WO2002046471A2 - Methods and compositions for the identification, assessment and therapy of human cancers - Google Patents

Methods and compositions for the identification, assessment and therapy of human cancers Download PDF

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WO2002046471A2
WO2002046471A2 PCT/US2001/041670 US0141670W WO0246471A2 WO 2002046471 A2 WO2002046471 A2 WO 2002046471A2 US 0141670 W US0141670 W US 0141670W WO 0246471 A2 WO0246471 A2 WO 0246471A2
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genes
cancer cells
expression
agent
cancer
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PCT/US2001/041670
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WO2002046471A3 (en
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Lajos Pusztai
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Board Of Regents, The University Of Texas System
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Publication of WO2002046471A3 publication Critical patent/WO2002046471A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • Cancers can be viewed as a breakdown in the communication between tumor cells and their environment, including their normal neighboring cells. Growth- stimulatory and growth-inhibitory signals are routinely exchanged between cells within a tissue. Normally, cells do not divide in the absence of stimulatory signals or in the presence of inhibitory signals. In a cancerous or neoplastic state, a cell acquires the ability to "override" these signals and to proliferate under conditions in which a normal cell would not.
  • tumor cells In general, tumor cells must acquire a number of distinct aberrant traits in order to proliferate in an abnormal manner. Reflecting this requirement is the fact that the genomes of certain well-studied tumors carry several different independently altered genes, including activated oncogenes and inactivated tumor suppressor genes.
  • cells In addition to abnormal cell proliferation, cells must acquire several other traits for tumor progression to occur. For example, early on in tumor progression, cells must evade the host immune system. Further, as tumor mass increases, the tumor must acquire vasculature to supply nourishment and remove metabolic waste. Additionally, cells must acquire an ability to invade adjacent tissue. In many cases cells ultimately acquire the capacity to metastasize to distant sites.
  • a therapeutic agent that is initially effective for a given patient becomes, overtime, ineffective or less effective for that patient.
  • the very same therapeutic agent may continue to be effective over a long period of time for a different patient.
  • a therapeutic agent that is effective, at . least initially, for some patients can be completely ineffective or even harmful for other patients. Accordingly, it would be useful to identify genes and/or gene products that represent prognostic markers with respect to a given therapeutic agent or class of therapeutic agents. It then may be possible to determine which patients will benefit from particular therapeutic regimen and, importantly, determine when, if ever, the therapeutic regime begins to lose its effectiveness for a given patient. The ability to make such predictions would make it possible to discontinue a therapeutic regime that has lost its effectiveness well before its loss of effectiveness becomes apparent by conventional measures
  • the present invention is directed to the identification of markers that can be used to determine whether cancer cells are sensitive to a therapeutic agent.
  • the present invention is also directed to the identification of therapeutic targets.
  • the invention features a number of "sensitivity genes.” Nucleic acid arrays were used to identify the sensitivity genes of the present invention. Table 1 sets forth genes whose expression is increased by at least five-fold in a relatively TAXOL sensitive cell line treated with TAXOL. Table 2 sets forth the genes whose expression is increased by at least five-fold in a relatively TAXOL resistant cell line treated with TAXOL. Table 3 sets forth genes that are relatively highly expressed in a relatively TAXOL resistant cell line treated with TAXOL compared to a relatively TAXOL sensitive cell line treated with TAXOL.
  • Table 4 sets forth genes that are relatively highly expressed in a relatively TAXOL sensitive cell line treated with TAXOL, compared to a relatively TAXOL resistant cell line, treated with TAXOL.
  • the genes set forth in Tables 1-4 are thus referred to herein as "sensitivity genes".
  • the present invention provides: 1) methods for determining whether a particular therapeutic agent will be effective in stopping or slowing tumor progression; 2) methods for monitoring the effectiveness of therapeutic agents used for the treatment of cancer; 3) methods for developing new therapeutic agents for the treatment of cancer; and 4) methods for identifying combinations of therapeutic agents for the treatment of cancer.
  • examining the expression of one or more of the identified sensitivity genes in a sample of cancer cells it is possible to determine which therapeutic agent or combination of agents will be most likely to reduce the growth rate of the cancer and can further be used in selecting appropriate treatment agents.
  • By examining the expression of one or more of the sensitivity genes in a sample of cancer cells it may also be possible to determine which therapeutic agent or combination of agents will be the least likely to reduce the growth rate of the cancer.
  • By examining the expression of one or more of the sensitivity genes it is possible to eliminate inappropriate therapeutic agents. By examining the expression of one or more sensitivity genes when cancer cells or a cancer cell line is exposed to a potential anti-cancer agent, it is possible to identify new anti-cancer agents.
  • the sensitivity genes in a sample of cancer cells taken from a patient during the course of therapeutic treatment, it is possible to determine whether the therapeutic treatment is continuing to be effective or whether the cancer has become resistant (refractory) to the therapeutic treatment. Importantly, these determinations can be made on a patient by patient basis or on an agent by agent (or combination of agents) basis. Thus, one can determine whether or not a particular therapeutic treatment is likely to benefit a particular patient or group/class of patients, or whether a particular treatment should be continued.
  • the present invention further provides previously unknown or unrecognized targets for the development of anti-cancer agents, such as chemotherapeutic compounds.
  • the identified sensitivity genes of the present invention can be used as targets in developing treatments (either single agent or multiple agent) for cancer.
  • the present invention is based, in part, on the identification of genes that can be used to determine whether cancer cells are sensitive to a therapeutic agent. Based on these identifications, the present invention provides: 1) methods for determining whether a therapeutic agent (or combination of agents) will or will not be effective in stopping or slowing tumor growth; 2) methods for monitoring the effectiveness of a therapeutic agent (or combination of agents) used for the treatment of cancer; 3) methods for identifying new therapeutic agents for the treatment of cancer; 4) methods for identifying combinations of therapeutic agents for use in treating cancer; and 5) methods for identifying specific therapeutic agents and combinations of therapeutic agents that are effective for the treatment of cancer in specific patients.
  • one or more of the sensitivity genes that are expressed by cancer cell lines that are sensitive to treatment with an agent can be used as markers (or surrogate markers) to identify cancer cells that can be successfully treated by that agent.
  • these genes can be used as markers to identify cancers that have become or at risk for becoming refractory to treatment with the agent.
  • a loss of expression of one or more of the sensitivity genes can be used as an indication that the cancer is or is at risk of becoming refractory to treatment.
  • One or more of the genes that are expressed by cancer cell lines resistant to treatment with an agent can be used as markers (or surrogate markers) to identify cancer cells that cannot be successfully treated by that agent.
  • these genes can be used as markers (or surrogate markers) to identify cancers that have become or are at risk of becoming refractory to treatment with the agent.
  • the expression level of the identified sensitivity genes, or the proteins encoded by the identified sensitivity genes may be used to: 1) determine if a cancer can be treated by an agent or combination of agents; 2) determine if a cancer is responding to treatment with an agent or combination of agents; 3) select an appropriate agent or combination of agents for treating a cancer; 4) monitor the effectiveness of an ongoing treatment; and 5) identify new cancer treatments (either single agent or combination of agents).
  • the identified sensitivity genes may be utilized as markers (surrogate and/or direct) to determine appropriate therapy, to monitor clinical therapy and human trials of a drug being tested for efficacy, and to develop new agents and therapeutic combinations.
  • the present invention provides methods for determining whether an agent, e.g., a chemotherapeutic agent, can be used to reduce the growth rate of cancer cells comprising the steps of: a) obtaining a sample of cancer cells; b) determining the level of expression in the cancer cells of one or more genes selected from the group consisting of the sensitivity genes (Tables 1-4); and c) identifying that an agent can or cannot be used to treat the cancer when one or more of the sensitivity genes is expressed.
  • an agent is said to reduce the rate of growth of cancer cells when the agent can reduce at least 50%, preferably at least 75%, most preferably at least 95% of the growth of the cancer cells.
  • Such inhibition can further include a reduction in survivability and an increase in the rate of death of the cancer cells.
  • the amount of agent used for this determination will vary based on the agent selected. Typically, the amount will be a predefined therapeutic amount.
  • agents are defined broadly as anything that cancer cells may be exposed to in a therapeutic protocol.
  • agents include, but are not limited to, chemotherapeutic agents, such as antimitotic agents, e.g., TAXOL, inblastine and vincristine, alkylating agents, e.g., melphanlan, BCNU and nitrogen mustard, Topoisomerase II inhibitors, e.g., VW-26, topotecan and Bleomycin, strand-breaking agents, e.g., doxorubicin and DHAD, cross-linking agents, e.g., cisplatin and CBDCA, anti-metabolic agents, e.g., Ara AC, 5-FU and methotrexate radiation and ultraviolet light.
  • chemotherapeutic agents such as antimitotic agents, e.g., TAXOL, inblastine and vincristine
  • alkylating agents e.g., melphanlan, BCNU and nitrogen mustard
  • chemotherapeutic agent is intended to include chemical reagents which inhibit the growth of proliferating cells or tissues wherein the growth of such cells or tissues is undesirable.
  • Chemotherapeutic agents are well known in the art (see e.g., Gilman A.G., et aL, The Pharmacological Basis of Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)), and are typically used to treat neoplastic diseases.
  • the chemotherapeutic agents generally employed in chemotherapy treatments are listed below in Table A.
  • the agents tested in the present methods can be a single agent or a combination of agents.
  • the present methods can be used to determine whether a single chemotherapeutic agent, such as TAXOL, can be used to treat a cancer or whether a combination of two or more agents can be used.
  • Preferred combinations will include agents that have different mechanisms of action, e.g., the use of an anti- mitotic agent in combination with an alkylating agent.
  • cancer cells refer to cells that divide at an abnormal
  • Cancer cells include, but are not limited to, carcinomas, such as squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region; sarcomas, such as f ⁇ brosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordosarcom
  • the source of the cancer cells used in the present method will be based on how the method of the present invention is being used. For example, if the method is being used to determine whether a patient's cancer can be treated with an agent, or a combination of agents, then the preferred source of cancer cells will be cancer cells obtained from a cancer biopsy from the patient. Alternatively, a cancer cell line similar to the type of cancer being treated can be assayed. For example if breast cancer is being treated, then a breast cancer cell line can be used. If the method is being used to monitor the effectiveness of a therapeutic protocol, then a tissue sample from the patient being treated is the preferred source. If the method is being used to identify new therapeutic agents or combinations, any cancer cells, e.g., cells of a cancer cell line, can be used.
  • cancer cell lines sources such as The National Cancer Institute, NCI-60 cells, are preferred.
  • standard biopsy methods such as a needle biopsy, can be employed.
  • the level or amount of expression of one or more genes selected from the group consisting of the genes identified in Tables 1- 4 is determined.
  • the level or amount of expression refers to the absolute level of expression of an mRNA encoded by the gene or the absolute level of expression of the protein encoded by the gene (i.e., whether or not expression is or is not occurring in the cancer cells).
  • the expression of two or more of the identified sensitivity genes more preferably, three or more of the identified sensitivity genes, most preferably all of the identified sensitivity genes.
  • determinations may be based on the normalized expression levels.
  • Expression levels are normalized by correcting the absolute expression level of a sensitivity gene by comparing its expression to the expression of a gene that is not a sensitivity gene, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene. This normalization allows one to compare the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-cancer sample, or between samples from different sources. Alternatively, the expression level can be provided as a relative expression level.
  • the level of expression of the gene is determined for 10 or more samples, preferably 50 or more samples, prior to the determination of the expression level for the sample in question.
  • the mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the gene(s) in question.
  • the expression level of the gene determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that gene. This provides a relative expression level and aids in identifying extreme cases of sensitivity.
  • the samples used will be from similar tumors or from non- cancerous cells of the same tissue origin as the tumor in question. The choice of the cell source is dependent on the use of the relative expression level data.
  • a mean expression score allows for the identification of extreme cases of sensitivity.
  • Using expression found in normal tissues as a mean expression score aids in validating whether the sensitivity gene assayed is tumor specific (versus normal cells). Such a later use is particularly important in identifying whether a sensitivity gene can serve as a target gene.
  • the mean expression value can be revised, providing improved relative expression values based on accumulated data.
  • the expression level can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the selected genes; measuring the amount of protein encoded by the selected genes; and measuring the activity of the protein encoded by the selected genes.
  • the mRNA level can be determine in in situ and in in vitro formats using methods known in the art. Many of such methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from the cancer cells (see, e.g., Ausubel et al., eds., 1987-1997, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Patent No. 4,843,155).
  • the isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays.
  • One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected.
  • the mRNA is immobilized on a solid surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose.
  • the probes are immobilized on a solid surface and the mRNA is contacted with the probes, for example in an Affymetrix gene array.
  • a skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by one or more of the sensitivity genes of the present invention.
  • An alternative method for determining the level of mRNA in a sample that is encoded by one of the sensitivity genes of the present invention involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci.
  • RNA does not need to be isolated from the cancer cells prior to detection.
  • a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the sensitivity gene being analyzed.
  • Hybridization with the probe indicates that the gene in question is being expressed.
  • a hybridization probe or a set of amplification primers are used.
  • a probe is defined as a nucleic acid molecule of at least 10 nucleotides, preferably at least 20 nucleotides, most preferably at least 30 nucleotides, that is complementary to the coding sequence of a sensitivity gene.
  • a probe will hybridize, preferably selectively hybridize, to the sensitivity gene that it is obtained from.
  • a skilled artisan can readily determine appropriate probes (both nucleotide sequence and length) for detecting the sensitivity genes of the present invention using art known methods and the nucleotide sequences of the sensitivity genes of the present invention.
  • amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands respectively or visa- versa) and contain a short region in between.
  • amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length.
  • Amplification primers can be used to produce a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
  • a skilled artisan can, readily determine appropriate primers (both nucleotide sequence and length) for amplifying and detecting the sensitivity genes of the present invention using art known methods and the nucleotide sequence of the sensitivity genes of the present invention.
  • a variety of methods can be used to determine the level of protein encoded by one or more of the sensitivity genes of the present invention. In general, these methods involve the use of a compound that selectively binds to the protein, for example an antibody.
  • Proteins from cancer cells can be isolated using techniques that are well known to those of skill in the art.
  • the protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
  • a variety of formats can be employed to determine whether a sample contains a protein or fragment thereof, that binds to a given antibody.
  • Example of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (ELISA).
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • ELISA enzyme linked immunoabsorbant assay
  • antibodies, or antibody fragments can be used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins.
  • Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody.
  • Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • suitable carriers for binding antibody or antigen and will be able to adapt such support for use with the present invention.
  • protein isolated from cancer cells can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose.
  • the support can then be washed with suitable buffers followed by treatment with the detectably labeled sensitivity gene product specific antibody.
  • the solid phase support can then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on the solid support can then be detected by conventional means.
  • Another embodiment of the present invention includes a step of detecting whether an agent stimulates the expression of one or more of the sensitivity genes of the present invention. Although some of the present sensitivity genes were identified as being expressed in non-treated cancer cells, treatment with an agent may, or may not, alter expression.
  • Alterations in the expression level of the sensitivity genes of the present invention can provide a further indication as to whether an agent will or will not be effective at reducing the growth rate of the cancer cells.
  • the present invention provides methods for determining whether an agent, e.g., a chemotherapeutic agent, can be used to reduce the growth rate of cancer cells comprising the steps of: a) obtaining a sample of cancer cells; b) exposing the sample of cancer cells to one or more test agents; c) determining the level of expression in the cancer cells of one or more genes selected from the group consisting of the genes identified in Tables 1-4 in the sample exposed to the agent and in a sample of cancer cells that is not exposed to the agent; and d) identifying that an agent can (or cannot) be used to treat the cancer when the expression of one or more of the genes is increased in the presence of said agent and/or when the expression of one or more of the genes is not increased in the presence of said agent.
  • an agent e.g., a chemotherapeutic agent
  • This embodiment of the methods of the present invention involves the step of exposing the cancer cells to an agent.
  • the method used for exposing the cancer cells to the agent will be based primarily on the source and nature of the cancer cells and the agent being tested.
  • the contacting can be performed in vitro or in vivo, in a patient being treated/evaluated or in animal model of a cancer.
  • exposing the cancer cells involves contacting the cancer cells with the compound, such as in tissue culture media.
  • a skilled artisan can readily adapt an appropriate procedure for contacting cancer cells with any particular agent or combination of agents.
  • the identified sensitivity genes can also be used as markers to assess whether a tumor has become refractory to an ongoing treatment (e.g., a chemotherapeutic treatment). When a tumor is no longer responding to a treatment the expression profile of the tumor cells will change.
  • the invention provides methods for determining whether an anti-cancer treatment should be continued in a cancer patient, comprising the steps of: a) obtaining two or more samples of cancer cells from a patient undergoing anti-cancer therapy; b) determining the level of expression of one or more genes selected from the group consisting of the sensitivity genes in the sample exposed to the agent and in a sample of cancer cells that is not exposed to the agent; and c) discontinuing treatment when the expression of one or more sensitivity genes decreases.
  • a patient refers to any subject undergoing treatment for cancer.
  • the preferred subject will be a human patient undergoing chemotherapy treatment.
  • This embodiment of the present invention relies on comparing two or more samples obtained from a patient undergoing anti-cancer treatment.
  • a baseline of expression prior to therapy is determined and then changes in the baseline state of expression is monitored during the course of therapy.
  • two or more successive samples obtained during treatment can be used without the need of a pre-treatment baseline sample.
  • the first sample obtained from the subject is used as a baseline for determining whether the expression of a particular gene is increasing or decreasing.
  • two or more samples from the patient are examined.
  • three or more successively obtained samples are used, including at least one pretreatment sample.
  • kits comprising compartmentalized containers comprising reagents for detecting one or more, preferably two or more, of the sensitivity genes of the present invention.
  • a kit is defined as a pre- packaged set of containers into which reagents are placed.
  • the reagents may include probes/primers and/or antibodies for use in detecting sensitivity gene expression.
  • the kits of the present invention may preferably contain instructions which describe a suitable detection assay. Such kits can be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting symptoms of cancer.
  • Sensitivity genes can be further characterized by using techniques known to those skilled in the art to yield more information regarding potential targets for the therapeutic treatment of cancer and for identifying other sensitivity genes. For example, characterization of the identified sensitivity genes can yield information regarding the biological function of the identified genes. Specifically, any of the sensitivity genes whose further characterization indicates that a modulation of the gene's expression or a modulation of the gene product's activity can reduce symptoms of cancer are designated "target genes.” As used herein, a target gene is a gene (or gene product) that when modulated, can provide therapeutic treatment of the cancer. As such target genes and target gene products can be used to identify therapeutics agents.
  • Sensitivity genes whose further characterization indicates that it does not influence growth or viability of cancer cells, but whose expression pattern contributes to a gene expression pattern correlative of, for example, the effectiveness of a drug is designated a "sensitivity gene" and cannot serve as a target gene.
  • Such genes can be used as diagnostic markers and as markers for assessing the effectiveness or potential effectiveness of a therapeutic agent.
  • nucleotide sequence of the identified genes obtained by standard techniques well known to those of skill in the art, can be used to further characterize such genes.
  • sequence of the identified genes can reveal homologies to one or more known sequence motifs that can yield information regarding the biological function of the identified gene product.
  • an analysis of the tissue and/or cell type distribution of the mRNA produced by the identified genes can be conducted, utilizing standard techniques well known to those of skill in the art. Such techniques can include, for example, Northern analyses, RT-coupled PCR and RNase protection techniques. Such analyses can be used to determine whether the identified genes are expressed in tissues expected to contribute to cancer, whether the genes are highly regulated in tissues that can be expected to contribute to cancer, and whether cells within a given tissue express the identified gene. Such an analysis can provide information regarding the biological function of an identified gene in instances wherein only a subset of the cells within the tissue is thought to be relevant to cancer.
  • the sequences of the identified genes can be used, utilizing standard techniques, to place the genes onto genetic maps, e.g., mouse (Copeland and Jenkins 1991, Trends in Genetics 7:113-118) and human genetic maps (Cohen et al., 1993, Nature 366:698-701).
  • genetic maps e.g., mouse (Copeland and Jenkins 1991, Trends in Genetics 7:113-118) and human genetic maps (Cohen et al., 1993, Nature 366:698-701).
  • Such mapping information can yield information regarding the genes' importance to human disease by, for example, identifying genes that map within a genetic region to which predisposition to cancer also maps.
  • In vivo systems can include, but are not limited to, animal systems that naturally exhibit symptoms of cancer or ones that have been engineered to exhibit such symptoms.
  • the role of identified gene products can be determined by transfecting cDNAs encoding these gene products into appropriate cell lines, such as, for example, cancer cell lines and analyzing the effect of the gene product on cell growth.
  • the expression of these genes can be modulated within the in vivo and/or in vitro systems, i.e., either over-expressed or under-expressed, and the subsequent effect on the system then assayed.
  • the activity of the product of the identified gene can be modulated by either increasing or decreasing the level of activity in the in vivo and/or in vitro system of interest, and assessing the effect of such modulation.
  • the information obtained through such characterizations can suggest relevant methods for the treatment of cancer.
  • treatment can include a modulation of gene expression and/or gene product activity. Characterization procedures such as those described herein can indicate where such modulation should involve an increase or a decrease in the expression or activity of the gene or gene product of interest.
  • the following assays are designed to identify compounds that bind to target gene products, compounds that bind to other cellular proteins that interact with a target gene product, and compounds that interfere with the interaction of the target gene product with other cellular proteins.
  • Such compounds can include, but are not limited to, other cellular proteins, natural products and small chemical molecules. Specifically, such compounds can include, but are not limited to, peptides, soluble peptides, Ig-tailed fusion peptides, extracellular portions of target gene product transmembrane receptors, members of random peptide libraries (see, e.g., Lam et al., 1991, Nature 354:82-84; Houghton et al, 1991, Nature 354:84-86) made of D-and/or L-configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate phosphopeptide libraries; see, e.g., Songyang et al., 1993, Cell 72:767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti- idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epi
  • Compounds identified via assays such as those described herein can be useful, for example, in elaborating the biological function of the target gene product, and for ameliorating symptoms of cancer.
  • compounds that interact with the gene product of the sensitivity gene can be used to treat the cancer.
  • In vitro systems can be designed to identify compounds capable of binding the target gene products of the invention. Compounds thus identified can be used to modulate the activity of target gene products in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.
  • the preferred target genes/products used in this embodiment are the sensitivity genes of the present invention.
  • the principle of the assays used to identify compounds that bind to the target gene product involves preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture.
  • These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring target gene product or the test substance onto a solid phase and detecting target gene product/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the target gene product can be anchored onto a solid surface, and the test compound, which is not anchored, can be labeled, either directly or indirectly.
  • microtiter plates can conveniently be utilized as the solid phase.
  • the anchored component can be immobilized by non-covalent or covalent attachments.
  • Non-covalent attachment can be accomplished by simply coating the solid surface with a solution of the protein and drying.
  • an immobilized antibody preferably a monoclonal antibody, specific for the protein to be immobilized, can be used to anchor the protein to the solid surface.
  • the surfaces can be prepared in advance and stored.
  • the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any specific complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
  • a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for the target gene or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.
  • any method suitable for detecting protein-protein interactions can be employed for identifying novel target product-cellular or extracellular protein interactions.
  • the target gene serves as the known "bait" gene.
  • the target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins.
  • cellular and extracellular macromolecules are referred to as "binding partners.”
  • binding partners Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product.
  • Such compounds can include, but are not limited to, molecules such as antibodies, peptides, and small molecules.
  • the basic principle of the assay systems used to identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner or partners involve preparing a reaction mixture containing the target gene product and the binding partner under conditions and for a time sufficient to allow the target gene product and its binding partner to interact and bind, thus forming a complex.
  • the reaction mixture is prepared in the presence and absence of the test compound.
  • the test compound can initially be included in the reaction mixture, or can be added at a time subsequent to the addition of target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected.
  • complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.
  • the assay for compounds that interfere with the interaction of the target gene products and binding partners can be conducted in a heterogeneous or homogeneous format.
  • Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction.
  • homogeneous assays the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested.
  • test compounds that interfere with the interaction between a selected target gene product and its binding partners can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the target gene product and its binding partner.
  • test compounds that disrupt preformed complexes e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed.
  • the various formats are described briefly below.
  • either the target gene product or its binding partner is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly.
  • the anchored species can be immobilized by non-covalent or covalent attachment.
  • Non- covalent attachment can be accomplished simply by coating the solid surface with a solution of the target gene product or binding partner and drying.
  • an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.
  • the surfaces can be prepared in advance and stored.
  • the partner of the immobilized species is exposed to the coated surface in the presence and absence of the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways.
  • the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes immobilized on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
  • test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.
  • the reaction can be conducted in a liquid phase in the presence or absence of the test compound.
  • the reaction products separated from unreacted components and any complexes detected, e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution and a labeled antibody specific for the other partner to detect anchored complexes.
  • test compounds that inhibit complex or that disrupt preformed complexes can be identified.
  • a preformed complex of the target gene product and binding partner is prepared such that either the target gene product or its binding partner is labeled and the signal generated by the label is quenched by complex formation (see, e.g., U.S. Patent No.
  • test substances that disrupt target gene product- cellular or extracellular binding partner interaction can be identified.
  • the present invention further provides methods for identifying new anti- cancer agents or combinations that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more of the sensitivity genes of the present invention.
  • the activity of the proteins encoded by the genes of the present invention can be used as a basis for identifying agents for overcoming agent resistance.
  • by blocking the activity of one or more of the proteins cancer cells will become sensitive to treatment with an agent that the unmodified cancer cells were resistant to.
  • Computer readable media comprising a sensitivity gene of the present invention is also provided.
  • “computer readable media” refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
  • optical storage media such as CD-ROM
  • electrical storage media such as RAM and ROM
  • hybrids of these categories such as magnetic/optical storage media.
  • recorded refers to a process for storing information on computer readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the sensitivity genes of the present invention.
  • a variety of data processor programs and formats can be used to store the sensitivity gene information of the present invention on computer readable medium.
  • the nucleic acid sequence corresponding to the sensitivity genes can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.
  • Any number of dataprocessor structuring formats e.g., text file or database
  • nucleotide or amino acid sequences of the present invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means.
  • Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.
  • the invention also includes an array comprising a sensitivity gene of the present invention.
  • the array can be used to assay expression of one or more genes in the array.
  • the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, up to about 7600 genes can be simultaneously assayed for expression. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.
  • the invention allows the quantitation of gene expression.
  • tissue specificity but also the level of expression of a battery of genes in the tissue is ascertainable.
  • genes can be grouped on the basis of their tissue expression jeer se and level of expression in that tissue.
  • tissue can be perturbed and the effect on gene expression in a second tissue can be determined.
  • the effect of one cell type on another cell type in response to a biological stimulus can be determined.
  • Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression.
  • the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect.
  • undesirable biological effects can be determined at the molecular level.
  • the effects of an agent on expression of other than the target gene can be ascertained and counteracted.
  • the array can be used to monitor the time course of expression of one or more genes in the array. This can occur in various biological contexts, as disclosed herein, for example development and differentiation, tumor progression, progression of other diseases, in vitro processes, such a cellular transformation and senescence, autonomic neural and neurological processes, such as, for example, pain and appetite, and cognitive functions, such as learning or memory.
  • the array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells. This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.
  • the array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes that could serve as a molecular target for diagnosis or therapeutic intervention.
  • Cancer can be treated by modulating the expression of a target gene or the activity of a target gene product.
  • the modulation can be of a positive or negative nature, depending on the specific situation involved, but in either case, the modulatory event results in amelioration of cancer symptoms.
  • Negative modulation refers to a reduction in the level and/or activity of target gene product relative to the level and/or activity of the target gene product in the absence of the modulatory treatment.
  • “Positive modulation” refers to an increase in the level and/or activity of target gene product relative to the level and/or activity of target gene product in the absence of modulatory treatment.
  • cancer can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of cancer symptoms.
  • cancer can be brought about, at least in part, by the absence or reduction of the level of gene expression, or a reduction in the level of a gene product's activity.
  • an increase in the level of gene expression and/or the activity of such gene products would bring about the amelioration of cancer symptoms.
  • a compound e.g., an agent identified using an assay described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of cancer.
  • Such molecules can include, but are not limited to, peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab') and FAb expression library fragments, and epitope-binding fragments thereof).
  • antisense and ribozyme molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity.
  • triple helix molecules can be utilized in reducing the level of target gene activity.
  • antisense RNA molecules
  • ribozyme RNA molecules
  • triple helix molecules RNA molecules
  • Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
  • antisense DNA oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest, are preferred.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage.
  • the composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA and must include the well-known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is incorporated by reference herein in its entirety.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding target gene proteins.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the molecule of interest for ribozyme cleavage sites that include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate sequences can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
  • Nucleic acid molecules to be used in triplex helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides.
  • the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, that generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Nucleotide sequences can be pyrimidine-based, that will result in TAT and CGC + triplets across the three associated strands of the resulting triple helix.
  • the pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
  • nucleic acid molecules can be chosen that are purine-rich, e.g., contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in that the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
  • Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • the technique utilized can also efficiently reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype.
  • nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy methods.
  • Anti-sense RNA and DNA, ribozyme and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyri- bonucleotides and oligoribonucleotides that are well known in the art such as, for example, solid phase phosphoramidite chemical synthesis.
  • RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • DNA molecules can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
  • Antibodies can be generated that are specific for target gene product and reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of cancer. Antibodies can be generated using standard techniques against the proteins themselves or against peptides corresponding to portions of the proteins.
  • the antibodies include but are not limited to polyclonal, monoclonal, Fab fragments, single chain antibodies, chimeric antibodies, and the like.
  • the target gene protein to which the antibody is directed is intracellular and whole antibodies are used, internalizing antibodies are preferred.
  • lipofectin or liposomes can be used to deliver the antibody or an antigen binding fragment thereof into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target protein in an effective manner is preferred.
  • peptides having an amino acid sequence corresponding to the domain of the variable region of the antibody that binds to the target gene protein can be used. Such peptides can be synthesized chemically or produced via recombinant DNA technology using methods well known in the art (e.g., see Creighton, 1983, supra; and Sambrook et al., 1989, supra).
  • single chain neutralizing antibodies that bind to intracellular target gene product epitopes can also be administered.
  • Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (1993, Proc. Natl. Acad. Sci. USA 90:7889-7893).
  • Therapeutic Treatment The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate cancer.
  • a therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of cancer.
  • Toxicity and therapeutic efficacy of therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 5 o.
  • Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in designing a dosage range for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms
  • levels in plasma can be measured, for example, by high performance liquid cbromatography.
  • compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates can be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • compositions for oral administration can be suitably formulated to give controlled release of the active compound.
  • buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, using a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions can, if desired, be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the active ingredient.
  • the pack can for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device can be accompanied by instructions for administration. Specific Examples
  • TAXOL is a chemical compound within a family of taxane compounds which are art- recognized as being a family of related compounds.
  • the language "taxane compound” is intended to include TAXOL, compounds which are structurally similar to TAXOL and/or analogs of TAXOL.
  • the language “taxane compound” can also include “mimics”. "Mimics” is intended to include compounds which may not be structurally similar to TAXOL but mimic the therapeutic activity of TAXOL or structurally similar taxane compounds in vivo.
  • the taxane compounds of this invention are those compounds which are useful for inhibiting tumor growth in subjects (patients).
  • taxane compound also is intended to include pharmaceutically acceptable salts of the compounds.
  • Taxane compounds have previously been described in U.S. Patent Nos. 5,641,803, 5,665,671, 5,380,751, 5,728,687, 5,415,869, 5,407,683, 5,399,363, 5,424,073, 5,157,049, 5,773,464, 5,821,263, 5,840,929, 4,814,470, 5,438,072, 5,403,858, 4,960,790, 5,433,364, 4,942,184, 5,362,831, 5,705,503, and 5,278,324, all of which are expressly incorporated by reference.
  • TAXOL The structure of TAXOL, shown below, offers many groups capable of being synthetically functionalized to alter the physical or pharmaceutical properties of TAXOL.
  • Taxotere a well known semi-synthetic analog of TAXOL, named Taxotere (docetaxel), has also been found to have good anti-tumor activity in animal models. Taxotere has t-butoxy amide at the 3' position and a hydroxyl group at the CIO position (U.S. 5,840,929).
  • Other examples of TAXOL derivatives include those mentioned in U.S. 5,840,929 which are directed to derivatives of TAXOL having the formula:
  • R 1 is hydroxy, -OC(O)R x , or-OC(O)OR x ;
  • R 2 is hydrogen, hydroxy, -OC(O)R x , or -OC(O)OR x ;
  • R 2 is hydrogen, hydroxy, or fluoro;
  • R 6 is hydrogen or hydroxy or R and R can together form an oxirane ring;
  • R is hydrogen, C ⁇ e alkyloxy, hydroxy, -OC(O)R x , -OC(O)OR x , -OCONR 7 R ⁇ ;
  • R 8 is methyl or R 8 and R 2 together can form a cyclopropane ring;
  • R 6 is hydrogen or R 6 and R 2 can together form a bond;
  • R 9 is hydroxy or -OC(O)R x ;
  • R 7 and R 11 are independently C ⁇ - 6 alkyl, hydrogen, aryl, or substituted aryl;
  • R 4 and R 5 are independently
  • R 10 is aryl, substituted aryl, C 3 . 6 cycloalkyl, C 2 . 6 alkenyl, C ⁇ _ 6 alkyl, all can be optionally substituted with one to six same or different halogen atoms or hydroxy;
  • R x is a radical of the formula:
  • D is a bond or C ⁇ _ 6 alkyl; and R a , R b and R c are independently hydrogen, amino, C ⁇ - 6 alkyl or C ⁇ _ 6 alkoxy.
  • R examples include methyl, hydroxymethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, chloromethyl, 2,2,2-trichloroethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, ethenyl, 2-propenyl, phenyl, benzyl, bromophenyl, 4- aminophenyl, 4-methylaminophenyl, 4-methylphenyl, 4-methoxyphenyl and the like.
  • R 4 and R 5 examples include 2-propenyl, isobutenyl, 3-furanyl (3-furyl), 3-thienyl, phenyl, naphthyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4- trifluoromethylphenyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, ethenyl, 2-propenyl, 2-propynyl, benzyl, phenethyl, phenylethenyl, 3,4- dimethoxyphenyl, 2-furanyl (2-furyl), 2-thienyl, 2-(2-furanyl)ethenyl, 2-methylpropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, cyclohexylethy
  • TAXOL derivatives can be readily made by following the well established paclitaxel chemistry.
  • C2, C6, C7, CIO, and/or C8 position can be derivatized by essentially following the published procedure, into a compound in which R 3 , R 8 , R 2 , R 2' , R 9 , R 6 ' and R 6 have the meanings defined earlier.
  • C4- acetyloxy group can be converted to the methoxy group by a sequence of steps.
  • C2-benzoyloxy see, S. H. Chen et al, Bioorganic and Medicinal Chemistry Letters, Vol. 4, No.
  • TAXOL derivatives include the sulfenamide taxane derivatives described in U.S. 5,821,263. These compounds are characterized by the C3' nitrogen bearing one or two sulfur substituents. These compounds have been useful in the treatment of cancers such as ovarian, breast, lung, gastic, colon, head, neck, melanoma, and leukemia.
  • U.S. 4,814,470 discusses TAXOL derivatives with hydroxyl or acetyl group at the CIO position and hydroxy or t-butylcarbonyl at C2' and C3' positions.
  • U.S. 5,438,072 discusses TAXOL derivatives with hydroxyl or acetate groups at the CIO position and a C2' substitutuent of either t-butylcarbonyl or benzoylamino.
  • U.S. 4,960,790 discusses derivatives of TAXOL which have, at the C2' and/or C7 position a hydrogen, or the residue of an amino acid selected from the group consisting of alanine, leucine, isoleucine, saline, phenylalanine, proline, lysine, and arginine, or a group of the formula:
  • n is an integer of 1 to 3 and R and R are each hydrogen on an alkyl radical having one to three carbon atoms or wherein R 2 and R 3 together with the nitrogen atom to which they are attached form a saturated heterocyclic ring having four to five carbon atoms, with the proviso that at least one of the substituents are not hydrogen.
  • R and R are each hydrogen on an alkyl radical having one to three carbon atoms or wherein R 2 and R 3 together with the nitrogen atom to which they are attached form a saturated heterocyclic ring having four to five carbon atoms, with the proviso that at least one of the substituents are not hydrogen.
  • Other similar water soluble TAXOL derivatives are discussed in U.S. 4,942,184,
  • TAXOL derivatives may also include protecting groups such as, for example, hydroxy protecting groups.
  • “Hydroxy protecting groups” include, but are not limited to, ethers such as methyl, t-butyl, benzyl, p-methoxybenzyl, p-nitrobenzyl, allyl, trityl, methoxymethyl, methoxyethoxymethyl, ethoxyethyl, tetrahydropyranyl, tetrahydrothiopyranyl, dialkylsilylethers, such as dimethylsilyl ether, and trialkylsilyl ethers such as trimethylsilyl ether, triethylsilyl ether, and t-butyldimethylsilyl ether; esters such as benzoyl, acetyl, phenylacetyl, for yl, mono-, di-, and trihaloacetyl such as chloroacetyl, dichloroace
  • Nucleic acid arrays were used to determine the level of expression of approximately 6500 nucleic acid sequences in a relatively TAXOL sensitive breast cancer cell line (MDA-MB-435, also referred to as MDA-435) and in a relatively TAXOL resistant human mammary epithelial cell primary cell line (HMEC), in the presence of TAXOL.
  • MDA-MB-435 relatively TAXOL sensitive breast cancer cell line
  • HMEC human mammary epithelial cell primary cell line
  • MDA-MB-435 also referred to as MDA-435
  • HMEC TAXOL resistant human mammary epithelial cell line
  • Procedures for growing cells and testing compounds have been described previously (Scudiero et al, Cancer Res. 1988, 48:4827-4833; Stinson et al, Anticancer Res.; Myers et al, Electrophoresis 1997, L8:647-653).
  • the HMEC cells were pooled cells from three individuals. Gene expression in HMEC cells and MDA-MB-435 cells was measured in the presence and absence of TAXOL.
  • the HMEC cells were exposed to 100 nm TAXOL for 12 hours prior to isolation of mRNA for expression analysis.
  • the MDA-435 cells were exposed to 100 nm TAXOL for 12 hours prior to isolation of mRNA for expression analysis.
  • the Affymetrix HUM6000 GeneChip system was used (Affymetrix, Inc.; Santa Clara, CA) to measure expression.
  • the HUM6000 chip design consisting of 65,000 features each containing 10 million oligonucleotides designed on the basis of sequence data available from GenBank, was employed.
  • the oligonucleotides on the arrays were designed at Affymetrix to cover the complementary strand at the 3' end of the human genes.
  • About 4000 known fully sequenced human gene cDNA's and more than 2000 human EST's displaying some similarity with known genes characterized in other organisms are represented on a set of four chips. Most genes are represented by 20 overlapping oligonucleotides.
  • a mismatch oligonucleotide is included for each probe design.
  • the sequence of the oligonucleotide probes on the arrays are selected based on a combination of sequence uniqueness-criteria and empirical rules developed at Affymetrix for the selection of oligonucleotides.
  • Double passed polyA RNA was prepared from the cell line pellets ( ⁇ 10 8 cells/pellet) using Invitrogen Fast Track 2.0 system.
  • the isolated polyA RNA (2 ⁇ g) was used to synthesize cDNA using Gibco BRL Superscript Choice System cDNA Synthesis Kit.
  • the following modified T7 RNA polymerase promoter -[T]24 primer was used:
  • double stranded cDNA was passed through a Phase Lock Gel (PLG, 5 Prime-3 Prime, Inc.; Boulder, CO) and precipitated with 0.5 vol. of 7.5M NH 4 OAc and 2.5 vol. of cold 100% EtOH.
  • IVT in vitro transcription reaction
  • T7 RNA polymerase T7 Megascript System: Ambion; Austin, TX
  • biotin-11-CTP and biotin-16- UTP EZO Diagnostics; Farmingdale, NY
  • the reaction was incubated for 6 h at 37°C. Products were cleaned over a RNeasy Kit (Qiagen; Chats worth, CA). About 45 ⁇ g of cRNA was fragmented by incubating at 94°C for 35 min in 40 mM Tris- Acetate pH 8.1, 100 mM potassium acetate and 30 mM magnesium acetate.
  • Hybridization solutions contained 1.0 M NaCl, 10 mM Tris-HCl (pH 7.6) and 0.005% Triton X-100, and 0.1 mg/ml unlabeled, sonicated herring sperm DNA (Promega). cRNA samples were heated in the hybridization solution to 99°C for 5 min followed by 45°C for 5 min before being placed in the hybridization cartridge. Hybridization was carried out at 40°C for 16 h with mixing on a rotisserie at 60 rpm.
  • the solutions were removed, the arrays were rinsed with 6X SSPE-T (0.9 M NaCl, 60 mM NaH 2 PO 4 , 6 mM EDTA, 0.005% Triton X-100 adjusted to pH 7.6), incubated with 6X SSPE-T for 1 hour at 50°C and then washed with 0.5X SSPE-T at 50°C for 15 min.
  • 6X SSPE-T 0.9 M NaCl, 60 mM NaH 2 PO 4 , 6 mM EDTA, 0.005% Triton X-100 adjusted to pH 7.6
  • 6X SSPE-T 0.9 M NaCl, 60 mM NaH 2 PO 4 , 6 mM EDTA, 0.005% Triton X-100 adjusted to pH 7.6
  • 6X SSPE-T 0.9 M NaCl, 60 mM NaH 2 PO 4 , 6 mM EDTA, 0.005% Triton X-100 adjusted to pH 7.6
  • RNA abundance was calculated (after discarding the maximum, minimum and any outliers beyond three standard deviations from the computed mean).
  • the sensitivity genes are summarized in Tables 1-4.
  • “gene ID” identifies the Genbank accession numbers for the sensitivity genes and "name” represents the name of the sensitivity gene.
  • the GenBank accession numbers (“AccNum”) for Tables 1 and 2 are cross-referenced as GI accession numbers ("GI Nbr") (see e.g. http:/www.ncbi.nlm.nih.gov/genbank/query__form.html").
  • Table 1 sets forth genes whose expression is increased by at least five-fold in a relatively TAXOL sensitive cell line treated with TAXOL.
  • Table 2 sets forth genes whose expression is increased by at least five-fold in a relatively TAXOL resistant cell line treated with TAXOL.
  • H+T represents HMEC cells treated with TAXOL.
  • H represents untreated HMEC cells
  • M+T represents MDA-MB-435 cells treated with TAXOL.
  • M represents untreated MDA-MB-435 cells.
  • Ratio C/D and “Ratio E/F” represents the ratios between the data.
  • Table 3 sets forth genes that are relatively highly expressed in the relatively TAXOL resistant HMEC cells treated with TAXOL, compared to the relatively highly TAXOL sensitive MDA-435 cell line, treated with TAXOL.
  • Table 4 sets forth genes that are relatively highly expressed in the relatively TAXOL sensitive MDA-435 cell line treated with TAXOL, compared to the relatively TAXOL resistant HMEC cells, treated with TAXOL.
  • CYTOSCELETON X52426 H-saplens mRNA (or cytokeraim 13 • Also Represents: X14640 152 76 273 30 2.01636 9.08935 136983 Homo Mpitn$ narmn (DNM) 'mRNA.' complete cds 94 212 .500 83 0.44554 6.05063 D38293 Human mRNA (or daihrin-like 'protein,* complete cds 39 69 252 30 0.56207 8.39464
  • HG3242 H Calcium “Channel,” “Voltage-Gated,” Alpha 1e “Subunit,” Alt Splice 3 - “Also 169 30 30 30 5.62003 1
  • Human omtgalght chain protein 14.1 (Ig lonbda dish rotated) gtn* M34518 30 30 284 Human lymphott «p*c «o Irw ⁇ frOon factor "mRNA,' complete odt • 'Al M36542 30 30 201 Huma atf»*1 eol ⁇ n typ* ll 1g*iM.* *xor» 'i l ' 2 *nd a ftb4M ⁇ M ⁇ 02 ⁇ 30 30 12 1 Human heavy chaw olttaaa IpA chein "gen*,* CH3 roglon wWi a 388 to ' M35220 30 30 178 Human ⁇ b*ta- ⁇ N «oat lgluco*emin lbwt(traM' "mRNA,* complete cdtM87347 30 30 124 Human htlott *t protein (HLP) "mRNA.” complete c

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Abstract

The present invention is directed to the identification of markers that can be used to determine whether cancer cells are sensitive to a therapeutic agent. Nucleic acid arrays were used to determine the level of expression of approximately 6500 nucleic acid sequences (genes) found in certain cell lines treated with a chemotherapeutic agent. Expression analysis was used to identify genes associated with sensitivity to certain chemotherapeutic agents.

Description

METHODS AND COMPOSITIONS FOR THE IDENTIFICATION, ASSESSMENT AND THERAPY OF HUMAN CANCERS
RELATED APPLICATIONS The present application claims priority to U.S. patent application serial no.
09/733,853, filed December 8, 2000, and is related to U.S. patent application serial no.
09/322,864, filed on May 28, 1999, U.S. patent application serial no. 09/374,554, filed on August 13, 1999 and U.S. provisional application serial no. 60/170,229, filed on
December 10, 1999, all of which are expressly incorporated by reference.
BACKGROUND OF THE INVENTION
Cancers can be viewed as a breakdown in the communication between tumor cells and their environment, including their normal neighboring cells. Growth- stimulatory and growth-inhibitory signals are routinely exchanged between cells within a tissue. Normally, cells do not divide in the absence of stimulatory signals or in the presence of inhibitory signals. In a cancerous or neoplastic state, a cell acquires the ability to "override" these signals and to proliferate under conditions in which a normal cell would not.
In general, tumor cells must acquire a number of distinct aberrant traits in order to proliferate in an abnormal manner. Reflecting this requirement is the fact that the genomes of certain well-studied tumors carry several different independently altered genes, including activated oncogenes and inactivated tumor suppressor genes. In addition to abnormal cell proliferation, cells must acquire several other traits for tumor progression to occur. For example, early on in tumor progression, cells must evade the host immune system. Further, as tumor mass increases, the tumor must acquire vasculature to supply nourishment and remove metabolic waste. Additionally, cells must acquire an ability to invade adjacent tissue. In many cases cells ultimately acquire the capacity to metastasize to distant sites.
It is apparent that the complex process of tumor development and growth must involve multiple gene products. It is therefore important to define the role of specific genes involved in tumor development and growth and identify those genes and gene products that can serve as targets for the diagnosis, prevention and treatment of cancers.
In the realm of cancer therapy it often happens that a therapeutic agent that is initially effective for a given patient becomes, overtime, ineffective or less effective for that patient. The very same therapeutic agent may continue to be effective over a long period of time for a different patient. Further, a therapeutic agent that is effective, at . least initially, for some patients can be completely ineffective or even harmful for other patients. Accordingly, it would be useful to identify genes and/or gene products that represent prognostic markers with respect to a given therapeutic agent or class of therapeutic agents. It then may be possible to determine which patients will benefit from particular therapeutic regimen and, importantly, determine when, if ever, the therapeutic regime begins to lose its effectiveness for a given patient. The ability to make such predictions would make it possible to discontinue a therapeutic regime that has lost its effectiveness well before its loss of effectiveness becomes apparent by conventional measures
SUMMARY OF THE INVENTION
The present invention is directed to the identification of markers that can be used to determine whether cancer cells are sensitive to a therapeutic agent. The present invention is also directed to the identification of therapeutic targets. The invention features a number of "sensitivity genes." Nucleic acid arrays were used to identify the sensitivity genes of the present invention. Table 1 sets forth genes whose expression is increased by at least five-fold in a relatively TAXOL sensitive cell line treated with TAXOL. Table 2 sets forth the genes whose expression is increased by at least five-fold in a relatively TAXOL resistant cell line treated with TAXOL. Table 3 sets forth genes that are relatively highly expressed in a relatively TAXOL resistant cell line treated with TAXOL compared to a relatively TAXOL sensitive cell line treated with TAXOL. Table 4 sets forth genes that are relatively highly expressed in a relatively TAXOL sensitive cell line treated with TAXOL, compared to a relatively TAXOL resistant cell line, treated with TAXOL. The genes set forth in Tables 1-4 are thus referred to herein as "sensitivity genes".
Various embodiments of the present invention are directed to uses of the sensitivity genes. In particular, the present invention provides: 1) methods for determining whether a particular therapeutic agent will be effective in stopping or slowing tumor progression; 2) methods for monitoring the effectiveness of therapeutic agents used for the treatment of cancer; 3) methods for developing new therapeutic agents for the treatment of cancer; and 4) methods for identifying combinations of therapeutic agents for the treatment of cancer.
By examining the expression of one or more of the identified sensitivity genes in a sample of cancer cells, it is possible to determine which therapeutic agent or combination of agents will be most likely to reduce the growth rate of the cancer and can further be used in selecting appropriate treatment agents. By examining the expression of one or more of the sensitivity genes in a sample of cancer cells, it may also be possible to determine which therapeutic agent or combination of agents will be the least likely to reduce the growth rate of the cancer. By examining the expression of one or more of the sensitivity genes, it is possible to eliminate inappropriate therapeutic agents. By examining the expression of one or more sensitivity genes when cancer cells or a cancer cell line is exposed to a potential anti-cancer agent, it is possible to identify new anti-cancer agents. In addition, by examining the expression of one or more of the sensitivity genes in a sample of cancer cells taken from a patient during the course of therapeutic treatment, it is possible to determine whether the therapeutic treatment is continuing to be effective or whether the cancer has become resistant (refractory) to the therapeutic treatment. Importantly, these determinations can be made on a patient by patient basis or on an agent by agent (or combination of agents) basis. Thus, one can determine whether or not a particular therapeutic treatment is likely to benefit a particular patient or group/class of patients, or whether a particular treatment should be continued. The present invention further provides previously unknown or unrecognized targets for the development of anti-cancer agents, such as chemotherapeutic compounds. The identified sensitivity genes of the present invention can be used as targets in developing treatments (either single agent or multiple agent) for cancer.
Other features and advantages of the invention will be apparent from the detailed description and from the claims. Although materials and methods similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred materials and methods are described below.
DETAILED DESCRIPTION OF THE INVENTION General Description
The present invention is based, in part, on the identification of genes that can be used to determine whether cancer cells are sensitive to a therapeutic agent. Based on these identifications, the present invention provides: 1) methods for determining whether a therapeutic agent (or combination of agents) will or will not be effective in stopping or slowing tumor growth; 2) methods for monitoring the effectiveness of a therapeutic agent (or combination of agents) used for the treatment of cancer; 3) methods for identifying new therapeutic agents for the treatment of cancer; 4) methods for identifying combinations of therapeutic agents for use in treating cancer; and 5) methods for identifying specific therapeutic agents and combinations of therapeutic agents that are effective for the treatment of cancer in specific patients. Specific Embodiments Identification Of Sensitivity Genes
The Examples provided below concern the identification of genes that are expressed in cancer cell lines that are sensitive to defined chemotherapeutic agents, namely taxane compounds.
Accordingly, one or more of the sensitivity genes that are expressed by cancer cell lines that are sensitive to treatment with an agent can be used as markers (or surrogate markers) to identify cancer cells that can be successfully treated by that agent. In addition, these genes can be used as markers to identify cancers that have become or at risk for becoming refractory to treatment with the agent.
A loss of expression of one or more of the sensitivity genes can be used as an indication that the cancer is or is at risk of becoming refractory to treatment. One or more of the genes that are expressed by cancer cell lines resistant to treatment with an agent can be used as markers (or surrogate markers) to identify cancer cells that cannot be successfully treated by that agent. In addition, these genes can be used as markers (or surrogate markers) to identify cancers that have become or are at risk of becoming refractory to treatment with the agent.
Determining Sensitivity To An Agent The expression level of the identified sensitivity genes, or the proteins encoded by the identified sensitivity genes, may be used to: 1) determine if a cancer can be treated by an agent or combination of agents; 2) determine if a cancer is responding to treatment with an agent or combination of agents; 3) select an appropriate agent or combination of agents for treating a cancer; 4) monitor the effectiveness of an ongoing treatment; and 5) identify new cancer treatments (either single agent or combination of agents). In particular, the identified sensitivity genes may be utilized as markers (surrogate and/or direct) to determine appropriate therapy, to monitor clinical therapy and human trials of a drug being tested for efficacy, and to develop new agents and therapeutic combinations. Accordingly, the present invention provides methods for determining whether an agent, e.g., a chemotherapeutic agent, can be used to reduce the growth rate of cancer cells comprising the steps of: a) obtaining a sample of cancer cells; b) determining the level of expression in the cancer cells of one or more genes selected from the group consisting of the sensitivity genes (Tables 1-4); and c) identifying that an agent can or cannot be used to treat the cancer when one or more of the sensitivity genes is expressed. As used herein, an agent is said to reduce the rate of growth of cancer cells when the agent can reduce at least 50%, preferably at least 75%, most preferably at least 95% of the growth of the cancer cells. Such inhibition can further include a reduction in survivability and an increase in the rate of death of the cancer cells. The amount of agent used for this determination will vary based on the agent selected. Typically, the amount will be a predefined therapeutic amount.
As used herein, the term "agent" is defined broadly as anything that cancer cells may be exposed to in a therapeutic protocol. In the context of the present invention, such agents include, but are not limited to, chemotherapeutic agents, such as antimitotic agents, e.g., TAXOL, inblastine and vincristine, alkylating agents, e.g., melphanlan, BCNU and nitrogen mustard, Topoisomerase II inhibitors, e.g., VW-26, topotecan and Bleomycin, strand-breaking agents, e.g., doxorubicin and DHAD, cross-linking agents, e.g., cisplatin and CBDCA, anti-metabolic agents, e.g., Ara AC, 5-FU and methotrexate radiation and ultraviolet light. Further to the above, the language "chemotherapeutic agent" is intended to include chemical reagents which inhibit the growth of proliferating cells or tissues wherein the growth of such cells or tissues is undesirable. Chemotherapeutic agents are well known in the art (see e.g., Gilman A.G., et aL, The Pharmacological Basis of Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)), and are typically used to treat neoplastic diseases. The chemotherapeutic agents generally employed in chemotherapy treatments are listed below in Table A.
TABLE A
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
The agents tested in the present methods can be a single agent or a combination of agents. For example, the present methods can be used to determine whether a single chemotherapeutic agent, such as TAXOL, can be used to treat a cancer or whether a combination of two or more agents can be used. Preferred combinations will include agents that have different mechanisms of action, e.g., the use of an anti- mitotic agent in combination with an alkylating agent. As used herein, cancer cells refer to cells that divide at an abnormal
(increased) rate. Cancer cells include, but are not limited to, carcinomas, such as squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region; sarcomas, such as fϊbrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synoviosarcoma and mesotheliosarcoma; leukemias and lymphomas such as granulocytic leukemia, monocytic leukemia, lymphocytic leukemia, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, or Hodgkins disease; and tumors of the nervous system including glioma, meningoma, medulloblastoma, schwannoma or epidymoma.
The source of the cancer cells used in the present method will be based on how the method of the present invention is being used. For example, if the method is being used to determine whether a patient's cancer can be treated with an agent, or a combination of agents, then the preferred source of cancer cells will be cancer cells obtained from a cancer biopsy from the patient. Alternatively, a cancer cell line similar to the type of cancer being treated can be assayed. For example if breast cancer is being treated, then a breast cancer cell line can be used. If the method is being used to monitor the effectiveness of a therapeutic protocol, then a tissue sample from the patient being treated is the preferred source. If the method is being used to identify new therapeutic agents or combinations, any cancer cells, e.g., cells of a cancer cell line, can be used. A skilled artisan can readily select and obtain the appropriate cancer cells that are used in the present method. For cancer cell lines, sources such as The National Cancer Institute, NCI-60 cells, are preferred. For cancer cells obtained from a patient, standard biopsy methods, such as a needle biopsy, can be employed.
In the methods of the present invention, the level or amount of expression of one or more genes selected from the group consisting of the genes identified in Tables 1- 4 is determined. As used herein, the level or amount of expression refers to the absolute level of expression of an mRNA encoded by the gene or the absolute level of expression of the protein encoded by the gene (i.e., whether or not expression is or is not occurring in the cancer cells).
Generally, it is preferable to determine the expression of two or more of the identified sensitivity genes, more preferably, three or more of the identified sensitivity genes, most preferably all of the identified sensitivity genes. Thus, it is preferable to assess the expression of a panel of sensitivity genes.
As an alternative to making determinations based on the absolute expression level of selected genes, determinations may be based on the normalized expression levels. Expression levels are normalized by correcting the absolute expression level of a sensitivity gene by comparing its expression to the expression of a gene that is not a sensitivity gene, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene. This normalization allows one to compare the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-cancer sample, or between samples from different sources. Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a gene, the level of expression of the gene is determined for 10 or more samples, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the gene(s) in question. The expression level of the gene determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that gene. This provides a relative expression level and aids in identifying extreme cases of sensitivity. Preferably, the samples used will be from similar tumors or from non- cancerous cells of the same tissue origin as the tumor in question. The choice of the cell source is dependent on the use of the relative expression level data. For example, using tumors of similar types for obtaining a mean expression score allows for the identification of extreme cases of sensitivity. Using expression found in normal tissues as a mean expression score aids in validating whether the sensitivity gene assayed is tumor specific (versus normal cells). Such a later use is particularly important in identifying whether a sensitivity gene can serve as a target gene. In addition, as more data is accumulated, the mean expression value can be revised, providing improved relative expression values based on accumulated data. In addition to detecting the level of expression of sensitivity and normalization genes, in some instances it will also be important to monitor the level of expression of genes that indicate cell viability. The expression of such genes can be used as markers of the specificity of any particular agent, or combination, tested.
The expression level can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the selected genes; measuring the amount of protein encoded by the selected genes; and measuring the activity of the protein encoded by the selected genes.
The mRNA level can be determine in in situ and in in vitro formats using methods known in the art. Many of such methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from the cancer cells (see, e.g., Ausubel et al., eds., 1987-1997, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Patent No. 4,843,155). The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. In one format, the mRNA is immobilized on a solid surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a solid surface and the mRNA is contacted with the probes, for example in an Affymetrix gene array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by one or more of the sensitivity genes of the present invention.
An alternative method for determining the level of mRNA in a sample that is encoded by one of the sensitivity genes of the present invention involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. For in situ methods, mRNA does not need to be isolated from the cancer cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the sensitivity gene being analyzed. Hybridization with the probe indicates that the gene in question is being expressed. In analyzing mRNA that encodes a particular sensitivity gene, either a hybridization probe or a set of amplification primers are used. As used herein, a probe is defined as a nucleic acid molecule of at least 10 nucleotides, preferably at least 20 nucleotides, most preferably at least 30 nucleotides, that is complementary to the coding sequence of a sensitivity gene. As such, a probe will hybridize, preferably selectively hybridize, to the sensitivity gene that it is obtained from. A skilled artisan can readily determine appropriate probes (both nucleotide sequence and length) for detecting the sensitivity genes of the present invention using art known methods and the nucleotide sequences of the sensitivity genes of the present invention.
As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands respectively or visa- versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Amplification primers can be used to produce a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. A skilled artisan can, readily determine appropriate primers (both nucleotide sequence and length) for amplifying and detecting the sensitivity genes of the present invention using art known methods and the nucleotide sequence of the sensitivity genes of the present invention.
A variety of methods can be used to determine the level of protein encoded by one or more of the sensitivity genes of the present invention. In general, these methods involve the use of a compound that selectively binds to the protein, for example an antibody.
Proteins from cancer cells can be isolated using techniques that are well known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
A variety of formats can be employed to determine whether a sample contains a protein or fragment thereof, that binds to a given antibody. Example of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (ELISA). A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cancer cells expresses a protein encoded or fragment of a protein, by one or more of the sensitivity genes of the present invention.
In one format, antibodies, or antibody fragments, can be used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins. In such uses, it is generally preferable to immobilize either the antibody or protein on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present invention. For example, protein isolated from cancer cells can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the detectably labeled sensitivity gene product specific antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support can then be detected by conventional means. Another embodiment of the present invention includes a step of detecting whether an agent stimulates the expression of one or more of the sensitivity genes of the present invention. Although some of the present sensitivity genes were identified as being expressed in non-treated cancer cells, treatment with an agent may, or may not, alter expression. Alterations in the expression level of the sensitivity genes of the present invention can provide a further indication as to whether an agent will or will not be effective at reducing the growth rate of the cancer cells. In such a use, the present invention provides methods for determining whether an agent, e.g., a chemotherapeutic agent, can be used to reduce the growth rate of cancer cells comprising the steps of: a) obtaining a sample of cancer cells; b) exposing the sample of cancer cells to one or more test agents; c) determining the level of expression in the cancer cells of one or more genes selected from the group consisting of the genes identified in Tables 1-4 in the sample exposed to the agent and in a sample of cancer cells that is not exposed to the agent; and d) identifying that an agent can (or cannot) be used to treat the cancer when the expression of one or more of the genes is increased in the presence of said agent and/or when the expression of one or more of the genes is not increased in the presence of said agent.
This embodiment of the methods of the present invention involves the step of exposing the cancer cells to an agent. The method used for exposing the cancer cells to the agent will be based primarily on the source and nature of the cancer cells and the agent being tested. The contacting can be performed in vitro or in vivo, in a patient being treated/evaluated or in animal model of a cancer. For cancer cells and cell lines and chemical compounds, exposing the cancer cells involves contacting the cancer cells with the compound, such as in tissue culture media. A skilled artisan can readily adapt an appropriate procedure for contacting cancer cells with any particular agent or combination of agents. Monitoring the Effectiveness of a Chemotherapeutic Agent
As discussed above, the identified sensitivity genes can also be used as markers to assess whether a tumor has become refractory to an ongoing treatment (e.g., a chemotherapeutic treatment). When a tumor is no longer responding to a treatment the expression profile of the tumor cells will change. In such a use, the invention provides methods for determining whether an anti-cancer treatment should be continued in a cancer patient, comprising the steps of: a) obtaining two or more samples of cancer cells from a patient undergoing anti-cancer therapy; b) determining the level of expression of one or more genes selected from the group consisting of the sensitivity genes in the sample exposed to the agent and in a sample of cancer cells that is not exposed to the agent; and c) discontinuing treatment when the expression of one or more sensitivity genes decreases. As used herein, a patient refers to any subject undergoing treatment for cancer. The preferred subject will be a human patient undergoing chemotherapy treatment.
This embodiment of the present invention relies on comparing two or more samples obtained from a patient undergoing anti-cancer treatment. In general, it is preferable to obtain a first sample from the patient prior to beginning therapy and one or more samples during treatment. In such a use, a baseline of expression prior to therapy is determined and then changes in the baseline state of expression is monitored during the course of therapy. Alternatively, two or more successive samples obtained during treatment can be used without the need of a pre-treatment baseline sample. In such a use, the first sample obtained from the subject is used as a baseline for determining whether the expression of a particular gene is increasing or decreasing.
In general, when monitoring the effectiveness of a therapeutic treatment, two or more samples from the patient are examined. Preferably, three or more successively obtained samples are used, including at least one pretreatment sample.
Kits Containing Reagents for Conducting the Methods of the Present Invention
The present invention further provides kits comprising compartmentalized containers comprising reagents for detecting one or more, preferably two or more, of the sensitivity genes of the present invention. As used herein a kit is defined as a pre- packaged set of containers into which reagents are placed. The reagents may include probes/primers and/or antibodies for use in detecting sensitivity gene expression. In addition, the kits of the present invention may preferably contain instructions which describe a suitable detection assay. Such kits can be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting symptoms of cancer.
Further Characterization of the Sensitivity Genes Sensitivity genes can be further characterized by using techniques known to those skilled in the art to yield more information regarding potential targets for the therapeutic treatment of cancer and for identifying other sensitivity genes. For example, characterization of the identified sensitivity genes can yield information regarding the biological function of the identified genes. Specifically, any of the sensitivity genes whose further characterization indicates that a modulation of the gene's expression or a modulation of the gene product's activity can reduce symptoms of cancer are designated "target genes." As used herein, a target gene is a gene (or gene product) that when modulated, can provide therapeutic treatment of the cancer. As such target genes and target gene products can be used to identify therapeutics agents. Sensitivity genes whose further characterization indicates that it does not influence growth or viability of cancer cells, but whose expression pattern contributes to a gene expression pattern correlative of, for example, the effectiveness of a drug is designated a "sensitivity gene" and cannot serve as a target gene. Such genes can be used as diagnostic markers and as markers for assessing the effectiveness or potential effectiveness of a therapeutic agent.
A variety of techniques can be utilized to further characterize the sensitivity genes herein identified. First, the nucleotide sequence of the identified genes, obtained by standard techniques well known to those of skill in the art, can be used to further characterize such genes. For example, the sequence of the identified genes can reveal homologies to one or more known sequence motifs that can yield information regarding the biological function of the identified gene product.
Second, an analysis of the tissue and/or cell type distribution of the mRNA produced by the identified genes can be conducted, utilizing standard techniques well known to those of skill in the art. Such techniques can include, for example, Northern analyses, RT-coupled PCR and RNase protection techniques. Such analyses can be used to determine whether the identified genes are expressed in tissues expected to contribute to cancer, whether the genes are highly regulated in tissues that can be expected to contribute to cancer, and whether cells within a given tissue express the identified gene. Such an analysis can provide information regarding the biological function of an identified gene in instances wherein only a subset of the cells within the tissue is thought to be relevant to cancer. Third, the sequences of the identified genes can be used, utilizing standard techniques, to place the genes onto genetic maps, e.g., mouse (Copeland and Jenkins 1991, Trends in Genetics 7:113-118) and human genetic maps (Cohen et al., 1993, Nature 366:698-701). Such mapping information can yield information regarding the genes' importance to human disease by, for example, identifying genes that map within a genetic region to which predisposition to cancer also maps.
Fourth, the biological function of the identified genes can be more directly assessed by utilizing relevant in vivo and in vitro systems. In vivo systems can include, but are not limited to, animal systems that naturally exhibit symptoms of cancer or ones that have been engineered to exhibit such symptoms.
The role of identified gene products can be determined by transfecting cDNAs encoding these gene products into appropriate cell lines, such as, for example, cancer cell lines and analyzing the effect of the gene product on cell growth.
In further characterizing the biological function of the identified genes, the expression of these genes can be modulated within the in vivo and/or in vitro systems, i.e., either over-expressed or under-expressed, and the subsequent effect on the system then assayed. Alternatively, the activity of the product of the identified gene can be modulated by either increasing or decreasing the level of activity in the in vivo and/or in vitro system of interest, and assessing the effect of such modulation. The information obtained through such characterizations can suggest relevant methods for the treatment of cancer. For example, treatment can include a modulation of gene expression and/or gene product activity. Characterization procedures such as those described herein can indicate where such modulation should involve an increase or a decrease in the expression or activity of the gene or gene product of interest.
Identification of Compounds that Interact with a Target Gene Product
The following assays are designed to identify compounds that bind to target gene products, compounds that bind to other cellular proteins that interact with a target gene product, and compounds that interfere with the interaction of the target gene product with other cellular proteins.
Such compounds can include, but are not limited to, other cellular proteins, natural products and small chemical molecules. Specifically, such compounds can include, but are not limited to, peptides, soluble peptides, Ig-tailed fusion peptides, extracellular portions of target gene product transmembrane receptors, members of random peptide libraries (see, e.g., Lam et al., 1991, Nature 354:82-84; Houghton et al, 1991, Nature 354:84-86) made of D-and/or L-configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate phosphopeptide libraries; see, e.g., Songyang et al., 1993, Cell 72:767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti- idiotypic, chimeric or single chain antibodies, and FAb, F(ab')2 and FAb expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules.
Compounds identified via assays such as those described herein can be useful, for example, in elaborating the biological function of the target gene product, and for ameliorating symptoms of cancer. For example, compounds that interact with the gene product of the sensitivity gene can be used to treat the cancer.
Screening Assays for Compounds and Cellular Proteins that Bind to a Target Gene Product
In vitro systems can be designed to identify compounds capable of binding the target gene products of the invention. Compounds thus identified can be used to modulate the activity of target gene products in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions. The preferred target genes/products used in this embodiment are the sensitivity genes of the present invention.
The principle of the assays used to identify compounds that bind to the target gene product involves preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring target gene product or the test substance onto a solid phase and detecting target gene product/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the target gene product can be anchored onto a solid surface, and the test compound, which is not anchored, can be labeled, either directly or indirectly.
In practice, microtiter plates can conveniently be utilized as the solid phase. The anchored component can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized, can be used to anchor the protein to the solid surface. The surfaces can be prepared in advance and stored. In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any specific complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for the target gene or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.
Any method suitable for detecting protein-protein interactions can be employed for identifying novel target product-cellular or extracellular protein interactions. In such a case, the target gene serves as the known "bait" gene.
Assays for Compounds that Interfere with the Binding of a Target Gene Product to a Second Cellular Protein The target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to as "binding partners." Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product. Such compounds can include, but are not limited to, molecules such as antibodies, peptides, and small molecules.
The basic principle of the assay systems used to identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner or partners involve preparing a reaction mixture containing the target gene product and the binding partner under conditions and for a time sufficient to allow the target gene product and its binding partner to interact and bind, thus forming a complex. In order to test an agent for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound can initially be included in the reaction mixture, or can be added at a time subsequent to the addition of target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and its binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.
The assay for compounds that interfere with the interaction of the target gene products and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between a selected target gene product and its binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the target gene product and its binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.
In a heterogeneous assay system, either the target gene product or its binding partner is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtitre plates are conveniently utilized. The anchored species can be immobilized by non-covalent or covalent attachment. Non- covalent attachment can be accomplished simply by coating the solid surface with a solution of the target gene product or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface. The surfaces can be prepared in advance and stored. In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface in the presence and absence of the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways.
Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes immobilized on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.
Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound. In this format, the reaction products separated from unreacted components and any complexes detected, e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified. In an alternate embodiment of the invention, a preformed complex of the target gene product and binding partner is prepared such that either the target gene product or its binding partner is labeled and the signal generated by the label is quenched by complex formation (see, e.g., U.S. Patent No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product- cellular or extracellular binding partner interaction can be identified.
Assays Based On Target Gene Product Activity
The present invention further provides methods for identifying new anti- cancer agents or combinations that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more of the sensitivity genes of the present invention. Specifically, the activity of the proteins encoded by the genes of the present invention can be used as a basis for identifying agents for overcoming agent resistance. Specifically, by blocking the activity of one or more of the proteins, cancer cells will become sensitive to treatment with an agent that the unmodified cancer cells were resistant to.
The choice of assay format will be based primarily on the nature and type of sensitivity protein being assayed. A skilled artisan can readily adapt protein activity assays for use in the present invention with the genes identified herein. For example, DNA ligase activity can be measured using art known methods. Computer Readable Means and Arrays
Computer readable media comprising a sensitivity gene of the present invention is also provided. As used herein, "computer readable media" refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. The skilled artisan will readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a sensitivity gene of the present invention.
As used herein, "recorded" refers to a process for storing information on computer readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the sensitivity genes of the present invention.
A variety of data processor programs and formats can be used to store the sensitivity gene information of the present invention on computer readable medium. For example, the nucleic acid sequence corresponding to the sensitivity genes can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. Any number of dataprocessor structuring formats (e.g., text file or database) may be adapted in order to obtain computer readable medium having recorded thereon the sensitivity genes of the present invention. By providing the sensitivity genes of the invention in computer readable form, one can routinely access the sensitivity gene sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the present invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.
The invention also includes an array comprising a sensitivity gene of the present invention. The array can be used to assay expression of one or more genes in the array. In one embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, up to about 7600 genes can be simultaneously assayed for expression. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues. In addition to such qualitative determination, the invention allows the quantitation of gene expression. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertainable. Thus, genes can be grouped on the basis of their tissue expression jeer se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues. Thus, one tissue can be perturbed and the effect on gene expression in a second tissue can be determined. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined. Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.
In another embodiment, the array can be used to monitor the time course of expression of one or more genes in the array. This can occur in various biological contexts, as disclosed herein, for example development and differentiation, tumor progression, progression of other diseases, in vitro processes, such a cellular transformation and senescence, autonomic neural and neurological processes, such as, for example, pain and appetite, and cognitive functions, such as learning or memory.
The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells. This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.
The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes that could serve as a molecular target for diagnosis or therapeutic intervention.
Treatment of Cancer by Modulation of Sensitivity Genes or Gene Products
Cancer can be treated by modulating the expression of a target gene or the activity of a target gene product. The modulation can be of a positive or negative nature, depending on the specific situation involved, but in either case, the modulatory event results in amelioration of cancer symptoms. "Negative modulation" refers to a reduction in the level and/or activity of target gene product relative to the level and/or activity of the target gene product in the absence of the modulatory treatment.
"Positive modulation" refers to an increase in the level and/or activity of target gene product relative to the level and/or activity of target gene product in the absence of modulatory treatment.
It is possible that cancer can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of cancer symptoms.
Alternatively, it is possible that cancer can be brought about, at least in part, by the absence or reduction of the level of gene expression, or a reduction in the level of a gene product's activity. As such, an increase in the level of gene expression and/or the activity of such gene products would bring about the amelioration of cancer symptoms.
Negative Modulatory Techniques
As discussed, above, successful treatment of cancer can be brought about by techniques that serve to inhibit the expression or activity of one or more target gene products. For example, a compound e.g., an agent identified using an assay described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of cancer. Such molecules can include, but are not limited to, peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab') and FAb expression library fragments, and epitope-binding fragments thereof).
Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity.
Among the compounds that can exhibit the ability to prevent and/or ameliorate symptoms of cancer are antisense, ribozyme, and triple helix molecules. Such molecules can be designed to reduce or inhibit either wild type, or if appropriate, mutant target gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. With respect to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest, are preferred.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. (For a review, see, for example, Rossi, 1994, Current Biology 4:469- 471.) The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. The composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA and must include the well-known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is incorporated by reference herein in its entirety. As such within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding target gene proteins.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the molecule of interest for ribozyme cleavage sites that include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate sequences can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
Nucleic acid molecules to be used in triplex helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides. The base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, that generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences can be pyrimidine-based, that will result in TAT and CGC+ triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules can be chosen that are purine-rich, e.g., contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in that the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
Alternatively, the potential sequences that can be targeted for triple helix formation can be increased by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
In instances wherein the antisense, ribozyme, and/or triple helix molecules described herein are utilized to reduce or inhibit mutant gene expression, it is possible that the technique utilized can also efficiently reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, to ensure that substantially normal levels of target gene activity are maintained, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy methods. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.
Anti-sense RNA and DNA, ribozyme and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyri- bonucleotides and oligoribonucleotides that are well known in the art such as, for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
Various well-known modifications to the DNA molecules can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone. Antibodies can be generated that are specific for target gene product and reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of cancer. Antibodies can be generated using standard techniques against the proteins themselves or against peptides corresponding to portions of the proteins. The antibodies include but are not limited to polyclonal, monoclonal, Fab fragments, single chain antibodies, chimeric antibodies, and the like.
In instances where the target gene protein to which the antibody is directed is intracellular and whole antibodies are used, internalizing antibodies are preferred. However, lipofectin or liposomes can be used to deliver the antibody or an antigen binding fragment thereof into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target protein in an effective manner is preferred. For example, peptides having an amino acid sequence corresponding to the domain of the variable region of the antibody that binds to the target gene protein can be used. Such peptides can be synthesized chemically or produced via recombinant DNA technology using methods well known in the art (e.g., see Creighton, 1983, supra; and Sambrook et al., 1989, supra). Alternatively, single chain neutralizing antibodies that bind to intracellular target gene product epitopes can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (1993, Proc. Natl. Acad. Sci. USA 90:7889-7893).
Therapeutic Treatment The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate cancer. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of cancer.
Effective Dose
Toxicity and therapeutic efficacy of therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED5o. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in designing a dosage range for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid cbromatography.
Formulations And Use
Pharmaceutical compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates can be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
For oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, using a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The compositions can, if desired, be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the active ingredient. The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration. Specific Examples
At least some of the examples set forth below relate to sensitivity to TAXOL. TAXOL is a chemical compound within a family of taxane compounds which are art- recognized as being a family of related compounds. The language "taxane compound" is intended to include TAXOL, compounds which are structurally similar to TAXOL and/or analogs of TAXOL. The language "taxane compound" can also include "mimics". "Mimics" is intended to include compounds which may not be structurally similar to TAXOL but mimic the therapeutic activity of TAXOL or structurally similar taxane compounds in vivo. The taxane compounds of this invention are those compounds which are useful for inhibiting tumor growth in subjects (patients). The term taxane compound also is intended to include pharmaceutically acceptable salts of the compounds. Taxane compounds have previously been described in U.S. Patent Nos. 5,641,803, 5,665,671, 5,380,751, 5,728,687, 5,415,869, 5,407,683, 5,399,363, 5,424,073, 5,157,049, 5,773,464, 5,821,263, 5,840,929, 4,814,470, 5,438,072, 5,403,858, 4,960,790, 5,433,364, 4,942,184, 5,362,831, 5,705,503, and 5,278,324, all of which are expressly incorporated by reference.
The structure of TAXOL, shown below, offers many groups capable of being synthetically functionalized to alter the physical or pharmaceutical properties of TAXOL.
Figure imgf000031_0001
For example, a well known semi-synthetic analog of TAXOL, named Taxotere (docetaxel), has also been found to have good anti-tumor activity in animal models. Taxotere has t-butoxy amide at the 3' position and a hydroxyl group at the CIO position (U.S. 5,840,929). Other examples of TAXOL derivatives include those mentioned in U.S. 5,840,929 which are directed to derivatives of TAXOL having the formula:
Figure imgf000032_0001
wherein R1 is hydroxy, -OC(O)Rx, or-OC(O)ORx; R2 is hydrogen, hydroxy, -OC(O)Rx, or -OC(O)ORx; R2 is hydrogen, hydroxy, or fluoro; R6 is hydrogen or hydroxy or R and R can together form an oxirane ring; R is hydrogen, C^e alkyloxy, hydroxy, -OC(O)Rx, -OC(O)ORx, -OCONR7Rπ; R8 is methyl or R8 and R2 together can form a cyclopropane ring; R6 is hydrogen or R6 and R2 can together form a bond; R9 is hydroxy or -OC(O)Rx; R7 and R11 are independently Cι-6 alkyl, hydrogen, aryl, or substituted aryl; R4 and R5 are independently Cι.6 alkyl, C2.6 alkenyl, C2.6 alkynyl, or - Z-R10; Z is a direct bond, Cι_6 alkyl, or C .6 alkenyl; R10 is aryl, substituted aryl, C3.6 cycloalkyl, C2.6 alkenyl, Cι_6 alkyl, all can be optionally substituted with one to six same or different halogen atoms or hydroxy; Rx is a radical of the formula:
Figure imgf000032_0002
wherein D is a bond or Cι_6 alkyl; and Ra, Rb and Rc are independently hydrogen, amino, Cι-6 alkyl or Cι_6 alkoxy.
Further examples of R include methyl, hydroxymethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, chloromethyl, 2,2,2-trichloroethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, ethenyl, 2-propenyl, phenyl, benzyl, bromophenyl, 4- aminophenyl, 4-methylaminophenyl, 4-methylphenyl, 4-methoxyphenyl and the like. Examples of R4 and R5 include 2-propenyl, isobutenyl, 3-furanyl (3-furyl), 3-thienyl, phenyl, naphthyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4- trifluoromethylphenyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, ethenyl, 2-propenyl, 2-propynyl, benzyl, phenethyl, phenylethenyl, 3,4- dimethoxyphenyl, 2-furanyl (2-furyl), 2-thienyl, 2-(2-furanyl)ethenyl, 2-methylpropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, cyclohexylethyl and the like.
TAXOL derivatives can be readily made by following the well established paclitaxel chemistry. For example, C2, C6, C7, CIO, and/or C8 position can be derivatized by essentially following the published procedure, into a compound in which R3, R8, R2, R2', R9, R6' and R6 have the meanings defined earlier. Subsequently, C4- acetyloxy group can be converted to the methoxy group by a sequence of steps. For example, for converting C2-benzoyloxy to other groups see, S. H. Chen et al, Bioorganic and Medicinal Chemistry Letters, Vol. 4, No. 3, pp 479-482 (1994); for modifying ClO-acetyloxy see, J. Kant et al, Tetrahedron Letters, Vol. 35, No. 31, pp 5543-5546 (1994) and U.S. Pat. No. 5,294,637 issued Mar. 15, 1994; for making CIO and/or C7 unsubstituted (deoxy) derivatives see, European Patent Application 590 267 A2 published Apr. 6, 1994 and PCT application WO 93/06093 published Apr. 1, 1993; for making 7β,8β-methano, 6,7-α,α-dihydroxy and 6,7-olefinic groups see, R. A. Johnson, Tetrahedron Letters, Vol. 35, No 43, pp 7893-7896 (1994), U.S. Pat. No.
5,254,580, issued Oct. 19, 1993, and European Patent Application 600 517A1 published Jun. 8, 1994; for making C7/C6 oxirane see, U.S. Pat. No. 5,395,850 issued Mar. 7, 1995; for making C7-epi-fluoro see, G. Roth et al, Tetrahedron Letters, Vol 36, pp 1609-1612 (1993); for forming C7 esters and carbonates see, U.S. Pat. No. 5,272,171 issued Dec. 21, 1993 and S. H. Chen et al., Tetrahedron, 49, No. 14, pp 2805-2828 (1993).
In U.S. 5,773,464, TAXOL derivatives containing epoxides at the Cι0 position are disclosed as antitumor agents. Other C-10 taxane analogs have also appeared in the literature. Taxanes with alkyl substituents at C-10 have been reported in a published PCT patent application WO 9533740. The synthesis of C-10 epi hydroxy or acyloxy compounds is disclosed in PCT application WO 96/03394. Additional C-10 analogs have been reported in Tetrahedron Letters 1995, 36(12), 1985-1988; J. Org. Chem. 1994, 59, 4015-4018 and references therein; K. V. Rao et. al. Journal of Medicinal Chemistry 1995, 38 (17), 3411-3414; J. Kant et. al. Tetrahedron Lett. 1994, 35(31), 5543-5546; WO 9533736; WO 93/02067; U.S.' Pat. No. 5,248,796; WO 9415929; and WO 94/15599.
Other relevant TAXOL derivatives include the sulfenamide taxane derivatives described in U.S. 5,821,263. These compounds are characterized by the C3' nitrogen bearing one or two sulfur substituents. These compounds have been useful in the treatment of cancers such as ovarian, breast, lung, gastic, colon, head, neck, melanoma, and leukemia. U.S. 4,814,470 discusses TAXOL derivatives with hydroxyl or acetyl group at the CIO position and hydroxy or t-butylcarbonyl at C2' and C3' positions.
U.S. 5,438,072 discusses TAXOL derivatives with hydroxyl or acetate groups at the CIO position and a C2' substitutuent of either t-butylcarbonyl or benzoylamino.
U.S. 4,960,790 discusses derivatives of TAXOL which have, at the C2' and/or C7 position a hydrogen, or the residue of an amino acid selected from the group consisting of alanine, leucine, isoleucine, saline, phenylalanine, proline, lysine, and arginine, or a group of the formula:
Figure imgf000034_0001
wherein n is an integer of 1 to 3 and R and R are each hydrogen on an alkyl radical having one to three carbon atoms or wherein R2 and R3 together with the nitrogen atom to which they are attached form a saturated heterocyclic ring having four to five carbon atoms, with the proviso that at least one of the substituents are not hydrogen. Other similar water soluble TAXOL derivatives are discussed in U.S. 4,942,184,
U.S. 5,433,364, and in U.S. 5,278,324.
Many TAXOL derivatives may also include protecting groups such as, for example, hydroxy protecting groups. "Hydroxy protecting groups" include, but are not limited to, ethers such as methyl, t-butyl, benzyl, p-methoxybenzyl, p-nitrobenzyl, allyl, trityl, methoxymethyl, methoxyethoxymethyl, ethoxyethyl, tetrahydropyranyl, tetrahydrothiopyranyl, dialkylsilylethers, such as dimethylsilyl ether, and trialkylsilyl ethers such as trimethylsilyl ether, triethylsilyl ether, and t-butyldimethylsilyl ether; esters such as benzoyl, acetyl, phenylacetyl, for yl, mono-, di-, and trihaloacetyl such as chloroacetyl, dichloroacetyl, trichloroacetyl, triffuoroacetyl; and carbonates such as methyl, ethyl, 2,2,2-trichloroethyl, allyl, benzyl, and p-nitrophenyl. Additional examples of hydroxy protecting groups may be found in standard reference works such as Greene and Wuts, Protective Groups in Organic Synthesis, Id Ed., 1991, John Wiley & Sons, and McOmie; and Protective Groups in Organic Chemistry, 1975, Plenum Press. Methods for introducing and removing protecting groups are also found in such textbooks. Example 1 Identification of Sensitivity Genes
Nucleic acid arrays were used to determine the level of expression of approximately 6500 nucleic acid sequences in a relatively TAXOL sensitive breast cancer cell line (MDA-MB-435, also referred to as MDA-435) and in a relatively TAXOL resistant human mammary epithelial cell primary cell line (HMEC), in the presence of TAXOL. This analysis led to the identification of genes that are increased by at least five-fold in a relatively TAXOL sensitive cell line, genes that are increased by at least- five-fold in a relatively TAXOL resistant cell line, genes that are relatively highly expressed in the TAXOL resistant human mammary epithelial cell primary cell line compared to the relatively TAXOL sensitive breast cancer cell line, and genes that are relatively highly expressed in the relatively TAXOL sensitive breast cancer cell line compared to the relatively TAXOL resistant human mammary epithelial cell primary cell line (Tables 1-4).
Cell Line Preparation A TAXOL sensitive cell line, MDA-MB-435, also referred to as MDA-435, and a TAXOL resistant human mammary epithelial cell line, HMEC, were used in the study. Procedures for growing cells and testing compounds have been described previously (Scudiero et al, Cancer Res. 1988, 48:4827-4833; Stinson et al, Anticancer Res.; Myers et al, Electrophoresis 1997, L8:647-653). The HMEC cells were pooled cells from three individuals. Gene expression in HMEC cells and MDA-MB-435 cells was measured in the presence and absence of TAXOL. The HMEC cells were exposed to 100 nm TAXOL for 12 hours prior to isolation of mRNA for expression analysis. The MDA-435 cells were exposed to 100 nm TAXOL for 12 hours prior to isolation of mRNA for expression analysis.
Oligonucleotide Array Expression Monitoring Chip
The Affymetrix HUM6000 GeneChip system was used (Affymetrix, Inc.; Santa Clara, CA) to measure expression.
The HUM6000 chip design, consisting of 65,000 features each containing 10 million oligonucleotides designed on the basis of sequence data available from GenBank, was employed. The oligonucleotides on the arrays were designed at Affymetrix to cover the complementary strand at the 3' end of the human genes. About 4000 known fully sequenced human gene cDNA's and more than 2000 human EST's displaying some similarity with known genes characterized in other organisms are represented on a set of four chips. Most genes are represented by 20 overlapping oligonucleotides. A mismatch oligonucleotide is included for each probe design. The sequence of the oligonucleotide probes on the arrays are selected based on a combination of sequence uniqueness-criteria and empirical rules developed at Affymetrix for the selection of oligonucleotides.
RNA extraction and preparation for hybridization Double passed polyA RNA was prepared from the cell line pellets (~108 cells/pellet) using Invitrogen Fast Track 2.0 system.
The isolated polyA RNA (2 μg) was used to synthesize cDNA using Gibco BRL Superscript Choice System cDNA Synthesis Kit. The following modified T7 RNA polymerase promoter -[T]24 primer was used:
5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-[T]24-
3'
To prepare labeled cRNA, double stranded cDNA was passed through a Phase Lock Gel (PLG, 5 Prime-3 Prime, Inc.; Boulder, CO) and precipitated with 0.5 vol. of 7.5M NH4OAc and 2.5 vol. of cold 100% EtOH. The in vitro transcription reaction (IVT) was carried out using T7 RNA polymerase (T7 Megascript System: Ambion; Austin, TX) with the following modifications: biotin-11-CTP and biotin-16- UTP (ENZO Diagnostics; Farmingdale, NY) were added to the rNTP cocktail for the IVT reaction. The reaction was incubated for 6 h at 37°C. Products were cleaned over a RNeasy Kit (Qiagen; Chats worth, CA). About 45 μg of cRNA was fragmented by incubating at 94°C for 35 min in 40 mM Tris- Acetate pH 8.1, 100 mM potassium acetate and 30 mM magnesium acetate.
Array hybridization and scanning
Hybridization solutions contained 1.0 M NaCl, 10 mM Tris-HCl (pH 7.6) and 0.005% Triton X-100, and 0.1 mg/ml unlabeled, sonicated herring sperm DNA (Promega). cRNA samples were heated in the hybridization solution to 99°C for 5 min followed by 45°C for 5 min before being placed in the hybridization cartridge. Hybridization was carried out at 40°C for 16 h with mixing on a rotisserie at 60 rpm. Following hybridization, the solutions were removed, the arrays were rinsed with 6X SSPE-T (0.9 M NaCl, 60 mM NaH2PO4, 6 mM EDTA, 0.005% Triton X-100 adjusted to pH 7.6), incubated with 6X SSPE-T for 1 hour at 50°C and then washed with 0.5X SSPE-T at 50°C for 15 min. Following washing, the hybridized cRNA was flourescently labeled by incubating with 2 μg/ml streptavidine-phycoerythrin (Molecular Probes, Eugene, OR) and 1 mg/ml acetylated BSA (Sigma, St. Louis, MO) in 6XSSPE- T at 40°C for 10 min. Unbound streptavidine-phycoerythrin was removed by rinsing at room temperature prior to scanning. Scanning was done on a specially designed confocal scanner made for Affymetrix by Molecular Dynamics. The excitation source was an argon ion laser and the emission was detected by a photomultiplier tube through a 560 nm longpass filter.
Quantitative analysis of hybridization patterns and intensities
Following a quantitative scan of an array, a grid was aligned to the image using the known dimensions of the array and the corner and edge controls regions as markers. The pixels in each region (about 20) were averaged after discarding outliers and pixels near feature boundaries. The image was reduced to a text file containing position, oligonucleotide sequence, ORF or locus name and intensity information. To determine the quantitative RNA abundance, the average of the difference (PM minus MM) for each probe family was calculated (after discarding the maximum, minimum and any outliers beyond three standard deviations from the computed mean).
Summary of Data
The sensitivity genes are summarized in Tables 1-4. In the tables, "gene ID" identifies the Genbank accession numbers for the sensitivity genes and "name" represents the name of the sensitivity gene. In Table 1-1, the GenBank accession numbers ("AccNum") for Tables 1 and 2 are cross-referenced as GI accession numbers ("GI Nbr") (see e.g. http:/www.ncbi.nlm.nih.gov/genbank/query__form.html").
Table 1 sets forth genes whose expression is increased by at least five-fold in a relatively TAXOL sensitive cell line treated with TAXOL. Table 2 sets forth genes whose expression is increased by at least five-fold in a relatively TAXOL resistant cell line treated with TAXOL. In Tables 1 and 2, "H+T" represents HMEC cells treated with TAXOL. "H" represents untreated HMEC cells, "M+T represents MDA-MB-435 cells treated with TAXOL. "M" represents untreated MDA-MB-435 cells. "Ratio C/D" and "Ratio E/F" represents the ratios between the data.
Table 3 sets forth genes that are relatively highly expressed in the relatively TAXOL resistant HMEC cells treated with TAXOL, compared to the relatively highly TAXOL sensitive MDA-435 cell line, treated with TAXOL. Table 4 sets forth genes that are relatively highly expressed in the relatively TAXOL sensitive MDA-435 cell line treated with TAXOL, compared to the relatively TAXOL resistant HMEC cells, treated with TAXOL.
Other Embodiments The present invention is not to be limited in scope by the specific embodiments described that are intended as single illustrations of individual aspects of the invention and functionally equivalent methods and components are within the scope of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. All references cited herein, including journal articles and patents, are expressly incorporated by reference. The content of cell GenBank and Image Consortium database records cited throughout this application (including in Tables 1-4) are also hereby incorporated by reference.
GENES INDUCED (>5X) BY TAXOL IN MUΛ- U-» 3 *,cu.4 gene (D na t H+T H M+T M ralio C D ralio E F
SIGNALLING PATHWAYS M80629 Human edc2-re!atβd protein kiπase (CHED) "mRNA,* complete cds 95 134 251 30 0.71016 8.37254 L36463 Homo sapiens ras Inhibitor (Rin 1 ) 'mRNA,* complete cds 294 290 270 40 1.01299 6.70752 AB000450 Human mRNA (or *VRK2." complete cds 189 179 200 30 1.05275 6.68085 M92432 Homo sapiens retinal guanylyl cydase (retGC) 'mRNA,* complete cds 104 30 205 37 3.45466 5.47521
U07139 Human voltage-gated calcium channel beta subunlt "mRNA,* complete c 147 253 179 30 0.5836 5.97422
APOPTOSIS PATHWAYS 050840 Human mRNA (or ceramide 'glucosyllransterase,* complete cds 776 740 835 117 1.04852 7.16072
CYTOSCELETON X52426 H-saplens mRNA (or cytokeraim 13 • Also Represents: X14640 152 76 273 30 2.01636 9.08935 136983 Homo Mpitn$ narmn (DNM) 'mRNA.' complete cds 94 212 .500 83 0.44554 6.05063 D38293 Human mRNA (or daihrin-like 'protein,* complete cds 39 69 252 30 0.56207 8.39464
TRANSCRIPTION FACTORS HG2724- Oncogene Tls Chop." Fusion Activated 590 533 460 30 1.10752 15.3369
M36542 Human lymphoid-spedfic Iranscnplion (actor "mRNA," complete cds - "Al 30 30 201 30 1 6.69966 AF000560 Homo sapiens TTF-I inleraeiing peplide 20 "mRNA,* partial cds. 70 235 183 30 0.29684 6.09991
METABOLIC PATHWAYS
M76424 H.sapiens carbonic an drase VII (CA VII) gene 172 385 567 30 0.44783 18.8884
D37937 Human mRNA (or *8lpha(1^)(ucosyttrans(erasel' 5,UTR partial sequenc 395 406 368 30 0.97106 12.2579
D14530 Human ho olog of yeast ribosomal protein 'S28,* complete cds 6400 7331 10274 1848 0.87301 5.55841
U55764 Human estrogen sulfotransferase 'mRNA,' partial cds 176 161 165 30 1.09278 5.49239
U34252 Human gamma-aminobutyraldehyde dehydrogenase 'mRNA,' complete 84 164 161 30 0.51026 5.35842
L16991 Human thymidylate klnase (CDC8) 'mRNA,* complele cds 130 205 346 68 0.63527 5.10028
U22029 Human cytoehrome P450 (CYP2A7) *mRNA,' complete cds 30 102 220 34 0.29357 6.44686
MISCELLENIOUS
D13627 Human mRNA (or KIAA0OO2 'gene.' complete cds 871 835 1098 30 0.98499 36.5995 011428 Human mRNA'tor PMP-22(PAS-ll/SR13 Gas-3) of peripheral " yelin," c 30 30 997 30 1 33.23 L41143 TCTA (T cell leukemia gene) 127 157 512 30 0.8136 17.0507 050930 Human mRNA (or KIAA0140 "gene.' complele cds 178 211 456 30 0.84509 15.1917 021260 Human mRNA (or KIAA0034 "gene," complele cds 1258 1321 1115 74 0.95297 15.023 D49487 Human mRNA for obese "gene." complele cds • Also Represents: U436 109 65 352 30 1.68253 11.7351 AD000092 RAD23A gene (human RAD23A homolog) extracted (rom Homo sapiens 30 30 308 30 t 1 10.2537 M34S16 Human omega light chain protein 14.1 (Ig lambda chain related) gene 30 30 284 30 1 9.47341
L05628 Human multidrug resistance-associated protein (MRP) 'mRNA.' complel 54 120 301 34 0.44416 8.80821
HG907-H Mg44 30 85 242 30 0.35395 8.07512
083782 Human mRNA (or KIAA0199 "gene." partial cds 275 223 233 30 1.2306 7.7556
U57627 Human (elal brain ocutocerebrorertal syndrome (OCRL1) "mRNA," comp 80 122 220 30 0.65305 7.33976
013643 Human mRNA (or K1AA0018 "gene." complete cds 648 969 217 30 0.66859 7.23275
D31766 Human mRNA (or KIAA0060 "gene." complele cds 223 350 728 110 0.63588 6.60141
X62573 H^apiens RNA (or Fc "receptor." TC9 104 49 189 30 2.14689 6.30249 AB000115 Human 'mRNA.' complete cds 30 43 188 30 0.70036.6.27419
MS5220 Human heavy chain disease IgA chain "gene," CH3 region with a 369 bp 30 30 179 30 1 5.96125 M582S5 Human membrane-assooaied protein (HEM-1 ) 'mRNA,* complete cds 250 220 166 30 1.13581 5.52588 D86973 Human mRNAtor KIAA0219 'gene." partial cds 30 168 166 30 0.17857 5.51897
TABLE 1 AD000092 1905905
AF000560 2145059
D11428 220009
D13627 286010
D13643 6630631
D14530 414348
D14686 485807
D21260 434760
D31766 498157
D38293 807814
D49487 904211
D49817 1468914
D50840 1350551
D50930 1469202
D83782 1228046
D86973 1504019
D87937 1842168
J02871 180968
J03756 183176
J04615 338246 05628 1835658
L07592 190229 16896 292934
L16991 291899
L36463 1695232
L36818 556191
L36983 1196422 41143 736684 76927 1929894
M27830 337384
M34516 409735
M36542 339495
M58285 407955
M64231 338393
M76125 292869
M76424 179965
M80629 180491
M85085 181138
M85220 184776
M92432 623414
M95678 190039
S74445 241541
U00802 392889
U07139 463890
U20325 665578
U22029 1008465
U34252 1049218
U34301 1143201
U47931 1208751
U55764 1932728
U56244 3287304
U57627 1420919
U58032 3912939
X03794 32363 X98225 1707492
Y00083 31959
Z48519 695605
Z49254 1478199
GENES INDUCED (>5X) BY TAXOL IN HMEC CELLS gene ID name H+T H M+T M ratio C/D ratio E/F
SIGNALLING PATHWAYS
U47931 Human G-proteln beta-3 subunit alternatively spliced form mRNA sequence. 674 91 30512 7.41113 0.05881 95678 Homo sapiens phospholipase C-beta-2 "mRNA," complete cds 688 108 161520 6.36808 0.3093
M76125 Human tyrosine klnase receptor (axl) "mRNA," complete cds - "Also Represe 179 31 67 30 5.81305 2219
S74445 cellular retinolc acid-binding protein "(human," "skin," "mRNA," 735 nt] 167 30 62 75 5.5774 0.83202-
HG3242 H Calcium "Channel," "Voltage-Gated," Alpha 1e "Subunit," Alt Splice 3 - "Also 169 30 30 30 5.62003 1
L35818 Human 51C protein, (clone 51C-3),INPPL1, Inosytol phosphate phosphatase 168 30 519615 5.60146 0.84489
U58032 Human myotubularin related protein 1 (MTMR1) putative tyrosine phosphata 177 31 113 84 5.66939 1.33256
CYTOSCELETON
X81637 H.sap!ens dathrin light chain b gene 173 30 30 30 5.75464 1 U00802 Human drebrin E2 mRNA "(DBN1)," complete cds • Also Represents: D1 53 152 30 191 45 5.08158 4.24929 U34301 Human nonmusde myosin heavy chain IIB "gene," promoter region and exon 186 30 30 30 6.19213 1
TRANSCRIPTION FACTORS
L16896 Human zinc finger protein "mRNA," complete cds 258 30 127 100 8.591 1.27705
METABOLIC PATHWAYS L76927 Human galactokinase (GALK1 ) "gene," complete cds 214 30 61 30 7.14343 20333 049817 Human mRNA for fructose "6-phosphate,2-kinase/fructose" "2,6-bisphosphat 184 30 38 76 6.13643 0.50246 M64231 Human spermidine synthase "gene," complete cds 150 30 128 61 5.01428 2.08232 J02871 Human lung cytochrome P450 (IV subfamily) Bl "protein," complete cds - Als 154 30 30 60 5.14889 0.50397 M27830 28S ribosomal RNA 169 30 266 35 5.61902 7.64583
MISCELLENIOUS J04615 Human lupus autoantlgen (small nuclear "ribonuclepoprotein." "snRNP." SM- 568 30 581 435 18.9443 1.3376 Z49254 saplens L23-related mRNA 348 38 587552 9.06464 1.06382 D14686 Human gene for glydne deavage system T-protein 222 30 68125 7.39518 0.5435 U20325 Human cocaine and amphetamine regulated transcript CART (hCART) "gene 300 41 143144 7.35368 0.99322 X98225 H.sapiens mRNA for gastrin-binding protein. /gb=X98225 /ntype=RNA 207 30 30 30 6.89884 1 M85085 Human deavage stimulation "factor," (pre-mRNA processing factor) 179 30 49 42 5.952 1.-17813 U56244 Human HIG-1 "mRNA." complete cds 171 30 153 57 5.70227 2.6579
J03756 Human growth hormone-variant (GH1) and growth hormone-variant-2 (GH2) 168 30 326401 5.60146 0.81351 HG1067-H Mu n (Gb:M22406) 168 30 161 80 5.58638 2.01579
Z48519 H.sapiens XG gene (done RACE5). Blood group antigene, Also Represents: 252 46 64104 5.52345 0.61302 X03794 Human embryonic mRNA 3' end with homoβo box (done HHOdO) - Also Re 165 30 210264 5.48542 0.79389 Y00083 Human mRNA for glioblastoma-derived T-cell suppressor factor G-TsF (trans 164 30 30 30 5.45176 1 L07592 Human peroxisome proliferator activated receptor "mRNA," complete cds 159 30 98 30 5.28677 3.25329 HG3934-H G1 Phase-Spedfic Gene 151 30 36 30 5.03502 1.19093 AB006781 Homo sapiens mRNA for "galectin-4," complete cds. /gb=AB006781 /ntype 188 37 198129 5.02266 1.54044
TABLE 2 Name Gene ID HMEC«T«XOI WGC MDΛ+Taxol MCA
AvgOtn AvgDffl Avg Dift Avg Dffl
Human gen* for L*U0Λn» Maotiboxytase.* coπyM* od» 018583 135 30 30 iyroeto*al<lnae«,*R*0(ptor'AxL'A«.8pfee2 HQ182-HT318S 174 .3d 30
CaMum "Channel' *Ve«ag»βaM,* Alpha 1* "Subunit," AIL Spfc* a • "H03242-HT4231 168 30 30 OIPhfeOpadBoOeno HG3flS4-HT4204 in 30 38
Homo aapiane|yM*omal membrane glycoprelaln-1 (LA P1)"mRNA,"comJ04182 1126 74 583 • 'Alto RepmβentK 029(32, M21642* L00190 123 30 '42
Huma cMUP|iho*ρho6attenaa1mRNA.'3-*nd L12052 248 30 30
Human tholnger potato ImflN coαvlaleedt Llββββ 258 30 127
Human giliatotdhau QAIK1) Igena,* complete cd L78827 214 30 81
Human αfemβon WiMoiy tactowalated pmteh 14 (URP14) Iββnβ," ocn M21084 114 30 55 Humanpanomaflo twain 1 (mYI) ImRNA,* oompMa odt M22812 118 30 30
Human gammframhobutyrio actdA (QABA-A) raoeptor baCa-1 eubun*-AIM50216 127 30 30 Human damage βftMUIαn faolor,' eompltt* cda M85085 178 30 48 βeMarfeαnoteβclWlndto iwteto'Ihuman.' ^ 874445 187 30 82
Human Ktrø* VMM,* complete ed* • Atao RapraaentK H0β4βWfT25U100ββ 145 30 30 Human ateda telantf iθtaila (ATM) ImBWA.* ocπy * ode U33841 143 30 30
Human nonmuada nqpoaki heevy chain IS "gene,* promoter ragton and • U34301 188 30 30 Human Iraneoriptfon factor EtFI (E2F1) "gene,' promoter and U47877 144 30 30
Human OfraWn beca-3 aubunR afameβvely epfaed form mRNA eequenc U47931 874 81 30 Human done 161488-2-3 B eel enptmidmBNA ton chwmooomeX Uββ04β 314 30 83 HuπmBoHEUnd^eoπιρonar<«(b^'nιRNA,*eom|Maod Uβββ78 304 30' 181
Human mad protein homolog (hittOβ) ImRNA,* oompW* cda UββOlβ 178 30 73
Human fc yoμhorin beta 3 ImRNΛ,' co pWa cat. #»U727βl Λttyρ*-R U72781 232 38 50 HomoaaplanaFUlBana.'eompMaoda U80184 242 44 30
Human C» Mn>ael lMtemι1mRNA.,cotnptotocd».^b-4^16S4 typ«.U81554 380 40 225 Human unknown praMn •mRNA,* partial edt. /≠JϋβSΛU Myp«-RNA U82311 383 48 188 Hunan clone WA<^4503unfewιm protein "mRN pertW edt U87408 217 83 34
H«B«lroquol»<l^hom*>domΛpfθWhinX-4"mftNA*p«ιl«I<Λ.Λ)UβO30β 182 30 31 Hunan pteoeota mRNA (or ohMng homwo wltttlng hormone pracuwoXO OSfl 232 30 30 Human mRNA tor gamma tateritron hdudbto carry ΓMPOOM gene (wKh r X02530 168 30 30 Human Horftron alpha gin* FNelpha 5 X02056 118 30 30
HumninOOOttgtneeignaleequtnee X05323 108 30. 30
Hunan gen* far PP15 (ptoctnM protein 15) X0731S 238 30 101
Human Iyer mRNA for 3«»»*cytCαA Holes* X14813 123 SO 30
Human mRNA far *ain XS1S21 720 81 218 umteπanged tnmunoglobuβn V(H)S XS8401 113 30 30
UMptara mRNA for dteoylglyoerol Unas* X82S35 1057 313 87
HatptanaRONmRNAfortyrotkwlilnet* X70040 379 48 74
HJtpitnegarMkrtanltz-typ*protatM'lnhfcftø,'HKIB8 X77188 124 33 30
RaepttnecteM lght chain bgen* X81837 173 30 30
HaaphnaMUβOmHNA X82458 488 47 148
HJtpfcnt mRNA for riboaomtiSβMntM X85108 247 30 85
Heaptene mRNA far goabMMng protth. tø-Xβ822S Λιtyp*>RNA XB822S 207 30 30 Haapitne mRNA for ttcheae Iphotphoprataki,* mppβ X88283 407 115 135
Htapttne RNA far ztr»ftig*r "protein." H««11 X08833 tie 30 30
Kaapltne mRNA far hair •towβn.'hHbβ X89142 273 30 30
Human mRNA far gfebla*»om*<J*ι1»d T-e*l tuppra**or (actor G-TtF (W Y00083 184 30 30 Heptane mRNA tor CHDS protein Y12478 188 30 30
H4tρiamlrepomyotlnltoloαn\nRN coα*>tettCOS Z24727 481 85 147
Z31357 145 30 48
188 30 30 H*p|^ mRNA far novtlgluo<>c<>rtloc>l r^or. oc^ j∞W 148 30 30
KS ϊS b- ** I-." » «* ' «~ 153 30 30
TABLE 3 Name Gene ID HMεOTaxol HuB3 MDAVTtxo) MOA
Avg OKI Avg OKI Avg Dtff Avg Dirt
Human TwflNV campltte cda ABOOOitS 30 43 I ββ
RAOa gtn* (human RA023A homolog) extracted from Homo ttptant DAD000082 30 30 308 Homo aaptene dtteytd wcWtr pctaetlum tharwel (KVLQTHtoS) "mRNA AFΘ03743 50 34 234 Human mRNA for IVM2(PA84V8R13»ae4) cι( p«|ρh*nl Vπyri * com Di 1 28 30 30 887 HurMn mRNA far KUA005S "e«t*.Oonvtete edt 028956 65 38 112
Human mRNA for cteMvtta tprotoln,' eoπyMe cdt 038283 38 68 . 252
Human RflNA far KIAAO0β2 lBtne,*coπvtotecdt 042054 I β t 67 1 18
Human mRNA for 8«dtnceytnιMonh* qnBietatOb* eompitie cdt 048357 30 30 518 Human mRNA tor oboe* "Ben*," compute cot- Alto Rtprottnti: U43853 048487 108 65 352 Human mRNA tor IMtnt tø»Oβ062S My t^WA 050525 30 42 161
Human mRNA tor KUA0132 *B«M,* oompltte cdt 050822 30 64 130
Human mRNA tor MAA0140 "gene." complete cot O50830 178 21 1 456
Human mRNA tor NAAOIβl Tgtnβ,* oompteto cot D7B883 30 30 102
'Aφtee,* Na K* Tmntpcrtbig,* Afeha 1 Potyptpcid* HG1034-HTI034 30 51 143
Onattodhg Protein *Λ<>4." Al Spec* 3 Hα24βS-HT48 1 54 30 137
Mg44 HQ807-HT807 30 85 242
8CN .TRANSOUXR NDAC^ATOROF'nUN9CflFnON 1-ALPHAβETA HUMISOF3Λ/M8783! 30 37 ■ 100 Human mu dtug mhttnoa attu Jtiid protein (MRP) "mRNA," oompteto < LQ5628 54 120 301 ttSriboeo alRNAgene M27830 188 30 266
Human omtgalght chain protein 14.1 (Ig lonbda dish rotated) gtn* M34518 30 30 284 Human lymphott«p*c«o IrwαfrOon factor "mRNA,' complete odt • 'Al M36542 30 30 201 Huma atf»*1 eolφn typ* ll 1g*iM.* *xor» 'il' 2 *nd a ftb4M^ Mβ02ββ 30 30 12 1 Human heavy chaw olttaaa IpA chein "gen*,* CH3 roglon wWi a 388 to ' M35220 30 30 178 Human ■b*ta-ι N«oat lgluco*emin lbwt(traM' "mRNA,* complete cdtM87347 30 30 124 Human htlott *t protein (HLP) "mRNA." complete cdt U08877 30 30 260
Human 0 roton 'VnRNA.* portal odt Uiooai 47 7β 1 15
Humtn fortlw protei FREACl •mRNA," coπyW cot UI3218 52 β l 120
Human chromoeome 17q12-21 "mRNA," don* "pOV-2." partial odt Utββtβ 30 30 127
Humtn OOββ ImRNA,* comptote cdt U21048 32 80 I θ l
Human Ck-eeeodated RS cydophln CARS-Cyp •mRNA," corrptel* cdt - A U40783 30 30 n o Human ptpβdtørolyl ieomerat* and **t«nβ*l mHeOo ragutator (PIN1) "ml U48070 24 1 143 437 PIEXR g*n* (ptexn nteted protein) axtracted from Hunan Xq2β gwxxnlo U52111 30 30 233 Human nfooOnio aoatyfchoOn* rtotfXor *|pha2 tubu* *pr*cunor.* "mRWU8243l 85 82 142 Human muMtptmkig mambrant protein imRNΛ." comptote cdt. tø»Uβ<U84β3i 47 87 1 1 1 HtaρieMfm AforC)rtofc*t*li 13 - Alto tpro**πtt: X14β40 XS2428 152 76 273
KβtptontRNAIorFo Ytotptor.' TCβ X62573 104 48 I ββ
Hatptent mRNA for gtyotrol Hn*n - Abo Rtpmttntt: L13043 X68886 58 30 141
HΛtømmRNA for DNA prirnet* (tubur* ρ5β) X74331 30 4 1 126 ftatplen* mRNA for putetlm cMoridt channtl X83378 30 47 1 1 1
Haaplana mRNA for OAT? protein Xβiβoβ 82 78 183
HjMpiant mRNA tor NOVproWn X86584 30 SO 150
Hat itnt mRNA for hudrw-rich primary rtepont* protein 1 X87248 30 30 108
Human mBNA for leutooyte aetoctetιd πιoteoute-1 alpha »ubunK (LFA-1 alY00786 Sβ 83 138 Rttpltot mRNA tor Iπtertnt tptdlta ew fri Z11502 30 30 128
TABLE 4

Claims

What is claimed is:
1. A method for determining whether an agent can be used to reduce the growth of cancer cells, comprising the steps of: a) obtaining a sample of cancer cells; b) determining whether said cancer cells express one or more genes selected from the group consisting of the genes identified in Tables 1-4; and c) identifying that an agent can be used to reduce the growth of said cancer cells when one or more of said genes is expressed by said cancer cells.
2. A method for determining whether an agent cannot be used to reduce the growth of cancer cells, comprising the steps of: a) obtaining a sample of cancer cells; b) determining whether said cancer cells express one or more genes selected from the group consisting of the genes identified in Tables 1-4; and c) identifying that an agent cannot be used to reduce the growth of said cancer cells when one or more of said genes is not expressed by said cancer cells.
3. The method of claim 1 , wherein said level of expression is determined by detecting the amount of mRNA that is encoded by said one or more genes present in said sample.
4. The method of claim 2, wherein said level of expression is determined by detecting the amount of mRNA that is encoded by said one or more genes present in said sample.
5. The method of claim 1 , wherein said level of expression is determined by detecting the amount of protein that is encoded by said one or more genes present in said sample.
6. The method of claim 2, wherein said level of expression is determined by detecting the amount of protein present that is encoded by said one or more genes present in said sample.
7. The method of claim 1 , wherein said cancer cells are selected from the group consisting of cancer cell lines and cancer cells obtained from a patient.
8. The method of claim 1, wherein said cancer cells are selected from the group consisting of cancer cell lines and cancer cells obtained from a patient.
9. The method of claim 1, wherein said agent is a chemotherapeutic compound.
10. The method of claim 2, wherein said agent is a chemotherapeutic compound
11. A method for determining whether an agent can be used to reduce the growth of cancer cells, comprising the steps of: a) obtaining a sample of cancer cells; b) exposing the cancer cells to one or more agents; c) determining the level of expression in the cancer cells of one or more genes selected from the group consisting of the genes identified in Tables 1-4 in the sample exposed to the agent and in a sample of cancer cells that is not exposed to the agent; and d) identifying that an agent can be used to reduce the growth of said cancer cells when the expression of one or more of said genes is increased in the presence of said agent.
12. A method for determining whether an agent cannot be used to reduce the growth of cancer cells, comprising the steps of: a) obtaining a sample of cancer cells; b) exposing the cancer cells to one or more agents; c) determining the level of expression in the cancer cells of one or more genes selected from the group consisting of the sensitivity genes identified in Tables 1-4 in the sample exposed to the agent and in a sample of cancer cells that is not exposed to the agent; and d) identifying that an agent cannot be used to reduce the growth of said cancer cells when the expression of one or more of said genes is not increased in the presence of said agent.
13. The method of claim 11 , wherein said level of expression is determined by detecting the amount of mRNA that is encoded by said one or more genes present in said sample.
14. The method of claim 12, wherein said level of expression is determined by detecting the amount of mRNA that is encoded by said one or more genes present in said sample.
15. The method of claim 11 , wherein said level of expression is determined by detecting the amount of protein that is encoded by said one or more genes present in said sample.
16. The method of claim 12, wherein said level of expression is determined by detecting the amount of protein present that is encoded by said one or more genes present in said sample.
17. The method of claim 11, wherein said cancer cells are selected from the group consisting of cancer cell lines and cancer cells obtained from a patient.
18. The method of claim 12, wherein said cancer cells are selected from the group consisting of cancer cell lines and cancer cells obtained from a patient.
19. The method of claim 11 , wherein said agent is a chemotherapeutic compound.
20. The method of claim 12, wherein said agent is a chemotherapeutic compound.
21. A method for determining whether treatment with a chemotherapeutic compound should be continued in a cancer patient, comprising the steps of: a) obtaining two or more samples comprising cancer cells from a patient during the course of chemotherapeutic compound treatment; b) determining the level of expression in the cancer cells of one or more genes selected from the group consisting of the genes identified in Tables 1-4 in the two or more samples; and c) continuing treatment when the expression level of one or more of said genes does not decrease during the course of treatment.
22. A method for determining whether treatment with a chemotherapeutic compound should be continued in a cancer patient, comprising the steps of: a) obtaining two or more samples comprising cancer cells from a patient during the course of chemotherapeutic compound treatment; b) determining the level of expression in the cancer cells of one or more genes selected from the group consisting of the genes identified in Tables 1-4 in the two or more samples; and c) discontinuing treatment when the expression level of one or more of said genes increases during the course of treatment.
23. The method of claim 21 , wherein said level of expression is determined by detecting the amount of mRNA that is encoded by said one or more genes present in said sample.
24. The method of claim 22, wherein said level of expression is determined by detecting the amount of mRNA that is encoded by said one or more genes present in said sample
25. The method of claim 21 , wherein said level of expression is determined by detecting the amount of protein that is encoded by said one or more genes present in said sample.
26. The method of claim 22, wherein said level of expression is determined by detecting the amount of protein present that is encoded by said one or more genes present in said sample.
27. A method for reducing the growth rate of cancer cells in a patient, comprising the step of administering to a patient suffering from cancer an agent identified using the method of claim 1 as being able to reduce the rate of growth of said cancer cells.
28. A method for reducing the growth rate of cancer cells in a patient, comprising the step of administering to a patient suffering from cancer an agent identified using the method of claim 11 as being able to reduce the rate of growth of said cancer cells.
PCT/US2001/041670 2000-12-08 2001-08-09 Methods and compositions for the identification, assessment and therapy of human cancers WO2002046471A2 (en)

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