1. Relating Activating K-Ras Mutations to Small Molecule Sensitivity in Non- Small-Cell Lung Cancer Flavian D. Brown Carleton College Class of 2009

2. 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

3. Ras Signaling Schubbert et al. (2007) Hyperactive Ras in developmental disorders and cancer. Nature Review of Cancer, Vol. 7 295-307. Oncogenic Mutation ← ← ←

4. Hypothesis  NSCLC tumors are genetically sensitized due to changes in cellular state secondary to activating K- Ras mutations. - Different drug targets - Oncogene Addiction

5. Small Molecule Screens + DMSO Control 100nl Pin Transfer 250 500 1000 48Hrs 72Hrs 2-10Hrs cell adherence 24 Hrs

6. 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 Hits From Primary Screen

7. Assay Development

8. Small Molecule Sensitivity

11. Structural Activity Relationship 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

12. 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

13. Impact  Genotype specific inhibitors for K-Ras mutants  Paradigm for investigating genotype- phenotype relationships in other malignancies - WGAS for somatic alterations  Molecularly targeted cancer therapeutics.

14. 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 - Jordi Barretina, Ph.D - Damian W. Young, Ph.D Broad Institute Screening - Nicola Tolliday, Ph.D - Josh Bittker, Ph.D - Melanie de Silva - Kate Hartland

15. References 1. 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) 2. Bourne, H. R., Sanders, D. A. & McCormick, F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125–132 (1990). 3. Diaz et al. Complex effects of Ras proto-oncogenes in tumorigenesis. Carcinogenesis, Vol. 25, No. 4, 535- 539 (2004). 4. Downward, J. Targeting RAS signaling pathways in cancer therapy. Nature Rev. Cancer 3, 11–22 (2003). 5. Gibbs, J. B. & Oliff, A. The potential of farnesyltransferase inhibitors as cancer chemotherapeutics. Annu. Rev. Pharmacol. Toxicol. 143–166 (1997). 6. Herrmann, C. Ras–effector interactions: after one decade. Curr. Opin. Struct. Biol. 13, 122–129 (2003) 7. 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) 8. Paez, et al. EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib Therapy. Science. 304, 1497 (2004). 9. 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). 10. Schubbert et al. Hyperactive Ras in developmental disorders and cancer. Nature, Vol. 7 295-307. (2007) 11. Swanson et al.; Raymond, J. Hohl. Anti-Cancer Therapy: Targeting the Mevalonate. Current Cancer Drug Target 2006, 6, 15-37 12. Vetter, I. R. & Wittinghofer, A. The guanine nucleotidebinding switch in three dimensions. Science 294, 1299–1304 (2001). 13. 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) 14. 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)