WO2009065232A1 - Procédés de diagnostic et de thérapie du cancer qui ciblent plk4/sak - Google Patents

Procédés de diagnostic et de thérapie du cancer qui ciblent plk4/sak Download PDF

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WO2009065232A1
WO2009065232A1 PCT/CA2008/002087 CA2008002087W WO2009065232A1 WO 2009065232 A1 WO2009065232 A1 WO 2009065232A1 CA 2008002087 W CA2008002087 W CA 2008002087W WO 2009065232 A1 WO2009065232 A1 WO 2009065232A1
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patient
plk4
cancer
antagonist
sample
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PCT/CA2008/002087
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Guohua Pan
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University Health Network
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
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    • A—HUMAN NECESSITIES
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    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
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    • A—HUMAN NECESSITIES
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11021—Polo kinase (2.7.11.21)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
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    • G—PHYSICS
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    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general

Definitions

  • cancers are diverse in their cellular origin and vary widely in responsiveness to treatments.
  • cancers of a particular tissue can also be diverse in cellular origin and have varying responsiveness to treatments.
  • breast cancer has been diagnosed by clinicians for many decades, however only relatively recent studies have demonstrated that breast cancers have phenotypic diversity among different breast cancer sub-types as demonstrated by DNA microarray profiling (see, e.g., Kapp et ah, BMC Genomics (2006) 7:231, and references cited therein).
  • Breast cancer has at least five sub-types: basal, ERRB2-overexpressing, luminal A, luminal B, and normal breast-like (Sorlie et ah, PNAS (2003) 100: 8418-8423; and Kapp et al, BMC Genomics (2006) 7:231).
  • the prognosis and chemotherapy sensitivity of the different molecular subgroups are different.
  • the basal subtype breast cancer does not typically express the estrogen receptor (ER) nor overexpress HER2, and has been associated with poor a clinical outcome (Carey et al., JAMA. (2006) 295:2492-2502).
  • luminal A tumors Compared with luminal B tumors, luminal A tumors express higher levels of ER and GATA3 and show more favorable patient outcomes, whereas luminal B tumors more often express human epidermal growth factor receptor- 1 (HERl), HER2, and/or cyclin El (Carey et al., JAMA. (2006) 295:2492-2502, and references cited therein). It has been reported that patients with luminal A type tumors lived considerably longer before they developed metastatic disease, whereas basal and ERBB2-overexpressing patients have a much shorter disease-free time (Sorlie et al., PNAS (2003) 100: 8418-8423). The reasons for these differing prognoses among breast cancer subtypes is not understood.
  • the polo-like kinase (PLK) family of serine/threonine kinases comprises at least four known members: PLKl, PLK2 (also known as Snk), PLK3 (also known as Fnk or Prk) and PLK4 (also known as Sak).
  • PLKl is the best characterized member of the family, whereas PLK2, PLK3 and PLK4 are less well characterized.
  • PLKl is a mitotic kinase and has been the target of therapeutic treatment of cancer (Johnson et al., Biochemistry (2007) 46(33): 9551-9563, and references cited therein).
  • PLK4 is the least understood and most divergent PLK member of the PLK family.
  • the N-terminal catalytic domain of PLK4 has a different substrate specificity from that of PLK1-3.
  • PLK4 also has a divergent C-terminus comprising only a single polo-box sequence, not tandem PB sequences like in PLK 1-3, that appears to act as a homodimerization domain rather than a localization domain (Lowery et al, (2005) Oncogene 24: 248-259).
  • PLK4 is involved in the control of mitotic entry and exit. It is a regulator of centrosome duplication (Habedanck et al. Nature Cell Biology 7: 1140-1146, 2005).
  • PLK4 transcripts increase from S through M phase, and the protein is ubiquitylated and destroyed by the anaphase promoting complex (APC) (Hudson et al. Curr. Biol. 11: 441-446, 2001; Fode et al. MoI. Cell. Biol. 16: 4665-4672, 1996).
  • APC anaphase promoting complex
  • PLK4 is required for late mitotic progression (Fode et al. PNAS. 91: 6388-6392, 1994; Hudson et al. Curr. Biol. 11 : 441 ⁇ 46, 2001), cell survival and postgastrulation embryonic development (Hudson et al. Curr. Biol. 11: 441-446, 2001).
  • PLK4 knockout mice are embryonic lethal (E7.5), with a marked increase in mitotic and apoptotic cells (Hudson et al. Curr. Biol. 11: 441-446, 2001).
  • PLK4 is transcriptionally repressed by p53 (Li et al. Neoplasia 7: 312-323, 2005). This repression is likely mediated through the recruitment of histone deacetylase (HDAC) repressors and repression appears to contribute to p53-induced apoptosis (Li et al. Neoplasia 7: 312-323, 2005).
  • HDAC histone deacetylase
  • PLK4 has been reported to be overexpressed in colorectal tumors with expression reported as low in adjacent normal intestinal mucosa (Macmillian et al. Ann. Surg. Oncol. 8: 729-740, 2001).
  • PLK4 mRNA has been reported to be overexpressed in some tumor cell lines (Hitoshi, et al, U.S. Patent Application No. US 2003/0027756).
  • PLK4 overexpression has not been demonstrated in cancers other than colorectal tumors.
  • PLK4 overexpression has not been demonstrated in association with specific sub-types of cancers.
  • the invention provides diagnostic and therapeutic methods that target PLK4. Such methods are particularly useful for diagnosis and therapeutic treatments of patients with breast cancer, and in particular, basal sub-type breast cancer or luminal B sub-type breast cancer. The methods are also particularly useful for diagnosis and therapeutic treatments of metastatic disease and angiogenesis disorders. Furthermore, the methods are also particularly useful for diagnosis and therapeutic treatments of soft tissue cancers. The methods are also useful as prognostic indicators, and in particular, as a predictor of disease relapse.
  • One aspect of the invention is a method for identifying a cancer patient candidate for anti-cancer therapy using a PLK4 antagonist, wherein the cancer patient is not a colorectal cancer patient.
  • the method comprises providing a suitable sample from the patient (e.g., a tumor sample, a cancer tissue sample, a soft tissue cancer sample) and assessing expression of PLK4 in the sample.
  • Expression of PLK4 in the sample indicates that the cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist.
  • increased expression of PLK4 in the sample relative to a suitable control indicates that the cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist.
  • the cancer patient is a patient who has been diagnosed with breast cancer.
  • the breast cancer patient is a patient diagnosed with basal sub-type breast cancer or luminal B sub-type breast cancer.
  • the cancer patient is a patient who has been diagnosed with a soft tissue cancer.
  • the cancer patient is a patient who has been diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • Another aspect of the invention is a method for identifying a breast cancer patient candidate for anti-cancer therapy using a PLK4 antagonist.
  • the method comprises providing a suitable sample from the patient (e.g., a tumor sample, a breast cancer tissue sample) and assessing expression of PLK4 in the sample.
  • Expression of PLK4 in the sample indicates that the breast cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist.
  • increased expression of PLK4 in the sample relative to a suitable control indicates that the breast cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist.
  • the breast cancer patient is a patient who has been diagnosed with basal sub-type breast cancer or luminal B sub-type breast cancer.
  • a further aspect of the invention is a method for selecting a cancer patient, such as a breast cancer patient, a soft tissue cancer patient, for PLK4 therapy.
  • the method comprises providing a suitable sample from the patient (e.g., a tumor sample, a breast cancer tissue sample, a soft tissue cancer sample) and assessing expression of PLK4 in the sample.
  • Expression of PLK4 in the sample indicates selecting the patient for anti-cancer therapy using a PLK4 antagonist.
  • increased expression of PLK4 in the sample relative to a suitable control indicates selecting the patient for anti-cancer therapy using a PLK4 antagonist.
  • the cancer patient is a patient who has been diagnosed with basal sub-type breast cancer or luminal B sub-type breast cancer.
  • the cancer patient is a patient who has been diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • Another aspect of the invention is a method for treating a basal sub-type breast cancer, luminal B sub-type breast cancer or a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma in a patient.
  • the method comprises administering to the patient a therapeutically effective amount of a PLK4 antagonist.
  • the method comprises administering to the patient a pharmaceutical composition comprising a PLK4 antagonist in an amount that is sufficient to treat the basal sub-type breast cancer, luminal B sub-type breast cancer, or a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma, in a patient.
  • the method further comprises administering another therapeutic agent, such as a PLKl antagonist.
  • the PLK4 antagonist and the other therapeutic agent, e.g., PLKl antagonist can be administered simultaneously or sequentially.
  • a further aspect of the invention is a method for predicting cancer metastasis in a cancer patient.
  • the method comprises providing a suitable sample from the patient (e.g., a tumor sample, a cancer tissue sample) and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control is indicative of an increased likelihood of developing cancer metastasis in the patient.
  • the cancer patient has breast cancer.
  • the breast cancer patient has basal sub-type breast cancer or luminal B sub-type breast cancer.
  • the invention is a method for screening a cancer patient, such as a breast cancer patient, a soft tissue cancer sarcoma patient, as an aid for selecting aggressive cancer therapy for the patient.
  • the method comprises obtaining a suitable sample from the patient (e.g., a tumor sample, a breast cancer tissue sample, a soft tissue cancer sample) and determining PLK4 expression in the sample.
  • a suitable sample e.g., a tumor sample, a breast cancer tissue sample, a soft tissue cancer sample
  • Increased PLK4 expression in the sample as compared with a suitable control indicates the need or clinical desirability for aggressive cancer therapy.
  • the cancer patient has basal sub-type breast cancer or luminal B sub-type breast cancer.
  • the cancer patient has a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • Another aspect of the invention is a method for inhibiting angiogenesis in a patient. The method comprises administering to the patient a therapeutically effective amount of a PLK4 antagonist.
  • the method comprises administering to the patient a pharmaceutical composition comprising a PLK4 antagonist in an amount that is sufficient to inhibit angiogenesis.
  • the method further comprises administering another therapeutic agent, such as a PLKl antagonist.
  • the PLK4 antagonist and the other therapeutic agent, e.g., PLKl antagonist can be administered simultaneously or sequentially.
  • a further aspect of the invention is a method for treating a cancer metastasis disease (such as a metastatic tumor) in a patient.
  • the method comprises administering to the patient a therapeutically effective amount of a PLK4 antagonist.
  • the method comprises administering to the patient a pharmaceutical composition comprising a PLK4 antagonist in an amount that is sufficient to treat a cancer metastasis disease in a patient.
  • the method further comprises administering another therapeutic agent, e.g., a PLKl antagonist.
  • the PLK4 antagonist and the other therapeutic agent, e.g., PLKl antagonist can be administered simultaneously or sequentially.
  • the invention is a method for identifying a patient that is likely to be responsive to PLK4 antagonist therapy.
  • the method comprises providing a suitable sample (e.g., a tissue sample, a tumor sample) from the patient and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control indicates that the patient is likely to be responsive to PLK4 antagonist therapy.
  • the tissue sample is a breast tissue sample and the patient has a breast cancer.
  • the patient has a basal sub-type breast cancer or a luminal B sub-type breast cancer.
  • the patient has a soft tissue cancer.
  • the soft tissue cancer patient has a sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • the patient has a disorder associated with angiogenesis.
  • the patient has a cancer metastasis disease, such as a metastatic tumor.
  • the invention is a method for predicting whether a patient will be responsive to PLK4 antagonist therapy.
  • the method comprises providing a suitable sample (e.g., a tissue sample, a tumor sample) from the patient and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control indicates that the patient will be responsive to PLK4 antagonist therapy.
  • the tissue sample is a breast tissue sample and the patient has a breast cancer.
  • the patient has a basal sub-type breast cancer or a luminal B sub-type breast cancer.
  • the patient has a soft tissue cancer.
  • the soft tissue cancer is a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • the patient has a disorder associated with angiogenesis.
  • the patient has a cancer metastasis disease.
  • the invention is a method for treating a cancer patient.
  • the method comprises administering to the patient a therapeutically effective amount of a small molecule PLK4 antagonist, such as an antagonist selected from the group consisting of:
  • the cancer patient is diagnosed with breast cancer.
  • the cancer patient is diagnosed with or has a basal sub-type breast cancer.
  • the breast cancer patient is diagnosed with or has a luminal B sub-type breast cancer.
  • the cancer patient is diagnosed with a soft tissue cancer.
  • the cancer patient is diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • FIG. 1 is the nucleotide sequence of human PLK4 (SEQ ID NO: 1).
  • FIG. 2 is the amino acid sequence of human PLK4 (SEQ ID NO: 2).
  • FIG. 3 is a schematic diagram of PLKl and PLK4.
  • the consensus sequence for serine-threonine kinase activity of each PLK is shown below the corresponding schematic kinase domain (SEQ ID NOS: 3 and 4) (where ⁇ denotes a large hydrophobic residue and ⁇ is a charged residue dependent on the context of the surrounding sequence).
  • PB is an abbreviation for "Polo-Box" domain.
  • FIG. 4 is a table summarizing PLKl, PLK2, PLK3 and PLK4 expression levels in breast cancer tissue samples as compared with normal breast, correlation of PLK expression with breast cancer patient survival and growth inhibition induced by depletion of PLK expression using small interference RNA.
  • FIG. 5 are graphs showing the transcript levels of PLK4 expression in various normal tissues and various sarcoma tumor samples. As can be seen, PLK4 expression is increased in soft tissue sarcoma tumor cells. The data analysis indicates that the PLK4 mRNA expression is elevated in majority of the tumors tissue samples in comparison to those in normal tissues. The PLK4 expression analysis was done using the Gene Expression Omnibus database that contains datasets for levels of human gene expression in human cancers at the National Centre for Biotechnology information.
  • FIGS. 6A and 6B are graphs illustrating PLK4 overexpression in breast and lung cancer cell lines.
  • the mRNA transcript levels of PLK4 in breast cancer cell lines were measured by quantitative RT-PCR and compared to those in normal human mammary epithelial cells (HMECs) from three separate donors.
  • the mRNA transcript levels of PLK4 in lung cancer cell lines were measured by quantitative RT-PCR and compared to those in normal human bronchial epithelial cells (NHBEs) from three separate donors.
  • the primers used are: 5 '-CCACAGACAACAATGCCAAC-S' (forward) (SEQ ID NO: 5) and 5'- GGTCTGCAAATGGAAAAGGA-3' (reverse) (SEQ ID NO: 6).
  • FlG. 7 is a graph illustrating the efficiency of PLK4 knockdown with different PLK4 siRNAs. As can be seen from the graph and indicated by the red arrows, PLK4 knockdown efficiency of greater than 70% was achieved with multiple siRNAs.
  • the sequences for the siRNAs are: siRNA#l 5'- AAGCCATGTACAAAGCAGGAA-3' (SEQ ID NO: 7); siRNA#2 5'- ACTCCTTTCAGACATATAAG-3' (SEQ IDNO: 8); siRNA#35'- AACTATCTTGGAGCTTTATAA-3' (SEQ IDNO: 9); and siRNA#45'- CTGGTAGTACTAGTTCACCTA-3' (SEQ IDNO: 10).
  • FIG. 8 is a table of results illustrating that siRNA depletion of PLK4 reduces cell viability in breast cancer cell lines.
  • the sulforhodamine B (SRB) assay is used for cell viability determination.
  • Cells in 96-well plates were transfected with the indicated siRNAs (40 nM) using Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation) and cultured at 37 0 C. After 5 days, the cells were washed with PBS, fixed with 10% ice-cold trichloroacetic acid at 4°C and dried at room temperature. Proteins were stained with SRB in 1% acetic acid at room temperature, washed with 1% acetic acid and dried at 37°C.
  • SRB sulforhodamine B
  • FIG. 9 is a series of cell cycle profiles in FACS analysis graphs. As shown,
  • PLK4 knockdown using siRNA increases G2/M and/or sub-Gl cell cycle populations in breast cancer cells. Depletion of PLKl also causes a similar cellular phenotype.
  • Cells in 6-well plates were transfected with the indicated siRNAs (40 nM) using Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation) and cultured at 37°C.
  • FIG. 10 is a series of cell cycle profiles in FACS analysis graphs illustrating that PLK4 knockdown does not affect the cell cycle profile of normal breast cells. A similar observation was made for the cells depleted of PLKl expression.
  • FIG. 11 are graphs of two exemplary experiments illustrating siRNA depletion of PLK4 and PLKl induces apoptosis in breast cancer cell lines but not in normal cells.
  • FIGS. 12A and 12B illustrate that siRNA depletion of PLK4 inhibits colony formation in soft agar.
  • FIG. 12A graphs colony size with the indicated siRNA treatments.
  • FIG. 12B graphs colony number with the indicated siRNA treatments.
  • FIG. 13 illustrates the chemical structures of PLK inhibitors (compounds 1- 21). Adapted from Johnson et ah, Biochemistry (2007) 46(33): 9551-9563.
  • FIG. 14 is a Table demonstrating various Ki values for the PLK inhibitors (compounds 1-21) illustrated in FIG. 13 against PLKl -4. Adapted from Johnson et ah, Biochemistry (2007) 46(33): 9551-9563.
  • FIG. 15 is a graph illustrating that high PLK4 expression is associated with reduced patient survival.
  • the data analysis was done using the gene expression data from the references: Hu et ah, BMC Genomics (2006) 7:96; van de Vijver et ah, N. Engl. J. Med. (2002) 347(25): 1999-2009; and Miller et al, PNAS (2005) 102(38): 13550-13555.
  • FIG. 16 is a graph illustrating that high PLK4 expression is associated with an increased incidence of metastasis.
  • FIGS. 17A and 17B are two data sets illustrating that high PLK4 expression may be associated with an increased risk of relapse.
  • FIG. 18 is a table detailing PLK4 overexpression in basal-like and luminal B subtypes of breast cancer is significantly associated with poor prognosis.
  • FIGS. 19A and 19B illustrate that PLK4 knockdown suppresses breast tumor growth.
  • FIG. 19A demonstrates the PLK4 protein levels in infected MDA-MB-468 cells at the start of the study (day 1).
  • pSIREN-shPLK4 infected MDA-MB-468 cells have an approximately 60% knockdown of PLK4 expression as compared to control.
  • a "therapy” is the administration of one or more therapeutic agents to a subject.
  • a subject is any individual (e.g., a mammal, such as a primate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, guinea pig, rat, mouse or other bovine, ovine, equine, canine feline, rodent or murine species) in need of therapy.
  • Therapy can be the administration of a therapeutic agent in a single dose, in multiple doses, simultaneously with other agents, or sequentially with other agents.
  • the one or more therapeutic agents can be administered to a subject at a particular dose (e.g., level, amount, mass) and on a particular schedule or at particular intervals (e.g., in increments of minutes, days, weeks, months, etc.).
  • a particular dose e.g., level, amount, mass
  • particular intervals e.g., in increments of minutes, days, weeks, months, etc.
  • a “therapeutic agent” is an agent which is used in a medical treatment, such as a therapy, e.g., to treat or cure a disease or condition, and/or to treat or alleviate a symptom, and/or to prevent or mitigate a disease or condition.
  • a “therapeutically effective amount” is an amount sufficient to achieve the desired therapeutic effect (such as a curing effect, treatment, or a prophylactic effect) under the conditions of administration.
  • directly inhibit tumor cell growth refers to specifically inhibiting tumor cell growth (e.g., reducing tumor cell proliferation, promoting tumor cell death), but not necessarily inhibiting conditions that promote or permit tumor cell growth (e.g., not necessarily inhibiting growth of new blood vessels (angiogenesis) to or within a tumor mass).
  • tumor cell growth e.g., reducing tumor cell proliferation, promoting tumor cell death
  • conditions that promote or permit tumor cell growth e.g., not necessarily inhibiting growth of new blood vessels (angiogenesis) to or within a tumor mass.
  • an “anti-tumor effective amount” is an amount of an agent that is sufficient to directly inhibit tumor cell growth (e.g., inhibit tumor cell proliferation), inhibit tumor survival and/or promote tumor cell death.
  • an “anti-metastasis effective amount” is an amount of an agent that is sufficient to directly inhibit cancer metastasis (e.g., inhibit metastatic cancer cell proliferation, inhibit spread of metastatic cancer cells, inhibit formation of metastatic cancer cells), inhibit metastatic cancer cell survival and/or promote metastatic cancer cell death.
  • an "anti-angiogenic effective amount” is an amount sufficient to inhibit angiogenesis.
  • An agent administered as a "primary therapy” is an agent that is the principal therapeutic agent in a therapy.
  • An adjunct therapy is another (e.g., secondary) therapy used together with the primary therapy, wherein the combination provides the desired treatment.
  • Adjunct therapy is also known in the art as "adjunctive therapy”.
  • An adjuvant therapy is a therapy given after the primary therapy to increase the chances of a cure.
  • Adjuvant therapy may include chemotherapy, radiation therapy, hormone therapy, biological therapy and the like.
  • aggressive therapy is the administration of a therapeutic agent or agents at higher doses, more frequent doses, or a combination thereof, than is normally administered in a typical therapeutic regime.
  • Aggressive therapy can also be the administration of a combination of therapeutic agents that are not typically administered in the same therapeutic regime.
  • Aggressive therapy is often at or near the limit of tolerance for a subject receiving such therapy.
  • aggressive chemotherapy is sufficiently toxic that the subject's bone marrow is likely to fail (e.g., the bone marrow will no longer be able to produce hematopoeitic cells after aggressive therapy).
  • the subject may receive an autologous bone marrow transplant, or receive a tissue-type matched bone marrow transplant.
  • PLK4 expression means the expression (e.g., presence, absence, level, amount, etc.) of PLK4 nucleic acid sequence (e.g., mRNA), protein, and/or the activity of the protein (e.g., kinase activity, ability to bind substrate, ability to localize in the cell, etc.) in a sample. PLK4 expression can also mean the localization of the protein in a tissue sample (e.g., cellular localization).
  • An "antagonist” is an agent that binds the target molecule (e.g., PLK4,
  • a "PLK4 antagonist” is an agent (e.g., small molecule, protein, peptide, polypeptide, peptidemimetic, non-peptidic molecule, antibody, siRNA molecule, antisense oligonucleotide, chemical compound, or a combination thereof) which specifically and preferably selectively binds PLK4, and inhibits one or more activities of PLK4; or an agent that inhibits (e.g., reduces, prevents) the expression of PLK4 gene and/or protein.
  • an agent e.g., small molecule, protein, peptide, polypeptide, peptidemimetic, non-peptidic molecule, antibody, siRNA molecule, antisense oligonucleotide, chemical compound, or a combination thereof
  • a PLK4 antagonist selectively binds or inhibits expression of PLK4 and, therefore, does not substantially bind other PLK family members ⁇ e.g., PLKl, PLK2, PLK3, or a combination thereof) under physiological or therapeutic conditions.
  • a "PLKl antagonist” is an agent that specifically and preferably selectively binds PLKl , and inhibits one or more activities of PLKl; or an agent that inhibits ⁇ e.g., reduces, prevents) the expression of PLKl gene and/or protein.
  • a PLKl antagonist selectively binds or inhibits expression of PLKl and, therefore, does not substantially bind other PLK family members ⁇ e.g., PLK2, PLK3, PLK4, or a combination thereof) under physiological or therapeutic conditions.
  • peptide refers to a compound consisting of from about 2 to about 100 amino acid residues wherein the amino group of one amino acid is linked to the carboxyl group of another amino acid by a peptide bond. Such peptides are typically less than about 100 amino acid residues in length and preferably are about 10, about 20, about 30, about 40 or about 50 residues.
  • peptidomimetic or “peptide mimetic” refers to molecules which are not polypeptides, but which mimic aspects of their structures.
  • antibody is intended to encompass all types of polyclonal and monoclonal antibodies ⁇ e.g., human, chimeric, humanized, primatized, veneered, single chain, domain antibodies (dAbs)) and antigen-binding fragments of antibodies ⁇ e.g., Fv, Fc, Fd, Fab, Fab', F(ab'), dAb) (see e.g., Harlow et al, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
  • dAbs single chain, domain antibodies
  • Soft tissue cancer is an art-recognized term that encompasses tumors derived from any soft tissue of the body. Such soft tissue connects, supports, or surrounds various structures and organs of the body, including, but not limited to, smooth muscle, skeletal muscle, tendons, fibrous tissues, fatty tissue, blood and lymph vessels, perivascular tissue, nerves, mesenchymal cells and synovial tissues.
  • soft tissue cancers can be of fat tissue, muscle tissue, nerve tissue, joint tissue, blood vessels, lymph vessels, and fibrous tissues.
  • Soft tissue cancers can be benign or malignant. Generally, malignant soft tissue cancers are referred to as sarcomas, or soft tissue sarcomas.
  • soft tissue tumors including lipoma, lipoblastoma, hibernoma, liposarcoma, leiomyoma, leiomyosarcoma, rhabdomyoma, rhabdomyosarcoma, neurofibroma, schwannoma (neurilemoma), neuroma, malignant schwannoma, neurofibrosarcoma, neurogenic sarcoma, nodular tenosynovitis, synovial sarcoma, hemangioma, glomus tumor, hemangiopericytoma, hemangioendothelioma, angiosarcoma, Kaposi sarcoma, lymphangioma, fibroma, elastofibroma, superficial fibromatosis, fibrous histiocytoma, fibrosarcoma, fibromatosis, dermatofibrosarcoma protuberans (DFSP), malignant fibrous hist
  • the soft tissue cancer is a sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • the invention is a method for identifying a cancer patient candidate for anti-cancer therapy using a PLK4 antagonist, wherein the cancer patient is not a colorectal cancer patient.
  • the method comprises providing a suitable sample from the patient (e.g., a tumor sample, a cancer tissue sample) and determining the PLK4 expression in the sample.
  • Increased PLK4 expression in the sample relative to a suitable control indicates that the cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist.
  • a method for identifying a breast cancer patient candidate for anti-cancer therapy using a PLK4 antagonist comprises providing a suitable sample from the patient (e.g., a tumor sample, a breast cancer sample) and determining the PLK4 expression in the sample.
  • Increased PLK4 expression in the sample relative to a suitable control indicates that the breast cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist.
  • a method for identifying a soft tissue cancer patient candidate for anti-cancer therapy using a PLK4 antagonist is a method for identifying a soft tissue cancer patient candidate for anti-cancer therapy using a PLK4 antagonist.
  • the method comprises providing a suitable sample from the patient (e.g., a tumor sample, a soft tissue cancer sample) and determining the PLK4 expression in the sample.
  • a suitable sample from the patient e.g., a tumor sample, a soft tissue cancer sample
  • Increased PLK4 expression in the sample relative to a suitable control indicates that the soft tissue cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist.
  • a suitable sample can be obtained for example by cell or tissue biopsy.
  • a sample can also be obtained from other tissues, bodily fluids and products, e.g., from a blood sample, spinal tap, feces, tissue smear, tissue scrape, and the like.
  • the sample can be a biopsy specimen (e.g, tumor, polyp, mass (solid, cellular)), aspirate, smear, fecal sample and/or blood sample.
  • the sample can be from a tissue that has a tumor (e.g., cancerous growth) and/or tumor cells, or is suspected of having a tumor and/or tumor cells.
  • a tumor biopsy can be obtained in an open biopsy, a procedure in which an entire (excisional biopsy) or partial (incisional biopsy) mass is removed from a target area.
  • a tumor sample can be obtained through a percutaneous biopsy, a procedure performed with a needle-like instrument through a small incision or puncture (with or without the aid of a imaging device) to obtain individual cells or clusters of cells (e.g., a fine needle aspiration (FNA)) or a core or fragment of tissues (core biopsy).
  • FNA fine needle aspiration
  • the biopsy samples can be examined cytologically (e.g., smear), histologically (e.g., frozen or paraffin section) or using any other suitable method (e.g., molecular diagnostic methods).
  • a tumor sample can also be obtained by in vitro harvest of cultured human cells derived from an individual's tissue.
  • Tumor samples can, if desired, be stored before analysis by suitable storage means that preserve a sample's protein and/or nucleic acid in an analyzable condition, such as quick freezing, or a controlled freezing regime. If desired, freezing can be performed in the presence of a cryoprotectant, for example, dimethyl sulfoxide (DMSO), glycerol, or propanediol-sucrose.
  • DMSO dimethyl sulfoxide
  • glycerol glycerol
  • propanediol-sucrose propanediol-sucrose.
  • Determining or assessing PLK4 expression can be performed using any suitable method, such methods are routine in the art. For example, screening a BAC array data set, quantitative polymerase chain reaction (QPCR), including quantitative real-time PCR, in situ hybridization, western blot analysis, immunohistochemical staining, kinase assays, and the like.
  • QPCR quantitative polymerase chain reaction
  • Other such methods to detect a PLK4 protein or peptide can include immunological and immunochemical methods like flow cytometry (e.g., FACS analysis), enzyme-linked immunosorbent assays (ELISA), including chemiluminescence assays, radioimmunoassay, and immunohistology, or other suitable methods such as mass spectroscopy.
  • antibodies to PLK4 can be used to determine the presence and/or expression level of PLK4 in a sample directly or indirectly using, for instance, immunohistology.
  • paraffin sections can be taken from a biopsy, fixed to a slide and combined with one or more antibodies by suitable methods.
  • Methods to detect a PLK4 gene or expression thereof include PLK4 nucleic acid amplification and/or visualization.
  • nucleic acid can be isolated from an individual by suitable methods which are routine in the art (see, e.g., Sambrook et al. Molecular Cloning, 1989).
  • Isolated nucleic acid can then be amplified (by e.g., polymerase chain reaction (PCR) (e.g., direct PCR, quantitative real time PCR, reverse transcriptase PCR), ligase chain reaction, self sustained sequence replication, transcriptional amplification system, Q-Beta Replicase, or the like) and visualized (by e.g., labeling of the nucleic acid during amplification, exposure to intercalating compounds/dyes, probes).
  • PCR polymerase chain reaction
  • PLK4 gene or expression thereof can also be detected using a nucleic acid probe, for example, a labeled nucleic acid probe (e.g., fluorescence in situ hybridization (FISH)) directly in a paraffin section of a tissue sample taken from, e.g., a tumor biopsy, or using other suitable methods.
  • a labeled nucleic acid probe e.g., fluorescence in situ hybridization (FISH)
  • FISH fluorescence in situ hybridization
  • PLK4 gene or expression thereof can also be assessed by Southern blot or in solution (e.g., dyes, probes).
  • a gene chip, microarray, probe e.g., quantum dots
  • other such device e.g., sensor, nanonsensor/detector
  • PLK4 expression in a tissue sample can be compared with a suitable control.
  • suitable controls are well recognized in the art and include, for example, normal cells or a non-neoplastic tissue sample such as one isolated from the donor of the cancer tissue sample, non-cancerous cells, non-metastatic cancer cells, non-malignant (benign) cells or the like, or any other suitable known or determined standard.
  • the control can be a known or pre-determined typical, normal or normalized range or level of expression of a PLK4 protein or gene (e.g., an expression standard).
  • the methods of the invention do not require that expression of the gene and/or protein be separately assessed in a suitable control each time a suitable sample from a patient is assessed for PLK4 expression.
  • whether PLK4 expression in a sample is increased or not can be determined using prior knowledge or comparison with a known or pre-determined typical, normal or normalized range or level of expression of a PLK4 protein or gene, such as a standard.
  • the standard can be a lack or very low expression of PLK4 expression. It is noted that non-dividing cells (including, e.g., differentiated cells) have very low /nondetectable levels of PLK4 expression, and thus may be used as a suitable standard or referenced as the known standard.
  • PLK4 expression can be compared to its expression in a known or a determined standard or it can be determined whether PLK4 expression exceeds a threshold, typical, normal, or normalized level.
  • a suitable control is a normal breast tissue sample obtained from the same donor of the breast cancer tissue sample.
  • a method for selecting a breast cancer patient for PLK4 therapy wherein the patient is or has been diagnosed with a basal sub-type breast cancer. In another embodiment, the patient is or has been diagnosed with a luminal B sub-type breast cancer.
  • a method for selecting a soft tissue cancer patient for PLK4 therapy wherein the patient is or has been diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • the invention is a method for screening a breast cancer patient as an aid for selecting aggressive cancer therapy of said patient (i.e., a method for determining if aggressive cancer therapy is indicated for a patient).
  • the method comprises providing a suitable sample from the patient, e.g., a tumor sample, a breast cancer tissue sample, and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control indicates the need for aggressive cancer therapy.
  • the patient has basal sub-type breast cancer.
  • the patient has luminal B sub-type breast cancer.
  • the invention is a method for screening a soft tissue cancer patient as an aid for selecting aggressive cancer therapy of said patient (i.e., a method for determining if aggressive cancer therapy is indicated for a patient).
  • the method comprises providing a suitable sample from the patient, e.g., a tumor sample, a soft tissue cancer tissue sample, and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control indicates the need for aggressive cancer therapy.
  • the patient has a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • a method for identifying a patient that will be responsive to PLK4 antagonist therapy comprises providing a suitable sample, e.g., a tissue sample, a tumor sample, from the patient and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control indicates that the patient will be responsive to PLK4 antagonist therapy.
  • the tissue sample is a breast tissue sample and the patient has breast cancer.
  • the patient that has breast cancer can have a basal sub-type breast cancer or a luminal B sub-type breast cancer.
  • the patient has a soft tissue cancer.
  • the patient has a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • the patient has a cancer metastasis disease.
  • the patient has a disorder associated with angiogenesis.
  • One aspect of the invention is a prognostic method for predicting (or indicating) a clinical outcome, e.g., relapse, cancer metastasis, survival, of a patient.
  • the method comprises providing a suitable sample from the patient, e.g., a tumor sample, a cancer tissue sample, and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control is indicative of the presence or increased likelihood of developing cancer metastasis in the patient.
  • the method predicts cancer metastasis in a patient diagnosed with a basal sub-type breast cancer.
  • the method predicts cancer metastasis in a patient diagnosed with luminal B sub-type breast cancer.
  • the method predicts cancer metastasis in a patient diagnosed with a soft tissue cancer. In another embodiment, the method predicts cancer metastasis in a patient diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcom
  • the prediction of cancer metastasis can be either an aid to diagnosis, prognosis, and/or assisting in the selecting a patient for a particular therapy.
  • a further aspect of the invention is a method for predicting (or indicating) reduced survival in a patient.
  • the method comprises providing a suitable sample from the patient, e.g., a tumor sample, a cancer tissue sample, and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control predicts the reduced survival of the patient.
  • the method predicts reduced survival in a patient diagnosed with a basal sub-type breast cancer.
  • the method predicts reduced survival in a patient diagnosed with luminal B sub-type breast cancer.
  • the method predicts reduced survival in a patient diagnosed with a soft tissue cancer.
  • the method predicts reduced survival in a patient diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • Another aspect of the invention is a method for predicting
  • the method comprises providing a suitable sample from the patient, e.g., a tumor sample, a cancer tissue sample, and determining PLK4 expression in the sample.
  • Increased PLK4 expression in the sample as compared with a suitable control predicts an increased risk of relapse of the patient.
  • the method predicts an increased risk of relapse in a patient diagnosed with a basal sub-type breast cancer.
  • the method predicts an increased risk of relapse in a patient diagnosed with luminal B sub-type breast cancer.
  • the method predicts an increased risk of relapse in a patient diagnosed with a soft tissue cancer.
  • the method predicts an increased risk of relapse in a patient diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • a "PLK4 antagonist” is an agent ⁇ e.g., small molecule, protein, peptide, polypeptide, peptidemimetic, non-peptidic molecule, antibody, siRNA molecule, antisense oligonucleotide, chemical compound, or a combination thereof) which specifically and preferably selectively binds PLK4, and inhibits one or more activities of PLK4; or an agent that inhibits ⁇ e.g., reduces, prevents) the expression of PLK4 gene and/or protein.
  • a PLK4 antagonist can, for example, inhibit binding of a ligand or substrate ⁇ e.g., ATP) to PLK4.
  • a PLK4 antagonist can inhibit the activity of a PLK4 in response to ligand or substrate binding.
  • a PLK4 antagonist that inhibits the expression and/or activity of PLK4 can be, for example, a natural or synthetic nucleic acid or nucleic acid analog, antisense molecule, small interfering RNA (siRNA), short hairpin RNA (shRNA), protein, peptide, peptidomimetic, antibody, chemical compound or the like.
  • a PLK4 antagonist selectively binds or inhibits expression of PLK4 and, therefore, does not substantially bind other PLK family members ⁇ e.g., PLKl , PLK2, and/or PLK3) under physiological or therapeutic conditions.
  • a composition comprising a PLK4 can be used in a such a screen or binding assay to detect and/or identify agents that can bind to a PLK4.
  • Compositions suitable for use include, for example, cells which naturally express a PLK4.
  • Agents which bind PLK4 can be further evaluated for PLK4 antagonist activity.
  • An agent that binds a PLK4 can be identified in a competitive binding assay, for example, in which the ability of a test agent to inhibit the binding of a reference agent (e.g., a ligand or substrate) is assessed.
  • the reference agent can be labeled with a suitable label (e.g., radioisotope, epitope label, affinity label (e.g., biotin and avidin or streptavidin), spin label, enzyme, fluorescent group, chemiluminescent group, dye, metal (e.g., gold, silver), magnetic bead) and the amount of labeled reference agent required to saturate the PLK4 in the assay can be determined.
  • a suitable label e.g., radioisotope, epitope label, affinity label (e.g., biotin and avidin or streptavidin), spin label, enzyme, fluorescent group, chemiluminescent group, dye, metal (e.g., gold, silver), magnetic bead
  • a suitable control e.g., unlabeled agent, label alone.
  • the capacity of a test agent to inhibit formation of a complex between the reference agent and a PLK4 can be determined as the concentration of test agent required for 50% inhibition (IC 5 0 value) of specific binding of labeled reference agent.
  • Specific binding is preferably defined as the total binding (e.g., total label in complex) minus the non-specific binding.
  • Non-specific binding is preferably defined as the amount of label still detected in complexes formed in the presence of excess unlabeled reference agent.
  • An agent which binds a PLK4 can be further studied to assess the ability of that agent to antagonize (reduce, prevent, inhibit) one or more functions of the PLK4.
  • Functional characteristics of a PLK4 include binding activities (e.g., ligand or substrate binding), kinase activity (e.g., phosphorylation of a substrate) and/or an ability to stimulate a cellular response (e.g., mitosis).
  • binding activities e.g., ligand or substrate binding
  • kinase activity e.g., phosphorylation of a substrate
  • an ability to stimulate a cellular response e.g., mitosis.
  • Such assays are standard in the art (see, e.g., Johnson et ah, Biochemistry (2007) 46(33): 9551-9563 for a description of kinase assays for assessing activity of PLKl, PLK 2, PLK 3 or PLK 4).
  • the agent can be incubated with PLK4 (purified, recombinant, or the like), in the presence of a suitable substrate (such as a peptide substrate, which can be labeled with a suitable label, e.g., an epitope label, an affinity label, such as biotin, avidin, streptavidin, and the like, magnetic bead, etc) under conditions suitable for kinase activity.
  • a suitable substrate such as a peptide substrate, which can be labeled with a suitable label, e.g., an epitope label, an affinity label, such as biotin, avidin, streptavidin, and the like, magnetic bead, etc
  • suitable conditions include the presence of a kinase reaction buffer, with ATP.
  • the ATP can be suitably labeled, e.g., with a radioisotope, epitope label, affinity label (e.g., biotin, avidin, streptavadin), spin label, enzyme, fluorescent group, chemiluminescent group, dye, metal (e.g., gold, silver), magnetic bead, or the like.
  • a radioisotope e.g., with a radioisotope, epitope label, affinity label (e.g., biotin, avidin, streptavadin), spin label, enzyme, fluorescent group, chemiluminescent group, dye, metal (e.g., gold, silver), magnetic bead, or the like.
  • phosphorylation of the substrate can be assessed, e.g., by determining the amount of label from the ATP has transferred to the substrate.
  • a phosphorylated substrate can be detected using a phospho-specif ⁇ c antibody.
  • the substrate peptide: TPSDSLIYDDGLS SEQ
  • a PLK4 antagonist can be, for example, a small molecule, which can be found in nature (e.g., identified, isolated, purified) and/or artificially produced (e.g., synthesized). Small molecules can be tested for PLK4 binding specificity in a screen for example, a high-throughput screen of chemical compounds and/or libraries (e.g., chemical, peptide, nucleic acid libraries). Compounds or small molecules can be identified from numerous available libraries of chemical compounds from, for example, the Chemical Repository of the National Cancer Institute, the Molecular Libraries Small Molecules Repository (PubChem) and other libraries that are commercially available.
  • small molecule antagonists of PLK including PLK4, are described in Johnson et ah, Biochemistry (2007) 46(33): 9551-9563 (see also FIGS. 17A, 17B and 18).
  • some small molecule inhibitors exhibit PKL4 selectivity (see, e.g., compounds 1, 2, 4-9 and 14- 17 disclosed herein and in Johnson et ah, Biochemistry (2007) 46(33): 9551-9563; see also FIGS. 17A, 17B and 18).
  • Such libraries or collections of molecules can also be prepared using well-known chemical methods, such as well-known methods of combinatorial chemistry. The libraries can be screed to identify compounds that bind and inhibit PLK4.
  • Identified compounds can serve as lead compounds for further diversification using well-known methods of medicinal chemistry.
  • a collection of compounds that are structural variants of the lead can be prepared and screened for PLK4 binding and/or inhibiting activity. This can result in the development of an structure activity relationship that links the structure of the compounds to biological activity.
  • Compounds that have suitable binding and inhibitory activity can be further developed for in vivo use.
  • small molecule PLK4 antagonists have the general structure [I], or pharmaceutically acceptable salts or solvates thereof:
  • small molecule PLK4 antagonists have the general structure [II], or pharmaceutically acceptable salts or solvates thereof:
  • small molecule PLK4 antagonists of the present invention used for treating a disease include the following small molecule compounds or pharmaceutically acceptable salts or solvates thereof:
  • the small molecule PLK4 antagonist is used to treat a cancer patient.
  • the cancer patient is a breast cancer patient.
  • the breast cancer patient has or is diagnosed with a basal sub- type breast cancer.
  • the breast cancer patient has or is diagnosed with a luminal B sub-type breast cancer.
  • the cancer patient is a soft tissue cancer patient.
  • the soft tissue cancer patient has or is diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • the small molecule PLK4 antagonist used to treat a disease is compound 8.
  • a PLK4 antagonist can be a peptide (e.g., synthetic, recombinant, fusion or derivatized) which specifically binds to and inhibits (reduces, prevents, decreases) the activity of the PLK4.
  • the peptide can be linear, branched or cyclic, e.g., a peptide having a heteroatom ring structure that includes several amide bonds.
  • Peptides, including cyclic peptides, that are selective for binding to a particular domain (e.g., unique domain) of a PLK4 can be produced.
  • a peptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be synthesized by suitable methods, for example, solid phase peptide synthesis (e.g., Merrifield-type synthesis) (see, e.g., Bodanszky et al. "Peptide Synthesis," John Wiley & Sons, Second Edition, 1976).
  • Peptides that are PLK4 antagonists can also be produced, for example, using recombinant DNA methodologies or other suitable methods (see, e.g., Sambrook J. and Russell D. W., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).
  • PLK4 antagonists can also be fusion peptides fused, for example to a carrier protein (e.g., myc, his, glutathione sulfhydryl transferase) and/or tagged (e.g., radiolabeled, fluorescently labeled).
  • a carrier protein e.g., myc, his, glutathione sulfhydryl transferase
  • tagged e.g., radiolabeled, fluorescently labeled
  • a peptide can comprise any suitable L-and/or D-amino acid, for example, common ⁇ -amino acids (e.g., alanine, glycine, valine), non- ⁇ -amino acids (e.g., ⁇ - alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitruline, homoserine, norleucine, norvaline, ornithine).
  • the amino, carboxyl and/or other functional groups on a peptide can be free (e.g., unmodified) or protected with a suitable protecting group.
  • Suitable protecting groups for amino and carboxyl groups, and methods for adding or removing protecting groups are known in the art and are disclosed in, for example, Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, 1991.
  • the functional groups of a peptide can also be derivatized (e.g., alkylated) using art known methods.
  • Peptides can be synthesized and assembled into libraries comprising a few to many discrete molecular species. Such libraries can be prepared using methods of combinatorial chemistry, and can be screened using any suitable method to determine if the library comprises peptides with a desired biological activity. Such peptide antagonists can then be isolated using suitable methods.
  • the peptide can comprise modifications (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifiers (e.g., cyclizing modifications)), if desired.
  • the peptide can also contain chemical modifications (e.g., N-methyl- ⁇ -amino group substitution).
  • the peptide antagonist can be an analog of a known and/or naturally-occurring peptide, for example, a peptide analog having conservative amino acid residue substitution(s). These modifications can improve various properties of the peptide (e.g., solubility, binding), including its PLK4 antagonist activity.
  • Peptidomimetic antagonists can be prepared by conventional chemical methods (see e.g., lichwood J.R. "Peptide Mimetic Design with the Aid of Computational Chemistry” in Reviews in Computational Biology, 2007, Vol. 9, pp.1-80, John Wiley and Sons, Inc., New York, 1996; Kazmierski W.K., "Methods of Molecular Medicine: Peptidomimetic Protocols," Humana Press, New Jersey, 1999).
  • Peptidomimetics can be prepared that are PLK4 antagonists.
  • polysaccharides can be prepared that have the same functional groups as peptides.
  • Peptidomimetics can be designed, for example, by establishing the three dimensional structure of a peptide agent in the environment in which it is bound or will bind to a target molecule.
  • the peptidomimetic comprises at least two components, the binding moiety or moieties and the backbone or supporting structure.
  • the binding moieties are the chemical atoms or groups which will react or form a complex (e.g., through hydrophobic or ionic interactions) with a target molecule, for example, with the amino acid(s) at or near the ligand binding site.
  • a target molecule for example, with the amino acid(s) at or near the ligand binding site.
  • the binding moieties in a peptidomimetic can be the same as those in a peptide or protein antagonist.
  • the binding moieties can be an atom or chemical group which reacts with the PLK4 in the same or similar manner as the binding moiety in the peptide antagonist.
  • binding moieties suitable for use in designing a peptidomimetic for a basic amino acid in a peptide include nitrogen containing groups, such as amines, ammoniums, guanidines and amides or phosphoniums.
  • binding moieties suitable for use in designing a peptidomimetic for an acidic amino acid include, for example, carboxyl, lower alkyl carboxylic acid ester, sulfonic acid, a lower alkyl sulfonic acid ester or a phosphorous acid or ester thereof.
  • the supporting structure is the chemical entity that, when bound to the binding moiety or moieties, provides the three dimensional configuration of the peptidomimetic.
  • the supporting structure can be organic or inorganic. Examples of organic supporting structures include polysaccharides, polymers or oligomers of organic synthetic polymers (such as, polyvinyl alcohol or polylactide). It is preferred that the supporting structure possess substantially the same size and dimensions as the peptide backbone or supporting structure. This can be determined by calculating or measuring the size of the atoms and bonds of the peptide and peptidomimetic. In one embodiment, the nitrogen of the peptide bond can be substituted with oxygen or sulfur, for example, forming a polyester backbone.
  • the carbonyl can be substituted with a sulfonyl group or sulfinyl group, thereby forming a polyamide (e.g., a polysulfonamide).
  • Reverse amides of the peptide can be made (e.g., substituting one or more-CONH-groups for a-NHCO-group).
  • the peptide backbone can be substituted with a polysilane backbone.
  • polyester peptidomimetic can be prepared by substituting a hydroxyl group for the corresponding ⁇ -amino group on amino acids, thereby preparing a hydroxyacid and sequentially esterifying the hydroxyacids, optionally blocking the basic and acidic side chains to minimize side reactions. Determining an appropriate chemical synthesis route can generally be readily identified upon determining the chemical structure.
  • Peptidomimetics can be synthesized and assembled into libraries comprising a few to many discrete molecular species. Such libraries can be prepared using well- known methods of combinatorial chemistry, and can be screened to determine if the library comprises one or more peptidomimetics which have the desired activity. Such peptidomimetic antagonists can then be isolated by suitable methods.
  • PLK4 antagonists are also agents that inhibit (reduce, decrease, prevent) the expression of PLK4.
  • Agents molecules, compounds, nucleic acids, oligonucleotides which inhibit PLK4 gene expression (e.g., transcription, mRNA processing, translation) are effective PLK4 antagonists.
  • small interfering ribonucleic acids siRNAs
  • shRNAs short hairpin ribonucleic acids
  • siRNA molecules can be polynucleotides that are generally about 20 to about 25 nucleotides long and are designed to bind specific RNA sequence (e.g., PLK4 mRNA).
  • siRNAs silence gene expression in a sequence-specific manner, binding to a target RNA (e.g., an RNA having the complementary sequence) and causing the RNA to be degraded by endoribonucleases.
  • siRNA molecules able to inhibit the expression of the PLK4 gene product can be produced by suitable methods. There are several algorithms that can be used to design siRNA molecules that bind the sequence of a gene of interest (see e.g., Mateeva O. et al Nucleic Acids Res. 35(8):Epub, 2007; Huesken D.
  • siRNA/shRNA molecules Stable expression of siRNA/shRNA molecules is advantageous in the treatment of cancer as it enables long-term expression of the molecules, potentially reducing and/or eliminating the need for repeated treatments.
  • Antisense oligonucleotides can also be used as PLK4 antagonists to inhibit PLK4 expression.
  • Antisense oligonucleotides are generally short (-13 to -25 nucleotides) single-stranded nucleic acids which specifically hybridize to a target nucleic acid sequence (e.g., mRNA) and induce the degradation of the target nucleic acid (e.g., degradation of the RNA through RNase H-dependent mechanisms) or sterically hinder the progression of splicing or translational machinery.
  • a target nucleic acid sequence e.g., mRNA
  • antisense oligonucleotides that can be used as PLK4 antagonists including methylphosphonate oligonucleotides, phosphorothioate oligonucleotides, oligonucleotides having a hydrogen at the T- position of ribose replaced by an 0-alkyl group (e.g., a methyl), polyamide nucleic acid (PNA), phosphorodiamidate morpholino oligomers (deoxyribose moiety is replaced by a morpholine ring), PN (N3 1 — »P5' replacement of the oxygen at the 3' position on ribose by an amine group) and chimeric oligonucleotides (e.g., T-O- Methyl/phosphorothioate).
  • PNA polyamide nucleic acid
  • PN N3 1 — »P5' replacement of the oxygen at the 3' position on ribose by an amine group
  • Antisense oligonucleotides can be designed to be specific for PLK4 using predictive algorithms.
  • the antisense oligonucleotides can be produced by suitable methods; for example, nucleic acid (e.g., D ⁇ A, R ⁇ A, P ⁇ A) synthesis using an automated nucleic acid synthesizer (from, e.g., Applied Biosystems) (see also Martin, P., HeIv. Chim. Acta 78:486-504, 1995).
  • Antisense oligonucleotides can also be stably expressed in a cell containing an appropriate expression vector.
  • Antisense oligonucleotides can be taken up by target cells (e.g., tumor cells) via the process of adsorptive endocytosis.
  • target cells e.g., tumor cells
  • antisense PLK4 can be delivered to target cells (e.g., tumor cells) by, for example, injection or infusion.
  • purified oligonucleotides or siR ⁇ A/shR ⁇ A can be administered alone or in a formulation with a suitable drug delivery vehicle (e.g., liposomes, cationic polymers, (e.g., poly-L-lysine' PAMAM dendrimers, polyalkylcyanoacrylate nanoparticles and polyethyleneimine) or coupled to a suitable carrier peptide (e.g., homeotic transcription factor, the Antennapedia peptide, Tat protein of HIV-I, E5CA peptide).
  • a suitable drug delivery vehicle e.g., liposomes, cationic polymers, (e.g., poly-L-lysine' PAMAM dendrimers, polyalkylcyanoacrylate nanoparticles and polyethyleneimine) or coupled to a suitable carrier peptide (e.g., homeotic transcription factor, the Antennapedia peptide, Tat protein of HIV-I, E5CA peptide).
  • Antibodies or antibody fragments which selectively bind to and inhibit the activity of a PLK4 can be produced, constructed, engineered and/or isolated by conventional methods or other suitable techniques.
  • antigen-specific antibodies can be raised against an appropriate immunogen, such as a recombinant mammalian (e.g., human) PLK4, or portion thereof (including synthetic molecules, e.g., synthetic peptides).
  • an appropriate immunogen such as a recombinant mammalian (e.g., human) PLK4, or portion thereof (including synthetic molecules, e.g., synthetic peptides).
  • a variety of methods have been described (see e.g., Kohler et al., Nature, 256: 495 497 (1975) and Eur. J. Immunol. 6: 511 519 (1976); Milstein et al., Nature 266: 550 552 (1977); Koprowski et al., U.S.
  • Antibodies can also be raised by immunizing a suitable host (e.g., mouse) with cells that express PLK4 (e.g., cancer cells/cell lines) or cells engineered to express PLK4 (e.g., transfected cells) (see e.g., Chuntharapai et al, J.
  • a suitable host e.g., mouse
  • PLK4 e.g., cancer cells/cell lines
  • PLK4 e.g., transfected cells
  • a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0 or P3X63Ag8.653) with antibody producing cells.
  • the antibody producing cells can be obtained from the peripheral blood, or preferably, the spleen or lymph nodes, of humans or other suitable animals immunized with the antigen of interest.
  • the fused cells can be isolated using selective culture conditions, and cloned by limited dilution.
  • Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
  • Antibody fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab')2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab')2 fragments.
  • Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site.
  • a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CHl domain and hinge region of the heavy chain.
  • Single chain antibodies, and human, chimeric, humanized or primatized (CDR grafted), or veneered antibodies, as well as chimeric, CDR grafted or veneered single chain antibodies, comprising portions derived from different species, and the like are also encompassed by the present invention and the term "antibody".
  • the various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein.
  • Humanized antibodies can be produced using synthetic or recombinant DNA technology using standard methods or other suitable techniques.
  • Nucleic acid (e.g., cDNA) sequences coding for humanized variable regions can also be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al, Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al, Cancer Research, 53: 851 856 (1993); Daugherty, B.L.
  • variants can also be readily produced.
  • cloned variable regions e.g., dAbs
  • sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al, U.S. 5,514,548; Hoogenboom et al, WO 93/06213, published April 1, 1993).
  • Suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, for example, methods which select a recombinant antibody or antibody-binding fragment (e.g., dAbs) from a library (e.g., a phage display library), or which rely upon immunization of transgenic animals (e.g., mice).
  • a recombinant antibody or antibody-binding fragment e.g., dAbs
  • a library e.g., a phage display library
  • transgenic animals capable of producing a repertoire of human antibodies are well- known in the art (e.g., Xenomouse® (Abgenix, Fremont, CA)) and can be produced using suitable methods (see e.g., Jakobovits et al, Proc. Natl. Acad. Sci.
  • a "PLKl antagonist” is an agent (e.g., small molecule, protein, peptide, polypeptide, peptidemimetic, non- peptidic molecule, antibody, siRNA molecule, antisense oligonucleotide, chemical compound, or a combination thereof) which specifically and preferably selectively binds PLKl, and inhibits one or more activities of PLKl; or an agent that inhibits (e.g., reduces, prevents) the expression of PLKl gene and/or protein.
  • a PLKl antagonist can, for example, inhibit binding of a ligand or substrate (e.g., ATP) to PLKl .
  • a PLKl antagonist can inhibit the activity of a PLKl in response to ligand or substrate binding.
  • a PLKl antagonist that inhibits the expression and/or activity of PLKl can be, for example, a natural or synthetic nucleic acid or nucleic acid analog, antisense molecule, small interfering RNA (siRNA), short hairpin RNA (shRNA), protein, peptide, peptidomimetic, antibody, chemical compound or the like.
  • a PLKl antagonist selectively binds or inhibits expression of PLKl and, therefore, does not substantially bind other PLK family members (e.g., PLK2, PLK3, and/or PLK4) under physiological or therapeutic conditions.
  • compositions comprising a PLKl can be used in a such a screen or binding assay to detect and/or identify agents that can bind to a PLKl .
  • Compositions suitable for use include, for example, cells which naturally express a PLKl . Agents which bind PLKl can be further evaluated for PLKl antagonist activity.
  • An agent that binds a PLKl can be identified in a competitive binding assay, for example, in which the ability of a test agent to inhibit the binding of a reference agent (e.g., a ligand or substrate) is assessed.
  • the reference agent can be labeled with a suitable label (e.g., radioisotope, epitope label, affinity label (e.g., biotin and avidin or streptavadin), spin label, enzyme, fluorescent group, chemiluminescent group, dye, metal (e.g., gold, silver), magnetic bead) and the amount of labeled reference agent required to saturate the PLKl in the assay can be determined.
  • the specificity of the formation of the complex between the PLKl and the test agent can be determined using a suitable control (e.g., unlabeled agent, label alone).
  • the capacity of a test agent to inhibit formation of a complex between the reference agent and a PLKl can be determined as the concentration of test agent required for 50% inhibition (IC 50 value) of specific binding of labeled reference agent.
  • Specific binding is preferably defined as the total binding (e.g., total label in complex) minus the non-specific binding.
  • Non-specific binding is preferably defined as the amount of label still detected in complexes formed in the presence of excess unlabeled reference agent.
  • An agent which binds a PLKl can be further studied to assess the ability of that agent to antagonize (reduce, prevent, inhibit) one or more functions of the PLKl .
  • Functional characteristics of a PLK4 include binding activities (e.g., ligand or substrate binding), kinase activity (e.g., phosphorylation of a substrate) and/or an ability to stimulate a cellular response (e.g., mitosis).
  • binding activities e.g., ligand or substrate binding
  • kinase activity e.g., phosphorylation of a substrate
  • an ability to stimulate a cellular response e.g., mitosis.
  • Such assays are standard in the art (see, e.g., Johnson et al., Biochemistry (2007) 46(33): 9551-9563 for a description of kinase assays for assessing activity of PLKl, PLK 2, PLK 3 or PLK 4).
  • the agent can be incubated with PLKl (purified, recombinant, or the like), in the presence of a suitable substrate (such as a peptide substrate, which can be labeled with a suitable label, e.g., an epitope label, an affinity label, such as biotin, avidin, streptavidin, and the like, magnetic bead, etc.) under conditions suitable for kinase activity.
  • a suitable substrate such as a peptide substrate, which can be labeled with a suitable label, e.g., an epitope label, an affinity label, such as biotin, avidin, streptavidin, and the like, magnetic bead, etc.
  • suitable conditions include the presence of a kinase reaction buffer, with ATP.
  • the ATP can be suitably labeled, e.g., with a radioisotope, epitope label, affinity label (e.g., biotin, avidin, streptavadin), spin label, enzyme, fluorescent group, chemiluminescent group, dye, metal (e.g., gold, silver), magnetic bead, or the like.
  • a radioisotope e.g., with a radioisotope, epitope label, affinity label (e.g., biotin, avidin, streptavadin), spin label, enzyme, fluorescent group, chemiluminescent group, dye, metal (e.g., gold, silver), magnetic bead, or the like.
  • a phosphorylated substrate can be detected using a phospho-specific antibody.
  • the substrate peptide: LGEDQAEEISDDLLEDSLSDEDE SEQ ID NO: 11
  • a PLKl antagonist can be, for example, a small molecule, which can be found in nature (e.g., identified, isolated, purified) and/or artificially produced (e.g., synthesized).
  • Small molecules can be tested for PLKl binding specificity in a screen for example, a high-throughput screen of chemical compounds and/or libraries (e.g., chemical, peptide, nucleic acid libraries).
  • Examples of small molecule antagonists of PLK, including PLKl are described in Johnson et a!., Biochemistry (2007) 46(33): 9551-9563 (see also FIGS. 17A, 17B and 18).
  • some small molecule inhibitors are more selective at inhibiting PKLl -3 than PLK4 (see, e.g., compounds 20 and 21 disclosed herein and in Johnson etal, Biochemistry (2007) 46(33): 9551-9563; see also FIGS. 17A, 17B and 18).
  • One aspect of the invention relates to a method for inhibiting the growth of a tumor (e.g., by directly inhibiting tumor growth) that expresses a PLK4 comprising administering to a patient with the tumor a therapeutically effective amount (e.g., an anti-tumor effective amount) of a PLK4 antagonist.
  • a therapeutically effective amount is an amount that is sufficient to inhibit ⁇ e.g., reduce, prevent or retard) tumor cell growth (e.g., as measured by tumor cell proliferation, tumor size or mass, tumor differentiation or de-differentiation) and/or tumor progression (e.g., increased malignancy, tumor cell invasion, and/or cancer metastasis) for a particular cancer.
  • the PLK4 antagonist directly inhibits the growth of the tumor by inducing apoptosis of the tumor cells or by inhibiting proliferation of the tumor cells.
  • the PLK4 antagonist can inhibit PLK4 gene expression (e.g., using siRNA, antisense oligonucleotides) or PLK4 protein activity (e.g., using an antibody, peptide, peptide mimetic) of PLK4, thereby directly inhibiting the growth of the cells of the tumor.
  • Another aspect of the invention is a method for treating a basal sub-type breast cancer, luminal B sub-type breast cancer, or a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma, in a patient.
  • the method comprises administering to the patient a therapeutically effective amount of a PLK4 antagonist.
  • the administered PLK4 antagonist inhibits tumor growth directly by inducing the death (e.g., apoptosis) of the cells of the tumor or by inhibiting the growth (e.g., proliferation) of the cells of the tumor.
  • the method further comprises administering another therapeutic agent, such as a PLKl antagonist.
  • a PLKl antagonist As described above, the PLK4 antagonist and the PLKl antagonist can be administered simultaneously or sequentially.
  • the invention is a method for treating a patient with cancer metastasis. The method comprises administering to the patient a therapeutically effective amount of a PLK4 antagonist.
  • the administered PLK4 antagonist inhibits cancer metastasis cell growth directly by inducing the death (e.g., apoptosis) of the cells of the cancer metastasis or by inhibiting the growth (e.g., proliferation) of the cells of the cancer metastasis.
  • the method further comprises administering another therapeutic agent, such as a PLKl antagonist.
  • the PLK4 antagonist and the other therapeutic agent, such as a PLKl antagonist can be administered simultaneously or sequentially.
  • the invention is a method for inhibiting angiogenesis in a patient. In one embodiment, the method comprises administering to the patient a therapeutically effective amount of a PLK4 antagonist.
  • the method comprises administering to the patient an anti-angiogenic effective amount of a PLK4 antagonist.
  • the method further comprises administering another therapeutic agent, such as a PLKl antagonist.
  • the PLK4 antagonist and the other therapeutic agent, such as a PLKl antagonist can be administered simultaneously or sequentially, as described above.
  • Angiogenesis is associated with some cancers, more specifically it is associated with many solid tumors, but pathological angiogenesis can be unrelated to a cancer.
  • pathological angiogenesis contributes to diabetic retinopathy, rheumatoid arthritis and macular degeneration.
  • Anti-angiogenic therapies can block angiogenic growth factor signals (e.g., endostatin, tumstatin, angiostatin) or the response of endothelial cells to those signals (e.g., avastatin).
  • Anti-angiogenic therapies may indirectly affect (inhibit, reduce) tumor growth by blocking the formation of new blood vessels that supply tumors with nutrients needed to sustain tumor growth and enable tumors to metastasize.
  • This type of dosing can be referred to as an "anti-angiogenic” or “metronomic” schedule.
  • This anti-angiogenic dosing schedule is in contrast to the high dose, cyclic treatment (maximum tolerated dosing) regimen frequently used for therapies that directly inhibit tumor growth.
  • An anti-angiogenic treatment regimen has been used with a targeted inhibitor of angiogenesis (thrombospondin 1 and platelet growth factor-4 (TNP-470)) and the chemotherapeutic agent cyclophophamide. Every 6 days, TNP- 470 was administered at a dose lower than the maximum tolerated dose and cyclophophamide was administered at a dose of 170 mg/kg. This treatment regimen resulted in complete regression of the tumors.
  • anti-angiogenic treatments are most effective when administered in concert with other anti-cancer therapeutic agents, for example, those agents that directly inhibit tumor growth (e.g., chemotherapeutic agents).
  • Anti-angiogenic therapy can be used alone in the treatment of an angiogenesis disorder, or used in conjunction with anti-tumor therapy.
  • an anti-angiogenic effective amount of a PLK4 antagonist for example, a small molecule, can be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, every 1 to 7 days over a period of about 4 to about 6 months.
  • an anti-angiogenic effective amount of a PLK4 antagonist for example, an antibody
  • an anti-angiogenic effective amount of a PLK4 antagonist can be from about 0.01 mg/kg to about 300 mg/kg body weight per treatment and preferably from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg every 1 to 7 days over a period of about 4 to about 6 months.
  • the anti-angiogenic effective amount of a PLK4 antagonist can be administered alone, as an adjuvant therapy to a primary cancer therapy (surgery, radiation), with anti-angiogenic therapies (e.g., avastatin, endostatin, tumstatin, angiostatin) or as a primary therapy with other adjuvant therapies (e.g., chemotherapeutic, hormone).
  • a primary cancer therapy surgery, radiation
  • anti-angiogenic therapies e.g., avastatin, endostatin, tumstatin, angiostatin
  • other adjuvant therapies e.g., chemotherapeutic, hormone
  • the effectiveness of a therapy can be determined by any suitable method (e.g., in situ immunohistochemistry, imaging (MRI, NMR), 3H-thymidine incorporation).
  • the methods described herein comprise administering a PLK4 antagonist.
  • the PLK4 antagonist may be administered to the individual in need thereof as a primary therapy (e.g., as the principal therapeutic agent in a therapy or treatment regimen); as an adjunct therapy (e.g., as a therapeutic agent used together with another therapeutic agent in a therapy or treatment regime, wherein the combination of therapeutic agents provides the desired treatment; "adjunct therapy” is also referred to as "adjunctive therapy”); in combination with an adjunct therapy; as an adjuvant therapy (e.g., as a therapeutic agent that is given to the subject in need thereof after the principal therapeutic agent in a therapy or treatment regimen has been given); or in combination with an adjuvant therapy (e.g., chemotherapy (e.g., dacarbazine (DTIC), Cis-platinum, cimetidine, tamoxifen, cyclophophamide), radiation therapy, hormone therapy (e.g.
  • a primary therapy e.g., as the principal therapeutic agent in
  • anti-estrogen therapy anti-estrogen therapy, androgen deprivation therapy (ADT)
  • ADT anti-estrogen therapy
  • androgen deprivation therapy ADT
  • luteinizing hormone-releasing hormone (LH-RH) agonists luteinizing hormone-releasing hormone (LH-RH) agonists
  • aromatase inhibitors AIs, such as anastrozole, exemestane, letrozole
  • estrogen receptor modulators e.g., tamoxifen, raloxifene, toremifene
  • a PLK4 antagonist can be administered as an adjuvant therapy (e.g., with another primary cancer therapy or treatment).
  • the PLK4 antagonist can be administered before, after or concurrently with a primary therapy like radiation and/or the surgical removal of a tumor(s).
  • the method comprises administering a therapeutically effective amount of a PLK4 antagonist and one or more other therapies (e.g., adjuvant therapies, other targeted therapies).
  • a PLK4 antagonist e.g., an adjuvant therapy (e.g., a chemotherapeutic agent) and/or the one or more other targeted therapies (e.g., a PLKl antagonist) and the PLK4 antagonist can be co-administered simultaneously (e.g., concurrently) as either separate formulations or as a joint formulation.
  • the therapies can be administered sequentially, as separate compositions, within an appropriate time frame (e.g., a cancer treatment session/interval such as 1.5 to 5 hours) as determined by the skilled clinician (e.g., a time sufficient to allow an overlap of the pharmaceutical effects of the therapies).
  • the adjuvant therapy and/or one or more other targeted therapies (e.g., a PLKl antagonist) and the PLK4 antagonist can be administered in a single dose or multiple doses in an order and on a schedule suitable to achieve a desired therapeutic effect (e.g., inhibition of tumor growth, inhibition of angiogenesis, and/or inhibition of cancer metastasis).
  • One or more agents that are an PLK4 antagonist can be administered in single or multiple doses. Suitable dosing and regimens of administration can be determined by a clinician and are dependent on the agent(s) chosen, pharmaceutical formulation and route of administration, various patient factors and other considerations. With respect to the administration of a PLK4 antagonist with one or more other therapies or treatments (adjuvant, targeted, cancer treatment-associated, and the like) the PLK4 antagonist is typically administered as a single dose (by e.g., injection, infusion, orally), followed by repeated doses at particular intervals (e.g., one or more hours) if desired or indicated.
  • the amount of the PLK4 antagonist to be administered can be determined by a clinician using the guidance provided herein and other methods known in the art and is dependent on several factors including, for example, the particular agent chosen, the subject's age, sensitivity, tolerance to drugs and overall well-being.
  • suitable dosages for a small molecule can be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 1 mg/kg body weight per treatment.
  • Suitable dosages for antibodies can be from about 0.01 mg/kg to about 300 mg/kg body weight per treatment and preferably from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg body weight per treatment.
  • the PLK4 antagonist is a polypeptide (linear, cyclic, mimetic)
  • the preferred dosage will result in a plasma concentration of the peptide from about 0.1 ⁇ g/mL to about 200 ⁇ g/mL. Determining the dosage for a particular agent, patient and cancer is well within the abilities of one of skill in the art.
  • the dosage does not cause or produces minimal adverse side effects (e.g., immunogenic response, nausea, dizziness, gastric upset, hyperviscosity syndromes, congestive heart failure, stroke, pulmonary edema).
  • an "anti-tumor effective amount" of a PLK4 antagonist is administered to a patient in need thereof.
  • agents which directly inhibit tumor growth e.g., chemotherapeutic agents
  • chemotherapeutic agents are conventionally administered at a particular dosing schedule and level to achieve the most effective therapy (e.g., to best kill tumor cells).
  • about the maximum tolerated dose is administered during a relatively short treatment period (e.g., one to several days), which is followed by an off-therapy period.
  • the chemotherapeutic cyclophosphamide is administered at a maximum tolerated dose of 150 mg/kg every other day for three doses, with a second cycle given 21 days after the first cycle. (Browder et al. Can Res 60: 1878-1886, 2000).
  • An anti-tumor effective amount of PLK4 which directly inhibits the expression or activity of PLK4 in a tumor cell can be administered, for example, in a first cycle in which the maximum tolerated dose of the antagonist is administered in one interval/dose, or in several closely spaced intervals (minutes, hours, days) with another/second cycle administered after a suitable off-therapy period (e.g., one or more weeks).
  • a suitable off-therapy period e.g., one or more weeks.
  • suitable dosing schedules and amounts for a PLK4 antagonist can be readily determined by a clinician of ordinary skill.
  • an anti-tumor effective amount of a PLK4 antagonist is preferably administered on a dosing schedule that is similar to that of the other cancer therapy (e.g., chemotherapeutics), or on a dosing schedule determined by the skilled clinician to be more/most effective at inhibiting (reducing, preventing) tumor growth.
  • a treatment regimen for an anti-tumor effective amount of a small molecule PLK4 antagonist can be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, every 1 to 7 days over a period of about 4 to about 6 months.
  • a treatment regimen for an anti -tumor effective amount of an antibody PLK4 antagonist can be from about 0.01 mg/kg to about 300 mg/kg body weight per treatment and preferably from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg body weight per treatment, every 1 to 7 days over a period of about 4 to about 6 months.
  • a treatment regimen for an anti-angiogenesis effective amount of a small molecule PLK4 antagonist can be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, every 1 to 7 days over a period of about 4 to about 6 months.
  • a treatment regimen for an anti-angiogenesis effective amount of an antibody PLK4 antagonist can be from about 0.01 mg/kg to about 300 mg/kg body weight per treatment and preferably from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg body weight per treatment, every 1 to 7 days over a period of about 4 to about 6 months.
  • routes of administration can be used including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intravenous, intraaterial, intramuscular, subcutaneous injection, intradermal injection), intravenous infusion and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops) routes of administration, depending on the agent and the particular cancer to be treated.
  • parenteral e.g., intravenous, intraaterial, intramuscular, subcutaneous injection, intradermal injection
  • intravenous infusion and inhalation e.g., intrabronchial, intranasal or oral inhalation, intranasal drops
  • routes of administration can be used including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intravenous, intraaterial, intramuscular, subcutaneous injection, intradermal injection), intravenous infusion and inhalation (e.g., intra
  • the agent can be administered to a mammalian subject as part of a pharmaceutical or physiological composition.
  • the agent can be administered as part of a pharmaceutical composition for inhibition of PLK4 expression (e.g., inhibition of PLK4 gene expression and/or inhibition of PLK4 activity) and a pharmaceutically acceptable carrier.
  • Formulations or compositions comprising a PLK4 antagonist or compositions comprising a PLK4 antagonist and one or more other therapeutic agents will vary according to the route of administration selected (e.g., solution, emulsion or capsule).
  • Suitable pharmaceutical carriers can contain inert ingredients which do not interact with the PLK4 antagonist.
  • Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's lactate and the like. Formulations can also include small amounts of substances that enhance the effectiveness of the active ingredient (e.g., emulsifying, solubilizing, pH buffering, wetting agents). Methods of encapsulation compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art. For inhalation, the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer or nebulizer or pressurized aerosol dispenser).
  • a suitable dispenser for administration e.g., an atomizer or nebulizer or pressurized aerosol dispenser.
  • a nucleic acid-based PLK4 antagonist e.g., siRNA, shRNA, antisense oligonucleotide, natural or synthetic nucleic acids, nucleic acid analogs, aptamers
  • a nucleic acid-based PLK4 antagonist e.g., siRNA, shRNA, antisense oligonucleotide, natural or synthetic nucleic acids, nucleic acid analogs, aptamers
  • a nucleic acid-based PLK4 antagonist e.g., siRNA, shRNA, antisense oligonucleotide, natural or synthetic nucleic acids, nucleic acid analogs, aptamers
  • chemically synthesized or in vitro transcribed nucleic acids can be transfected into cells in cell culture by any suitable method (e.g., viral infection).
  • the nucleic acids may also be expressed endogenously from expression vectors or PCR products in host cells or packaged into synthetic or engineered compositions (e.g., liposomes, polymers, nanoparticles) that can then be introduced directly into the bloodstream of a mammalian subject (by, e.g., injection, infusion).
  • Anti-PLK4 nucleic acids or nucleic acid expression vectors e.g., retroviral, adenoviral, adeno- associated and herpes simplex viral vectors, engineered vectors, non-viral-mediated vectors
  • the agent can be administered via in vivo expression of recombinant protein.
  • In vivo expression can be accomplished by somatic cell expression according to suitable methods (see, e.g., U.S. Patent No. 5,399,346).
  • a nucleic acid encoding the polypeptide can also be incorporated into retroviral, adenoviral or other suitable vectors (often a replication deficient infectious vector) for delivery, or can be introduced into a transfected or transformed host cell capable of expressing the polypeptide for delivery.
  • the cells can be implanted (alone or in a barrier device), injected or otherwise introduced in an amount effective to express the polypeptide in a therapeutically effective amount.
  • EXAMPLE 1 PLK4 Identification and Association With Breast and Lung Cancer Gene expression profiling and overexpression ofPLK4 analysis (e.g., FIG. 1 ).
  • Amplification primers for PLK4 are:
  • Amplification primers for ⁇ -Actin are:
  • EXAMPLE 2 Identification of PLK4 Association with Soft Tissue Cancers (e.g. FIG. 5).
  • FIG. 5 illustrates that the box plots of PLK4 mRNA expression levels represented by signal intensity values in normal tissues and sarcoma tissues. It is shown that PLK4 is over-expressed in sarcomas compared to its levels in the normal tissues tested.
  • EXAMPLE 3 Validation of PLK4 as an Anti-Tumor Target (e.g., FIGS 7-12) siRNAs and reagents:
  • the sequences for the PLK4 siRNAs are: siRNA#l 5'-AAGCCATGTACAAAGCAGGAA-S' (SEQ ID NO: 7); siRNA#2 5'-ACTCCTTTCAGACATATAAG-S' (SEQ ID NO: 8); siRNA#3 5'-AACTATCTTGGAGCTTTATAA-S' (SEQ ID NO: 9); and siRNA#4 5'-CTGGTAGTACTAGTTCACCTA-S' (SEQ ID NO: 10).
  • Cell viability (e.g., FIG 8): Cells in 96-well plates were transfected with the indicated siRNAs (40 nM) using Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation) and cultured at 37 0 C. After 5 days, the cells were washed with PBS, fixed with 10% ice-cold trichloroacetic acid at 4 0 C and dried at room temperature. Proteins were stained with SRB in 1% acetic acid at room temperature, washed with 1% acetic acid and dried at 37°C. To dissolve the SRB bound proteins, 10 mM Tris base was added to each well and incubated at room temperature with mechanical agitation.
  • FIGS. 9 and 10 Cell cycle analysis assays: Cells in 6-well plates were transfected with the indicated siRNAs (40 nM) using Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation) and cultured at 37°C. After 3-4 days, the cells were trypsinized, washed in PBS and centrifuged. Cell pellets were resuspended in propidium iodide in Hepes buffer, mixed and incubated at room temperature in the dark. Samples were read in a FACSCalibur flow cytometer (Becton, Dickinson and Company) and data analyzed using FIoJo software (Tree Star, Inc.).
  • Apoptosis assay (e.g., FIG 11): Apoptosis was measured using Cell Death Detection ELISA PLUS Kit (Roche) according to the manufacturer's protocol. Cells in 96-well plates were transfected with the indicated siRNAs (40 nM) using
  • Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation) and cultured at 37 0 C. After varying amounts of time, the cells were washed in PBS, lysed and added to streptavidin-coated microplates containing anti-histone-biotin and anti- DNA-POD antibodies. Nucleosomes were photometrically detected by measuring POD activity with 2,2'-Azino-di[3-ethyl-benz-thiazolin-sulfonat] as a substrate using a SpectraPlus microplate reader (Molecular Devices Corporation).
  • Cell growth inhibition (e.g., FIG 12): Cells were transfected with the indicated siRNAs (40 nM) using Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation), mixed with culture medium containing 0.7% agar in 6- well plates and cultured at 37 0 C. After 3 weeks, the top layer of the culture was stained with 0.2% p-iodonitrotetrazolium violet and colonies were counted using a Sorcerer Colony Counter (Optomax).
  • EXAMPLE 4 PLK4 Overexpression is Associated with Reduced Patient Survival, Increased Incidence of Metastasis, Increased Risk of Relapse, and Poor Prognosis
  • Patient data sets e.g., FIGS. 15-18: All gene expression analyses were done in 3 individual datasets and in a combined dataset. The datasets were combined after normalizing expression values, e.g. logratios, of each gene by mean and standard deviation in each dataset. Regression models in the combined dataset contained a dataset term in addition to the logratio term. To verify that results did not depend on normality assumption, another combined dataset was assembled using logratio ranks, computed separately in each dataset and scaled to vary from 0 to 1. The same analyses were performed and general agreement with the results from the normalized logratio dataset was verified.
  • logratios normalizing expression values
  • Tumor gene expression profiles were classified into subtypes defined by Hu et al.
  • the classification of the Perou dataset was provided by its authors; NEJM295 and PNAS datasets were classified into Luminal A, Luminal B, Basal-like and HER2+/ER- subtypes using the intrinsic gene set defined by Hu et al. Different subtypes were compared to each other and, where available, to normal controls.
  • Perou dataset was the only one that contained a normal control group. All cancers as a group and individual subtypes were compared to the control group using t tests. Significantly higher than normal expression of PLK4, with p values ⁇ 0.05 and false discovery rates ⁇ 0.1, was detected in all cancers as a group and in Basal-like, Luminal B, and HER2+/ER- subtypes. The over-expression was the highest and most significant in the basal-like subtype, where it was, on average, 2- fold higher than normal. An additional analysis was performed on the Perou dataset to determine if a fraction of cancers expressed a gene significantly outside of the normal range. This analysis was designed to detect over-expression in a subset of a potentially heterogeneous population of cancers.
  • the normal range was defined as the mean +/- 3 standard deviations of the normal control group.
  • the fractions of tumor samples that fell above and below this range were recorded as percentages of the total number of tumors, in all cancers as a group and in individual subtypes.
  • PLK4 was found to be over-expressed in 26.4% of all tumors and in 48.5% of basal-like tumors.
  • EXAMPLE 5 PLK4 knockdown suppresses breast tumor growth: xenograft models (e.g., FIGS. 19A and l9B) Materiah and methods: A double-stranded oligonucleotide encoding a human PLK4 gene-specific shRNA (sense insert sequence 5'- GTTCTATCTTGGAGCTTTAT-3'; SEQ ID NO: 14) was ligated into the RNAi- Ready-pSIREN-RetroQ-ZsGreen retroviral vector (Clontech).
  • Amphotropic- Phoenix packaging cells (ATCC, Manassas, VA) were transiently transfected with either control RNAi-Ready-pSIREN-RetroQ-ZsGreen-shLUC (Clontech) or RNAi- Ready-pSIREN-RetroQ-ZsGreen-shPLK4 using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN). Culture supernatants were collected 2 days after transfection and filtered through 0.45- ⁇ m pore-size filters.
  • MDA-MB-468 breast cancer cells ATCC, Manassas, VA were infected with retroviruses by culturing the cells for 24 hours in 1 :1 Phoenix conditioned media (Dulbecco's Modified Eagle's Media, 10% FCS, supplemented with 8 ⁇ g/ml Polybrene; Sigma- Aldrich). This transfection process was repeated three times to increase the transfection efficiency.
  • Phoenix conditioned media Dulbecco's Modified Eagle's Media, 10% FCS, supplemented with 8 ⁇ g/ml Polybrene; Sigma- Aldrich.
  • RNAi-Ready-pSIREN- RetroQ-ZsGreen-shLUC and RNAi-Ready-pSIREN-RetroQ-ZsGreen-shPLK4 infected cancer cells were trypsinized, counted and injected subcutaneously into the left and right hindlimb, respectively, of nude mice at concentrations of 5 ⁇ lO 6 cells (5 mice per group).
  • the infected cells were also analyzed for PLK4 expression using Western blotting with an anti-PLK4 antibody. The tumors were measured and viable tumor area was calculated twice weekly for approximately 10 weeks.

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Abstract

L'invention porte sur des procédés d'évaluation de l'expression de PLK4, chez un patient atteint d'un cancer, qui sont à utiliser pour déterminer si le patient est un candidat pour une thérapie par antagoniste de PLK4, pour prédire une métastase du cancer et si le patient est un candidat pour une thérapie du cancer agressive. L'invention propose également l'utilisation d'antagonistes de PLK4 dans le traitement du cancer et d'une métastase. Les cancers préférés pour le traitement sont le cancer du sein (sous-type basal et sous-type luminal B) et les cancers de tissu mou.
PCT/CA2008/002087 2007-11-20 2008-11-20 Procédés de diagnostic et de thérapie du cancer qui ciblent plk4/sak WO2009065232A1 (fr)

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USRE47731E1 (en) 2009-04-06 2019-11-19 University Health Network Kinase inhibitors and method of treating cancer with same
US9796703B2 (en) 2010-04-06 2017-10-24 University Health Network Synthesis of chiral 2-(1H-indazol-6-yl)-spiro[cyclopropane-1,3′-indolin]-2′-ones
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WO2023159307A1 (fr) * 2022-02-23 2023-08-31 Repare Therapeutics Inc. Inhibiteurs de kinase 4 de type polo (plk4), compositions pharmaceutiques, leurs procédés de préparation et leurs utilisations

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