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Directed Analysis Genetics Biochemistry Cell Biology Global Analysis Functional Genomics Networks High throughput data set analysis C. Rieder If you want to understand cancer, you need answers to the many questions about the role genome instability plays. ---Bert Vogelstein, 2002 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 Budding yeast cell cycle START G1 M G2 S Chromosome cycle Metaphase Anaphase Improperly attached kinetochore Spindle Checkpoint Bub1, Bub3, Mad1, Mad2, Mad3 Cohesin Separase Securin APCCdc20 1998 hBUB1 Improperly attached kinetochore Spindle Checkpoint Bub1, Bub3, Mad1, Mad2, Mad3 Cohesin Separase Securin APCCdc20 2004 hZW10, hZwilch, hRod 1998 hBUB1 Improperly attached kinetochore Spindle Checkpoint Bub1, Bub3, Mad1, Mad2, Mad3 hMRE11 Cohesin Separase 2004 hCDC4 Securin hDING APCCdc20 2004 hZW10, hZwilch, hRod 1998 hBUB1 Improperly attached kinetochore Spindle Checkpoint Bub1, Bub3, Mad1, Mad2, Mad3 hMRE11 Cohesin Separase 2004 hCDC4 Securin hDING APCCdc20 <20% of CIN mutational spectrum in colon cancer Chromosome Transmission Fidelity (ctf) Screen Colony Sectoring Assay non-essential Chromosome Fragment M SUP11 + White colony (10-5) wt EMS mutagenesis ctf mutant Sectored colony (10-3) 138 mutants, ~50 genes Summary of the 24 Cloned ctf Mutants ctf 1 3 4 5 6 7 8 10 11 12 13 14 15 17 18 19 s3 s127 s138 s141 s143 s155 s165 s166 # alleles 30 11 8 5 5 5 3 3 3 3 1 1 1 2 3 2 1 1 1 1 1 1 1 1 Gene Name CTF1/CHL1 CTF3 CTF4/CHL15/POB1 CTF5/MCM21 CTF6/RAD61 CTF7/ECO1 CTF8 CDC6 PDS5 CTF12/SCC2 CTF13 CTF14/NDC10 CTF15/RPB4 CTF17/MCM17/CHL4 CTF18/CHL12 CTF19 BIM1 SIC1 SPT4 NUP170 MAD1 MCM16 SCC3 SMC1 Essential? Yes Yes Yes Yes Yes Yes Yes Yes Function Cohesion (helicase) Kinetochore protein Cohesion (establishment) Kinetochore protein Cohesion Cohesion (establishment) Cohesion (alternative RFC) DNA replication Cohesion Cohesion Kinetochore protein (CBF3) Kinetochore protein (CBF3) Subunit of RNA polymerase II Kinetochore protein Cohesion (alternative RFC) Kinetochore protein Microtubule binding Cdk inhibitor Chromatin structure Nucleoporin Spindle chkpt. / kinetochore Kinetochore protein Cohesin subunit Cohesin subunit Kinetochore proteins Cohesion DNA /RNA metabolism 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 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 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 96 well plate frozen glycerol stock pin 96 strains onto G418 plates condense 4 plates onto 1 Yeast as a tool to discover drugs and their mechanism of action Identifying “orphan drug” targets via drug induced haploinsufficiency in yeast heterozygotes Identification of natural compounds as potential anti-cancer agents Metastasis requires invasion of adjacent tissue by tumour cells Development of cell-based screens for antiinvasive compounds Michel Roberge, Raymond Andersen Lianne McHardy, Cal Roskelley N N H NH2 Motuporamine C N N H NH2 DihydroMotuporamine C Xestospongia exigua from outer reef off Motupore Island, Papua New Guinea Motuporamine C inhibits angiogenesis in vivo Photographs 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) 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 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 Genet 21, 278-83. (1999) Drug-Induced Haploinsufficiency Drug Y/Y y∆/Y Y Y Y Alive Alive Y Y Drug Y Alive Dead Can these techniques identify the target or targetted pathways of a drug with an unknown mechanism? Can they predict the target in human cells? 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 dhMotC affects yeast growth in liquid culture Screen with or without 60 µM dhMotC identification of strains showingincreasedsensitivit 8 strains in duplicate Treatment: DMSO dhMotC Heterozygous deletion strains sensitive to dhMotC ORF NAME Biological Process YCL034W LSB5 actin filament organization YNL314W DAL82 allantoin catabolism and transcription initiation from YML099CARG81 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 YHR147CMRPL6 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 Which are more relevant? * essential genes Ranking of strain sensitivity in liquid culture using low dhMotC concentration (20 µM) Supersensitive strains (Integrated Growth Curve Difference >2) Dihydrosphingosine rescues growth inhibition by dhMotC CSG2 deletion rescues growth inhibition by dhMotC dhMotC reduces cellular ceramide levels 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