 100 ⁇
  • fibrbronectin AMS Biotechnology
  • wells were incubated with Dulbecco's PBS only. After coating, the wells were washed with 1 * PBS three times, and 2% BSA in l x PBS was added to the wells and incubated for 1 h at 37 °C. The wells were then washed with l x PBS, and subsequently 1 x 105 cells that were pretreated with agents or vehicle control were added to the wells and incubated at 37 °C, 5% C02 for 6 h. The wells were then washed, and the adherent cells were collected and counted.
  • Fibronectin AMS Biotechnology
  • Emerling BM, et al. (2013) Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53-null tumors. Cell 155(4):844-857.
  • Type I phosphatidylinositol-4-phosphate 5-kinases are distinct members of this novel lipid kinase family.
  • Carver BS et al. (201 1) Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer. Cancer Cell 19(5): 575-586.

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Abstract

L'invention concerne de nouvelles compositions utiles sur le plan pharmaceutique, qui peuvent être utiles dans le traitement de maladies telles que le cancer. L'invention concerne également l'utilisation de ces compositions pour le diagnostic et le pronostic de maladies, la sélection de traitement et la surveillance de la progression d'une maladie.
PCT/US2015/042084 2014-07-24 2015-07-24 Compositions pour le traitement, le diagnostic et le pronostic de maladies WO2016014992A2 (fr)

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