A Genome-wide RNAi Screen Identifies Multiple Synthetic Lethal Interactions with the Ras Oncogene

1. A Genome-wide RNAi ScreenIdentifies Multiple Synthetic LethalInteractions with the Ras Oncogene Mauricio Velasco PriyaVasudeva

2. Targeting Cancer Cells • KRAS Oncogene (20-30% of human tumors in 90% of cancers) • Inhibit apoptosis (PI3K pathway). • Increase cell proliferation, differentiation, growth and survival (MAPK pathway).

3. Past approaches • Reversing oncoproteins VS attacking tumor-specific vulnerabilities. • Ras enzyme kinetics are hard to inhibit. • Substrate affinity • Catalyst proteins • Conformational changes • Farnesyl transferase - Post-translational modification on KRAS

4. Reverse Genetics, and the knockdown of KRAS-related factors. • Genome-wide loss-of-function analysis - Development of retroviral/lentiviralshRNA libraries.

5. Synthetic Lethality Screens • 74,905 retroviral shRNAstargetting 32,293 unique human transcripts

6. Multicolor Competition Assay • Infected RasMut GFP+ cells and Ras WT GFP- cells with RSL shRNAs • Found that 83 of RSL genes tested result in synthetic lethality of RasMut cells.

7. Molecules that sustain KRAS Oncogene expression • Genes involved in biosomal biogenesis, translation control, protein neddylation and sumoylation pathways, and RNA splicing.

8. Effect of KRAS depletion on cancer cells RNAi against KRAS - Looked at phenotypic expression. - Showed that KRAS is sufficient to suppress cancer-related phenotypes.

9. RAS Mut cells sensitive to mitotic stress • Genes used to regulate mitosis. -Their depletion phenotypes are likely to be on-target effects as multiple shRNAs gave similar results.

10. Mitotic inhibitors • Paclitaxel is inhibitor of mitotic spindle function • Rasoncogene causes cell to be hypersensitive to mitotic disruption

11. PLK1 inhibition reduces Mut viability • PLK1 (Polo-like kinase I) • Mitosis and cell division • BI-2536 • Binds to PLK1

12. Delay of Mitotic exit • Sub-G1 arrest is indicative of apoptosis. • Does PLK1 inhibition delay mitotic entry? • Nocodazole • RO-3306 • CDK1 inhibitor

13. APC inhibition leads to lethality • APC/C regulates mitotic progression by tagging cell cycle proteins for degradation after mitosis. • Showed that RasMut cells heavily depend on normal levels of APC/C for survival.

14. RAS Mut cells hypersensitive to proteasome inhibitors • Screening identified shRNAs involved in proteasome. • Inhibitors of proteasome showed synthetic lethality with RasMut cells • Sensitivity to inhibition is partly due to mitotic stress.

15. Mouse Xenograft Tumors • Using inhibitor of PLK1, BI-2536, results saw a decrease in tumor size of RasMut cells. • HTC116 cells more sensitive to BI-2536 then DLD-1

16. Mitotic Machinery is Achilles heel of RasMut Cells. • RasMut cells are dependent on key mitotic proteins for cellular progression. • APC/C function is critical for RasMut cell survival

17. Diagnostics • Kaplan-Meier Analysis • Gene Signature: Genes in a type of cell line whose expression is uniquely diagnostic of a condition/illness.

18. Gene targets with increased survival • Liam

19. Conclusions/Article Highlights • Synthetic lethality is a useful way to link gene/transcript/protein interactions. • KRAS affects mitotic progression, thus making cells more dependable on PLK1 for such process. • APC/C is likely a preferred target for KRAS-related cancers. • A similar approach could help illuminate potential targets for various types of cancers.

20. Further Reading • Downward, J. 2003. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer. 3:11-22. • Bos, J. 1989. Rasoncogenes in human cancer: a review. Cancer Res. 49: 4682-9. • Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D., Darnell, J. 2000. Chapter 25, Cancer. Molecular Cell Biology (4th ed.). San Francisco: W.H. Freeman.