Transcript Relating Activating K-Ras Mutations to Small Molecule Sensitivity in NonSmall-Cell Lung Cancer Flavian D.

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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Arcaro, A. The small GTP-binding protein Rac promotes the dissociation of gelsolin from actin filaments
in neutrophils. J. Biol.Chem. 273, 805–813 (1998)
Bourne, H. R., Sanders, D. A. & McCormick, F. The GTPase superfamily: a conserved switch for diverse
cell functions. Nature 348, 125–132 (1990).
Diaz et al. Complex effects of Ras proto-oncogenes in tumorigenesis. Carcinogenesis, Vol. 25, No. 4, 535539 (2004).
Downward, J. Targeting RAS signaling pathways in cancer therapy. Nature Rev. Cancer 3, 11–22 (2003).
Gibbs, J. B. & Oliff, A. The potential of farnesyltransferase inhibitors as cancer chemotherapeutics. Annu.
Rev. Pharmacol. Toxicol. 143–166 (1997).
Herrmann, C. Ras–effector interactions: after one decade. Curr. Opin. Struct. Biol. 13, 122–129 (2003)
Lynch et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of
non-small-cell lung cancer to gefitinib. The New England Journal of Medicine. Vol. 350 No.21, 2129-2139.
(2004)
Paez, et al. EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib Therapy.
Science. 304, 1497 (2004).
Repasky, G. A., Chenette, E. J. & Der, C. J. Renewing the conspiracy theory debate: does Raf function
alone to mediate Ras oncogenesis? Trends Cell Biol. 14, 639–647 (2004).
Schubbert et al. Hyperactive Ras in developmental disorders and cancer. Nature, Vol. 7 295-307. (2007)
Swanson et al.; Raymond, J. Hohl. Anti-Cancer Therapy: Targeting the Mevalonate. Current Cancer Drug
Target 2006, 6, 15-37
Vetter, I. R. & Wittinghofer, A. The guanine nucleotidebinding switch in three dimensions. Science 294,
1299–1304 (2001).
Zhang et al. Knockdown of Mutant K-ras Expression by Adenovirus-Mediated siRNA Inhibits the In Vitro
and in Vivo Growth of Lung Cancer Cells. Cancer Biology and Therapy 1481-1486 (2006)
Zhang et al. Silencing the epidermal growth factor receptor gene with RNAi may be developed as a
potential therapy for non small lung cancer. Genetic Vaccines and Therapy 3:5 (2005)