Section14:RNAi Synthetic Lethality Screens

From Assay Guidance Wiki

A variation of a LOF screen is a synthetic lethality (or, synthetic lethal) screen which combines the use of RNAi and a drug (at single concentration or multiple concentrations) to identify knockdown events that would modulate drug response such as sensitizers that enhance drug effect. This offers a powerful approach to identify genetic determinants of drug response, especially in cancer. Most of the assay optimization and follow-up assays for si/shRNA described in part B apply here. The extra optimization and differences in data analysis will be discussed below.

Assay Optimization

In synthetic lethality screens, the incubation time of the drug, its potency and stability also need to be evaluated. Drug dose and time response (DDTR) experiments can be carried out to optimize these conditions in either 96-well or 384-well plates. For instance (see Figure7), in a 96-well plate, 10-point drug dilutions with NT and negative controls (at fixed si/shRNA concentration) can be applied along each row excluding the two edge columns. Assay readouts need to be monitored over a period of time, say from day 1 to day 6. The result of such an experiment is mentioned below.

Figure 7. 96-well plate layout for DDTR. NT: non-transfected; NS: non-silencing siRNA (negative control)

Example: A DDTR experiment for one drug was done from Day 1 to Day6 to determine the appropriate drug incubation time for subsequent siRNA synthetic lethality screens. Data from Day 3 to Day 6 in Figure 8 (data from Day 1 and Day 2 data was not informative for curve fitting). Sigmoidal dose response curves of NT and NS were obtained from the experimental plates designed as above and IC50 values were estimated for each day. From Figure 8, we can see that:

NS and NT produce almost exactly the same dose response curves over the various concentrations sampled
IC50 values of the drug (either from NT or NS curves, see vertical drop lines in Figure 8) tend to stabilize from Day 4 (for NT, Day 3: 44.59nM, Day 4: 35.78nM, Day5: 36.54nM, Day 6: 31.77nM)
Signal window between the zero concentration and the highest concentration of the drug tends to stabilize from Day 5 (Z prime factor calculated using NT at concentration zero and 200nM: Day 3-0.42; Day 4-0.89; Day 5-0.73; Day 6-0.73).

Therefore 5 days of drug treatment would be recommended.

Figure 8. DDTR example of one experiment with four replicates from Day 1 to Day 6 (Data from Day 1 and 2 was not for curve fitting and is not shown); black solid lines and solid points are for NT, red dash lines and bullet points for NS.

Design and Data Analysis

Design of screens

There are two main designs for synthetic lethality screens: single and multiple concentrations of drug. The hit selection strategy will vary accordingly.

Single-concentration experiment - Typically drug concentrations < IC50 are chosen (say IC10 and/or IC30). At each point including zero, we recommend at least three replicates (may reduce to duplicates in a high-throughput screen).
Multiple-concentration experiment - A full dose response curve of the drug is used. We recommend 7 doses with duplicates as a minimum. For larger scale screens where number of points and replicates are an issue, we would suggest increased dose points, provided they are chosen carefully to cover the full range of dose response.

Note: Several advantages exist with a RNAi synthetic lethality screen run with multiple concentrations. Non-linear curve fitting to identify biologically more relevant hits that demonstrate a ‘shift’ in DDR is made possible. Replicates are not as major an issue and achieving exact dose effect is not a concern due to curve fitting. In our experience, it is likely to produce more robust screen actives (less false-positives) and reduce follow-up steps.

In synthetic lethality screens, other necessary considerations are:

Monitoring drug dose response in a large scale screen, such as control charting on drug potency (Section III, Quantitative Biology Manual Section).
Choice of sensitizer control (positive control) which may be targets related to drug MOA.
Inclusion of extra control plates (see Appendix) along with other library plates in the screen to assess the quality of the screen especially HTS.

Signal window

In RNAi based synthetic lethality assays, Z prime factor and Signal Window can be calculated using the negative and positive controls for the untreated and lower dose conditions and is not necessary for the high dose conditions.

Hit Selection in Synthetic Lethality Screens

Normalization methods basically are the same as described earlier for LOF screens. The basic idea of synthetic lethality experiments is to identify hits that result in maximum chemosensitization. Therefore, we suggest the following hit selection process:

When using a cell growth or death assay, we suggest excluding si/shRNA hits that are result in high cytotoxicity without drug. This is to prevent confounding interpretation around drug potentiation (These hits can be tested separately for any sensitization effect). As an example, in our experience, we have excluded hits that cause >60% loss of viability from the following analysis. Other threshold values can also be obtained by using population-based methods suggested by statisticians (such as 2 or 3 standard deviations from the mean).
After the first step,
- In a single-concentration experiment with sufficient replicates, one can use statistical models (such as linear models) to pick statistically significant hits that demonstrate significant interaction of drug and siRNA. Furthermore, to rank hits, we suggest a non-parametric metric based on the interaction between RNAi, drug and the combination, called “potentiation score”, based on the idea of independent events, calculated as shown below for inhibition assays, such as cell viability:
  
  Or, for activation assays, such as cell apoptosis:
  
  "UT" here refers to the untreated condition; the “drug only” and “combination” are at the same drug dose point. P >1 indicates that the combination effect is more than the product of two individual effects. The threshold values can be determined using population-based methods. An example of hits in single-concentration experiment is illustrated in Figure 9.
  
  Figure 9. A hit from single-dose synthetic lethality screen in a cell proliferation assay. The black round points are for negative controls (NS), showing not much different effect w/ or w/o drug; the off-diagonal red triangle points are for the hit, which does not have much of an effect on its own but has significantly more effect with drug.
- In a multiple-concentration experiment, sigmoidal curve comparison is done between RNAi with and without small molecule. Using cell viability assay as an example, hits that demonstrate a significant left shift of dose response curves (Figure 10) would be of interest. One should first exclude those response curves above the negative controls (to avoid transfection artifacts) and then look for a decrease of IC50/EC50 values. Statistical tests like t-test between IC50/EC50 estimates, F test for two curve fittings, or information criteria can be used for testing significance (GraphPad Prism Manual). Guidelines on curve fitting quality can be found in Section III, Quantitative Biology Manual. In general, we recommend hits that show at least a 2-fold EC50 shift with respect to the negative control.
  Figure 10. A hit in multiple-dose synthetic lethality siRNA screen in a cell viability assay. The decrease of IC50 value of the red dose response curve (the siRNA with the drug) compared to the black curve (negative control, NS with the drug) is observed (the dropping lines indicate the positions of IC50 values).
  
  Apart from the follow-up mentioned above (B.5) we recommend confirmation of sensitization in a multiple dose format (10-point with replicates). If available, testing related compounds for specificity is suggested.