1. Chris Chander & Verna Vu

2. Ras: family of small GTPase
Ubiquitously expressed in all cell lineages & organs
Cell growth, differentiation and proliferation
Mutations are prevalent in most cancer
GTP-bound is active
Point-mutations lead to:
Interference with Ras GAP binding
Constitutively active GTP bound state
KRAS most frequently mutated
Found at higher frequencies in pancreatic, thyroid, colon, lung, liver cancer
Poor prognosis
KRAS mutations are observed in 40-50% of human colorectal adenomas and
carcinomas; up to 80% in relapse patients
Ras is the prominent cancer drug target
(GAP responsible for GTP hydrolysis)
KRAS: oncogene

3. Important Genes in Ras pathway
Anaphase-promoting complex/cyclosome APC/C
Proteasome
Mitotic kinase PLK1

4. Challenges for cancer therapeutics:
Lack of understanding of the vulnerabilities of these cancers
Goal: inhibit cellular drug targets to selectively kill cancer cells
Exploiting oncogene addiction:
Use inhibitors to block oncoproteins
Exploiting nononcogene addiction:
Inhibiting proteins that are not oncoproteins
but ultimately reverses oncogenic state
Vulnerabilities: aren’t obvious & can’t be predicted so genetic exploration is the most direct approach to their discovery
Oncogene addiction: the phenomenon that some tumours exhibit exquisite dependence on a single oncogenic protein (or pathway) for sustaining growth and proliferation
nononcogene addiction: tumors dependent on proteins that aren’t oncoproteins

5. Genome-wide RNAi synthetic lethal screen
against KRAS oncogene
Find genes whose loss of function constitutes
synthetic lethality with RAS oncogene
Synthetic lethality: mutations in 2+ genes
leads to cell death, but a mutation in only one
of these genes does not
Found a set of proteins whose depletion selectively
impaired the viability of RAS mutant cells
RNAi: is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules
Synthetic lethality: arises when a combination of mutations in two or more genes leads to cell death, whereas a mutation in only one of these genes does not, and by itself is said to be viable
scbt.com

6. KRAS WT/G13D (Mut) screen against isogenic
KRAS WT/- shRNA (WT) library
DLD-1: colorectal cancer cell line that was used
Carries G13D mutation: endogenous point mutation that exhibits addiction to KRAS oncogene
DLD-1 Mut and WT cells were infected with 6 pools of ~13k shRNAs in triplicates
shRNA: short hairpin RNA
Mut= mutant
WT= wild type
carries an endogenous activating KRAS G13D point mutation required for maintaining their oncogenic state exhibit addiction to KRAS oncogene and malignant phenotype depends on mutant KRAS
Library of 74,905 retroviral shRNA
Target: 32,293 unique human transcripts
19,542 RefSeq transcripts

7. Identify Ras synthetic lethal (RSL) candidates
RSL candidates: shRNAs that show selective depletion (dropout) in the Ras Mut but not Ras WT cells.
Analyzed relative abundance of each shRNA over time by microarray hybridization to identify antiproliferative shRNA.
shRNA were PCR-recovered and labeled with dye
Relaxed stats: 1,742 RSL shRNAs targeting 1,613 genes
Stringent cut off: 379 shRNAs targeting 368 genes
elledgelab.med.harvard.edu

8. Depleting identified proteins can impair fitness of mutant relative to WT in competition assay
To rule out off-target effects: tested multiple shRNA against several of these genes
This indicates that Ras requires additional support from many genes to maintain oncogenic state
HCT116: human colon cancer cells
Many of these are essential genes under circumstances of complete depletion. Partially reducing activity leads to enhanced growth defects in mut cells

9. Many mitotic genes are identified as RSL candidates
Ras mutants exhibit characteristics of increased mitotic stress
When released from mitotic block higher fraction of Ras Mut exhibited lagging chromosomes and abnormal anaphases
Ras mutants have 50% higher mitotic index indicating slower mitotic progression. Mitotic index in asynchronous DLD-1 cells growing in log-phase as measured by phospho-H3 Ser10 staining
Mitotic index is defined as the ratio between the number of cells in a population undergoing mitosis to the number of cells not undergoing mitosis.
In addition, when released from mitotic block higher fraction of Ras mut cells exhibit lagging chromosomes and abnormal anaphases

10. Ras Mut are hypersensitive to mitotic stress
Paclitaxel is a microtubule stabilizer
Prometaphase block caused by paclitaxel in mut cells but not in WT
increased mitotic stress renders the cell hypersensitive to perturbation of the mitotic machinery
paclitaxel inhibits the normal breakdown of microtubules

11. Ras Mutant Cells Display Hypersensitivity to PLK1 Function Inhibition
Several shRNA to PLK1 demonstrate increased toxicity to Ras mutant cells compared to WT
BI-2536 selectively inhibits PLK1.
Increasing concentrations of BI-2536 demonstrate a significant decrease in mutant cell viability

13. Inhibition of APC/C and Proteasome Results in Ras Mutant Hypersensitivity
APC/C targets key mitotic proteins for degradation through its activity as an E3 ubiquitin ligase
Ras oncogene causes heightened dependence on APC/C making cells sensitive to any reduction in the activity of this complex
CDC=subunits of APC/C complex

15. Inhibition of APC/C and Proteasome Results in Ras Mutant Hypersensitivity
Proteasome activity is required for APC/C activation in addition to APC/C targeted degradation of mitotic proteins (MG132 & Bortezomib inhibit the proteasome)
PS: protease subunit

16. NSCLC cancer cell line
Impaired APC/C function or an enhanced requirement for APC/C function may underlie critical oncogenic stress associated with Ras mutation
Tested sensitivity, of non- small lung cancer cell lines (NSCLC) with and without Ras mutations, to shRNA mediated knockdown of either APC1/APC4
APC1/APC4 (subunits of APC/C complex)
NSCLC cells with Ras mutations more sensitive to shRNA agaisnt supporting theory of ras asssociated with mitotic stress

17. In vivo BI-2536 decreases tumor growth
Mice treated with PLK1 inhibitor BI-2536 demonstrate significantly subcutaneous tumor growth

18. Human Lung Adenocarcinomas
Patients with lung tumors having a (+) ras signature show enhanced survival in correlation with genes associated with decreasing APC/C activity
3 genes associated with increased survival in Ras+ signature tumor
Simultaneous investigation of all 3 genes, expression profiles with -CDC16, -COP9, +EVI5 show near 100% survival
predicted ras mutation status based on transcription profile of smaller set of lung tumors whos ras expression was known(validated with two other cohorts of lung tumor samples)

19. CDC 16 = APC/C activity
(APC/C subunit)
COPS3 = APC/C activity
EVI5 = APC/C activity

20. Benefits & Implications:
Genome-wide RNAi synthetic lethal screen can be done by combining microarray and shRNA
Highly parallel format makes it cost-effective & is flexible in assay design of this approach
Signals are highly reproducible in replicate PCR
Highly specific
Provide additional gene targets for therapeutic exploration
Discover larger set of oncogenic and nononcogenic targets that cancer cells rely on
Shed new light on Ras mechanisms of action
Potentially provide new biomarkers for patient stratification
Future studies should look for other drugs that could be used to in target selected genes→ clinical trials
Stratification of clinical trials, is the partitioning of subjects and results by a factor other than the treatment given.

21. Further Reading
Russo, M.A., Kang, K.S., Di Cristofano, A The PLK1 inhibitor GSK461364A is effective in poorly differentiated and anaplastic thyroid carcinoma cells, independent of the nature of their driver mutations. Thyroid. 23:
Schlabach, M.R., Luo, J., Solimini, N.L., Hu, G., Xu, Q., Li, M.Z., Zhao, Z., Smogorzewska, A., Sowa, M.E., Ang, X.L., Westbrook, T.F., Liang, A.C., Chang, K., Hackett, J.A., Harper, J.W., Hannon, G.J., Elledge, S.J Cancer proliferation gene discovery through functional genomics. Science : 620–624.

22. Critiques and Limitations
Extensive use of different pathways. Difficult to determine what each gene affected without going back through the mechanism or looking up the protein
A few figures were difficult to interpret based on how they plotted the data