1. Francisco Rodriguez William Zhao

2. Purpose How are cancers caused? Oncogenes Ras subfamily of the small GTPases Ras function Ras synthetic lethality (RSL) candidates

3. Important Terms KRas (GTPase) PLK1 Anaphase-promoting complex/cyclosome (APC/C) COP9 BI-2536 Paclitaxel Bortezomib MG132 EVI5 EMI1

4. Techniques Used RNAi Microarray Deconvolution Multicolor Competitive Assay (MCA) Fluorescence-activated cell sorting (FACS)

5. Known and Unknown At the time, mutations in small GTPase Ras were known to be found in many cancer types. However, little was known about how the mutated gene interacted with various biological processes. Additionally, while Ras mutant cells were known to be known to almost always be pre-cancerous or cancer cells, nothing was known about their sensitivities to common cancer treatments, such as treating abnormal mitotic division via the PLK1 pathway and APC/C activity.

6. Experiment setup Genome-wide loss of function shRNA library in lentivirus Kras Oncogene Microarray Deconvolution to create multiplex platform Allowed for high parallel screening of >10,000 shRNA

7. RNAi Screening Colorectal cancer cells cell line DLD-1 with endogenous activating KRAS G13D point mutation Disrupt KRAS G13D allele reduces MAPK activity in isogenic clones (Figures 1B-1D ) From Figure 1

8. KRAS WT/G13D (Ras Mut) DLD-1 cells vs. isogenic KRAS WT/- (Ras WT) DLD-1 control cells From Figure 1

9. Results of Primary screening Screened KRAS WT/G13D (Ras Mut) DLD-1 cells and the isogenic KRAS WT/- (Ras WT) DLD-1 control cells RSL candidates identified Identified 1741 RSL shRNAs targeting 1613 genes Stringent cutoff identified a subset 379 RSL shRNAs targeting 368 genes

10. Validation using MCA Multicolor Competitive Assay + FACS analysis –320 candidate RSL shRNAs – Used RSL shRNA and Firefly luciferase (FF) shRNA From Figure1

11. Validation using MCA (cont’d) 83 shRNA out of the 320, which target 77 genes, decrease the fitness of Ras Mut cells compared to Ras WT cells Second MCA performed on isogenic pair of colorectal cancer cell lines: HCT116 KRAS WT/G13D and HCT116 KRAS WT/- 50 of 68 RSL shRNA showed synthetic lethality

12. shRNA’s Recovered Against KRAS – Only suppresses Few against MAP Kinase and PI-3 Kinase – Used MEK inhibitor U0126 and PI3K inhibitor LY294002 – No selective toxicity PANTHER used to find biological processes enriched in screen Several subunits of COP9 signalsome Depleting COPS3 &COPS4 impaired fitness Ras Mut Depleting COPS4 impaired growth on soft adherent surface and agarose

13. Functional Diversity of RSL Candidates From Figure 2

14. To rule out off-target effects, several shRNAs were tested against several of the genes identified in the primary screen

15. Sensitivity due to Mitotic Stress A number RSL genes involved in mitotic regulation Mitotic Stress Faster doubling in mutant (Figure 1C) Depletion phenotype is an on-target effect From Figure 3

16. Observation of mitotic events Using the Eg5 kinesin inhibitor monastrol to arrest cells, then released The usage of nocodazole and paclitaxel, mitotic spindle inhibitors From Figure 3

17. Testing for sensitivity No sensitivity to Nocodazole in either DLD-1 or HCT116 Ras Mut cells Nocodazole a depolymerizer Paclitaxel a stabilizer HCT116 higher sensitivity to Paclitaxel From Figure 3

18. Cell cycle analysis G2/M and Mitotic accumulation of Ras Mut cells (DLD-1 cells) Paclitaxel causes prometaphase block Indicates increased mitotic stress From Figure 3

19. PLK1 inhibition PLK1 deregulated in cancers Multiple shRNAs against PLK1 identified as causing toxicity in Ras Mut cells siRNAs against PLK1 also yielded the same result PLK1 inhibitor, BI-2536 used

20. PLK1 inhibition (Cont’d) From Figure 4

21. Additional effects on mitosis G2/M accumulation of DLD-1 Ras Mut Prometaphase block From Figure 4

22. Moving forward Does inhibition of PLK1 cause delayed mitotic entry? Cells arrested at G2/M with RO-3306, a CDK1 inhibitor (Figure 4E) No effect on mitotic entry, so the ability to complete Mitosis with BI-2536 was tested (figure 4F) 48 hr treatment with BI-2536 caused a high accumulation of sub G1 (figure 4G)

23. From Figure 4

24. APC/C & Proteasome Inhibition Several APC/C subunits found in the screening, e.g. Cdc16, and Cdc27 APC/C role in mitosis, degradation of Cyclins EMI1 inhibits APC/C, unless phosphorylated by PLK1 EVI5 prevents EMI1 phosphorylation Lentivirus used to over express both From Figure 5

26. Silencing the proteosome shRNAs against proteosome subunits Using proteosome inhibitors MG132 and Bortezomib From Figure 5

27. Cell cycle analysis From Figure 5

28. Mitotic inhibitors in vivo Mouse xenograft models used, they had DLD-1 or HCT116 tumors treated with PLK1 inhibitor BI-2536. Tumor growth to be significantly attenuated Agreed with in vivo, also HCT116 more sensitive compared to DLD-1 (Figure 4B) From Figure 6

29. The mitotic stress of Ras Tested to see if mitotic stress is in other cancers with diff oncogene mutations PIK3CA oncogene in DLD-1 and HCT116 cells tested with paclitaxel, BI-2536, bortezomib, or MG132. PIK3CA Mut more resistant Test non-small cell-lung cancer (NSCLC) with or without Ras mutations (Figure D) As a group Ras mutants were sensitive From Figure 6

30. RAS expression Signature From Figure 7 -Ras expression from mutant KRAS gene used to probe for increased, decreased, or equivalent Ras signatures -116 negative signatures and 143 positive signatures

31. From Figure 7 Observations of cell survival rates given either negative or positive Ras mutations

32. Discussion Given the results of the experiment, the Ras oncogene was found to only be able to maintain a steady state if it interacted with several cellular functions, mainly concerning mitotic events. After taking mitotic events into account, the authors proposed that the dependence of mutant Ras oncogenes oninfluencing key mitotic proteins could be used as a red flag for the Ras oncogenetic state, a characteristic that could be used for novel methods of oncologic treatment.

33. Discussion (cont’d) One possible future question could examine the difference between Ras+ and Ras- mutants in terms of interactions with mitotic protein factors and how that influences the survival rate. Because the differences in survival rate have already been shown in this paper, the next logical step is to examine why the Ras+ signature offers an enhanced prognosis and survival rate in patients Another possible future approach would be to perform RNA-seq on the + and – mutants to determine specifically what protein expressions are affected by having a positive or negative Ras signature.

34. Additional Readings KRAS Mutations and Primary Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib, Pao W, Wang T, Riely G, Miller V, Pan Q, Ladanyi M, Zakowzki M, Heelan R, Kris M, Varmus H. This article examined the resistance and mechanism of lung cancers to two types of kinase inhibitors via the KRAS mutation Prevalence of ras gene mutations in human colorectal cancers, Bos J, Fearon E, Hamilton S, Verlaan-De Vries M, Van Boom J, Van Der Eb A, Vogelstein B. Further examination of ras in cancers and highlights why the mutation remains so virulent and successful.