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WO2010030865A2 - Perte de fbxw7, un biomarqueur de la sensibilité au traitement de tumeurs par des inhibiteurs du mtor - Google Patents

Perte de fbxw7, un biomarqueur de la sensibilité au traitement de tumeurs par des inhibiteurs du mtor Download PDF

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WO2010030865A2
WO2010030865A2 PCT/US2009/056641 US2009056641W WO2010030865A2 WO 2010030865 A2 WO2010030865 A2 WO 2010030865A2 US 2009056641 W US2009056641 W US 2009056641W WO 2010030865 A2 WO2010030865 A2 WO 2010030865A2
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mtor
fbxw7
cancer
determining
gene
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WO2010030865A3 (fr
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Jian-hua MAO
Allan Balmain
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • G01N33/575—
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Cancer is a disease marked by the uncontrolled growth of abnormal cells. Cancer cells have overcome the barriers imposed on normal cells, which have a finite lifespan, to grow indefinitely. As cancerous cell growth continues, genetic alterations may mount until a cancerous cell has achieved a more aggressive growth phenotype. If left untreated, metastasis, which is the spread of cancer cells to distant areas of the body by way of the lymph system or bloodstream, may ensue. Metastasis can result in the destruction of healthy organs and tissues when cancerous cells infiltrate different areas of the body and continue growing to form new tumors.
  • mTOR mimalian target of rapamycin
  • S6-K, 4E-BP signaling mechanisms that operate upstream
  • the present invention demonstrates that mTOR is targeted for ubiquitination and consequent degradation by binding to the tumor suppressor protein FBXW7 (also known as hCDC4, FBWl and MGO).
  • FBXW7 binds directly to mTOR, leading to increased ubiquitination and protein turnover, both in mouse and in human cells. Since deletion or mutation of human PTEN (phosphatase and tensin homologue) also leads to activation of mTOR, the genetic relationship between FBXW7 and PTEN in human cancers was also investigated.
  • this invention provides a method of determining if a subject at risk for or diagnosed with cancer should receive mTOR inhibitor therapy, the method involving a step of determining if one or more copies of the Fbxw7 gene is mutated (includes deletion) in a patient sample.
  • this invention provides a method of determining the risk of cancer.
  • the method involves the step of determining the amounts of mTOR, p-mTOR and/or one or more downstream components relative to one or more upstream components, wherein the downstream and upstream components are components of the mTOR signaling pathway.
  • this invention provides a method of determining if a subject at risk for or diagnosed with cancer should receive mTOR inhibitor therapy, the method involves a step of determining the amounts of mTOR, p-mTOR and/or one or more downstream components relative to one or more upstream components in a patient sample, wherein said downstream and upstream components are components of the mTOR signaling pathway.
  • a method of assaying for compounds that treat cancer in a subject comprising the steps of contacting a cell with one or more candidate compounds, and monitor the effect of said one or more candidate compounds on Fbxw7 gene or protein expression; thereby indentifying said one or more candidate compounds that alters mTOR expression or activity.
  • Figure 1 illustrates identification of Fbxw7 novel targets by genome-wide search using CPD sequences. Comparison of minimal consensus CPD sequences found in mTOR with those of the known human target proteins cyclin E (CCNE) and C-Myc.
  • CCNE cyclin E
  • Figure 2 illustrates that CPD sequences of mTor are evolutionarily conserved in different species ranging from humans to zebra fish.
  • Figure 3 shows that depletion of FBXW7 increases mTOR and p-mTOR levels.
  • Figure 3 A shows that the levels of mTor and p-mTor, as well as the downstream mTor target S6-kinase (P-S6), are upregulated in Fbxw7-/- MEFs, whereas the levels of Akt and p-Akt (lower panels) were not appreciably affected. Two independent preparations of MEFs were used, with similar results in both cases.
  • Figure 3B illustrates elevation of mTor in tissues from Fbxw7 knockout mice.
  • Lane 1 contains extracts from wild-type mice, lane 2 from Pten+I- mice, and lane 3 from Fbxw7+/- mice. Comparing lane 1 with lane 3, mTor and p-mTor protein levels are increased in the Fbxw7+I- tissues. In contrast, levels of Akt and p-Akt are not significantly altered in the same Fbxw7+/- lysates. While loss of one copy of Pten does not significantly change mTor protein levels, both Akt and p-Akt are elevated.
  • Figure 3C shows that the levels of mTOR and p-mTOR, as well as the downstream mTOR target S6-kinase (P-S6), are upregulated by depletion FBXW7 in human colon cancer cell lines HCTl 16 and DLDl, whereas the levels of AKT and p-AKT (lower panels) were not appreciably affected.
  • Figure 3D illustrates downregulation of mTOR by expression of a dominant negative truncated FBXW7 protein.
  • Overexpression of full length FBXW7 (HA-FBXW7) in 293T cells decreases the mTOR and p-mTOR levels (lane 2) in comparison to cells containing only the empty vector (lane 1), in contrast, overexpression of a dominant-negative form of FBXW7 (HA-FBXW7- ⁇ F) increases the mTOR and p-mTOR levels (Iane3).
  • Figure 4 shows that mTOR interacts directly with FBXW7.
  • Figure 4 A illustates immunoprecipitation of HA tagged FBXW7 identifies mTOR as an interacting protein.
  • Human 293 cells were transfected with the full length or truncated (FBXW7 or FBXW7- ⁇ F) constructs, followed by immunoprecipitation of the HA-tagged proteins and immunoblotting with antibodies against mTOR (top panel).
  • immunoprecipitation of mTOR shows that the region of FBXW7 that interacts is the WD40 domain which is retained in the FBXW7 ⁇ F construct (middle panel).
  • Lower panel shows the input levels of mTOR and FBXW7 proteins in the lysates.
  • Figure 4B shows that the wild type fragment of human mTOR was cloned into p3XFL AG-CMV- 10 vector.
  • the DelT631 and T631G fragments were generated by in vitro site-directed mutagenesis.
  • Figure 4C shows that FBXW7 binds the wild-type fragment of mTOR, but binding to the mTOR(delT631) and mTOR(T631G) mutants was dramatically reduced by about 80%.
  • Figure 4D shows that MCF7 breast cancer cells show increased mTOR levels upon treatment with MG-132, but this is not seen in SUM149PT cells which have no functional FBXW7 gene.
  • FIG. 4E illustrates that the regulation of mTOR by FBXW7 is through the proteasome-dependent degradation pathway.
  • 293 T and SUM 149PT cells were transfected with a vector containing CMV promoter-driven HA-ubiquitin. Immunoprecipitation of mTOR followed by Western blot analysis of the HA-ubiquitin showed that in 293T cells which have functional FBXW7 protein, the immunoprecipitated mTOR is ubiquitinated, whereas SUM 149PT cells, which lack FBXW7, show no mTOR ubiquitination. The two right hand lanes show overall ubiquitin levels in the same cell lysates.
  • FIG. 4F HCTl 16_WT and HCTl 16_FBXW7-/- cells were transfected with HA- ubiquitin.
  • Immunoprecipitation of mTOR followed by Western blot analysis of the HA- ubiquitin showed that ubiquitination of mTOR was only seen in the HCTl 16_WT cells, and not in HCTl 16_FBXW7-/- cells.
  • the vector lane shows HCTl 16_FBXW7-/- cells transfected with empty vector construct.
  • FIG. 4G illustrates that ubiquitination of mTOR is restored by exogenous FBXW7 expression.
  • HCTl 16_FBXW7-/- cells were transfected with an FBXW7-expressing construct and HA-tagged ubiquitin.
  • Immunoprecipitation of mTOR showed increased ubiquitination compared to controls.
  • the increased ubiquitination had only a small effect on mTOR levels in these experiments ( Figure 8), probably because of the pleiotropic effects of over-expressing this potent tumor suppressor in tumor cells, which leads to growth arrest and apoptosis.
  • Figure 5 illustrates genetic interaction between FBXW7 and PTEN in human breast cancers.
  • the panels show concordance of gene copy number changes in breast cancer cell lines (Figure 5A) and in 3 independent sets of primary breast tumors ( Figures 5B-D).
  • the lighter-gray bar indicates loss, the darker-gray bar indicates gain, and the black bar indicates no changes in gene copy number.
  • Figure 5A illustrates the data from 53 human breast cancer cell lines, ordered in the vertical axis from 1-53 (Neve R.M. et al., Cancer Cell 10:515-27 (2006)).
  • Figures 5B-D illustrate three independent sets of human primary breast cancers, the DNAs of which have been analyzed by the same BAC CGH microarray platform.
  • the copy number of FBXWl and PTEN was determined based on the published CGH data (FBXW7 was based on BAC RPl 1-73G16, PTEN on BAC RPl 1-380G5). Loss is defined as Iog 2 (ratio) ⁇ -0.25 and gain as Iog 2 (ratio) >0.25.
  • FIG. 6 RNA expression of FBXW7 and PTEN in breast cancer cell lines was analyzed by RT-PCR.
  • the arrows indicate cell lines carrying mutations as determined by sequence analysis (see Table 1).
  • Figure 7 illustrates the relationship between loss of FBXW7 and sensitivity to mTOR inhibitor (rapamycin) treatment.
  • Figure 7A shows that the breast cancer SUM 149PT cells which have homozygous mutations in FBXW7 were killed at a rapamycin concentration of 10OnM, whereas MDA- MB453 cells with wild type FBXW7 were resistant to this treatment.
  • Figure 7B deals with the treatment of nude mouse xenografts with rapamycin.
  • the SUM 149PT cells showed a relative decrease in size followed by stable tumor growth, whereas the MDAMB453 cells were unaffected by treatment.
  • Figure 7C shows that the deletion or mutation of FBXW7 increases sensitivity to mTOR inhibitor (rapamycin) treatment in human breast cancers.
  • Tumor cells with deletion or mutation of FBXW7 are in Group 1, those with deletion or mutation of PTEN are Group 2, and cells with wild type copies of both genes are in Group 3.
  • Figures 7D-F shows that downregulation of FBXW7 using specific shRNA in rapamycin resistant MDA-MB453 (D), LY2 (E) and MCF7 (F) cells increases the sensitivity to this treatment.
  • Figure 8 shows that the exogenous FBXW7 expression in SUM 149PT cells reduces mTOR protein levels (lane 2), but increased mTOR levels upon treatment with MG-132 (lane 4).
  • Figure 9 HA-FBXW7 co-immunoprecipitates with mTOR fragment, HA-HtrA2 does not.
  • Lane 1 vector;
  • Lane 2 HA-FBXW7 and Flag-mTOR fragment (wild type);
  • Lane 3 HA-HtrA2 and Flag-mTOR fragment (wild type).
  • FIG. 10 HA-FBXW7 co-immunoprecipitates with endogenous mTOR, HA- HtrA2 does not. Lane 1 : vector; Lane 2: HA-FBXW7; and Lane 3: HA-HtrA2. DETAILED DESCRIPTION OF THE INVENTION INTRODUCTION
  • the present invention provides methods of determining whether or not a cancer patient has a mutated Fbxw7 gene (point mutations, deletions, or additions, including the absence of the gene by complete deletion and promoter silencing) and thereby determining whether or not the patient is a candidate for mTOR inhibitor therapy.
  • Polymorphism may also be considered as a mutation for the purposes of this application.
  • the determination involves detecting Fbxw7 DNA, RNA, or protein and determining whether or not the molecule is mutated, thereby determining whether or not the gene is mutated.
  • PCR e.g., Taqman
  • sequencing techniques Southern, western, and northern blots
  • microarrays e.g., DNA sequencing techniques
  • immunohistochemical techniques e.g., ELISA
  • mass spectroscopy e.g., mass spectroscopy
  • This invention also presents a method of determination of risk of cancer by comparing the relative amounts of components of the mTOR signaling pathway that are downstream to mTOR versus the amounts of components of the mTOR signaling pathway that are upstream to mTOR. Relatively higher levels of mTOR and downstream components as compared to a control are indicative of absence of the Fbxw7 protein or presence of the Fbxw7 mutated gene.
  • Mammalian target of rapamycin protein inhibitor or “mTOR inhibitor” includes drugs such as rapamycin, deforolimus, sirolimus, temsirolimus, and everolimus that selectively inhibit the mammalian target of rapamycin (mTOR).
  • cancer refers to a disease involving cells that have the potential to metastasize to distal sites and exhibit phenotypic traits that differ from those of non-cancer cells. Cancer cells acquire a characteristic set of functional capabilities during their development, albeit through various mechanisms. Such capabilities include evading apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion/metastasis, limitless replicative potential, and sustained angiogenesis.
  • cancer cell is meant to encompass both pre-malignant and malignant cancer cells.
  • cancers include carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, head and neck cancer, e.g. , oral cavity, pharyngeal and tongue cancer, kidney, breast, lung, kidney, bladder, colon, endometrium, T- cell acute lymphoblastic leukemia (T-ALL), ovarian, prostate, pancreas, stomach, brain, skin, melanoma, basal cell, uterine, testicular, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's lymphoma, leukemia, and multiple myeloma.
  • non-Hodgkin's lymphomas e.g., Burkitt's, Small Cell, and Large Cell lympho
  • the term "marker” refers to a molecule (typically protein, nucleic acid, carbohydrate, or lipid) that is expressed in the cell, expressed on the surface of a cancer cell, secreted by a cancer cell or over or under expressed as compared to a non-cancer cell, and which is useful for the diagnosis of cancer, for providing a prognosis, and for preferential targeting of a pharmacological agent to the cancer cell.
  • a molecule typically protein, nucleic acid, carbohydrate, or lipid
  • markers are molecules that are differentially expressed, e.g., overexpressed or underexpressed in a melanoma or other cancer cell in comparison to a normal cell, for instance, 1 -fold over/under expression, 2-fold over/under expression, 3 -fold over/under expression or more in comparison to a noncancerous cell, or a primary cancer (vs. mets).
  • a marker can be a molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a non-cancerous cell.
  • markers may be used singly or in combination with other markers for any of the uses, e.g., determination of the suitability of a subject for mTOR inhibitor therapy, disclosed herein.
  • Biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, hair, nails, skin tissue, lymph and tongue tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc.
  • a biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, Mouse; rabbit; or a bird; reptile; or fish.
  • a "biopsy” refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the diagnostic and prognostic methods of the present invention. The biopsy technique applied will depend on the tissue type to be evaluated (e.g., skin, colon, prostate, kidney, bladder, lymph node, liver, bone marrow, blood cell, etc.), the size and type of the tumor (e.g., solid or suspended, blood or ascites), among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy.
  • An “excisional biopsy” refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it.
  • An “incisional biopsy” refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor.
  • a diagnosis or prognosis made by endoscopy or fluoroscopy can require a "core-needle biopsy” of the tumor mass, or a “fine-needle aspiration biopsy” which generally obtains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, for example, in Harrison 's Principles of Internal Medicine, Kasper, et al, eds., 16th ed., 2005, Chapter 70, and throughout Part V.
  • this invention provides a method of determining if a subject at risk for or diagnosed with cancer should receive mTOR inhibitor therapy, the method involving a step of determining if the Fbxw7 gene is mutated in a patient sample.
  • this invention provides a method of determining if a subject is at risk for cancer.
  • the method involves determining the amounts of mTOR, p- mTOR or one or more downstream components relative to one or more upstream components in a patient sample and comparing the amounts to those obtained in a known sample.
  • the known sample could either be a cancerous sample or a cancer-free sample.
  • upstream and downstream are used in this application are with reference to mTOR in the mTOR signaling pathway. Relatively higher levels of mTOR and /or downstream components could be interpreted as indicative of the subject being at risk for cancer (i.e. through disruption of the Fbxw7 pathway).
  • a method of determining if a subject is at risk for cancer should receive mTOR inhibitor therapy involves determining the amounts of mTOR, p-mTOR or one or more downstream components relative to one or more upstream components in a patient sample and comparing the amounts to those obtained in a known sample.
  • the known sample could either be a cancerous sample or a cancer-free sample. Relatively higher levels of mTOR and/or downstream components may be used as diagnostic tool for presenting mTOR inhibitors.
  • a ratio (quantity of the protein mTOR or its downstream components to amount of upstream components) in a patient sample appears to be greater that that found in a known non-cancerous sample, then the subject may be at a risk for cancer.
  • the ratio in a positive patient sample are at least 1.5 times those found in a known non-cancerous sample. More preferably they are at least 2.0 times as large and most preferably they are 5.0 times as large.
  • the patient sample includes any biological sample as described above in the definitions section.
  • the sample is a biopsy or blood, or saliva.
  • the biological sample may be collected by any of the multiple means known to one of skilled in the art, few of such collection methods are discussed above.
  • the upstream component(s) is/are preferably selected from p-Akt, Akt, and PDK and the downstream component(s) is/are preferably selected from p-S6K, 4E-BP and downstream markers of protein translation activity, such as Eif4 family members, Mnkl or Mnk2 .
  • a method of assaying for compounds that treat cancer in a subject comprising the steps of contacting a cell with one or more candidate compounds, and monitor the effect of said one or more candidate compounds on Fbxw7 gene or protein expression; thereby indentifying said one or more candidate compounds that alters mTOR expression or activity.
  • test molecules may be chosen from a compound libraries or from known anticancer agents. Combinations may also be used in the above disclosed screening method.
  • typical mTOR inhibitors can be selected preferably from rapamycin, deforolimus, sirolimus, temsirolimus, everolimus, polymorphs, prodrugs and derivatives thereof.
  • mTOR inhibitors could be as discussed in US Patent Publication no. 2007/0167478.
  • Suitable derivatives of rapamycin could also be as discussed in WO 94/09010, WO 95/16691, WO 96/41807, U.S. Pat. No. 5,362,718 or WO 99/15530. All of the documents listed above are incorporated herein by reference.
  • the subject may receive additional treatments if such a treatments are deemed necessary. Additional treatments may involve the use of surgery, anti-cancer agents, radiation, hormonal therapy or immunotherapy.
  • anti -cancer agents acceptable for use in the present invention include, without limitation, alkylating agents, anti-metabolites, plant alkaloids and terpenoids, topoisomerase inhibitors, antineoplastics, hormone therapeutics, photosensitizers, kinase inhibitors, etc.
  • alkylating agents include, without limitation, cisplatin, caroplatin, oxaliplatin, mechlorethamine, cyclophophamide, chlorambucil, busulfan, hexamethylmelamine, thiotepa, cyclophohphamine, uramustine, melphalan, ifosfamide, carmustine, streptozocin, dacarbazine, temozolomide, etc.
  • anti-metabolite agents include, without limitation, Aminopterin, Methotrexate, Pemetrexed, Raltitrexed, Cladribine, Clofarabine, Fludarabine, Mercaptopurine, Pentostatin, Thioguanine, Capecitabine, Cytarabine, Decitabine, Fluorouracil, Floxuridine, Gemcitabine, etc.
  • plant alkaloids and terpenoids include, without limitation, Docetaxel, Larotaxel, Paclitaxel, Vinblastine, Vincristine, Vindesine, Vinorelbine, etc.
  • topoisomerase inhibitors include, without limitation, Camptothecin, Topotecan, Irinotecan, Rubitecan, Etoposide, Teniposide, etc.
  • antineoplastics include, without limitation, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Pixantrone, Valrubicin, Actinomycin, Bleomycin, Mitomycin, Plicamycin, etc.
  • Examples of photosensitizer agents include, without limitation, Aminolevulinic acid, Methyl aminolevulinate, Porfimer sodium, Verteporfin, etc.
  • Examples of kinase inhibitors include, without limitation, Axitinib, Bosutinib, Cediranib, Dasatinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, Lestaurtinib, Nilotinib, Semaxanib, Sorafenib, Sunitinib, Vandetanib, Seliciclib, etc.
  • anti-cancer agents include, without limitation, Alitretinoin, Tretinoin, Aflibercept, Altretamine, Amsacrine, Anagrelide, Arsenic trioxide, Pegaspargase, Bexarotene, Bortezomib, Celecoxib, Denileukin diftitox, Elesclomol, Estramustine, Irofulven, Ixabepilone, Masoprocol, Mitotane, Oblimersen, Testolactone, Tipifarnib, Trabectedin.
  • determining Fbxw7 gene mutation can be performed in multiple methods, preferred methods could involve methods such as analysis of the patient DNA, RNA and protein.
  • RNA and protein can be carried out by various means.
  • Preferred means include PCR (e.g., Taqman), sequencing techniques, Southern, western, and northern blots, microarrays, immunohistochemical techniques, ELISA, mass spectroscopy, enzymatic, binding or functional assays.
  • Preferred methods for the detection and quantification of mTOR, upstream and downstream proteins include western blot, immunohistochemical techniques, ELISA and mass spectroscopy.
  • PCR assays such as Taqman allelic discrimination assay, available from Applied Biotech.
  • Biosystems can be used to identify RNA.
  • mass spectroscopy can be used to detect either nucleic acid or protein.
  • Any antibody-based technique for determining a level of expression of a protein of interest can be used.
  • immunoassays such as ELISA, Western blotting, flow cytometry, immunofluorescence, and immunohistochemistry can be used to detect protein in patient samples. Combinations of the above methods, such as
  • Analysis of a protein or nucleic acid can be achieved, for example, by high pressure liquid chromatography (HPLC), alone or in combination with mass spectrometry (e.g., MALDI/MS, MALDI-TOF/MS, tandem MS, etc.).
  • HPLC high pressure liquid chromatography
  • mass spectrometry e.g., MALDI/MS, MALDI-TOF/MS, tandem MS, etc.
  • PCR amplification techniques are described in, e.g., Ausubel et al and Innis et al , supra.
  • General nucleic acid hybridization methods are described in Anderson, "Nucleic Acid Hybridization," BIOS Scientific Publishers, 1999.
  • Amplification or hybridization of a plurality of nucleic acid sequences can also be performed from mRNA or cDNA sequences arranged in a microarray.
  • Microarray methods are generally described in Hardiman, “Microarrays Methods and Applications: Nuts & Bolts,” DNA Press, 2003; and Baldi et al, “DNA Microarrays and Gene Expression: From Experiments to Data Analysis and Modeling,” Cambridge University Press, 2002.
  • sequence analysis can be performed using techniques known in the art including, without limitation, sequence analysis, and electrophoretic analysis.
  • sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al, Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al, Methods MoI Cell Biol, 3:39-42 (1992)), single base extension sequencing (SBE), pyrosequencing (Ronaghi et al, Science, 281(5375):363-365 (1998)), sequencing with mass spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al, Nat.
  • MALDI-TOF/MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • Non-limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis.
  • antibody reagents can be used in assays to detect expression levels of protein biomarkers of the invention in patient samples using any of a number of immunoassays known to those skilled in the art. Immunoassay techniques and protocols are generally described in Price and Newman, “Principles and Practice of Immunoassay,” 2nd Edition, Grove's Dictionaries, 1997; and Gosling, "Immunoassays: A Practical Approach,” Oxford University Press, 2000. A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used (see, e.g., Self et al., Curr. Opin. BiotechnoL, 7:60-65 (1996)).
  • immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated.
  • EIA enzyme multiplied immunoassay technique
  • ELISA enzyme-linked immunosorbent assay
  • MAC ELISA IgM antibody capture ELISA
  • MEIA microparticle enzyme immunoassay
  • CEIA capillary electrophoresis immunoassays
  • RIA radioimmunoassays
  • IRMA immuno
  • Immunoassays can also be used in conjunction with laser induced fluorescence (see, e.g., Schmalzing et al., Electrophoresis, 18:2184-93 (1997); Bao, J. Chromatogr. B. Biomed. ScL, 699:463-80 (1997)).
  • Liposome immunoassays such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention (see, e.g., Rongen et al., J. Immunol. Methods, 204:105-133 (1997)).
  • nephelometry assays in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the methods of the present invention.
  • Nephelometry assays are commercially available from Beckman Coulter (Brea, CA; Kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biochem., 27:261-276 (1989)).
  • Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody.
  • An antibody labeled with iodine- 125 is an antibody labeled with iodine- 125.
  • a chemiluminescence assay using a chemiluminescent antibody specific for the protein marker is suitable for sensitive, non-radioactive detection of protein levels.
  • An antibody labeled with fluorochrome is also suitable.
  • fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R- phycoerythrin, rhodamine, Texas red, and lissamine.
  • Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), y- galactosidase, urease, and the like.
  • a horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
  • TMB chromogenic substrate tetramethylbenzidine
  • An alkaline phosphatase detection system can be used with the chromogenic substrate p- nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm.
  • a ⁇ -galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl- ⁇ -D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm.
  • An urease detection system can be used with a substrate such as urea- bromocresol purple (Sigma Immunochemicals; St. Louis, MO).
  • a signal from a direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength.
  • a quantitative analysis can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer's instructions.
  • the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
  • the antibodies can be immobilized onto a variety of solid supports, such as polystyrene beads, magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), pieces of a solid substrate material or membrane (e.g., plastic, nylon, paper), and the like.
  • An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
  • Immunohistochemical (IHC) methods which are well known by those skilled in the art, may be used . See, for example, U.S. Pat. No. 6,441,143 to Koski et al., U.S. Pat. No. 6,376,201 to Miron et al., U.S. Pat. No. 5,876,712 to Cheever et al., U.S. Pat. No. 5,854,009 to Klug, and U.S. Pat. No. 5,843,684 to Levine et al., U.S. Pat. No. 4,968,603 to Slamon et al.
  • DAKO anti-Her2 IHC System for Immunoenzymatic Staining Package Insert
  • direct and indirect assays According to the first assay, binding of an antibody to the target antigen is determined directly.
  • This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction.
  • the fluorescent tag or label can be fluorescein.
  • the enzymatic label can be horseradish peroxidase or alkaline phosphatase.
  • unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody.
  • a labeled secondary antibody binds to the primary antibody.
  • a chromagenic or fluorogenic substrate can be added to provide visualization of the antigen.
  • Signal amplification may occur because several secondary antibodies may react with different epitopes on the primary antibody.
  • the primary and/or secondary antibody used for immunohistochemistry typically can be labeled with a detectable moiety. IHC techniques are further described in Immunohistochemical Staining Methods. Thomas Boenisch, ed. (3rd ed. 2001).
  • Useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different biomarkers.
  • Such formats include protein microarrays, or "protein chips” (see, e.g., Ng et al., J. Cell MoI. Med., 6:329- 340 (2002)) and certain capillary devices (see, e.g., U.S. Pat. No. 6,019,944).
  • each discrete surface location may comprise antibodies to immobilize one or more protein markers for detection at each location.
  • Surfaces may alternatively comprise one or more discrete particles (e.g., microparticles or nanoparticles) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one or more protein markers for detection.
  • Analysis of the level of a biomarker can be carried out in a variety of physical formats. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate diagnosis or prognosis in a timely fashion.
  • the antibodies or nucleic acid probes of the invention can be applied to sections of patient biopsies immobilized on microscope slides.
  • the resulting antibody staining or in situ hybridization pattern can be visualized using any one of a variety of light or fluorescent microscopic methods known in the art.
  • the Fbxw7 gene has been identified as a p53-de ⁇ endent tumor suppressor gene that undergoes deletion and/or mutation in tumors from a wide range of human tissues, including breast, colon, endometrium, stomach, lung, ovary, pancreas, and prostate (Welcker M. et al., Nat Rev Cancer. 8:83-93 (2008); and Akhoondi S. et al, Cancer Res. 67:9006-12 (2007)).
  • Point mutations in FBXW7 were reported to be the most common genetic lesions in human T-cell acute lymphoblastic leukemia (T-ALL), indicating a wide role for this protein in suppression of most forms of human malignancies.
  • T-ALL T-cell acute lymphoblastic leukemia
  • the overall point mutation frequency of FBXW7 is about 6% (Akhoondi S. et al., Cancer Res. 67:9006-12 (2007)), but since Fbxw7 is a haplo-insufficient tumor suppressor (Mao J. H. et al., Nature 432:775-9 (2004)) and deletions at 4q are relatively frequent in human cancers (Mitelman F. et al., Mitelman Database of Chromosome Aberrations in Cancer (2007)), the impact of loss of FBXW7 in human cancers may have been underestimated.
  • FBXW7 encodes an F-box protein essential for ubiquitination of several well-defined oncoproteins including Myc, Cyclin E, c-Jun, Notch and Aurora-A (Welcker M et al., Nat Rev Cancer. 8:83-93 (2008)). While loss 0 ⁇ FBXW7 has been associated with increased genetic instability through deregulation of Cyclin E (Rajagopalan H. et al., Nature 428: 77-81 (2004)), c-Myc (Yada M. et al., EMBO J. 23: 2116- 25 (2004); and Welcker M.
  • mTOR is a central component of several complex signaling networks that regulate cell growth, metabolism and proliferation. Deregulation of mTOR is emerging as common theme in diverse human conditions including cancer, cardiovascular disease and ageing, and several drugs targeting this pathway are presently in clinical trials. The possibility that Fbxw7 may directly regulate levels of mTor was tested by analyzing two independent preparations of mouse embryonic fibroblasts (MEFs) from Fbxw7+/- mice ( Figure 3A).
  • MEFs mouse embryonic fibroblasts
  • a fragment of mTOR (from 1 to 898 AAs) that contains the putative CPD was cloned, two mutants were generated, one carrying a deletion of T631 [mTOR(delT631)], and the other a point mutation converting T631 to G [mTOR(T63 IG)] (all with Flag tags) (Figure 4B).
  • the wild type and mutant versions of this mTOR fragment were co-expressed with HA-FBXW7 in 293T cells, followed by immunoprecipitation with antibodies against one protein and Western blot analysis of the second.
  • MCF7 and SUM149PT cells were treated with the proteasome inhibitor MG-132.
  • MCF7 cells are widely used breast epithelial cells that express a wild type FBXWl gene, while SUM 149PT cells were derived from a breast carcinoma and have undergone homozygous mutation of FBXWl (Strohmaier H. et al., Nature. 413, 316-22 (2001) and Table 1).
  • RNA expression levels for FBXW7 and PTEN in 25 breast cancer cell lines were analyzed and sequence analysis of the complete coding regions of both genes in these cell lines was also carried out. In all cases, the FBXW7 gene continued to be expressed (Figure 6), indicating that no gene silencing had occurred, although very low levels were found in 5 cells lines (lanes 10, 13, 14, 16, 20; Figure 6). All of these lines had lost one copy of the FBXW7 gene except one (SUM 149PT, lane 16), in which a point mutation was detected (table 1).
  • the PTEN gene was found to be silent in two cell lines ( Figure 6, lane 11 and 12), and both had lost one copy of the PTEN gene. Three mutations in PTEN were found (in cell lines MDA-MB-415, BT549 and MDA-MB-468, lanes 15, 17 and 24, Figure 6, and table 1). All of these data, taken together, suggest that gene silencing (for example by promoter methylation) or point mutations in FBXW7 and PTEN axe relatively rare mechanisms of inactivation of these genes, in comparison to single copy deletions. These data are further compatible with the identification of both genes as haplo-insufficient tumor suppressors (Mao J. H., Nature 432:775-9 (2004); and Di Cristofano A. et al., Nat Genet. 19:348-55 (1998)) that generally do not require complete inactivation of both gene copies.
  • Rapamycin treatment On the basis of the observation that deletion or mutation of FBXW7 in human breast cancer cells leads to increased levels of mTOR, the possibility that cells harboring these deletions may show increased sensitivity to the mTOR inhibitor rapamycin was tested.
  • Two breast cancer cell lines, SUM 149PT cells (homozygous FBXWl mutations) and MDA-MB453 cells (wild type FBXW7) were treated with rapamycin and counted numbers of viable cells using the CellTiter-Glo Luminescent assay. SUM 149PT cells proved to be very sensitive to this treatment (IC 50 ⁇ 200 nM), whereas MDA-MB453 cells were relatively resistant (IC 50 > 2 ⁇ M).
  • FBXW7 is a haploinsufficient tumor suppressor that undergoes heterozygous loss in a substantial proportion of human tumours
  • the data suggest new approaches to reduce mTOR levels in cancers by the use of drugs that may re-activate the remaining copy of Fbxw7 in a similar way that nutlins have been shown to activate wild type copies of p53 in human tumors (Buolamwini J. K. et al., Curr Cancer Drug Targets. 5:57-68 (2005)).
  • Loss of FBXW7 may also be a useful biomarker for sensitivity of human tumors to inhibitors of the mTOR pathway.
  • Total protein extracts were prepared from MEFs, cancer cells, and tissues with RIPA lysis buffer.
  • 50 ⁇ g of protein extracts per lane were electrophoresed, transferred to PVDF membranes (Millipore), and immunoblotted with AKT (Cell Signaling Technology), p-AKT (Cell Signaling Technology), mTOR (Cell Signaling Technology), p- mTOR (Cell Signaling Technology), p-S6 (Cell Signaling Technology), and HA (Covance) antibodies; as control, the same membranes were stripped and immunoblotted again with anti- ⁇ -actin antibody (AC-15, Sigma).
  • AKT Cell Signaling Technology
  • p-AKT Cell Signaling Technology
  • mTOR Cell Signaling Technology
  • p-S6 Cell Signaling Technology
  • HA Covance
  • the membranes were washed and treated with rat anti- species IgG ⁇ -chain secondary antibody conjugated to horseradish peroxidase (Amersham Pharmacia).
  • the antigen -antibody reactions were visualized by using an enhanced chemiluminesence assay ECL (Amersham Pharmacia) and exposed to enhanced chemiluminesence film.
  • 293T cells were transiently transfected with pcDNA3.1 expressing HA tagged FBXW7 and FBXW7- ⁇ F respectively using Fugene (Roche).
  • the cells were harvested 24 hours after the transfection and lysed in lysis buffer (50 mM Hepes pH 7.5, 250 mM NaCl, 0.5% NP40 or 0.25% dodecyl beta D maltoside in Ix PBS with protease inhibitors) incubated on ice for 60 min and centrifuged at 13,000g for 15 min. The supernatant was collected and protein concentration was estimated with the Pierce BCA assay reagent.
  • lysis buffer 50 mM Hepes pH 7.5, 250 mM NaCl, 0.5% NP40 or 0.25% dodecyl beta D maltoside in Ix PBS with protease inhibitors
  • Probes are FAM probes designed specifically for TaqMan (Applied Biosystems). All primer sets were used to perform amplifications in triplicate on the ABI 7700 instrument (Applied Biosystems, Foster City, CA, USA). Reactions (50 ⁇ l) were performed in 1 xTaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 1.6 nM primer, 0.4 nM probe, 12.5 ng DNA. Cycling parameters were as follows: 95°C for 12 min ⁇ l cycle, (95°C for 20 s, 60°C for 20 s, 72°C for 60 s) ⁇ 40 cycles.
  • Copy number was determined from the PCR cycle number (CT) at which DNAs reach a threshold amount of fluorescence above background.
  • CT PCR cycle number
  • amplification at a reference locus is performed once per plate in triplicate for each individual DNA.
  • the CT values for each set of triplicates are averaged.
  • ⁇ Ct values are determined for locus in tumor samples and a set of six normal genomic DNAs.
  • ⁇ CT- ⁇ Ct tumor-Average ⁇ CT (normal). If ⁇ CT>0.5, it means gain; If ⁇ CT ⁇ - 0.5, it means loss.
  • Cell number was measured using the CellTiter-Glo Luminescent assay (Promega) according to the manufacturer's instructions, and luminescence was recorded with a luminometer (BioTek FLx800, BioTek Instruments, Inc.). The cells are seeded in 96 well plates and exposed to mTOR inhibitor rapamycin (100 nM to 2 ⁇ M) for 72 hours. The number of cells used per experiment is determined empirically. Each cell line and drug concentration will be set up in 6 replicate wells and repeated at least 3 times. shRNA constructs and retrovirus production
  • a shRNA with high FBXW7 knockdown efficiency was cloned into pSuper retrovirus cassettes (OligoEngine).
  • the shRNA sequences for FBXW7 are
  • the breast cancer cell lines (MDA-MB453, LY2 and MCF7) were infected with high-titre retroviral stocks produced by the transient transfection of 293T ecotropic Phonix cells. After infection with the pSuper retrovirus allowing the expression of shRNA molecules, cells were selected with l-2 ⁇ g /ml of puromycin in the culture medium. The control of the experiment was pSuper vector without RNAi.
  • the fragment of human mTOR was amplified from human cDNA by PCR using the primers (CCCAAGCTTGAACCTCAGGGCAAGATGCT) and
  • the fragment was digested with HindIII and Xbal, and cloned into the Hindlll-Xbal site of the p3XFLAG-CMV-10 expression vector.
  • the two mutants (delT631 and T631G) were generated by in vitro site- directed mutagenesis using QuickChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, USA). The primers for delT631 mutagenesis are
  • TCCCGCCTGCTCCCCTCCATCCAC and GTGGATGGAGGGGAGCAGGCGGGA for T631G
  • CTCCCGCCTGCTCGGACCCTCCATCCAC and GTGGATGGAGGGTCCGAGCAGGCGGGAG All constructs were verified by sequencing.
  • REFERENCE 1 bases 1 to 3896
  • REFERENCE 2 bases 1 to 3896
  • AUTHORS Grim J. E., Gustafson, M. P. , Hirata,R.K., Hagar,A.C, Swanger,J., Welcker,M., Hwang, H. C, Ericsson, J., Russell, D. W. and
  • TITLE Isoform- and cell cycle-dependent substrate degradation by the Fbw7 ubiguitin ligase
  • E-CDK2 -specific activity during the cell cycle regulate the timing of cyclin E degradation.
  • REFERENCE 3 (bases 1 to 3896) AUTHORS Lee , J . W . , Jeong , E . G . , Lee , S . H . , Yoo , N . J . and Lee , S . H . TITLE hCDC4 gene mutation is rare in colorectal carcinomas in Korean patients
  • PUBMED 17992009 REMARK GeneRIF There were no mutations in exons 8 or 9 in 160 acute leukemia samples from Korea, in contrast to earlier reports suggesting a role in T-ALL.
  • REFERENCE 6 bases 1 to 3896
  • TITLE A family of mammalian F-box proteins
  • This gene encodes a member of the F-box protein family which is characterized by an approximately 40 amino acid motif, the
  • the F-box proteins constitute one of the four subunits of ubiquitin protein ligase complex called SCFs (SKPl-cullin-F- box) , which function in phosphorylation-dependent ubiquitination.
  • SCFs ubiquitin protein ligase complex
  • the F-box proteins are divided into 3 classes: Fbws containing WD-
  • FbIs containing leucine-rich repeats and Fbxs containing either different protein-protein interaction modules or no recognizable motifs.
  • the protein encoded by this gene was previously referred to as FBX30, and belongs to the Fbws class; in addition to an F-box, this protein contains 7 tandem WD40 repeats. This protein binds directly to cyclin E and probably targets cyclin E for ubiguitin-mediated degradation. Mutations in this gene are detected in ovarian and breast cancer cell lines, implicating the gene ' s potential role in the pathogenesis of human cancers .
  • Transcript Variant This variant (1) represents the longest transcript and encodes the longest isoform (1) .
  • CDS 150..2273 /gene " FBXW7"
  • transcript variant 1 is encoded by transcript variant 1; F-box protein SEL-10; homolog of C elegans sel-10; archipelago,
  • Drosophila homolog of; F-box protein FBW7; F-box and

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Abstract

La présente invention concerne des méthodes permettant de déterminer si un patient atteint d’un cancer présente ou non un gène Fbxw7 muté (mutations ponctuelles, polymorphismes, délétions ou additions, y compris absence du gène par délétion complète et silençage du promoteur) et ainsi de déterminer si le patient est éligible ou non à un traitement par inhibiteur du mTOR. La présente invention porte aussi sur une méthode de détermination du risque de cancer par comparaison des quantités relatives des constituants en amont et en aval du mTOR dans la voie de signalisation du mTOR.
PCT/US2009/056641 2008-09-11 2009-09-11 Perte de fbxw7, un biomarqueur de la sensibilité au traitement de tumeurs par des inhibiteurs du mtor Ceased WO2010030865A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012119091A1 (fr) * 2011-03-02 2012-09-07 Beth Israel Deaconess Medical Center, Inc. Mcl-1 en tant que cible thérapeutique dans un néoplasme carencé en scffbw7
WO2012130720A3 (fr) * 2011-03-28 2012-12-20 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh Prédiction de la sensibilité vis-à-vis d'inhibiteurs de pi3k/mtor
WO2013130093A1 (fr) 2012-03-02 2013-09-06 Genentech, Inc. Biomarqueurs pour un traitement à base de composés chimiothérapeutiques anti-tubuline
CN105112515A (zh) * 2015-08-14 2015-12-02 深圳市众偱精准医学研究院 Fbxw7基因及表达产物在肾癌检测、肾癌药物制备中的用途
WO2016104777A1 (fr) * 2014-12-26 2016-06-30 国立大学法人九州大学 Procédé de traitement d'un cancer
WO2021108927A1 (fr) * 2019-12-06 2021-06-10 The Governing Council Of The University Of Toronto Procédés et compositions pour traiter des cancers ayant des altérations dans la protéine f-box et à répétition wd (fbxw7) et/ou un gain ou une amplification de la cycline l1 (ccnl1)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE527385T1 (de) * 2006-11-16 2011-10-15 Genentech Inc Mit tumoren assoziierte genetische variationen

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012119091A1 (fr) * 2011-03-02 2012-09-07 Beth Israel Deaconess Medical Center, Inc. Mcl-1 en tant que cible thérapeutique dans un néoplasme carencé en scffbw7
US10006089B2 (en) 2011-03-02 2018-06-26 Beth Israel Deaconess Medical Center, Inc. MCL-1 as a therapeutic target in SCFFBW7 deficient neoplasm
WO2012130720A3 (fr) * 2011-03-28 2012-12-20 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh Prédiction de la sensibilité vis-à-vis d'inhibiteurs de pi3k/mtor
WO2013130093A1 (fr) 2012-03-02 2013-09-06 Genentech, Inc. Biomarqueurs pour un traitement à base de composés chimiothérapeutiques anti-tubuline
WO2016104777A1 (fr) * 2014-12-26 2016-06-30 国立大学法人九州大学 Procédé de traitement d'un cancer
CN105112515A (zh) * 2015-08-14 2015-12-02 深圳市众偱精准医学研究院 Fbxw7基因及表达产物在肾癌检测、肾癌药物制备中的用途
WO2021108927A1 (fr) * 2019-12-06 2021-06-10 The Governing Council Of The University Of Toronto Procédés et compositions pour traiter des cancers ayant des altérations dans la protéine f-box et à répétition wd (fbxw7) et/ou un gain ou une amplification de la cycline l1 (ccnl1)

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