US20090186951A1 - Identification of novel pathways for drug development for lung disease - Google Patents

Identification of novel pathways for drug development for lung disease Download PDF

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US20090186951A1
US20090186951A1 US12/234,588 US23458808A US2009186951A1 US 20090186951 A1 US20090186951 A1 US 20090186951A1 US 23458808 A US23458808 A US 23458808A US 2009186951 A1 US2009186951 A1 US 2009186951A1
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activation
pi3k
pathway
individual
biomarkers
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Jerome S. Brody
Avrum Spira
Adam Gustafson
Andrea Bild
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University of Utah Research Foundation UURF
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    • A61K31/5375—1,4-Oxazines, e.g. morpholine
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    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • G01N2333/91—Transferases (2.)
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    • G01N2333/91205—Phosphotransferases in general
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2800/7023—(Hyper)proliferation
    • G01N2800/7028—Cancer

Definitions

  • Cigarette smoke is the dominant cause of lung cancer in the United States, accounting for an estimated 90% of all cases [1].
  • the damage caused by cigarette smoke is not limited solely to the lung, but rather constitutes a ‘field of injury’ throughout the entire respiratory tract [2-6].
  • An important product of the field of injury hypothesis is the ability to glean clinically relevant information from cells collected in regions of the respiratory tract, such as the bronchial airway, that can be obtained in a less invasive manner than is typical of collecting primary lung tissue.
  • a gene expression-based biomarker measured in the cytologically normal bronchial airway epithelium that can distinguish smokers with and without lung cancer has been developed [7]. This airway gene expression biomarker achieved 83% accuracy in predicting whether a smoker had a lung tumor in a prospective test set, and 94% accuracy when combined synergistically with clinical variables [7, 8].
  • lung cancer Beyond serving as an early diagnostic tool for lung cancer, gene expression changes in the cytologically normal airway epithelium have the potential to improve our understanding of the signaling events deregulated during early stages of lung cancer.
  • Lung cancer development in humans is a complex process involving multiple aberrant events that, when accumulated, lead to deregulation of crucial cell functions, including cell survival and proliferation.
  • signaling pathways In primary tumors resected from patients with lung cancer, many signaling pathways have previously been found to be deregulated, such as p53, RAS and phosphatidylinositol 3-kinase (PI3K) [9-11].
  • the invention provides biomarkers for oncogenic pathways activated in cytologically normal airway epithelial cells of individuals with lung disease. These biomarkers and pathways may provide prognostic and/or diagnostic indicators of lung disease, e.g., lung cancer. Additionally, these pathways and biomarkers may provide therapeutic targets for the treatment of lung disease, as well as markers for the assessment of treatment efficacy.
  • the invention relates to the use of gene expression profiling methods to identify gene expression signatures of oncogenic pathways activated in lung disease, e.g., lung cancer. These gene expression signatures may provide prognostic or diagnostic indicators for lung disease, e.g., lung cancer. Moreover, oncogenic pathways which are activated in lung disease may be targets for therapeutic intervention. In one embodiment the oncogenic pathway can be, for example, one or more of the PI3K and Np63 pathways.
  • the invention also relates to a method of identifying an individual at increased risk of lung disease, comprising determining the activation status of the Np63 and/or PI3K pathway in a cytologically normal airway epithelial cell from said individual, wherein activation of the Np63 and/or PI3K pathway is indicative that said individual is at increased risk of lung disease as compared with an individual in whom the Np63 and/or PI3K pathway is not activated.
  • the individual is a smoker or a non-smoker.
  • the lung disease is lung cancer.
  • the activation status of the PI3K pathway is determined using gene expression data for one or more biomarkers of the PI3K pathway.
  • at least one of said one or more biomarkers is a gene which is increased upon PI3K activation, and in other embodiments at least one of said one or more biomarkers is a gene which is decreased upon PI3K activation. Combinations of biomarkers which are increased and decreased upon PI3K activation may also be used.
  • at least one of said one or more biomarkers is a gene which is upstream of PI3K activation, while in other embodiments at least one of said one or more biomarkers is a gene which is downstream of PI3K activation.
  • expression data for said one or more biomarkers of the PI3K pathway is obtained using an oligonucleotide microarray.
  • the activation status of the PI3K pathway is determined using one or more gene expression products of one or more biomarkers of the PI3K pathway.
  • Said gene expression products may be nucleotide or amino acid products and can be detected using methods known in the art.
  • the activation status of the PI3K pathway is determined by assessing the activation of IGF1R, wherein activation of IGF1R is indicative of activation of the PI3K pathway. In other embodiments of the invention the activation status of the PI3K pathway is determined by assessing the activation of PKC, wherein activation of PKC is indicative of activation of the PI3K pathway.
  • the invention also relates to a method of identifying an individual at increased risk of lung disease, comprising determining the activation status of PKC in a cytologically normal airway epithelial cell from said individual, wherein activation of PKC is indicative that said individual is at increased risk of lung disease as compared with an individual in whom PKC is not activated.
  • the invention further relates to a method of identifying an individual at increased risk of lung disease, comprising determining the activation status of IGF1R in a cytologically normal airway epithelial cell from said individual, wherein activation of IGF1R is indicative that said individual is at increased risk of lung disease as compared with an individual in whom IGF1R is not activated.
  • the invention provides an oligonucleotide array having immobilized thereon one or more probes for one or more biomarkers of the PI3K pathway, and wherein said array does not have immobilized thereon probes for other biomarkers.
  • said one or more biomarkers of the PI3K pathway are selected from the group consisting of IGF1R, PKC, the biomarkers disclosed in [29], and combinations thereof.
  • the invention also relates to a method of reducing the risk of lung disease in an individual comprising administering to an individual at risk of lung disease one or more agents (e.g., one or more agents, regimens or treatments or combinations thereof) which inhibit the PI3K pathway.
  • one or more agents e.g., one or more agents, regimens or treatments or combinations thereof
  • the PI3K pathway is activated in said individual prior to administration of said one or more agents.
  • the lung disease is lung cancer.
  • said one or more agents are administered to said individual prophylactically before the development of lung disease.
  • the invention in another embodiment relates to a method of differentially classifying a cytologically normal test airway epithelial cell, comprising identifying a gene expression signature associated with activation of a biological pathway of interest in a normal airway epithelial cell; assessing gene expression in differentially classified airway epithelial cells to identify one or more correlations between classification of an airway epithelial cell and activation of a biological pathway of interest; and assessing gene expression in a cytologically normal test airway epithelial cell, wherein the gene expression profile of the cytologically normal airway epithelial cell to be classified indicates whether the biological pathway of interest is activated and thus differentially classifies the cell.
  • the biological pathway of interest is an oncogenic pathway.
  • the differential classification is increased risk of disease versus decreased risk of disease, while in other embodiments the differential classification is response to treatment versus non-response to treatment.
  • the invention provides a method for identifying the activation of an oncogenic pathway in a mammal having or at risk of having lung disease, e.g., lung cancer.
  • the method may include: (a) providing a biological sample, e.g., a biological sample from an airway passage of the mammal, wherein the biological sample comprises a gene expression product (e.g., mRNA or protein) from at least one gene that is indicative of activation of said pathway, and (b) detecting the expression of said gene.
  • the pathway can be one or more of the following: Ras, Myc, E2F3, beta-catenin, Src, Np63, PI3K and combinations thereof.
  • the mammal can be, for example, a human.
  • Bio samples may be provided, for example, from bronchial, nasal or buccal epithelium, or from biopsied tissue samples.
  • detection of gene expression is accomplished using an oligonucleotide array having immobilized thereon one or more nucleotide sequences or fragments thereof which are probes for the relevant gene(s).
  • Identification of activation of an oncogenic pathway may indicate that the mammal is a candidate for treatment to inhibit activation of said pathway or for additional or more frequent screening to identify development of disease, e.g., cancer.
  • the invention provides a method of screening candidate therapeutic agents which may be useful in the treatment of lung disease, e.g., lung cancer.
  • candidate agents may be screened for their ability to modulate (e.g., inhibit) the activation of an oncogenic pathway identified by methods described herein as associated with lung disease.
  • the agent's ability to modulate activation of the pathway may be assessed, for example, by its ability to alter a gene expression signature of an oncogenic pathway from a signature which is associated with disease to a signature which is not associated with disease, e.g., is normal.
  • the candidate therapeutic agent may be assessed for its ability to modulate a specific functional effect or readout of the pathway.
  • Agents identified as having the ability to inhibit an activated oncogenic pathway associated with lung disease may be suitable for treatment of lung disease in a mammal.
  • the efficacy of a treatment regimen can be evaluated by assessing the gene expression signature of a mammal at various time points over the course of treatment.
  • a shift in the gene expression signature of an oncogenic pathway from one associated with disease to one not associated with disease, e.g., to a normal signature is indicative of efficacious treatment.
  • absence of a shift in the gene expression signature toward a normal signature is indicative that treatment is not efficacious and that perhaps alternative treatment regimens are indicated.
  • FIGS. 1A-1C show that PI3K and ⁇ Np63 are differentially activated in smokers with lung cancer.
  • pathway activation probabilities were calculated in samples obtained from the cytologically normal airway. Pathway levels are summarized using box plots, where the bar represents the median value, the box denotes the range of the data points from the 25th to 75th percentile, and the whiskers specify the range of the remaining 1st and 4th quartile. As shown in FIG. 1A , when grouping the activation levels by lung cancer status (blue for no lung cancer, red for lung cancer), two pathways were found to be statistically different after random permutation tests: PI3K (p ⁇ 0.001), and ⁇ Np63 (p ⁇ 0.001).
  • pathway activation probabilities were also calculated for healthy never (green), former (brown) and current smokers (gray) ( FIG. 1B ), as well as current smokers with (orange) or without (gray) chronic obstructive pulmonary disease (COPD) ( FIG. 1C ). Neither of the potential confounding variables showed a statistically significant difference in pathway activation.
  • FIG. 2 shows oncogenic pathway activity in lung tumor and adjacent normal tissue.
  • Oncogenic pathway activity was calculated for a dataset of lung adenocarcinoma and adjacent normal tissue [32].
  • FIGS. 3A-3B show the biochemical validation of PI3K activity in prospectively collected airway samples. Airway brushings were collected prospectively from patients under suspicion of having lung cancer in Boston and Utah. As shown in FIG. 3A , kinase assays were used to measure in vivo levels of PI3K pathway activity. Patients with lung cancer generally had higher levels of PI3K activity than those without lung cancer. A subset of the Boston cohort had extra sample run on microarray so that computational predicted PI3K activity could be correlated to in vivo activity. The probability of pathway activity is shown below the patients that had extra samples. Pearson correlation of the computationally predicted PI3K activity and the biochemically measured activity was 0.48. As shown in FIG.
  • FIG. 3B Western blots querying proteins both upstream and downstream of PI3K were quantified and then correlated with PI3K kinase levels measured in FIG. 3A . Correlations are presented in a heatmap manner, where blue represents negative correlation, and red represents positive correlation. Correlation analysis is also broken down into all samples, only samples with lung cancer, and only control samples. Both p-IGF1R and p-PKC are positively correlated with PI3K activity in patients with lung cancer, suggesting possible sub-pathways driving the increased PI3K pathway activity in the airway of smokers with lung cancer.
  • FIG. 4 demonstrates that smokers with dysplasia have an increased activation of the PI3K pathway.
  • Genes that increase when the PI3K pathway is activated, as defined by in vitro perturbation, are displayed in a heatmap. Blue represents low expression of a gene, while red represents higher expression of a gene.
  • GSEA was used to quantify the enrichment of this gene set (p ⁇ 0.001, FDR Q ⁇ 0.001).
  • FIG. 5A-5C show that Myo-inositol inhibits the PI3K pathway in vitro. Insulin was used to activate the PI3K pathway in three different cell lines: ( FIG. 5A ) BEAS-2B (bronchial airway cell line), ( FIG. 5B ) BT549 (breast cancer cell line), and ( FIG. 5C ) HEK293 (human embryonic kidney cell line). The cell lines were then treated with varying doses of myo-inositol and LY-294002. PIP3 levels were measured to quantify the activation levels of the PI3K pathway (y-axis).
  • pathway activity uses gene expression data to link in vitro activation of an isolated signaling pathway to predict status of that pathway in patient samples. This approach has been successful at predicting pathway status in cell lines as well as tumors where the initiation event is known [13].
  • a strength of in vitro defined pathway signatures is that they are capable of identifying pathway activity at the gene expression level, allowing the measurement of multiple pathways using a single microarray experiment.
  • gene expression based predictions of pathway activity have been found to correlate significantly to drugs that target the specific pathway [13-20]. Numerous studies have also found correlation of predicted pathway status and therapeutic responsiveness in clinical trials with targeted therapies [22-25].
  • PI3K and ⁇ Np63 pathways have an increased activation in the normal airway of smokers with lung cancer ( FIG. 1A ). This is an interesting finding, because a priori cytologically normal cells are not expected to show signs of oncogenic pathway deregulation. Additionally, it is important to note that non-lung cancer controls used as described herein have an extensive range of alternative pathologies that could also impact the PI3K pathway, and are not just healthy volunteers. However, the increased activity is not correlated to smoking status or COPD ( FIG. 1B , 1 C).
  • PI3K is required for malignant progression in lung cancer, and that inhibition of this pathway blocks tumorigenesis [40].
  • Increased activity of the PI3K pathway has also previously been observed in many different cancers, including lung cancer [41].
  • Some of the common causes of deregulation that confer constitutive activation in tumors include a mutation in the tyrosine kinase domain of EGFR; a mutation, deletion or suppression of the tumor suppressor PTEN; increased PI3K gene copy number [42] or a mutation in p110 ⁇ , the catalytic subunit of PI3K [41].
  • PI3K activity in the normal airway epithelium of lung cancer patients is positively correlated with activation of IGF1R (upstream) and PKC (downstream), and is not positively correlated with HER2 (upstream) and AKT (downstream) ( FIG. 3 ).
  • IGF1R upstream
  • PKC downstream
  • HER2 upstream
  • AKT downstream
  • FIG. 3 results suggest a specific signaling cascade leading to PI3K activation and subsequent downstream effects in these cells.
  • Increased levels of IGF signaling have been associated with lung cancer in some studies.
  • current inhibitors of the IGF pathway have been found to have significant responses in lung cancer patients.
  • PKC is a kinase downstream of PI3K, and has previously been found to have increased levels in dysplastic lesions and lung cancer [33, 34].
  • results described herein implicate the IGFR1/PI3K/PKC pathway as central to lung cancer development, even at a pre-malignant state.
  • Airway gene expression profiling on these subjects post-treatment may also help identify a subset of patients that would benefit from long-term therapy. More broadly, these results suggest that a smoker's pattern of airway gene-expression reflects perturbation of specific oncogenic pathways, potentially allowing for personalized chemoprophylaxis and therapy.
  • the invention provides a general approach to identifying pathway status (e.g., oncogenic pathway status) in cytologically normal cells of the airway which may be useful as an early predictor of lung disease and/or which may provide targets for therapeutic intervention (e.g., early intervention).
  • oncogenic or other pathways of interest are activated in a cell (e.g., human epithelial cell, human primary epithelial cell culture) in vitro to identify gene expression signatures or patterns which are associated with pathway activation.
  • a cell e.g., human epithelial cell, human primary epithelial cell culture
  • cells can be perturbed using an adenovirus expressing an activating or necessary component of the pathway of interest (e.g., an adenovirus expressing p110 or other suitable agent).
  • Differentially classified samples e.g., a sample from a lung cancer patient v. sample from an individual without lung cancer, a sample from a treatment responsive patient v. a sample from a treatment non-responsive patient, etc.
  • a sample from a lung cancer patient v. sample from an individual without lung cancer e.g., a sample from a treatment responsive patient v. a sample from a treatment non-responsive patient, etc.
  • pathway status e.g., activation
  • phenotype e.g., disease state, treatment response, etc.
  • histologically normal airway cells can be tested to identify a gene expression pattern associated with activation of a particular pathway, and based on the correlation between pathway status and phenotype, the phenotype (e.g., disease state such as cancerous, non-cancerous) of the individual from whom the cell sample is obtained can be predicted.
  • the phenotype e.g., disease state such as cancerous, non-cancerous
  • associations between disease state and pathway status (as indicated by gene expression) can be identified, and these associations can be leveraged in therapeutic and prognostic applications.
  • the impact of candidate agents and treatment regimens on activation of one or more pathways can be assessed by monitoring gene expression associated with said pathway(s) to identify agents and/or regimens having a desired effect.
  • the invention also relates to a method of identifying an individual at increased risk of lung disease, comprising determining the activation status of an oncogenic pathway, e.g., the Np63 and/or PI3K pathway, in a cytologically normal airway epithelial cell from said individual.
  • Activation of, e.g., the Np63 and/or PI3K pathway is indicative that said individual is at increased risk of lung disease as compared with an individual in whom the Np63 and/or PI3K pathway is not activated.
  • the individual is a smoker or a non-smoker.
  • the lung disease is lung cancer. I some embodiments the activation status of multiple pathways (e.g., oncogenic pathways) is assessed simultaneously.
  • the activation status of the PI3K pathway is determined using gene expression data for one or more (i.e., 1, 2, 3, 4, 5 or more than 5) biomarkers of the PI3K pathway.
  • at least one of said one or more biomarkers is a gene which is increased upon PI3K activation, and in other embodiments at least one of said one or more biomarkers is a gene which is decreased upon PI3K activation. Combinations of biomarkers which are increased and decreased upon PI3K activation may also be used.
  • at least one of said one or more biomarkers is a gene which is upstream of PI3K activation, while in other embodiments at least one of said one or more biomarkers is a gene which is downstream of PI3K activation.
  • the isolated nucleic acid is obtained from a cytologically normal airway epithelial cell and used to evaluate expression of a gene or multiple genes using any method known in the art for measuring gene expression, including analysis of mRNA transcripts as well as analysis of DNA methylation.
  • gene expression can be determined by detection of RNA transcripts, for example by Northern blotting, for example, wherein a preparation of RNA is run on a denaturing agarose gel, and transferred to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Labeled (e.g. radiolabeled) cDNA or RNA is then hybridized to the preparation, washed and analyzed using methods well known in the art, such as autoradiography.
  • Northern blotting for example, wherein a preparation of RNA is run on a denaturing agarose gel, and transferred to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Labeled (e.g. radiolabeled) cDNA or RNA is then hybridized to the preparation, washed and analyzed using methods well known in the art, such as autoradiography.
  • RNA transcripts can further be accomplished using known amplification methods. For example, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, or reverse transcribe mRNA into cDNA followed by symmetric gap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall, et al., PCR Methods and Applications 4: 80-84 (1994).
  • RT-PCR polymerase chain reaction
  • RT-AGLCR symmetric gap ligase chain reaction
  • amplification methods which can be utilized herein include but are not limited to the so-called “NASBA” or “3SR” technique described in PNAS USA 87: 1874-1878 (1990) and also described in Nature 350 (No. 6313): 91-92 (1991); Q-beta amplification as described in published European Patent Application (EPA) No. 4544610; strand displacement amplification (as described in G. T. Walker et al., Clin. Chem. 42: 9-13 (1996) and European Patent Application No. 684315; and target mediated amplification, as described by PCT Publication WO 9322461.
  • NASBA so-called “NASBA” or “3SR” technique described in PNAS USA 87: 1874-1878 (1990) and also described in Nature 350 (No. 6313): 91-92 (1991); Q-beta amplification as described in published European Patent Application (EPA) No. 4544610; strand displacement amplification (as described in G. T. Walker et al
  • In situ hybridization visualization may also be employed, wherein a radioactively labeled antisense RNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography.
  • the samples may be stained with haematoxylin to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows the developed emulsion.
  • Non-radioactive labels such as digoxigenin may also be used.
  • RNA expression can be detected on a DNA array, chip or a microarray. Oligonucleotides corresponding to a gene(s) of interest are immobilized on a chip which is then hybridized with labeled nucleic acids of a test sample obtained from a patient. Positive hybridization signal is obtained with the sample containing transcripts of the gene of interest.
  • Methods of preparing DNA arrays and their use are well known in the art. (See, for example U.S. Pat. Nos. 6,618,6796; 6,379,897; 6,664,377; 6,451,536; 548,257; U.S. 20030157485 and Schena et al.
  • Serial Analysis of Gene Expression can also be performed (See for example U.S. Patent Application 20030215858).
  • the methods of the present invention can employ solid substrates, including arrays in some preferred embodiments.
  • Methods and techniques applicable to polymer array synthesis have been described in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos.
  • Patents that describe synthesis techniques in specific embodiments include U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189, 5,889,165, and 5,959,098.
  • Nucleic acid arrays that are useful in the present invention include, but are not limited to those that are commercially available from Affymetrix (Santa Clara, Calif.) under the brand name GeneChip7. Example arrays are shown on the website at affymetrix.com.
  • the present invention also contemplates many uses for polymers attached to solid substrates. These uses include gene expression monitoring, profiling, library screening, genotyping and diagnostics. Examples of gene expression monitoring, and profiling methods are shown in U.S. Pat. Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248 and 6,309,822. Examples of genotyping and uses therefore are shown in U.S. Ser. No. 60/319,253, Ser. No. 10/013,598, and U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659, 6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other examples of uses are embodied in U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506.
  • mRNA is extracted from the biological sample to be tested, reverse transcribed, and fluorescent-labeled cDNA probes are generated.
  • the microarrays capable of hybridizing to the gene of interest are then probed with the labeled cDNA probes, the slides scanned and fluorescence intensity measured. This intensity correlates with the hybridization intensity and expression levels.
  • gene expression is measured using quantitative real time PCR.
  • Quantitative real-time PCR refers to a polymerase chain reaction which is monitored, usually by fluorescence, over time during the amplification process, to measure a parameter related to the extent of amplification of a particular sequence. The amount of fluorescence released during the amplification cycle is proportional to the amount of product amplified in each PCR cycle.
  • the present invention also contemplates sample preparation methods in certain preferred embodiments.
  • the nucleic acid sample may be amplified by a variety of mechanisms, some of which may employ PCR. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (Eds.
  • LCR ligase chain reaction
  • LCR ligase chain reaction
  • Landegren et al. Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)
  • transcription amplification Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88/10315
  • self-sustained sequence replication Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO90/06995
  • selective amplification of target polyhucleotide sequences U.S. Pat. No.
  • CP-PCR consensus sequence primed polymerase chain reaction
  • AP-PCR arbitrarily primed polymerase chain reaction
  • NABSA nucleic acid based sequence amplification
  • Other amplification methods that may be used are described in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317, each of which is incorporated herein by reference.
  • Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al. Molecular Cloning: A Laboratory Manual (2.sup.nd Ed. Cold Spring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying out repeated and controlled hybridization reactions have been described, for example, in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which are incorporated herein by reference.
  • the present invention also contemplates signal detection of hybridization between ligands in certain preferred embodiments. See, for example, U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, in provisional U.S. Patent application 60/364,731 and in PCT Application PCT/US99/06097 published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.
  • Computer software products of the invention typically include computer readable medium having computer-executable instructions for performing the logic steps of the method of the invention.
  • Suitable computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc.
  • the computer executable instructions may be written in a suitable computer language or combination of several languages. Basic computational biology methods are described in, e.g.
  • the present invention also makes use of various computer program products and software for a variety of purposes, such as probe design, management of data, analysis, and instrument operation. See, for example, U.S. Pat. Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.
  • the present invention may have preferred embodiments that include methods for providing genetic information over networks such as the Internet as shown in, for example, U.S. patent application Ser. No. 10/063,559, 60/349,546, 60/376,003, 60/394,574, 60/403,381.
  • the activation status of the PI3K pathway is determined using one or more gene expression products of one or more biomarkers of the PI3K pathway.
  • Said gene expression products may be nucleotide or amino acid products and can be detected using methods known in the art.
  • the activation status of the PI3K pathway is determined by assessing the activation of IGF1R, wherein activation of IGF1R is indicative of activation of the PI3K pathway. In other embodiments of the invention the activation status of the PI3K pathway is determined by assessing the activation of PKC, wherein activation of PKC is indicative of activation of the PI3K pathway. In some embodiments of the invention the activation status of the PI3K pathway is determined by assessing the expression of one or more biomarkers for the PI3K pathway disclosed in [29], the teachings of which are incorporated herein by reference.
  • the activation status of the Np63 pathway is determined using gene expression data for one or more biomarkers of the Np63 pathway.
  • at least one of said one or more biomarkers is a gene which is increased upon Np63 activation, and in other embodiments at least one of said one or more biomarkers is a gene which is decreased upon Np63 activation. Combinations of biomarkers which are increased and decreased upon Np63 activation may also be used.
  • at least one of said one or more biomarkers is a gene which is upstream of Np63 activation, while in other embodiments at least one of said one or more biomarkers is a gene which is downstream of Np63 activation.
  • expression data for said one or more biomarkers of the Np63 pathway is obtained using an oligonucleotide microarray.
  • the activation status of the Np63 pathway is determined using one or more gene expression products of one or more biomarkers of the Np63 pathway.
  • Said gene expression products may be nucleotide or amino acid products and can be detected using methods known in the art.
  • the invention also relates to a method of identifying an individual at increased risk of lung disease, comprising determining the activation status of PKC in a cytologically normal airway epithelial cell from said individual, wherein activation of PKC is indicative that said individual is at increased risk of lung disease as compared with an individual in whom PKC is not activated.
  • the invention further relates to a method of identifying an individual at increased risk of lung disease, comprising determining the activation status of IGF1R in a cytologically normal airway epithelial cell from said individual, wherein activation of IGF1R is indicative that said individual is at increased risk of lung disease as compared with an individual in whom IGF1R is not activated.
  • the invention provides an oligonucleotide array having immobilized thereon one or more probes for one or more biomarkers of the PI3K pathway, and wherein said array does not have immobilized thereon probes for other biomarkers.
  • said one or more biomarkers of the PI3K pathway are selected from the group consisting of IGF1R, PKC, the biomarkers disclosed in [29], and combinations thereof.
  • the invention also relates to a method of reducing the risk of lung disease in an individual comprising administering to an individual at risk of lung disease one or more agents (e.g., one or more agents, regimens or treatments or combinations thereof) which inhibit the PI3K pathway.
  • one or more agents e.g., one or more agents, regimens or treatments or combinations thereof
  • the PI3K pathway is activated in said individual prior to administration of said one or more agents.
  • the lung disease is lung cancer.
  • said one or more agents are administered to said individual prophylactically before the development of lung disease.
  • Airway epithelial brushings were collected from current and former smokers under suspicion of lung cancer who were undergoing diagnostic flexible bronchoscopy from four institutions—Boston University Medical Center, Boston Veterans Administration, Lahey Clinic and St. James's Hospital (previously described in [7], see demographics in Table 1 for samples used in this study). Additional brushes were collected from volunteer healthy current, former and never smokers, as well as smokers with COPD, who were undergoing bronchoscopy (some previously published in [31], demographics in Table 1 and 2).
  • Cytologically normal airway epithelial samples from patients with dysplastic airway lesions at the University of British Columbia were collected before and after 2-3 months of treatment with myo-inositol (n 20, 10 patients with two samples each), as well as six additional samples collected pre-treatment with myo-inositol as part of a dose response study.
  • bronchial brushing was performed in three separate 6-8th generation bronchial airways using a 1.7 mm diameter bronchial cytology brush (Hobbs Medical, Stafford Springs, Conn.). The brush was retrieved and immediately immersed in RNALater and kept frozen at ⁇ 80° C. until assayed.
  • Epithelial cell content of representative bronchial brushing samples has been quantitated by cytocentrifugation (ThermoShandon Cytospin, Pittsburgh, Pa.) of the cell pellet and staining with a cytokeratin antibody (Signet, Dedham Mass.).
  • the cells in the bronchial brush contained >90% bronchial epithelial cells. At least 1 ⁇ g of each sample were later hybridized to Affymetrix Human Exon ST microarrays according to the manufacturer's protocol. Data from exon arrays were normalized using RMA-sketch in the Affymetrix Expression Console software.
  • Airway samples collected prospectively for biochemical validation were snap frozen in liquid nitrogen. For a subset of patients, additional brushes were collected and hybridized to Affymetrix HG U133A 2.0 chips. Microarrays were MAS5.0 normalized in Affymetrix Expression Console.
  • adenovirus constructs for key members of a specific pathway (e.g., p110, the catalytic subunit of PI3K) in order to activate the pathway of interest.
  • a gene signature for each pathway was defined by selecting 200 probesets based on correlation with the class variable (e.g., perturbed vs. GFP control). Training of the metagene model was accomplished using the perturbed and GFP control samples, by first summarizing the pathway signature in the training data using the most dominant component from singular value decomposition (SVD), and then using Bayesian fitting of a probit regression model. This was done for each of the pathways, and each model was applied to samples of interest. Resulting pathway probabilities were scaled between zero and one. To determine whether an oncogenic pathway was differentially activated, first a rank-sum test was performed, and for p-values less than 0.05, a random permutation analysis was performed.
  • class variable e.g., perturbed vs. GFP control
  • gene identifiers in the dataset of interest were randomized, and a p-value from a Wilcoxon rank sum test was calculated to measure differential activation between class variables (e.g., lung cancer vs. no lung cancer). This was repeated 1,000 times.
  • Microarray data was initially preprocessed by RMA normalizing, and then corrected for batch effects using DWD [43]. Specifically, to standardize expression data in the development of metagene models, DWD was applied to correct batch effects between the oncogenic pathway signature microarray samples, and bronchial airway microarray samples.
  • Gene Set Enrichment Analysis [30] was calculated using GSEA v2.
  • ⁇ 0 is the intercept
  • ⁇ 1 measures lung cancer risk (whether the sample has dysplasia or not)
  • ⁇ 2 is the cumulative cigarette smoke exposure for each person (pack-years)
  • b is a random effect correcting for batch differences
  • ⁇ is the error term.
  • the coefficient of ⁇ 1 was used to rank the genes for GSEA.
  • the GSEA analysis done in the U133A airway dataset used the default signal to noise ranking. 100 gene set permutations were used to calculate FDR.
  • BEAS-2B, BT549, or HEK293 cells were starved in BEBM, RPMI, or DMEM medium, respectively (Clonetics, GibcoBRL).
  • This media contained either 0.1% added supplements for the BEAS-2B cells or 0.1% fetal bovine serum for BT549 and HEK293 cells for 24 hrs.
  • Cells were then pretreated with increased concentrations of myo-inositol (Sigma), or LY294002 (Sigma) for 16 hrs. at 37° C. Prior stimulation, the cells were treated with fresh drugs for another 30 min, then 500 uM of insulin (SIGMA) were added for 15 minutes at 37° C.
  • SIGMA serum uM of insulin
  • the cells were lysed in RIPA buffer (20 mM TRIS (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 1 mM EDTA, 0.1% SDS) containing 0.1 mM sodium orthovanadate, 2 mM PMSF, 100 uM protease inhibitors (Sigma). Lysates were centrifuged at 14000 rpm for 20 minutes at 4° C. and incubated with monoclonal anti-p85 PI3K (Santa Cruz) antibody for 1 hr at 4° C.
  • RIPA buffer 20 mM TRIS (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 1 mM EDTA, 0.1% SDS
  • Lysates were centrifuged at 14000 rpm for 20 minutes at 4° C. and incubated with monoclonal anti-p85 PI3K (Santa Cruz) antibody for 1 hr at
  • the bounded proteins were precipitated with 50 ul of 50% slurry protein G Sepharose (Sigma) and washed three times with lysis buffer, three times with buffer containing 0.1 mM Tris (pH 7.4), 5 mM LiCl, 0.1 mM sodium orthovanadate, and two times with buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.1 mM sodium orthovanadate.
  • the beads were washed in kinase buffer (50 mM Tris (pH 7.4), 10 mM MgCl 2 ) containing 20 uM cold ATP (Sigma), and resuspended in 45 ul of kinase buffer containing 5 ul of L-a-phosphatidylinositol-4,5-bisphosphate (Avanti Polar Lipids) (1 mg/ml), and 20 uCi ATP (32-P) for 20 minutes at RT.
  • the reactions were stopped by addition of 100 ul 1N HCl, and the lipids were extracted with 160 ul of CHCl3/MeOH (1:1).
  • the phosphorylated products were separated by TLC on Silica 60 plates pretreated with potassium oxalate in a CHCl3/MeOH/NH4 solution (45:35:1.5).
  • the production of PIP3 was evaluated by autoradiography and quantified by densitometry analysis and scintillation analysis. All experiments for each cell line were repeated at least twice with similar results.
  • Cell extracts from bronchoscopy brushes were prepared by adding 200 ul of RIPA buffer. To facilitate the detachment of the cells from the brushes, the tubes were vortexed three times for 5 seconds. Both cell and bronchoscopy brushing extracts were centrifuged at 14000 rpm for 20 minutes at 4° C. and the pellets discarded. The protein yield was quantified by Bradford assay, and equivalent amount of protein was loaded to 7% SDS-PAGE gels.
  • the membrane were blocked for 1 h in blocking buffer (Tris buffer saline containing 0.1% Tween20 and 2.5% BSA, or Tris buffer saline containing 0.1% of Tween 20 and 5% low fat milk), and placed in primary antibody (Tris buffer saline containing 0.1% Tween 20 and 2.5% BSA, 0.02% sodium azide) overnight at 4° C.
  • blocking buffer Tris buffer saline containing 0.1% Tween20 and 2.5% BSA, or Tris buffer saline containing 0.1% of Tween 20 and 5% low fat milk
  • primary antibody Tris buffer saline containing 0.1% Tween 20 and 2.5% BSA, 0.02% sodium azide
  • the primary antibodies used in this study are the followed: rabbit phospho-PKC (pan)( ⁇ II Ser660) ratio 1:100 (Cell Signaling Techn.); rabbit phospho-IGF-I Receptor ⁇ (Tyr1131)/Insulin Receptor ⁇ (Tyr 1146) ratio 1:100 (Cell Signaling Techn.); rabbit phospho-PLC ⁇ 1 (Tyr783), ratio 1:500 (Cell Signaling Techn.); rabbit phospho-AKT (Ser473) ratio 1:100 (Cell Signaling Techn.); goat PI3-Kinase p110 ⁇ (C17) ratio 1:50 (Santa Cruz), rabbit phospho-ERK ratio 1:100 (Cell Signaling) rabbit GAPDH, ratio 1:1000 (AbCam).
  • Nitrocellulose were washed three times in Tris buffer saline containing 0.1% Tween 20 and/or 0.1% NP-40. Primary antibody was detected using horseradish peroxidase-linked secondary antibody and visualized with the ECL Plus Western Blot Detection system (GE Healthcare).
  • Oncogenic pathway signatures [13] were experimentally derived by activating a pathway via expression of a specific oncogene in primary human epithelial cells. A gene expression signature was then defined by identifying which genes are altered following pathway activation, and used to predict pathway activity in other in vivo samples.
  • oncogenic pathway activation probabilities for seven signaling pathways were calculated for the bronchial airway epithelial of current and former smokers with suspicion for lung cancer[7]. It is important to note that although approximately half of these patients were ultimately diagnosed with primary lung cancer (the remainder were found to have alternate lung pathologies), the brushings collected from the proximal mainstem bronchus (i.e. not adjacent to the tumor or lung lesion), were cytologically normal and were >90% epithelial. Thus, a priori one would not expect differential oncogenic pathway activity in the normal airway of smokers with lung cancer.
  • PI3K Activation is not Significantly Correlated to Cumulative Smoke Exposure or COPD
  • PI3K is Activated in Lung Cancer Tissue
  • PI3K enzymatic activity was measured in a prospectively collected cohort of cytologically-normal airway epithelial samples from subjects undergoing bronchoscopy for clinical suspicion of lung cancer. Samples were independently obtained from Boston University Medical Center and University of Utah Hospital between October 2007 and June 2008. Subjects were followed post-bronchoscopy until a final diagnosis of lung cancer or an alternate lung pathology was made. Importantly, subjects without lung cancer had a range of other pathologies, including metastatic cancer of non-lung origin, sarcoidosis, septic emboli, and pneumonia. Following protein extraction from the airway brushings, a PI3K kinase assay was performed. Based on our genomic predictions, we would expect a majority of samples from patients with cancer to have high PI3K activity and only a minority of the samples from patients with alternative pathologies to have high PI3K activity.
  • PI3K showed increased activation in the majority of patients with lung cancer (70% of the lung cancer samples in the genomic analysis were in the top half of PI3K activity), as compared to patients without lung cancer (30%).
  • the high-risk cohort was hybridized to Affymetrix Human Exon arrays. Due to platform differences with the oncogenic pathway signatures, we were unable to use the metagene model to calculate pathway activity. Instead, GSEA was used to compare PI3K activity between the two groups, and the PI3K gene signature defined by the in vitro experiments was split into two gene sets defined by the genes that go up or down with PI3K activation (PI3K_Up, PI3K_Down). Given that there were significant differences in cumulative tobacco exposure between the two groups, a linear model incorporating both batch effects and pack-years was used to rank the genes for use in GSEA.
  • GSEA analysis was again used to measure changes in the expression of genes in the PI3K pathway.
  • the decrease in airway PI3K activity seen in patients who respond to myo-inositol demonstrates that regression of dysplasia is correlated to the activity level of this pathway.
  • PI3K activity was then quantified using a standard kinase assay protocol [38, 39].
  • three different cell lines were analyzed in replicate: BEAS-2B (airway), BT549 (breast cancer) and HEK293 (embryonic kidney).
  • myo-inositol inhibited PI3K activity levels in a dose-dependent manner ( FIG. 5 ).
  • myo-inositol is an inhibitor of PI3K, and has chemoprophylactic properties associated with regression of dysplasia in airway epithelial cells.

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