Identification of Synthetic Lethal Interactions Using High-Throughput, Arrayed CRISPR/Cas9-Based Platforms.

Over the past two decades, the concept of synthetic lethality (SL) that queries genetic relationships between gene pairs has gradually emerged as one of the best strategies to selectively eliminate cancer cells. Some of the most successful approaches to identify synthetic lethal interactions (SLIs) were largely dependent on pooled screening formats that require heavy validation in order to mitigate false positives. Here, we describe a high-throughput method to identify SLIs using CRISPR-based strategy that covers, high-throughput production of plasmid DNA preparations, lentiviral production, and subsequent cellular transduction using single guide RNAs (sgRNAs). This method could be adopted to query hundreds of SLIs. As an example, we describe the methods associated with building an interaction map for DNA damage and repair (DDR) genes. The use of multiwell plates and image-based quantification allows a comparative measurement of SLIs at a high-resolution on a one-by-one basis. Furthermore, this scalable, arrayed CRISPR screening method can be applied to multiple cancer cell types, and genes of interest, resulting in new functional discoveries that can be exploited therapeutically.

The CINs of Polo-Like Kinase 1 in Cancer.

Polo-like kinase 1 (PLK1) is overexpressed near ubiquitously across all cancer types and dysregulation of this enzyme is closely tied to increased chromosomal instability and tumor heterogeneity. PLK1 is a mitotic kinase with a critical role in maintaining chromosomal integrity through its function in processes ranging from the mitotic checkpoint, centrosome biogenesis, bipolar spindle formation, chromosome segregation, DNA replication licensing, DNA damage repair, and cytokinesis. The relation between dysregulated PLK1 and chromosomal instability (CIN) makes it an attractive target for cancer therapy. However, clinical trials with PLK1 inhibitors as cancer drugs have generally displayed poor responses or adverse side-effects. This is in part because targeting CIN regulators, including PLK1, can elevate CIN to lethal levels in normal cells, affecting normal physiology. Nevertheless, aiming at related genetic interactions, such as synthetic dosage lethal (SDL) interactions of PLK1 instead of PLK1 itself, can help to avoid the detrimental side effects associated with increased levels of CIN. Since PLK1 overexpression contributes to tumor heterogeneity, targeting SDL interactions may also provide an effective strategy to suppressing this malignant phenotype in a personalized fashion.

Targeting the CINful genome: Strategies to overcome tumor heterogeneity.

Genomic instability, and more specifically chromosomal instability (CIN), arises from a number of processes that are defective in cancer, such as aberrant mitotic cell division, replication stress, defective DNA damage repair, and ineffective telomere maintenance. CIN is an emerging hallmark of cancer that contributes to tumor heterogeneity through increased rates of genetic alterations. As genetic heterogeneity within a single tumor and between tumors is a key challenge leading to treatment failures, this brings to question, whether therapeutic approaches should aim at the genetic diversity or a specific mutation present within these tumors. Answering this question will determine the future of personalized targeted therapies. Here we discuss, how the genetic diversity associated with CIN in tumor cells can be used as a therapeutic advantage and targeted by exploiting the genetic concepts of synthetic lethality and synthetic dosage lethality. Given that a number of CIN-related pathways work together to fix the DNA damage within our genome and ensure proper segregation of chromosomes, we specifically focus on the genetic interactions amongst these pathways and their potential therapeutic applicability in cancer. We also discuss, how tumor genetic heterogeneity can be targeted in emerging immunotherapeutic approaches.