WO2011070148A1 - Diagnostic et thérapie différentiels pour inhibiteurs de kinase - Google Patents
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- WO2011070148A1 WO2011070148A1 PCT/EP2010/069367 EP2010069367W WO2011070148A1 WO 2011070148 A1 WO2011070148 A1 WO 2011070148A1 EP 2010069367 W EP2010069367 W EP 2010069367W WO 2011070148 A1 WO2011070148 A1 WO 2011070148A1
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- ros1
- nek9
- nme4
- tbk1
- bmp2k
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- the present invention relates to novel methods and compositions for prognosing the effectiveness of kinase inhibitors such as sunitinib in the therapy of hyperproliferative and particularly metastatic diseases.
- the invention allows the development of individually adapted strategies for treating conditions associated with and/or caused by the occurrence of a given type of hyperproliferating and/or metastatic cells.
- hyperproliferative diseases A major problem in the treatment of hyperproliferative diseases is the large variety of diseases and conditions resulting from or being caused by the occurrence of hyperproliferating cells in a multicellular organism.
- the variety of hyperproliferating diseases both results from the multiplicity of various molecular causes and trigger events, and from the multiplicity of different cell types existing in an organism such as a mammal.
- a specific diagnosed hyperproliferative disease such as a specific cancer disease having a specific molecular profile, consequently not any drug and not any method having antiproliferative effects can be used.
- hyperproliferative diseases require on the one hand the development of new medicaments having specific antiproliferative effects and differential methods of treatment.
- an attending physician has to decide or predict which of the available drugs and methods of treatment may be successfully applied for the treatment or prevention of a specific diagnosed hyperproliferative disease.
- agents are used for their therapy or prevention, which modulate mechanisms being equally changed in a lot of hyperproliferative cells and thus are more or less effective in the treatment of various hyperproliferative diseases or conditions.
- an approach is not suitable for treating of a subject suffering from a specific kind of a hyperproliferative disease.
- Protein kinases e.g tyrosine kinases
- tyrosine kinases are part of intracellular signal cascades which regulate cell division, differentiation and/or apoptosis and thus are involved directly or indirectly in the control of cell proliferation.
- cell receptor associated tyrosine kinases are involved in the transmission of growth signals from receptors on the cell surface to the nucleus, where the cell division is started.
- These signal cascades are often disturbed in hyperproliferating cells such as cancer cells, for example due to mutations in the sequence of tyrosine kinase encoding genes involved in the cell cycle regulation.
- hyperproliferating cells may exhibit a decreased or increased level of one or more tyrosine kinases. Therefore, genes encoding tyrosine kinases which are involved in the intracellular transmission of growth signals and the expression of which is altered in hyperproliferating cells, belong to the group of proto-oncogenes.
- ROS1 V-ros UR2 sarcoma virus oncogene homolog 1
- ROS1 belongs to the "Sevenless" subfamily of tyrosine kinase insulin receptor genes.
- the protein encoded by this gene is a type 1 integral membrane protein with tyrosine kinase activity. Studies have shown that ROS1 protein may function as a growth or differentiation factor receptor.
- Sunitinib also known as SU11248 is an indolin-2-one compound, which is the active ingredient of the therapeutic agent Sutent ® is an orally administered, multi-targeted receptor tyrosine kinase inhibitor for the treatment of renal cell carcinoma (RCC) and imatinib-resistant gastrointestinal stromal tumor (GIST).
- RRC renal cell carcinoma
- GIST imatinib-resistant gastrointestinal stromal tumor
- Sunitinib was the first cancer drug simultaneously approved for two different indications and has become a standard of care for both of these cancers.
- Administration of SU11248 inhibits intracellular signal transmission of multiple receptor tyrosine kinases leading to both reduced tumor vascularization and cancer cell death, and ultimately tumor shrinkage. In some cases, however, administration of SU11248 and related compounds has been less effective. The reason for this observation was not known.
- WO 2007/109571 discloses methods of predicting and monitoring tyrosine kinase inhibitor therapy.
- Response to Sunitinib-therapy is predicted by genotyping a polymorphic site in c-Kit, PDGFR and/or VEGFR, and additionally measuring VEGFR and/or PDGFR expression.
- Subjects negative for these biomarkers are genotyped at a polymorphic site in K-Ras to determine the presence or absence of a K-Ras activating mutation.
- a protein expression profile can be determined by PTEN expression and a protein activation profile can be determined by assessing Erk (MAPK) activation and Akt activation. From these measurements a cumulative index value is determined, which is used to predict Sunitinib-sensitivity or an increased likelihood of responding to Sunitinib.
- MAPK Erk
- WO 2008/138578 discloses methods, kits and devices for identifying biomarkers of treatment response and use thereof to predict treatment efficacy. Biomarkers for treatment with Sunitinib are not disclosed.
- the present inventors have identified specific polypeptides as novel targets of anti-cancer drugs, particularly of kinase inhibitor such as indolin-2-one compounds.
- the presence of these polypeptides, particularly the presence of an increased amount and/or activity of these polypeptides is an indicator that a therapy comprising administration of said anti-cancer drugs is effective. It was shown for the first time that these novel targets are expressed differently in individual cancer cell lines. In other words, an increased or decreased expression level of at least one of the novel targets in a type of cancer cells as compared to the expression level in a non- hyperproliferating wild-type or normal cells, e.g.
- these polypeptides or nucleic acids encoding said polypeptides represent excellent prognostic markers if a drug, particularly a kinase inhibitor as defined herein, for example sunitinib, is effective in the treatment of a subject suffering from a hyperproliferative disease.
- the present invention relates to a method of determining if a subject suffering or suspected to suffer from a hyperproliferative disorder is susceptible to treatment with a kinase inhibitor, particularly Sunitinib, comprising measuring the expression level and/or activity of (i) a ROS1 , BMP2k, NEK9, NME4 and/or TBK1 polypeptide and/or (ii) a ROS1 , BMP2k, NEK9, NME4 and/or TBK1 nucleic acid in a sample comprising diseased cells from said subject, wherein an increased ROS1 , BMP2k, NEK9, NME4 and/or TBK1 expression level and/or activity is indicative for susceptibility to said treatment.
- a kinase inhibitor particularly Sunitinib
- the present invention relates to a diagnostic composition or kit comprising a detection reagent for (i) a ROS1 , BMP2k, NEK9, NME4 and/or TBK1 polypeptide or (ii) a ROS1 , BMP2k, NEK9, NME4 and/or TBK1 nucleic acid for stratifying of subjects suffering or suspected to suffer from a hyperproliferative disorder with regard to their susceptibility to treatment with a kinase inhibitor, particularly Sunitinib.
- the present invention relates to a method of treating a subject suffering or suspected to suffer from a hyperproliferative disorder with a kinase inhibitor, particularly Sunitinib, comprising measuring the expression level and/or activity of (i) a ROS1 , BMP2k, NEK9, NME4 and/or TBK1 polypeptide and/or (ii) a ROS1 , BMP2k, NME4 and/or TBK1 NEK9 nucleic acid in a sample comprising diseased cells from said subject, wherein an increased ROS1 , BMP2k, NEK9, NME4 and/or TBK1 expression level and/or activity is indicative for susceptibility to said treatment, selecting a susceptible subject, and administering to said subject a pharmaceutically effective amount of said kinase inhibitor, particularly Sunitinib.
- a kinase inhibitor particularly Sunitinib
- the present invention relates to a method of treating a subject suffering from a hyperproliferative disorder associated with, accompanied by or caused by an increased expression level of ROS1 , BMP2k, NEK9, NME4 and/or TBK1 , comprising administering to said subject a pharmaceutically effective amount of a kinase inhibitor.
- the target polypeptides of the invention are selected from ROS1 , particularly human ROS1 , as defined in SEQ ID NO:1 , BMP2k, particularly human BMP2k, as defined in SEQ ID NO:2 or 3, NEK9, particularly human NEK9, as defined in SEQ ID NO:4, NME4, particularly human NME4, as defined in SEQ ID NO:5 or TBK1 , particularly human TBK1 , as defined in SEQ ID NO:6, allelic variants and homologs thereof having at least 70-80%, preferably at least 80-90%, more preferably at least 90-95%, and most preferably at least 95-99.9% or more identity with the sequences of SEQ ID NO:1 , 2, 3, 4, 5 or 6 over the entire length, provided that said allelic variants and homologs exhibit the characteristics of a target polypeptide as defined above.
- the target polypeptide is ROS1 as defined in SEQ ID NO:1.
- the target nucleic acids encode a target polypeptide selected from ROS1 (SEQ ID NO:1), BMP2k (SEQ ID NO:2 or 3), NEK9 (SEQ ID NO:4), NME4 (SEQ ID NO:5) or TBK1 (SEQ ID NO:6) allelic variants and homologs thereof as defined above. More preferably, the target nucleic acid is a ROS1 nucleic acid having the sequence of SEQ ID NO:7.
- the target nucleic acid may be selected from a BMP2k nucleic acid having the sequence of SEQ ID NO:8 or 9 and a NEK9 nucleic acid having the sequence of SEQ ID NO:10, a NME4 nucleic acid having the sequence of SEQ ID NO:11 or a TBK1 nucleic acid having the sequence of SEQ ID NO:11.
- the target polypeptides or nucleic acids of the invention may be over- or under-expressed in a variety of hyperproliferating (e.g. tumor) cell types as compared to the corresponding non-hyperproliferating wild-type cells.
- hyperproliferating e.g. tumor
- over- or under-expression means a degree of polypeptide synthesis leading to an amount of said polypeptide within or at the surface of the hyperproliferating cell, which differs from that of the wild-type or normal cell by at least factor 1.5, preferably at least factor 2, more preferably by at least factor 5.
- the expression profile of the target structures according to the invention may be tumor-type specific.
- Many of the investigated cancer cell lines exhibit an over-expression of a target structure as defined above.
- ROS1 is highly expressed in NSCLC cell lines, glioblastoma, pancreas cancer and metastatic renal cancer carcinoma (mRCC), whereas ROS1 is rarely expressed in certain cancer cells of breast and colon tissue.
- the activity of the target polypeptide according to the invention may be increased or decreased in a variety of hyperproliferating (e.g. tumor) cell types as compared to that of the corresponding non-hyperproliferating wild-type cells.
- an increased or decreased enzymatic activity means an alteration in the activity by at least factor 1.5, preferably by at least factor 2, more preferably by at least factor 5.
- the targets as defined above can be used for the prognosis if a kinase inhibitor and in particular a tyrosine kinase inhibitor, would be antiproliferatively and/or pro-apoptotically effective and thus suitable for the treatment of a subject suffering from a diagnosed hyperproliferative disease as, for example, a cancer disease, in a given case.
- the invention comprises the step of detecting and/or measuring the expression and/or the activity level of at least one target molecule as defined above in a sample comprising diseased cells from said subject, wherein, e.g. as compared to a sample of non-diseased wildtype cells, an increased expression and/or activity level is indicative for susceptibility to a treatment with a kinase inhibitor.
- the subject may be an animal, and in particular a mammal.
- the subject is a human being.
- the subject is a cancer patient, e.g. a human cancer patient.
- the subject is a patient who has undergone surgery and/or radiation therapy.
- hyperproliferative disorder refers to conditions associated with, accompanied by or caused by the occurrence of cellular hyperproliferation, particularly associated with invasiveness, migration and/or metastasis formation. More particularly, a hyperproliferative disorder is associated with metastasis formation.
- a hyperproliferative disorder may be associated with an increased or decreased expression of at least one target molecule as described herein. Further, a hyperproliferative disorder may be associated with an increased or decreased activity, e.g. a kinase activity, particularly a tyrosine kinase activity of at least one target molecule as described herein.
- the hyperproliferative disorder may be cancer, e.g. an invasive, migratory and/or metastatic cancer or a non-metastatic cancer.
- the cancer is metastatic cancer.
- the cancer may be bone, brain, breast, cervix, colon, gastric, kidney, liver, lung, ovary, pancreas, prostate, skin cancer, and more preferably brain cancer, e.g. glioblastoma or lung cancer, e.g. non-small cell lung cancer (NSCLC), gastric cancer, e.g. GIST or renal cancer, e.g. RCC.
- NSCLC non-small cell lung cancer
- the kinase inhibitor is a tyrosine kinase inhibitor, e.g. an indolin-2-one compound, such as sunitinib.
- the expression level and/or activity of a target molecule as defined above may be determined by any appropriate method known to a person skilled in the art.
- the expression level and/or activity of a target molecule may be determined on protein level or on the nucleic acid level.
- a determination of the expression level and/or activity of a target molecule may comprise the steps of
- sample comprising diseased cells or tissue from a subject suffering or suspected to suffer from a hyperproliferative disease, wherein the sample may be a tissue sample or a body fluid sample,
- step (ii) contacting the cell sample of step (i) with a detection reagent for a target molecule as described above, and determining the expression level and/or activity of the target molecule, and
- control sample e.g. from a sample of non-diseased cells or tissue.
- the test sample may be a tissue sample, e.g. a biopsy or a tissue section, or a body fluid sample, e.g. blood, serum, plasma, urine, lymph fluid, cerebrospinal fluid, saliva etc.
- the sample may be subjected to an immunoassay, wherein the detection reagent is used which has the ability to specifically bind to the target polypeptide.
- the detection reagent may comprise an antibody, an antibody fragment or an aptamer directed against ROS1 , BMP2k, NEK9, NME4 or TBK1.
- Quantitative or semiquantitative immunoassays which can be used herein are known in the art.
- An example for an applicable test format is an enzyme linked immunosorbent assay (ELISA).
- the phosphorylation status of the target polypeptide may be determined using antibodies specifically directed against phosphorylated amino acid residues.
- a target polypeptide e.g. a tyrosine kinase
- catalytic/enzymatic activity of a target polypeptide may be determined by methods known in the art.
- the determination of the target molecule may also be carried out on nucleic acid level. Determining the expression level of a target nucleic acid can be, for example, carried out by a hybridization assay, optionally combined with an amplification step. Suitable hybridization/amplification protocols are known in the art, such as PCR-based protocols.
- the detection reagent may comprise a hybridization probe and optionally an amplification primer.
- the probe may be single- stranded DNA, RNA, PNA or a nucleic acid analogue having a length of at least 10, preferably at least 15 nucleotides, which is sufficiently complementary to at least one strand of the target nucleic acid that may specifically hybridize with the target nucleic acid under stringent conditions as described, for example, in Sambrook et al. (1989) Molecular Cloning. A laboratory manual, second edition, Cold Spring Harbor Laboratory Press.
- Stringent conditions may, e.g., comprise a washing step of 30 minutes in 0.1 x SSC, buffer, 0.5% SDS at 55°C, 62°C or 68°C.
- the reagent may comprise at least one hybridization probe directed to a nucleic acid encoding a ROS1 , NEK9, BMP2k, NME4 or TBK1 nucleic acid as described above, e.g. an mRNA or cDNA. Further, the reagent may comprise at least one amplification primer.
- the method according to the invention comprises measuring the expression level and/or activity of at least one target molecule.
- the method also comprises the measurement of the expression level and/or activity of two or more, preferably 2, 3, 4 or 5 target molecules.
- the method according to the invention may avoid application of kinase inhibitors to subjects which are not susceptible or substantially non-susceptible to treatment.
- the efficacy of cancer treatment may be increased and specific treatment protocols for individual patients may be provided.
- a diagnostic composition or kit For detecting or measuring the expression level of a target molecule selected from ROS1 , BMP2k, NEK9, NME4 and/or TBK1 polypeptides and/or nucleic acids, a diagnostic composition or kit may be used.
- a further aspect of the present invention relates to a diagnostic composition or kit comprising a detection reagent for detecting or measuring at least one target molecule as defined above.
- the detection reagent may comprise an agent which specifically binds to said target molecule under defined conditions.
- the agent may be an anti-ROS1 , anti-BMP2k, anti-NEK9, anti-NME4 or anti-TBK1 antibody, or an antigen binding fragment, or an aptamer.
- an agent may be a hybridization probe directed a ROS1 , BMP2k, NEK9, NME4 and/or TBK1 nucleic acid.
- the agent as described herein may be coupled with an appropriate labelling group, in order to facilitate detection. Examples for suitable labelling groups, such as radioactive groups, fluorescent groups or other labelling groups are known in the art.
- the diagnostic composition or kit according to the invention can be used for stratifying of subjects suffering or suspected to suffer from a hyperproliferative and/or metastatic disorder with regard to their susceptibility to a treatment with a kinase inhibitor.
- Another aspect of the present invention relates to a method of treating a subject suffering from a hyperproliferative and/or metastatic disease comprising determining if said subject is susceptible to a treatment with a kinase inhibitor, e.g. sunitinib.
- This method may comprise the steps of: measuring the expression level and/or the activity of at least one target molecule as defined above in a test sample comprising diseased cells or tissue from said subject, wherein, an increase in the expression level and/or activity is indicative for susceptibility to said treatment, selecting a susceptible subject, and
- a further aspect of the present invention relates to a method for treating a hyperproliferative and/or metastatic disease, comprising administering to a subject in need thereof, a therapeutically effective amount of a kinase inhibitor, wherein said disorder associated with, accompanied by or caused by cells showing an increased expression level of at least one target molecule selected from ROS1 , BMP2k, NEK9, NME4 and/or TBK1.
- the kinase inhibitor is preferably an indolin-2-one compound, and in particular sunitinib.
- the subject is preferably selected for treatment by being diagnosed for an increased target molecule expression level and/or activity before the treatment. In other embodiments, the subject may be monitored during and/or after the treatment with respect to the target molecule expression level and/or activity.
- the subject is an animal, particularly a mammal such as a human being.
- the hyperproliferative disorder may be cancer, e.g. a cancer as described above, particularly a cancer associated with metastatis formation.
- the treatment may comprise administration of a kinase inhibitor, e.g. an indolin-2-one compound, such as sunitinib alone or in combination with other medicaments, e.g. antiproliferative agents, i.e. cytostatic, cytotoxic, chemotherapeutic and/or immunotherapeutic agents, e.g. further kinase inhibitors selected from Lapatinib, Gefitinib, Eriotinib, and/or an anti-tumour antibody, such as Herceptin or Erbitux, and combinations thereof.
- a kinase inhibitor e.g. an indolin-2-one compound, such as sunitinib alone or in combination with other medicaments
- antiproliferative agents i.e. cytostatic, cytotoxic, chemotherapeutic and/or immunotherapeutic agents, e.g. further kinase inhibitors selected from Lapatinib, Gefitinib, Eriotinib, and/or an anti-t
- the kinase inhibitor may also be administered in combination with other types of therapy such as radiation and/or surgery.
- Figure 1 (A) is a schematic depiction of a method for the identification of target structures molecules in a cell.
- the compound SU11248 was coupled to Sepharose and contacted with a total cell lysate under in vitro association conditions. Cellular components bound to SU11248 were identified.
- Figure 1 (B) shows the identification of ROS1 as a target polypeptide for SU11248.
- Figure 2 shows the results of an ROS1 -expression profiling within different cancer cell lines.
- Figure 3 shows the effect of siRNA molecules directed against ROS1 , BMP2k, NEK9, NME4 and TBK1 mRNA on the viability (A), caspase activity (B1 ), apoptosis (B2), cell proliferation (C) and cell morphology (D) of different cancer cell lines.
- Figure 4 shows the effect of siRNA molecules directed against ROS-1 , BMP2k, NME4 and TBK1 mRNA on SU11248 treatment efficacy in different cancer cell lines.
- Figure 5 shows the nucleotide (A) and amino acid (B) sequences of ROS1 , BMP2k, NEK9, NME4 and TBK1 mRNAs or polypeptides.
- Inhibition Rate (%) [1-(ODtreated- ODtreated (day0)/ODcontrol- ODcontrol (day0))]x100%.
- the IC 50 i.e. the drug concentration that reduced the absorbance observed in untreated cells by 50% was calculated by using Hill three parameter log fit in SIGMA Plot 10.
- Inhibition Rate (%) [1- (ODtreated- ODtreated (day0)/ODcontrol- ODcontrol (day0))]x100%.
- the IC 50 i.e. the drug concentration that reduced the absorbance observed in untreated cells by 50% was calculated by using Hill threeparameter log fit in SIGMA Plot 10.0.
- the assay was performed as described in the manufacturer ' s protocol (Roche). Briefly, in a 96- well flat-bottomed plate, 1000- 2000 cells/100 ⁇ cell suspension was seeded. After 24 h, cells were exposed to different concentrations of compound. Each treatment was tested in triplicate wells. At the end of exposure (24 h, 48 h and 72 h) cells were incubated with BrdU for 6- 18 h at 37°C. The labeled cells were fixed with ethanol and prior to incubation with a monoclonal antibody to BrdU, the DNA was partially digested with nucleases to allow the antibody to access BrdU.
- the anti- BrdU antibody labeled with peroxidase (POD)
- POD peroxidase
- Transfected or compound- treated cells were trypsinized after 72h of siRNA transfection or drug application and collected by centrifugation. For fixation, cells were washed once with PBS, resuspended in 1ml cold 70% ethanol and stored overnight at 4°C. Cells were then collected by centrifugation, washed once with PBS and incubated with 0.01% Triton, 0.1 % sodium citrate, 0.02mM propidium iodide (Sigma) in the dark for 2h at 4°C. Cells were analyzed by flow cytometry (FACS Calibur, BD Bioscience). Using the CellQuestPro software, each of the three peaks (representing cells in Gi, S, and G 2 /M phases, respectively) obtained in the flow cytometry profile of fluorescence plotted against cell number was gated and quantified.
- Caspase 3/7 activity of siRNA transfected or compound- treated cells was measured using the Caspase 3/7 GIo- Assay from Promega according to manufacturer ' s instruction.
- Sunitinib maleate (SU11248) was purchased from ACC Corporation (San Diego, USA).
- SU11248 was chemically modified and kindly provided by VICHEM CHEMIE Ltd., Budapest, Hungary.
- Drained epoxy-activated Sepharose 6B was resuspended in 2 vol of 0.1 , 0.3, 1 or 3 mM Sunitinib dissolved in 50% DMSO/50% 0.05M Na 2 C0 3 (pH1 ). After adding of 10 mM NaOH, coupling was performed overnight at 30°C in the dark. After three washes with 50% DMSO/50% 0.05M Na 2 C0 3 (pH11), remaining reactive groups were blocked with 1 M ethanolamine. Subsequent washing steps were performed according to the manufacturer's instructions. To generate the control matrix, epoxy-activated Sepharose 6B was incubated with 1 M ethanol-amine and equally treated as described above. The beads were stored at 4°C in the dark as a suspension in 20% ethanol.
- Cells (80 mg protein) were lysed in 30 ml of buffer containing 20 mM Hepes (pH 7.5), 150 mM NaCI, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM DTT plus additives (10 mM sodium fluoride/1 mM orthovanadate/10 pg/ml aprotinin/10 pg/ml leupeptin/1 mM phenylmethylsulfonylfluoride/10% glycerol), cleared by centrifugation, and adjusted to 1 M NaCI.
- the filtrated lysate was loaded with a flow rate of 100 ⁇ /min on an HR 5/2 chromatography column (Amersham Biosciences) containing 600 ⁇ of SU 11248 matrix equilibrated to lysis buffer without additives containing 1 M NaCI.
- the column was washed with 15 column volumes and equilibrated to lysis buffer without additives containing 150 mM NaCI, and bound proteins were eluted in the same buffer containing 1 mM SU11248, 10 mM ATP, and 20 mM MgCI 2 with a flow rate of 50 ⁇ /min.
- the volume of protein-containing elution fractions was reduced to 1/10 in a Centrivap concentrator (Labonco, Kansas City, MO) before precipitation according to Wessel and Fluegge ((Wessel and Flugge, 1984)). Protein pellets were desolved in 1.5x SDS- sample buffer, pooled and subjected to 1 D- SDS- Gel electrophoresis.
- Protein pellets after Wessel-Fluegge precipitation were boiled in 1.5 x LDS- sample buffer with 0.5 mM DTT for 10 min at 70°C and subsequently separated by one-dimensional SDS-PAGE using NuPage Novex Bis- Tris gels and NuPage MOPS SDS running buffer (Invitrogen) according to the manufacturer's instruction.
- the gel was stained with Coomassie Blue using the colloidal blue staining kit (Invitrogen).
- Gel bands were cut into 1 mm 3 cubes and washed with 50 mM ammonium bicarbonate, 50 % ethanol.
- gel pieces were incubated with 10 mM DTT in 50 mM ammonium bicarbonate for 1 h at 56°C.
- Alkylation of cysteines was performed by incubating the samples with 55 mM iodoacetamide in 50 mM ammonium bicarbonate for 45 min at 25°C in the dark.
- Gel pieces were washed two times with 50 mM ammonium bicarbonate and incubated with 12.5 ng/ ⁇ Trypsin in 50 mM ammonium bicarbonate for 16h at 37°C for protein digestion.
- Supernatants were transferred to fresh tubes and remaining peptides were extracted by incubating the gel pieces two times in 30% acetonitrile in 3% TFA followed by dehydration with 100% acetonitrile.
- the extracts were combined and used for direct peptide identification by mass spectrometry after desalting the samples using RP-C18 stage tip columns.
- the peptide mixture was injected onto the column with a flow of 0.5 ⁇ /min and subsequently eluted with a 5-40 % acetonitrile gradient in 0.5 % acetic acid with a flow of 0.25 ⁇ /min.
- the gradient was 140 min.
- Mass spectra were acquired in the positive ion mode applying a data-dependent automatic switch between survey scan and tandem mass spectra (MS/MS) acquisition.
- the hybrid linear ion trap/orbitrap instrument was used with the lock mass option in both MS and MS/MS mode.
- the polydimethylcyclosiloxane (PCM) ions generated in the electrospray process from ambient air were used for internal mass recalibration in real time.
- Mass spectrometric data were analyzed with the in-house developed software MaxQuant (version 1.0.12.0 for the proteome and version 1.0.11.5 for the phosphoproteome analysis). MS/MS spectra were searched by Mascot (version 2.2.2, Matrix Science) against the Human IPIBase (version 3.37) combined with common contaminants and concatenated with the reversed versions of all sequences. The following parameters were set for the Mascot searches. Trypsin allowing for cleavage N-terminal to proline and cleavage between aspartic acid and proline was chosen as enzyme specificity.
- Cysteine carbamidomethylation eas selected as a fixed modification, while protein N-terminal acetylation, methionine oxidation and serine, threonine and tyrosine phosphorylation were selected as variable modifications. Maximally three missed cleavages and up to three labeled amino acids according to SILAC or non- SILAC study were allowed. Initial mass deviation of precursor ion and fragment ions were up to 5 ppm and 0.5 Da, respectively. MaxQuant automatically identified and quantified SI LAC peptides and proteins. SILAC protein ratios were calculated as the median of all peptide ratios assigned to the protein.
- a false discovery rate (FDR) of 0.01 was required for proteins and peptides with a minimum length of 6 amino acids. Furthermore, a posterior error probability (PEP) for each MS/MS spectrum below or equal to 0.01 was required. In case the identified peptides of two proteins were the same or the identified peptides of one protein included all peptides of another protein, these proteins (e.g. isoforms and homologs) were combined by MaxQuant and reported as one protein group.
- FDR false discovery rate
- PEP posterior error probability
- A498 kidney cancer cells were seeded at a density of 150.000 cells/well in 6- well flat-bottom cell culture dishes. 24h prior to SU 1248 treatment, cells were starved for 24h in medium containing 0% FCS. Drug incubation was performed for 2h, followed by pervanadate stimulation for 5min at 37°C. Cells were lysed and subjected to immunoprecipitation with an antiROSI antibody over night.
- RNAi transfection 15.000 cells/ml were seeded into 6-well cell culture plates. At 30% confluency cells were transfected with 30 pmol of validated or pre-designed siRNA from Ambion using RNAiMax (Invitrogen) according to the manufacturer's instruction. GI2 siRNA was taken as control. 5d after transfection cells were used for cell cycle analysis by flow cytometry, MTT-, SRB-, BrdU-assay and western blotting. The knock-down efficiency was monitored by RT-PCR and Western Blotting. 1.3.3 Western Blotting
- Membranes were washed three times with TBST and incubated with horseradish peroxidase-conjugated antimouse, antirabbit or antigoat secondary antibody in TBST + 4% nonfat dry milk for 1h at room temperature. Membranes were washed three times with TBST and visualized by ECL (Western Lightning, Perkin Elmer) on X-ray films.
- Binding partners of the indolin-2-one compound sunitinib were identified using affinity chromatography and mass spectrometry.
- SU11248 Binding partners of the indolin-2-one compound sunitinib
- For FPLC- affinity chromatography an SU11248 matrix by coupling SU11248 to ECH/epoxy-Sepharose was prepared. Whole cell lysate was incubated with the SU11248 Sepharose matrix under in vitro association conditions. Bound polypeptides were eluted and subjected to LC/MS analysis after in-gel trypsin digestion of PAGE-separated protein bands (Fig. 1A). By this method, the polypeptides ROS1 , BMP2k, NEK9, NME4 and TBK1 could be identified as binding partners of SU1 248 (cf. Fig. 1B for ROS1 ).
- Fig. 3A MTT colorimetric assay
- Fig. 3B1 caspase assay
- Fig. 3B2 FACS-PI assay
- Fig. 3C The effect of siRNA molecules specific for ROS1 , BMP2k and NEK9 mRNA on the proliferation of different cancer cell lines is shown in Fig. 3C (BrdU- Assay). In several cancer cell lines a significant decrease of cell proliferation was observed.
- siRNA specific for ROS1 mRNA, BMP2k mRNA, or NEK9 mRNA versus control (ctrl) siRNA is shown in Fig. 3D (microscopic picture).
- Administration of siRNA leads to morphological changes and a reduction in cell growth.
- Target depletion was achieved by transient RNAi experiments and the SU11248 efficacy was measured using a standard MTT colorimetric method. Cancer cells were either transfected with control or target-specific siRNA, grown for two days under serum conditions and treated with increasing SU11248 concentrations for 72h. SU11248 efficacy is shown at 5 ⁇ and expressed in percent relative to mock-transfected, SU11248 treated cells. Results were averaged over three to five independent experiments (error bars: SEM). In Fig. 4, it is shown that depletion of ROS1 , B P2k, NME4 and TBK1 by siRNA leads to a dramatic decrease in SU11248 treatment efficacy.
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Abstract
L'invention concerne de nouveaux procédés et compositions qui permettent de pronostiquer l'efficacité d'inhibiteurs de kinase tels que le sunitinib dans la thérapie des maladies hyperprolifératives et, en particulier, métastatiques. L'invention permet par conséquent la mise au point de stratégies individuellement adaptées pour le traitement d'états associés à l'apparition, et/ou causés par l'apparition, d'un type donné de cellules hyperprolifératives et/ou métastatiques.
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WO2014152777A2 (fr) * | 2013-03-15 | 2014-09-25 | Insight Genetics, Inc. | Procédés et compositions pour le diagnostic et le traitement de cancers résistants aux inhibiteurs de ros1 |
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AU2012332424A1 (en) | 2011-11-02 | 2014-06-05 | Synta Pharmaceuticals Corp. | Combination therapy of Hsp90 inhibitors with platinum-containing agents |
WO2013074594A1 (fr) | 2011-11-14 | 2013-05-23 | Synta Pharmaceuticals Corp. | Association thérapeutique d'inhibiteurs de hsp90 et d'inhibiteurs de braf |
EP2849751A1 (fr) * | 2012-05-10 | 2015-03-25 | Synta Pharmaceuticals Corp. | Traitement du cancer avec des composés inhibiteurs de hsp90 |
CN114277141B (zh) * | 2020-03-30 | 2022-09-02 | 中国医学科学院肿瘤医院 | 外泌体cda、mboat2等在肺癌诊断中的应用 |
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US6333194B1 (en) * | 1999-01-19 | 2001-12-25 | The Children's Hospital Of Philadelphia | Hydrogel compositions for controlled delivery of virus vectors and methods of use thereof |
WO2007109571A2 (fr) | 2006-03-17 | 2007-09-27 | Prometheus Laboratories, Inc. | Procédés de prédiction et de suivi de la thérapie par l'inhibiteur de la tyrosine kinase |
WO2008138578A2 (fr) | 2007-05-11 | 2008-11-20 | Medical Prognosis Institute | Procédés, coffrets et dispositifs pour identifier des biomarqueurs de réponse à un traitement et utilisation de ceux-ci pour prédire l'efficacité du traitement |
EP2105511A1 (fr) * | 2008-03-28 | 2009-09-30 | Fraunhofer-Gesellschaft zur Förderung der Angewandten Forschung e.V. | Compositions de médicament et substances pour le traitement et l'identification de l'adénocarcinome pulmonaire |
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WO1987007305A1 (fr) | 1986-05-21 | 1987-12-03 | Cold Spring Harbor Laboratory | Sequence adn codant l'oncogene ros, polypeptides codes a partir de celle-ci, et diagnostic et autres procedes bases sur cette sequence |
US6333194B1 (en) * | 1999-01-19 | 2001-12-25 | The Children's Hospital Of Philadelphia | Hydrogel compositions for controlled delivery of virus vectors and methods of use thereof |
WO2007109571A2 (fr) | 2006-03-17 | 2007-09-27 | Prometheus Laboratories, Inc. | Procédés de prédiction et de suivi de la thérapie par l'inhibiteur de la tyrosine kinase |
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Cited By (2)
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WO2014152777A2 (fr) * | 2013-03-15 | 2014-09-25 | Insight Genetics, Inc. | Procédés et compositions pour le diagnostic et le traitement de cancers résistants aux inhibiteurs de ros1 |
WO2014152777A3 (fr) * | 2013-03-15 | 2014-11-13 | Insight Genetics, Inc. | Procédés et compositions pour le diagnostic et le traitement de cancers résistants aux inhibiteurs de ros1 |
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