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Directed Analysis

Directed Analysis. Global Analysis. Functional Genomics Networks High throughput data set analysis. Genetics Biochemistry Cell Biology. C. Rieder. If you want to understand cancer, you need answers

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Directed Analysis

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  1. Directed Analysis Global Analysis Functional Genomics Networks High throughput data set analysis Genetics Biochemistry Cell Biology

  2. C. Rieder If you want to understand cancer, you need answers to the many questions about the role genome instability plays. ---Bert Vogelstein, 2002

  3. Genetic Instability in Human Cancers MIN: Microsatellite instability (increased mutation rate) CIN: Chromosome instability (increased aneuploidy rate) Yeast as an experimental model CIN biology CIN candidate genes, CIN cancer genes Therapeutics

  4. START G1 M S G2 Budding yeast cell cycle Chromosome cycle

  5. Metaphase Anaphase

  6. Spindle Checkpoint Improperly attached kinetochore Bub1, Bub3, Mad1, Mad2, Mad3 Cohesin APCCdc20 Separase Securin

  7. 1998 hBUB1 Spindle Checkpoint Improperly attached kinetochore Bub1, Bub3, Mad1, Mad2, Mad3 Cohesin APCCdc20 Separase Securin

  8. 2004 hZW10, hZwilch, hRod 1998 hBUB1 Spindle Checkpoint Improperly attached kinetochore Bub1, Bub3, Mad1, Mad2, Mad3 hMRE11 Cohesin APCCdc20 Separase Securin 2004 hDING hCDC4

  9. <20% of CIN mutational spectrum in colon cancer 2004 hZW10, hZwilch, hRod 1998 hBUB1 Spindle Checkpoint Improperly attached kinetochore Bub1, Bub3, Mad1, Mad2, Mad3 hMRE11 Cohesin APCCdc20 Separase Securin 2004 hDING hCDC4

  10. Chromosome Transmission Fidelity (ctf) Screen White colony (10-5) wt EMS mutagenesis Sectored colony ctf mutant (10-3) Colony Sectoring Assay non-essential Chromosome Fragment SUP11 M + 138 mutants, ~50 genes

  11. Summary of the 24 Cloned ctf Mutants Kinetochore proteins Cohesion DNA /RNA metabolism

  12. What are all the genes “mutable” to CIN? Essential vs. non-essential Dominant vs. recessive Redundant vs. non-redundant Karen Yuen UBC Forrest Spencer Johns Hopkins Cheryl Dunbar Warren

  13. Global screens for candidate CIN proteins Gene deletion set Genetic interaction screening Synthetic lethals Synthetic dosage lethals Haploinsufficiency modifiers Direct phenotype screening Genome instability assays Systematic Two-hybrid Protein complexes/ mass spectrometry

  14. S. cerevisiae Genome Deletion Project • “Complete” set of yeast nonessential deletion mutants • ~4,700 haploid strains • ~4,700 homozygous diploid strains • nonessential genes deleted with kanMX = fifty 96 well plate • ~6,000 heterozygous diploid strains pin 96 strains onto G418 plates condense 4 plates onto 1 96 well plate frozen glycerol stock

  15. Yeast as a tool to discover drugs and their mechanism of action Identifying “orphan drug” targets via drug induced haploinsufficiency in yeast heterozygotes

  16. Identification of natural compounds as potential anti-cancer agents Metastasis requires invasion of adjacent tissue by tumour cells Development of cell-based screens for anti-invasive compounds Michel Roberge, Raymond Andersen Lianne McHardy, Cal Roskelley

  17. N N N H 2 H Motuporamine C N N N H 2 H DihydroMotuporamine C Xestospongia exigua from outer reef off Motupore Island, Papua New Guinea

  18. Motuporamine C inhibits angiogenesis in vivoPhotographs of developing CAMs incubated for 2 days with VEGF (A) or VEGF and motuporamine C at 2.5 µM (B), 5 µM (C) or 10 µM (D)

  19. How to identify the mechanism of action of motuporamines? • Motuporamines are anti-invasive and anti-angiogenic compounds with apparent in vivo activity • they are attractive drug candidates • Invasion is a complex process, incompletely understood • Structure of motuporamines gives no clue to function

  20. Yeast “chemical genomics” approaches to identify drug targets • Drug-induced haploinsufficiency screen: • Collection of heterozygous diploid yeast strains in which one allele of every gene is individually deleted • Lowering the dosage of a gene from two copies to one • usually results in a strain that is sensitized to drugs • that act on the product of this gene • Proof of principle study: • Giaever et al.. Nat Genet21, 278-83. (1999)

  21. Drug-Induced Haploinsufficiency Drug Y Y Y Y Alive Alive Y/Y Drug Alive Dead y∆/Y Y Y Can these techniques identify the target or targetted pathways of a drug with an unknown mechanism?Can they predict the target in human cells?

  22. Steps of drug-induced haploinsufficiency screen • 1- selection of a drug-induced phenotype • 2- systematic high-throughput drug-induced phenotypic screen of yeast heterozygous deletion diploid set • 3- quantitative ranking of drug sensitivity - PRIORITIZATION • 4- confirmation of drug mode of action in yeast • 5- assessment of cognate mode of action in the mammalian system

  23. dhMotC affects yeast growth in liquid culture

  24. Screen with or without 60 µM dhMotC identification of strains showingincreasedsensitivity 8 strains in duplicate Treatment: DMSO dhMotC

  25. Heterozygous deletion strains sensitive to dhMotC ORF NAME Biological Process YCL034W LSB5 actin filament organization YNL314W DAL82 allantoin catabolism and transcription initiation from YML099C ARG81 arginine metabolism YBR078W ECM33 cell wall organization and biogenesis YNL267W* PIK1 * cytokensis, post Golgi transport and signal transduction YLR286C CTS1 cytokinesis, completion of separation YDL192W ARF1 ER to golgi transport and intra-golgi transport YBR290W BSD2 heavy metal ion transport and protein-vacuolar targeting YLR025W SNF7 late endosome to vacuole transport YHR147C MRPL6 protein biosynthesis YOL040C* RPS15 * protein biosynthesis YAL005C SSA1 protein folding and protein-nucleus import, translocation YIL047C SYG1 signal transduction YBR265W* TSC10 * sphingolipid biosynthesis YMR296C* LCB1 * sphingolipid biosynthesis YJR007W* SUI2 * translation initiation YML092C* PRE8 * ubiquitin-dependent protein catabolism YER140W YER140W unknown YER188W YER188W unknown YGR205W YGR205W unknown YLR294C YLR294C unknown * essential genes Which are more relevant?

  26. Ranking of strain sensitivity in liquid culture using low dhMotC concentration (20 µM)

  27. Supersensitive strains (Integrated Growth Curve Difference >2)

  28. Dihydrosphingosine rescues growth inhibition by dhMotC

  29. CSG2 deletion rescues growth inhibition by dhMotC dhMotC reduces cellular ceramide levels

  30. Drug-induced haploinsufficiency Determining drug mode of action in yeast Predicting the target/ target pathway in human cells Advantages Systematic, unbiased and genome-wide Adaptable to other phenotypes. Pathway conservation = physiological phenotype Development of chemical probes See: Baetz et al PNAS 2004 Also: Lum et al 2004 Cell Giaver et al 2004 PNAS

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