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High throughput functional
genomics approaches to identifying novel drivers of melanoma
Brian Gabrielli
Cell Cycle Group
ARVEC
Functional genomics screens
Unbiased approaches to identify new genes and pathways
gene expression phenotype gene function
(↓siRNA, shRNA) (↑cDNA, ORF) (Cell-based assays) (therapeutic targets)
sequence function
cells
Gene vectors: ORF, siRNA
ASSAY: add drugs, change growth conditions, etc.
LABEL: (antisera, dyes)
COLLECT AND ANALYSE DATA : Plate reader, high content imager
X ~23 000
HIGH CONTENT IMAGE ANALYSIS
ARVEC arrayed libraries Gene overexpression
~ 17 033 ORFs as Gateway entry clones
Cloned into a lentiviral expression vector by ARVEC 96-well plates
Screens
•G1 phase progression factors
•Bypass of p16-induced senescence
•ER response genes in BC (Liz Musgrove, Garvan)
•EMT genes in BC (Rik Thompson, SVI)
•Drivers of resistance to Brafi + MEKi in melanoma (Helen Rizos, WMI)
•Inhibitors of p53 induction in DBA (Ross Hannan, Peter Mac)
•Driver mutations in melanoma
•UV-induced G2 phase checkpoint and repair
ARVEC arrayed libraries Gene silencing
siRNA libraries: Dharmacon Smart Pool whole-genome
Lentiviral shRNA libraries: (Open Biosystems) whole genome
Screens
•Ca signaling in BC (Greg Monteith, UQ)
•Synthetic lethality with HPV (Nigel McMillan, UQ)
•Synthetic lethality with checkpoint defect
•G2 phase regulators (Andrew Burgess CNRS)
•miR/anti-miRs of EGFRi resistance/sensitivity (Nicole Cloonan, QIMR)
•Synthetic lethality with RA in neuroblastoma (Bellamy Cheung, CCIA)
•UV-induced G2 phase checkpoint and repair
Melanoma
• 10% Australian cancers
• 3% Australian cancer-related fatalities
• Highest incident cancer in 19-40 year old Australians
• Risk factors: UVR exposure, B-Raf, MiTF, MC1R, p16INK4A
• Treatment: chemo ineffective, B-Rafi-MEKi, immune checkpoint
drugs?
• Early diagnosis has >90% long term survival
Genome wide over expression screen to identify genes that bypass p16
induced senescence
Chuaire-Noack et al., nt. J. Morphol., 28(1):37-50, 2010
Naevus
p16 Ki-67
Melanoma
p16-induced Senescence is a Barrier to Melanoma
Experimental Approach
2 days
5 days
IPTG
p16 ↑
GFP-only lentivirus
Potential hORF “hit”
or +ve control lentivirus •CCNE1 •CDK4
• CDK4-R24C
IPTG
Imaging-based assay for: • Gene transduction (GFP) • Proliferation (Ki-67) •Cell size (-tubulin) • S and G2/M DNA content
p16 ↑
DNA (DAPI) GFP Ki-67 (Cy3) -tubulin (Cy5)
pLV
41
1 +
IPTG
C
DK
4-R
24
C +
IPTG
Note: same 10 x magnification for all fields
Assay Development
Screened a human ORFeome library (~17,000 unique ORFs) using HCS in collaboration with UQDI ARVEC Facility (www.di.uq.edu.au/arvechcsfacility).
~200 x
Primary Screen Hits
Unstained wells
HPV18E7
Secondary Screen Validated Hits
HMGB2 and CDK6 increased in melanoma
CDK6
0
3
6
9
12
H-s
co
re
HMGB2
0
3
6
9
12
H-s
co
re
How can we stratify the validated hits?
0
10
20
30
40
50
60
70
80
90
100
%RB positive (GFP cells only)
Do hits deplete RB?
Does depletion of hits affect proliferation/viability?
CDK6 Hi Lo Hi p16 Wt Methylated Mut
ACKNOWLEDGEMENTS
Gab Lab Won Jae Lee
Kelly Brooks
Sandra Pavey
Kee Ming Chia
Carly Fox
Weili Wang
Alex Pinder
Loredana Spoerri
Vanessa Oakes
Brooke Edwards
James Chen
Sheena Daignault
Alex Stevenson
Weili Wang
Fawzi Bokhari
ARVEC Facility
Tom Gonda
Duka Skalamera
Max Ranall
Mareike Dahmer
Bioinformatics
Konstantin Shakhbazov
Pamela Mukhopadhyay
Paul Leo
Collaborators
Nigel McMillan, Griffith
Nick Hayward, QIMR
Chris Schmidt, QIMR
Rick Sturm IMB
Peter Soyer UQ SOMS
Duncan Lambie PAH
Graeme Walker QIMR
Richard Scolyer MIA
Helen Rizos, Westmead
Grant McArthur PMCI
Petranel Ferrao PMCI
Functional genomics screens
- populating pathways
Unbiased approach to identify
new genes and pathways