Cancer drug targets 2013
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Cancer drug targets 2013 Cancer drug targets 2013 Presentation Transcript

  • Cancer Drug Targets Jesse Liang, Ph.D. 1 Sample & Assay Technologies
  • Drug – Body Interactions When we talk about drug targets (direct or indirect), we not only talk about the gene expression, but also the mutation and the epigenetic status of these targets. All of these factors, together with the drug activity itself, affect the efficacy of a therapeutics. -2- Sample & Assay Technologies
  • Today’s Agenda 1. Overview the actively investigated cancer drug target genes . . . 2. Profile key cancer-related gene expressions 3. Detect gene mutations 4. Analyze the histone modifications of key cancer drug target genes . -3- Sample & Assay Technologies
  • Overview the Key Cancer Drug Target Genes (17 Groups) Growth Factors & Receptors: EGFR, ERBB2, ERBB3, ERBB4, FIGF, FLT1, FLT4, IGF1, IGF1R, IGF2, KDR, KIT, PDGFRA, PDGFRB. Hormone Receptors: ESR1, ESR2, PGR. Protein Kinases: AURKA, AURKB, AURKC, PLK1, PLK2, PLK3, PLK4, PRKCA, PRKCB, PRKCD, PRKCE. Receptor Tyrosine Kinase Signaling: AKT1, AKT2, GRB2. PI3K Pathway: MTOR, PIK3C2A, PIK3C3, PIK3CA. G Protein Signaling: RHOA, RHOB. Ras: HRAS, KRAS, NRAS. Apoptosis: BCL2, BIRC5. Cell Cycle: CDK1, CDC25A, CDK2, CDK4, CDK5, CDK7, CDK8, CDK9, MDM2, MDM4, TERT. Cathepsins: CTSB, CTSD, CTSL1, CTSS. Heat Shock Proteins: HSP90AA1, HSP90B1. Topoisomerases, Type II: TOP2A, TOP2B. Transcription Factors: ATF2, HIF1A, IRF5, NFKB1, TP53. Histone Deacetylases (HDAC): HDAC1, HDAC11, HDAC2, HDAC3, HDAC4, HDAC6, HDAC7, HDAC8. Poly ADP-Ribose Polymerases (PARP): PARP1, PARP2, PARP4, TNKS. Drug Metabolism: ABCC1, GSTP1, PTGS2 (COX2), TXN, TXNRD1. Structural Protein: NTN3. . . -4- Sample & Assay Technologies
  • Our Way of Profiling Gene Expressions – Using RT2 Profiler PCR Array 84 Pathway-Specific Genes of Interest 5 Housekeeping Genes Genomic DNA Contamination Control Reverse Transcription Controls (RTC) n=3 Positive PCR Controls (PPC) n=3 -5- Sample & Assay Technologies
  • Simple Workflow of PCR Arrays cDNA Synthesis (kit) 45 minutes Load Plates (Use 8-Channel Pipettors) 2 minutes Run 40 cycle qPCR Program 2 to 2.5 hours compatible with all major real-time PCR instruments Upload and Analyze Data 15 minutes PCR Array System = PCR Array Plate + cDNA Synthesis Kit + Master Mix -6- Sample & Assay Technologies
  • Profile Key Cancer Drug Target Genes We provide over 150 different PCR Arrays. . . Cancer Drug Targets PCR Array http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-507A.html Growth Factors & Receptors: EGFR, ERBB2, ERBB3, ERBB4, FIGF, FLT1, FLT4, IGF1, IGF1R, IGF2, KDR, KIT, PDGFRA, PDGFRB. Hormone Receptors: ESR1, ESR2, PGR. Protein Kinases: AURKA, AURKB, AURKC, PLK1, PLK2, PLK3, PLK4, PRKCA, PRKCB, PRKCD, PRKCE. Receptor Tyrosine Kinase Signaling: AKT1, AKT2, GRB2. PI3K Pathway: MTOR, PIK3C2A, PIK3C3, PIK3CA. G Protein Signaling: RHOA, RHOB. Ras: HRAS, KRAS, NRAS. Apoptosis: BCL2, BIRC5. Cell Cycle: CDK1, CDC25A, CDK2, CDK4, CDK5, CDK7, CDK8, CDK9, MDM2, MDM4, TERT. Cathepsins: CTSB, CTSD, CTSL1, CTSS. Heat Shock Proteins: HSP90AA1, HSP90B1. Topoisomerases, Type II: TOP2A, TOP2B. Transcription Factors: ATF2, HIF1A, IRF5, NFKB1, TP53. Histone Deacetylases: HDAC1, HDAC11, HDAC2, HDAC3, HDAC4, HDAC6, HDAC7, HDAC8. Poly ADP-Ribose Polymerases (PARP): PARP1, PARP2, PARP4, TNKS. Drug Metabolism: ABCC1, GSTP1, PTGS2 (COX-2), TXN, TXNRD1. Structural Protein: NTN3. . . -7- Sample & Assay Technologies
  • 3 Purposes of Using Cancer Drug Targets PCR Array 1. Screen Profile the key cancer drug target genes before and after the treatment (drugs, chemicals, siRNA, shRNA, plasmids…) to identify the targets. . . . . . . 2. Validate – After getting the results of microarrays; We also provide primers for individual genes. 3. Find other novel co-targets. -8- Sample & Assay Technologies
  • Profile Key Cancer-Related Genes with Other PCR Arrays . Cancer Pathway Finder PCR Array http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-033A.html . Cell Cycle Control & DNA Damage Repair: ATM, BRCA1, CCNE1 (cyclin E1), CDC25A, CDK2, CDK4, CDKN1A (p21Waf1), CDKN2A (p16Ink4), CHEK2 (chk2 / Rad53), E2F1, MDM2, RB1, S100A4, TP53 (p53). Apoptosis and Cell Senescence: APAF1, BAD, BAX, BCL2, BCL2L1 (bcl-X), CASP8, CFLAR (CASPER), FAS, GZMA, HTATIP2, TERT (telomerase), TNFRSF1A (TNF-a receptor), TNFRSF10B (DR5), TNFRSF25 (DR3). Signal Transduction Molecules and Transcription Factors: AKT1, ERBB2, ETS2, FOS, JUN, MAP2K1 (MEK), MYC, NFKB1 (NFκB), NFKBIA (IκBα), PIK3R1 (PI3K p85α), RAF1, SNCG. Adhesion: ITGA1 (integrin α1), ITGA2 (integrin α2), ITGA3 (integrin α3), ITGA4 (integrin α4), ITGAV (integrin αV), ITGB1 (integrin β1), ITGB3 (integrin β3), ITGB5 (integrin β5), MCAM, MTSS1, PNN, SYK, EPDR1. Angiogenesis: ANGPT1 (angiopoietin-1), ANGPT2 (angiopoietin-2), COL18A1 (endostatin), FGFR2, IFNA1 (IFNα), IFNB1 (IFNβ), IGF1, IL8, PDGFA, PDGFB, TEK (tie-2), TGFB1, TGFBR1 (ALK-5), THBS1 (thrombospondin-1), TNF, VEGFA. Invasion and Metastasis: MET, MMP1 (collagenase-1), MMP2 (gelatinase A), MMP9 (gelatinase B), MTA1, MTA2, NME1, NME4, PLAU, PLAUR, S100A4, SERPINB5 (maspin), SERPINE1 (PAI1), TIMP1, TIMP3, TWIST1. . . . . . . . . . . . . -9- Sample & Assay Technologies
  • Profile Key Cancer-Related Genes with Other PCR Arrays . Oncogenes and Tumor Suppressor Genes PCR Array http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-502A.html . Oncogenes: BAX, BCL2L1, CASP8, CDK4, ELK1, ETS1, HGF, JAK2, JUNB, JUND, KIT, KITLG, MCL1, MET, MOS, MYB, NFKBIA, NRAS, PIK3CA, PML, PRKCA, RAF1, RARA, REL, ROS1, RUNX1, SRC, STAT3, ZHX2. Tumor Suppressor Genes: ATM, BRCA1, BRCA2, CDH1, CDKN2B, CDKN3, E2F1, FHIT, FOXD3, HIC1, IGF2R, MEN1, MGMT, MLH1, NF1, NF2, RASSF1, RUNX3, S100A4, SERPINB5, SMAD4, STK11, TP73, TSC1, VHL, WT1, WWOX, XRCC1. Both Oncogenic & Tumor Suppressor Properties: BCR, EGF, ERBB2, ESR1, FOS, HRAS, JUN, KRAS, MDM2, MYC, MYCN, NFKB1, PIK3C2A, RB1, RET, SH3PXD2A, TGFB1, TNF, TP53. Transcription Factors: ABL1, BRCA1, BRCA2, CDKN2A, CTNNB1, E2F1, ELK1, ESR1, ETS1, FOS, FOXD3, HIC1, JUN, JUNB, JUND, MDM2, MEN1, MYB, MYC, MYCN, NF1, NFKB1, PML, RARA, RB1, REL, RUNX1, RUNX3, SMAD4, STAT3, TGFB1, TNF, TP53, TP73, TSC1, VHL, WT1, ZHX2. Epithelial-to-Mesenchymal Transition (EMT): BRCA2, CDKN2B, CTNNB1, ERBB2, HGF, JAK2, KIT, MCL1, NF1, RUNX3, S100A4, SMAD4, TGFB1, VHL. Angiogenesis: AKT1, CTNNB1, EGF, ERBB2, NF1, PML, RUNX1, TGFB1. Apoptosis: BAX, BCL2, BCL2L1, BRCA1, CASP8, E2F1, MCL1, MGMT, TNF, VHL. Cell Adhesion: APC, CDH1, CDKN2A, CTNNB1, KITLG, NF1, NF2, TGFB1. Cell Cycle: ATM, BRCA1, BRCA2, CCND1, CDK4, CDKN1A, CDKN2A, CDKN2B, CDKN3, E2F1, HGF, MEN1, STK11, TP53. . . . . . . . . . - 10 - Sample & Assay Technologies
  • Profile Cancer Type-Specific Genes . 1. Breast Cancer http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-131A.html . . 2. Lung Cancer http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-134A.html . . 3. Liver Cancer http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-133A.html . . 4. Leukemia http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-137A.html . . 5. Lymphoma http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-139A.html . . 6. Prostate Cancer http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-135A.html . - 11 - Sample & Assay Technologies
  • Study Gene Mutations 1. In many cases, cancer drug targets are mutated genes; or else mutated genes affect cell response to a specific drug, how to identify these gene mutations? QIAGEN® works in partnership with Amgen, Merck Serono, and BMS/Lilly/Imclone to develop global companion diagnostics for detecting KRAS mutations to support the antiEGFR therapies — Vectibix® and Erbitux® —in patients with metastatic colorectal cancer. 2. If you have known a mutant gene as a drug target, is this mutant gene expressed in your samples? - 12 - Sample & Assay Technologies
  • Principle of Cancer Mutation PCR Arrays – DNA Technology * Real-time PCR is the most sensitive and reliable method for the detection of DNA . mutations. * Our scientists have developed real-time PCR assays to detect lower than 1% somatic . mutations in the background of wild-type DNA templates. * Amplification Refractory Mutation System (ARMS®) technology is a strategy in which . primers with a mismatched 3’-residue will not function as primers in a PCR reaction. In other words, ARMS-designed primers will preferentially amplify the mutant sequence. The single nucleotide mutations are selected from comprehensive somatic mutation databases (COSMIC) and peer-reviewed scientific literature based on their clinical or functional relevance and frequency of occurrence. - 13 - Sample & Assay Technologies
  • An Example of ARMS® Template: Wild-Type AGTCAGTC Primer TCAGTAAA AGTCAGTC Won’t Work . . . Template: Mutation AGTCAGTT Primer TCAGTAAA AGTCAGTT Will Work . . . - 14 - Sample & Assay Technologies
  • Cancer Mutation PCR Array Design Design: . Cancer Mutation PCR Array is a collection of mutation specific assays for multiple mutations in a single gene, as well as mutations present in genes in downstream signaling activities. The mutations are selected from comprehensive databases (such as the COSMIC database) and literature reviews based on their clinical or functional relevance and frequency of occurrence. . . Example: EGFR Pathway Somatic Mutation PCR Array – 96 well plate • EGFR Mutations: 20 • PI3K Mutations: 7 • AKT1 Mutations: 1 • PTEN Mutations: 6 • KRAS Mutations: 16 • HRAS Mutations: 11 • NRAS Mutations: 12 • BRAF Mutations: 8 • MEK1 Mutations: 4 . - 15 - Sample & Assay Technologies
  • Different Cancer Mutation PCR Arrays and Assays – Designed to Discover and Verify Drug Target Biomarkers Custom available! (New!) We have just added esophageal cancer and head & neck cancer. We also provide over 900 individual gene assays: http://www.sabiosciences.com/mutationproductlist.php?tab=browsebygene - 16 - Sample & Assay Technologies
  • Study the Epigenetic Status of the Genes Histone modifications define chromatin structure, and correlate closely with the transcriptional activity of the associated genes. . Adding HDAC (histone deacetylase) inhibitors to a drug regimen can greatly change the cell response (e.g. The 4 . FDA-approved epigenetic drugs for clinical use include 2 DNA demethylating agents, 5azacytidine and decitabine, and 2 histone deacetylase (HDAC) inhibitors, vorinostat and valproic acid. So far, epigenetic drugs have been approved mainly for the treatment of blood cancers). . . . - 17 - Sample & Assay Technologies
  • Introduction to Histone Modification Five major families of histone exist: H1, H2A, H2B, H3, H4 and H5. Histones H2A, H2B, H3 and H4 are known as the core histones, while histones H1 and H5 are known as the linker histones. . Major modifications of histone include acetylation, methylation, phosphorylation, ubiquitination, and sumoylation. Combinations of histone modifications constitute a code, the so-called “histone code”. . H3K4me1 means the monomethylation of the 4th residue (a lysine) from the start (i.e., the Nterminal) of the histone H3 protein. . The common nomenclature of histone modifications is: 1. The name of the histone (e.g., H3) 2. The single-letter amino acid abbreviation (e.g., K for lysine) and the amino acid position in the protein 3. The type of modification (me: methyl, p: phosphate, ac: acetyl, ub: ubiquitin) . . . . - 18 - Sample & Assay Technologies
  • An Example Experiment with ChIP PCR Array Together with our easy and fast EpiTect One-Day ChIP Kit (http://www.sabiosciences.com/chipqpcr_product/HTML/334471.html), ChIP-Grade Antibody Kit (http://www.sabiosciences.com/chipgradeantibody.php), one million cells per assay as starting material, ChIP PCR Arrays provide 100% effective call rates. . . P19 mouse embryonic carcinoma cells were prepared for ChIP Assay using the EpiTect OneDay ChIP Kit and anti-H3K4me3 Antibody Kit. One million cells were used as starting material for each ChIP Assay. The purified ChIP DNA samples were characterized using Mouse Stem Cell Transcription Factor ChIP PCR Array with 1/100th of the ChIP DNA as template in each well. The Real-Time PCR results demonstrate 100 % effective call rates for the Input Fraction (Ct < 30). The difference of Ct value between the anti-H3K4me3 antibody and the control IgG fractions indicates the specific enrichment of the antibody, whereas the high Ct value of the control IgG fraction indicates the low background of the assay. . - 19 - Sample & Assay Technologies
  • Analyze Cancer Drug Target Genes with ChIP PCR Array Growth Factors & Receptors: EGFR, ERBB2, ERBB3, ERBB4, FIGF, FLT1, FLT4, IGF1, IGF1R, IGF2, KDR, KIT, PDGFRA, PDGFRB. Hormone Receptors: ESR1, ESR2, PGR. Protein Kinases: AURKA, AURKB, AURKC, PLK1, PLK2, PLK3, PLK4, PRKCA, PRKCB, PRKCD, PRKCE. Receptor Tyrosine Kinase Signaling: AKT1, AKT2, GRB2. PI3K Pathway: MTOR, PIK3C2A, PIK3C3, PIK3CA. G Protein Signaling: RHOA, RHOB. Ras: HRAS, KRAS, NRAS. Apoptosis: BCL2, BIRC5. Cell Cycle: CDK1, CDC25A, CDK2, CDK4, CDK5, CDK7, CDK8, CDK9, MDM2, MDM4, TERT. Cathepsins: CTSB, CTSD, CTSL1, CTSS. Heat Shock Proteins: HSP90AA1, HSP90B1. Topoisomerases, Type II: TOP2A, TOP2B. Transcription Factors: ATF2, HIF1A, IRF5, NFKB1, TP53. Histone Deacetylases: HDAC1, HDAC11, HDAC2, HDAC3, HDAC4, HDAC6, HDAC7, HDAC8. Poly ADP-Ribose Polymerases: PARP1, PARP2, PARP4, TNKS. Drug Metabolism: ABCC1, GSTP1, PTGS2, TXN, TXNRD1. Structural Protein: NTN3. . . - 20 - Sample & Assay Technologies
  • Other ChIP PCR Arrays Please Note: Our cataloged ChIP PCR Arrays (http://www.sabiosciences.com/chipqpcrarrays.php) are for histone modification study only. If you are interested in transcription factor binding sites or other nuclear factor binding sites, please order custom-designed ChIP PCR Arrays from us. - 21 - Sample & Assay Technologies
  • We Provide Service – Send Samples to Us & Receive Results! Whole Genome Illumina Gene Expression Profiling Illumina Genotyping . Pathway / Focused Panel PCR Array miRNA PCR Array (special miRNome Profiling service for US customers in May!) http://www.sabiosciences.com/promotion/servicecoremirnapromo.php Mutation Profiling Methylation . . . . . . Individual Gene / Locus Real-time PCR Mutation Detection Methylation . . . Sample Preparation – DNA, RNA Extraction and Purification Cells, Tissue or Biofluids Fixed Tissue Small Sample . . . . - 22 - Sample & Assay Technologies
  • Contact Information . . . . . Jesse Liang Email: jesse.liang@qiagen.com Technical Support: 1-888-503-3187 Email: support@sabiosciences.com Check Webinar Calendar: http://www.sabiosciences.com/seminarlist.php . - 23 - Sample & Assay Technologies