Directed Analysis
GeneticsBiochemistryCell Biology
Functional GenomicsNetworks High throughput data set analysis
Global Analysis
C. Rieder
If you want to understand cancer, you need answersto 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
G1
SG2
M
START
Budding yeast cell cycle Budding yeast cell cycle Chromosome cycle Chromosome cycle
Metaphase Anaphase
Spindle Checkpoint
APCCdc20SecurinSeparase
Cohesin
Improperly attached kinetochore
Bub1, Bub3, Mad1, Mad2, Mad3
Spindle Checkpoint
APCCdc20SecurinSeparase
Cohesin
Improperly attached kinetochore
Bub1, Bub3, Mad1, Mad2, Mad3
hBUB11998
Spindle Checkpoint
APCCdc20SecurinSeparase
Cohesin
Improperly attached kinetochore
Bub1, Bub3, Mad1, Mad2, Mad3
hZW10, hZwilch, hRod
hDING
hMRE11
hBUB1
hCDC4
1998
2004
2004
Spindle Checkpoint
APCCdc20SecurinSeparase
Cohesin
Improperly attached kinetochore
Bub1, Bub3, Mad1, Mad2, Mad3
hZW10, hZwilch, hRod
hDING
hMRE11
hBUB1
hCDC4
1998
2004
2004<20% of CIN mutationalspectrum in colon cancer
non-essential Chromosome Fragment
M SUP11
+
Colony Sectoring Assay
Chromosome Transmission Fidelity (ctf) Screen
EMS mutagenesis
ctf mutant Sectored colony
(10-3)
wt
White colony
(10-5)
138 mutants, ~50 genes
ctf # alleles Gene Name Essential? Function1 30 CTF1/CHL1 Cohesion (helicase)3 11 CTF3 Kinetochore protein4 8 CTF4/CHL15/POB1 Cohesion (establishment)5 5 CTF5/MCM21 Kinetochore protein6 5 CTF6/RAD61 Cohesion7 5 CTF7/ECO1 Yes Cohesion (establishment)8 3 CTF8 Cohesion (alternative RFC)
10 3 CDC6 Yes DNA replication11 3 PDS5 Yes Cohesion12 3 CTF12/SCC2 Yes Cohesion13 1 CTF13 Yes Kinetochore protein (CBF3)14 1 CTF14/NDC10 Yes Kinetochore protein (CBF3)15 1 CTF15/RPB4 Subunit of RNA polymerase II17 2 CTF17/MCM17/CHL4 Kinetochore protein18 3 CTF18/CHL12 Cohesion (alternative RFC)19 2 CTF19 Kinetochore proteins3 1 BIM1 Microtubule binding
s127 1 SIC1 Cdk inhibitors138 1 SPT4 Chromatin structures141 1 NUP170 Nucleoporins143 1 MAD1 Spindle chkpt. / kinetochores155 1 MCM16 Kinetochore proteins165 1 SCC3 Yes Cohesin subunits166 1 SMC1 Yes Cohesin subunit
Kinetochore proteins
Cohesion
DNA /RNA metabolism
Summary of the 24 Cloned ctf Mutants
What are all the genes “mutable” to CIN?
Essential vs. non-essentialDominant vs. recessiveRedundant vs. non-redundant
Karen Yuen UBC
Forrest Spencer Johns HopkinsCheryl Dunbar Warren
Global screens for candidate CIN proteins
Gene deletion setGenetic interaction screening Synthetic lethals
Synthetic dosage lethalsHaploinsufficiency 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 platefrozen glycerol stock
condense 4 plates onto 1
pin 96 strains onto G418 plates
Yeast as a tool to discover drugs and their mechanism of action
Identifying “orphan drug” targets via drug induced haploinsufficiency in yeast heterozygotes
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
Identification of natural compounds as potential anti-cancer agents
N NH
NH2
Motuporamine C
Xestospongia exiguafrom outer reef offMotupore Island, Papua New Guinea
N NH
NH2
DihydroMotuporamine C
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)
– 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
How to identify the mechanism of action of motuporamines?
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
Y/Y
y∆/Y
Y Y Y Y
YY
Alive
Alive
Alive
Dead
Drug
Drug
Can these techniques identify the target or targetted pathways of a drug with an unknown mechanism?
Can they predict the target in human cells?
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
Steps of drug-induced haploinsufficiency screen
dhMotC affects yeast growth in liquid culture
Screen with or without 60 µM dhMotC identification of strains showingincreasedsensitivity
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Treatment: DMSO dhMotC
8 strainsin duplicate
Heterozygous deletion strains sensitive to dhMotC
ORF NAME Biological Process
YCL034W LSB5 actin filament organizationYNL314W DAL82 allantoin catabolism and transcription initiation from YML099C ARG81 arginine metabolismYBR078W ECM33 cell wall organization and biogenesisYNL267W* PIK1 * cytokensis, post Golgi transport and signal transduction YLR286C CTS1 cytokinesis, completion of separationYDL192W ARF1 ER to golgi transport and intra-golgi transportYBR290W BSD2 heavy metal ion transport and protein-vacuolar targeting YLR025W SNF7 late endosome to vacuole transportYHR147C MRPL6 protein biosynthesis YOL040C* RPS15 * protein biosynthesis YAL005CSSA1 protein folding and protein-nucleus import, translocation YIL047C
SYG1 signal transductionYBR265W* TSC10 * sphingolipid biosynthesisYMR296C* LCB1 * sphingolipid biosynthesisYJR007W* SUI2 * translation initiationYML092C* PRE8 * ubiquitin-dependent protein catabolismYER140W YER140W unknownYER188W YER188W unknownYGR205W YGR205W unknownYLR294C YLR294C unknown
* essential genes Which are more relevant?
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 yeastPredicting the target/ target pathway in human cells
AdvantagesSystematic, unbiased and genome-wideAdaptable to other phenotypes.Pathway conservation = physiological phenotypeDevelopment of chemical probes
See: Baetz et al PNAS 2004Also: Lum et al 2004 Cell Giaver et al 2004 PNAS