Hartwell, Friend and colleagues30
were the first to propose the concept of using synthetic lethality screening to identify new anticancer drugs. Recognizing that one of the limiting steps impeding drug discovery was the identification of tumour-selective characteristics, they suggested that loss-of-function mutations — such as those found in DNA repair genes or tumour suppressor genes — could be exploited. In the ideal setting, the first mutation would be a cancer-driving defect and highly conserved evolutionarily from model organisms to humans. Thus, by screening in yeast, synthetic lethal interactions could be identified, either by candidate selection or by genome-wide screening. Choosing putative targets requires both prior knowledge of involved pathways and the ability to generate specific mutations to investigate the effects of various combinations of mutations on these pathways. By contrast, genome-wide screening is a blind, unbiased search that requires large-scale genetic screening technology.
As proof-of-concept, Hartwell et al.
performed a small-scale screen of a panel of 70 different isogenic strains from budding yeast with deletions in DNA damage response genes against US Food and Drug Administration (FDA)-approved chemotherapies. For validation of the feasibility of such a screen, the focus was on genetic instability as a basis for drug discovery. The rationale was that genetic instability is a common feature of many tumours, and that the genetic changes that underlie this genetic instability of tumour cells — in particular, defects in DNA damage response and repair pathways — could make tumour cells more sensitive to the effects of some drugs than normal cells. Hartwell, Friend and colleagues30
were able to determine the drug sensitivities of two anticancer agents: cisplatin and mitoxantrone. Cisplatin demonstrated increased specificity for yeast strains that were defective in post-replication repair, whereas mitoxantrone — which functions as a topoisomerase II poison — resulted in increased sensitivity of yeast strains that were defective in double-stranded DNA break repair. This work demonstrated the feasibility of using large genetic screens to identify synthetic lethal interactions.
The value and applicability of synthetic lethality in the context of mammalian cells, especially in the cancer setting, is now more fully recognized. Efforts are no longer limited to model organisms nor are they limited to known essential cancer-driving pathways, such as those involved in genomic instability or DNA damage repair. Considerable technological advances have made it possible to screen for genes involved in synthetic lethal interactions in a mammalian setting (). Notably, the advent of libraries of either siRNAs or short hairpin RNAs (shRNAs), as well as combinatorial and diversity-oriented libraries of small molecules, enables genome-wide investigation of specific mutations in a rapid manner in mammalian cells.
Mammalian synthetic lethality screens for anticancer efficacy
There are fundamental similarities between screening either siRNA/shRNA libraries or small-molecule compounds to identify synthetic lethal interactions — both approaches frequently use matched, isogenic lines (in which an essential cancer gene has been identified) and a functional readout to assess whether an agent is cytotoxic, cytostatic or has no effect (). Both types of unbiased screens can also reveal unexpected connections that can directly and indirectly advance drug development efforts as well as basic research into our understanding of cancer biology. However, these two types of screens also have distinct although not necessarily mutually exclusive goals. The screening of RNA interference (RNAi)-based libraries can identify genes that are important in a pathway context and thus provide a better understanding of the fundamental biology behind interactions. By contrast, the goal of screening a small-molecule library is typically to obtain candidate compounds for the treatment of a given cancer genotype. Along with differing aims, there are advantages and disadvantages of high-throughput screening of either RNAi-based libraries or small-molecule compound libraries to identify synthetic lethal interactions (BOX 1
Box 1 | Advantages and disadvantages of RNAi libraries or small-molecule compound libraries
There are certain considerations when screening for synthetic lethal interactions using RNA interference (RNAi) libraries or small-molecule compound libraries. Below, we list some advantages and disadvantages of these two approaches for the identification of synthetic lethal interactions.
- A top-down approach allows direct target identification
- An interaction may not necessarily lead to a therapeutic; that is, a compound to inhibit or activate the identified interaction target may not exist
- Nonspecific toxicity related to RNAi may lead to false negatives
Small-molecule compound libraries
- Directly provide potential candidates for optimization into lead compounds
- A bottom-up approach means that a target must be identified; therefore it does not immediately provide any new information on the biology of the disease or genetic interactions
- Amenable to structure–activity relationship analysis, which could help in optimizing compounds and identifying approaches to combat drug resistance
RNAi-based screens allow for the direct discovery of unknown gene–gene interactions and pathways. However, the identification of such interactions and pathways does not necessarily lead readily to potential therapeutic candidates.
One approach for RNAi-based genome-wide screening requires a reverse transfection step, in which cells are plated onto pre-seeded plates containing the RNAi library along with a transfection reagent. Following incubation to allow for expression, the cells are then assayed for changes in viability. Off-target toxicity that is inherent to RNAi-based screening may be a source of false readouts. Alternative approaches for screening RNAi-based libraries include using plasmid vectors, in which cells are transfected with DNA encoding shRNA48
. Viral methods, such as infection with retroviruses containing targeting sequences, can also be used for conditional inactivation of genes49
. These plasmid-based approaches offer another advantage — the inclusion of a DNA ‘bar code’, which is a unique sequence for each shRNA-encoding plasmid that can be amplified from a mixed population. The abundance of each bar code within a pool under different conditions reveals the effect of an individual shRNA on survival and growth without the need to assay each plasmid individually.
Another important consideration is that the primary hits obtained from an RNAi library screen require a second screen to identify the agents that target the identified genes or their products50
. If the synthetic lethal interactor is a known gene, then compounds to inhibit its activity may already exist51
. Although knowing what the genetic target is for a synthetic lethal interaction could indicate the mechanism of action and aid in the development of small-molecule therapeutics, focusing on the known function of the target gene could also be misleading, as other mechanisms that are independent of the known function of the gene could be responsible for the synthetic lethality. Identifying a pathway involved in synthetic lethal interactions also provides additional targets for small-molecule inhibitors.
In contrast to RNAi libraries, identification of small molecules that demonstrate synthetic lethality in a screen directly provides potential candidates for optimization into lead compounds (for example, by improving potency or pharmacokinetic properties) without additional steps. In addition, the identification of the target of the small molecule can be valuable in structural modelling or for investigating structure–activity relationships to generate additional analogues with improved characteristics. However, this can require substantial additional effort compared to the RNAi-based screens for which the target is already known. A second screen using RNAi may be necessary to reveal the target (or targets).
It should be noted that using isogenic cell lines is not without challenges and is not the only approach that can be taken for synthetic lethality screens. Although this approach is technically and conceptually attractive for identifying synthetic lethal interactions with common, known cancer mutations, the single genetic variation being studied may not actually be the only difference in ‘isogenic cells’, which is a confounding factor in these types of studies. Rather, genetic drift between pairs of isogenic cell lines may result in multiple differences that can alter responses to RNAi or drug treatment. The problem of genetic drift may be especially acute when the mutation of interest results in a defect in DNA repair or genomic instability.
An alternative to isogenic lines has been established by Canaani and colleagues52–54
; in this strategy, a single human cancer cell line that is deficient in a gene of interest is used. Complementation of this gene is provided by a low-copy unstable episome expressing this gene. In the context of a drug or RNAi screen, retention of this episome is selected under synthetic lethal conditions, thus revealing novel interactions. Although isogenic lines often provide a valuable avenue for synthetic lethality screens, this work demonstrates that other approaches also have distinct advantages.
Conditional synthetic lethality screens
To date, synthetic lethality screens have focused on specific, fixed genetic mutations and ignored transient yet unique features that can also be exploited. Conditional synthetic lethality screening demonstrates great potential by using interactions based on temporary situations to further increase the therapeutic index and the selectivity for cancer cells. Conditional synthetic lethality can develop in several different contexts — for example, in response to ionizing radiation, cytotoxic chemotherapeutic agents, or changes in the cellular microenvironment. Recent studies by Bindra et al.
and Chan et al.
have demonstrated that tumour hypoxia decreases the expression of homologous recombination proteins such as the DNA repair protein RAD51. Therefore, by suppressing the expression of DNA repair proteins, hypoxia conditionally transforms cells into a recombinational-deficient state and consequently they are sensitive to PARP inhibitors57
The impact of this conditional state of recombination deficiency induced by hypoxia should be common to most solid tumours, and could potentially be enhanced by agents that selectively increase tumour hypoxia by altering their metabolism. For example, treatment of transplanted tumours in mice with dichloroacetate (DCA) increases pyruvate consumption in the mitochondria and total oxygen consumption, which increases tumour hypoxia58
. As DCA is already in clinical trials, it represents a practical approach for increasing tumour hypoxia, decreasing the expression of recombination proteins and sensitizing tumour cells to agents such as PARP inhibitors that will induce synthetic lethality59
. This approach represents one example of how conditional synthetic lethal interactions can potentially be manipulated and exploited for cancer therapy ().
Example of a conditional synthetic lethality opportunity