Transcript Presentation - Broad Institute

Relating Activating K-Ras Mutations
to Small Molecule Sensitivity in NonSmall-Cell Lung Cancer
Flavian D. Brown
Carleton College
Class of 2009
Lung Cancer
 Leading cause of death from cancer in the world
 Over 90% of NSCLC contain mutations in
EGFR, BRAF and K-Ras
 Discovery of Gefitnib
Ras Signaling
←
Oncogenic
Mutation
←
Oncogenic
Mutation
←
Schubbert et al. (2007) Hyperactive Ras in developmental disorders and cancer. Nature Review of Cancer, Vol. 7 295-307.
Hypothesis
 NSCLC tumors are genetically sensitized due to
changes in cellular state secondary to activating KRas mutations.
- Different drug targets
- Oncogene Addiction
Small Molecule Screens
Pin Transfer
250
500
1000
100nl
cell adherence
24 Hrs
2-10Hrs
+ DMSO Control
48Hrs
72Hrs
Hits From Primary Screen
A549: Hits Highlighted
Color coding on the images:
Red = unbiased commercial compound Forma set
Green = bioactives (including kinase inhibiting drugs)
Magenta = HDAC biased DOS
Blue = commercial kinase biased (CBkinase)
Yellow = analyticon purified natural products
Black = DMSO control plate
Gray = +con dose plate
Assay Development
H1792
Fluorescence (535/595nm)
70000000
60000000
50000000
2hr
40000000
4hr
30000000
6hr
8hr
20000000
10000000
0
0
200
400
600
Cells/Well
800
1000
1200
Small Molecule Sensitivity
16
14
IC50 (µmol)
12
10
8
6
4
2
0
A549 (K-Ras)
H460 (K-Ras)
H1792 (K-Ras)
H1975 (EGFR)
Cell Lines
H1650 (EGFR)
H1395 (BRAF)
Small Molecule Sensitivity
18
16
IC50 (µmol)
14
12
10
8
6
4
2
0
A549 (K-Ras)
H460 (K-Ras)
H1792 (K-Ras)
H1975 (EGFR)
Cell Lines
H1650 (EGFR)
H1395 (BRAF)
Small Molecule Sensitivity
16
14
IC50 (umol)
12
10
8
6
4
2
0
A549 (K-Ras)
H460 (K-Ras)
H1792 (K-Ras)
H1975 (EGFR)
Cell Lines
H1650 (EGFR)
H1395 (BRAF)
Structural Activity Relationship
Br
O
H
N
OH
N
N
H
O
I
O
O
N
OH
N
H
N
H
O
OH
NH
N
HN
O
Aromatic group at the
opposite end of
structures
Carbon spacer can be
rigid or flexible
Hydroxamic acids
attached to a 4 or 5
carbon chain
Future Investigations
 Analyze signaling downstream of the activating
mutation
-Immunofluorescence
-Western Blotting
Target Identification
- Pull down assay
 Correlate phenotypic data with genetic data
- SNP copy number
Impact
 Genotype specific inhibitors for K-Ras
mutants
 Paradigm for investigating genotypephenotype relationships in other malignancies
- WGAS for somatic alterations
 Molecularly targeted cancer therapeutics.
Acknowledgements
Principle Investigator
-
Stuart L.Schreiber, Ph.D
Mentor
-
Gopal S. Ramachandran, Ph.D
Summer Research Program in Genomics
-
Shawna Young
Lucia Vielma
Maura L. Silverstein
Bruce Birren, Ph.D
Collaborators
Broad Institute Screening
-
-
Jordi Barretina, Ph.D
Damian W. Young, Ph.D
Nicola Tolliday, Ph.D
Josh Bittker, Ph.D
Melanie de Silva
Kate Hartland
References
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