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