Synthetic genetic targeting of genome instability in cancer

Abstract

Cancer is a leading cause of death throughout the World. A limitation of many current chemotherapeutic approaches is that their cytotoxic effects are not restricted to cancer cells, and adverse side effects can occur within normal tissues. Consequently, novel strategies are urgently needed to better target cancer cells. As we approach the era of personalized medicine, targeting the specific molecular defect(s) within a given patient's tumor will become a more effective treatment strategy than traditional approaches that often target a given cancer type or sub-type. Synthetic genetic interactions are now being examined for their therapeutic potential and are designed to target the specific genetic and epigenetic phenomena associated with tumor formation, and thus are predicted to be highly selective. In general, two complementary approaches have been employed, including synthetic lethality and synthetic dosage lethality, to target aberrant expression and/or function associated with tumor suppressor genes and oncogenes, respectively. Here we discuss the concepts of synthetic lethality and synthetic dosage lethality, and explain three general experimental approaches designed to identify novel genetic interactors. We present examples and discuss the merits and caveats of each approach. Finally, we provide insight into the subsequent pre-clinical work required to validate novel candidate drug targets.

Figure 1

Synthetic Genetic Approaches in Model Organisms and Cancer. (a) Synthetic lethality is a rare genetic interaction that occurs when two independent and viable mutations or deletions (gene1 [blue yeast] or gene2 [orange yeast]) result in death when combined (red yeast). If a slow growth phenotype is observed, a synthetic growth defect or synthetic sickness is defined. (b) A cancer-associated hypomorphic mutation or deletion in a gene (e.g., GENE1 is a deleted tumor suppressor gene) is selectively killed through a synthetic lethal approach by silencing or inhibiting the protein product encoded by GENE2. (c) A cancer-associated hypermorphic mutation or amplification (e.g., GENE3 is an amplified oncogene) is selectively killed through a synthetic dosage lethal interaction by silencing or inhibiting the protein product encoded by GENE4.

Figure 2

Conceptual Models Depicting the Underlying Mechanisms that Account for Synthetic Lethal Interactions. Conceptual models detailing three mechanisms that produce a synthetic lethal interaction—circles represent genes, cancer-associated mutations, or deletions are identified by a red “X”, and synthetic lethal interactors (i.e., drug targets) are identified by blue arrows. (a) Partial ablation of two functions encoded within a single essential pathway (e.g., epistasis group), such that the pathway is no longer functional. (b) Ablation of two functions encoded within two distinct parallel pathways. For example, defects in DNA replication would lead to DNA errors requiring repair, and small molecule inhibitors preventing accurate repair will cause lethality. (c) Ablation of two functions encoded within two separate pathways that together impinge on a single essential process. For example, DNA double strand breaks can only be repaired through two pathways, namely homologous recombination repair and non-homologous end joining—defects and inhibition of both pathways will cause cellular cytotoxicity.

Figure 3

Fundamental Approaches to Identify Synthetic Genetic Interactors. A flowchart depicting the three fundamental experimental approaches designed to identify synthetic genetic interactors. Knowledge-based direct tests, cross-species approaches or whole genome approaches can be employed to screen for candidate interactors (i.e., drug targets) of a given cancer query gene. Depending on the scale of the screen either siRNA duplexes or shRNA constructs can be employed so that direct comparisons can be made between the experimental query line and an isogenic control line. In general, gene knockout cells or gene amplification cells are employed as the query lines for tumor suppressor genes (synthetic lethal interactors) and oncogenes (synthetic dosage lethal interactors), respectively. Targeted killing of the query cell line relative to an isogenic control is typically evaluated using any one of a number of experimental assays—six common assays are listed. Once a putative interactor is identified, subsequent validation is required before it is confirmed as a novel candidate drug target. Depending on the target, a chemical screen may be initiated, which may yield candidates requiring subsequent validation, optimization and pre-clinical study. Alternatively, if known inhibitors exist they can be evaluated in either pre-clinical studies, or, if the compound has already received approval for use in other human diseases, clinical studies may be initiated with appropriate approval.