Exploiting Cancer Synthetic Lethality in Cancer-Lessons Learnt from PARP Inhibitors.

PARP inhibitors now have proven utility in the treatment of homologous recombination (HR) defective cancers. These drugs, and the synthetic lethality effect they exploit, have not only taught us how to approach the treatment of HR defective cancers but have also illuminated how resistance to a synthetic lethal approach can occur, how cancer-associated synthetic lethal effects are perhaps more complex than we imagine, how the better use of biomarkers could improve the success of treatment and even how drug resistance might be targeted. Here, we discuss some of the lessons learnt from the study of PARP inhibitor synthetic lethality and how these lessons might have wider application. Specifically, we discuss the concept of synthetic lethal penetrance, phenocopy effects in cancer such as BRCAness, synthetic lethal resistance, the polygenic and complex nature of synthetic lethal interactions, how evolutionary double binds could be exploited in treatment as well as future horizons for the field.

Paralog dispensability shapes homozygous deletion patterns in tumor genomes.

Genomic instability is a hallmark of cancer, resulting in tumor genomes having large numbers of genetic aberrations, including homozygous deletions of protein coding genes. That tumor cells remain viable in the presence of such gene loss suggests high robustness to genetic perturbation. In model organisms and cancer cell lines, paralogs have been shown to contribute substantially to genetic robustness-they are generally more dispensable for growth than singletons. Here, by analyzing copy number profiles of > 10,000 tumors, we test the hypothesis that the increased dispensability of paralogs shapes tumor genome evolution. We find that genes with paralogs are more likely to be homozygously deleted and that this cannot be explained by other factors known to influence copy number variation. Furthermore, features that influence paralog dispensability in cancer cell lines correlate with paralog deletion frequency in tumors. Finally, paralogs that are broadly essential in cancer cell lines are less frequently deleted in tumors than non-essential paralogs. Overall, our results suggest that homozygous deletions of paralogs are more frequently observed in tumor genomes because paralogs are more dispensable.

Gene essentiality in cancer is better predicted by mRNA abundance than by gene regulatory network-inferred activity.

Gene regulatory networks (GRNs) are often deregulated in tumor cells, resulting in altered transcriptional programs that facilitate tumor growth. These altered networks may make tumor cells vulnerable to the inhibition of specific regulatory proteins. Consequently, the reconstruction of GRNs in tumors is often proposed as a means to identify therapeutic targets. While there are examples of individual targets identified using GRNs, the extent to which GRNs can be used to predict sensitivity to targeted intervention in general remains unknown. Here we use the results of genome-wide CRISPR screens to systematically assess the ability of GRNs to predict sensitivity to gene inhibition in cancer cell lines. Using GRNs derived from multiple sources, including GRNs reconstructed from tumor transcriptomes and from curated databases, we infer regulatory gene activity in cancer cell lines from ten cancer types. We then ask, in each cancer type, if the inferred regulatory activity of each gene is predictive of sensitivity to CRISPR perturbation of that gene. We observe slight variation in the correlation between gene regulatory activity and gene sensitivity depending on the source of the GRN and the activity estimation method used. However, we find that there is consistently a stronger relationship between mRNA abundance and gene sensitivity than there is between regulatory gene activity and gene sensitivity. This is true both when gene sensitivity is treated as a binary and a quantitative property. Overall, our results suggest that gene sensitivity is better predicted by measured expression than by GRN-inferred activity.

Complex synthetic lethality in cancer.

The concept of synthetic lethality has been widely applied to identify therapeutic targets in cancer, with varying degrees of success. The standard approach normally involves identifying genetic interactions between two genes, a driver and a target. In reality, however, most cancer synthetic lethal effects are likely complex and also polygenic, being influenced by the environment in addition to involving contributions from multiple genes. By acknowledging and delineating this complexity, we describe in this article how the success rate in cancer drug discovery and development could be improved.

Genetic interactions under the microscope.

Traditional genetic interaction screens profile phenotypes at aggregate level, missing interactions that may influence the distribution of single cells in specific states. Here, Heigwer and colleagues use an imaging approach to generate a large-scale high-resolution genetic interaction map in Drosophila cells and demonstrate its utility for understanding gene function.

Antibody reliability influences observed mRNA-protein correlations in tumour samples.

Reverse phase protein arrays (RPPA) have been used to quantify the abundance of hundreds of proteins across thousands of tumour samples in the Cancer Genome Atlas. By number of samples, this is the largest tumour proteomic dataset available and it provides an opportunity to systematically assess the correlation between mRNA and protein abundances. However, the RPPA approach is highly dependent on antibody reliability and approximately one-quarter of the antibodies used in the the Cancer Genome Atlas are deemed to be somewhat less reliable. Here, we assess the impact of antibody reliability on observed mRNA-protein correlations. We find that, in general, proteins measured with less reliable antibodies have lower observed mRNA-protein correlations. This is not true of the same proteins when measured using mass spectrometry. Furthermore, in cell lines, we find that when the same protein is quantified by both mass spectrometry and RPPA, the overall correlation between the two measurements is lower for proteins measured with less reliable antibodies. Overall our results reinforce the need for caution in using RPPA measurements from less reliable antibodies.

Targeting synthetic lethal paralogs in cancer.

Synthetic lethal interactions, where mutation of one gene renders cells sensitive to inhibition of another gene, can be exploited for the development of targeted therapeutics in cancer. Pairs of duplicate genes (paralogs) often share common functionality and hence are a potentially rich source of synthetic lethal interactions. Because the majority of human genes have paralogs, exploiting such interactions could be a widely applicable approach for targeting gene loss in cancer. Moreover, existing small-molecule drugs may exploit synthetic lethal interactions by inhibiting multiple paralogs simultaneously. Consequently, the identification of synthetic lethal interactions between paralogs could be extremely informative for drug development. Here we review approaches to identify such interactions and discuss some of the challenges of exploiting them.

Experimental reproducibility limits the correlation between mRNA and protein abundances in tumor proteomic profiles.

Large-scale studies of human proteomes have revealed only a moderate correlation between mRNA and protein abundances. It is unclear to what extent this moderate correlation reflects post-transcriptional regulation and to what extent it reflects measurement error. Here, by analyzing replicate profiles of tumors and cell lines, we show that there is considerable variation in the reproducibility of measurements of transcripts and proteins from individual genes. Proteins with more reproducible measurements tend to have a higher mRNA-protein correlation, suggesting that measurement reproducibility accounts for a substantial fraction of the unexplained variation between mRNA and protein abundances. The reproducibility of individual proteins is somewhat consistent across studies, and we exploit this to develop an aggregate reproducibility score that explains a substantial amount of the variation in mRNA-protein correlations across multiple studies. Finally, we show that pathways previously reported to have a higher-than-average mRNA-protein correlation may simply contain members that can be more reproducibly quantified.

Comprehensive prediction of robust synthetic lethality between paralog pairs in cancer cell lines.

Pairs of paralogs may share common functionality and, hence, display synthetic lethal interactions. As the majority of human genes have an identifiable paralog, exploiting synthetic lethality between paralogs may be a broadly applicable approach for targeting gene loss in cancer. However, only a biased subset of human paralog pairs has been tested for synthetic lethality to date. Here, by analyzing genome-wide CRISPR screens and molecular profiles of over 700 cancer cell lines, we identify features predictive of synthetic lethality between paralogs, including shared protein-protein interactions and evolutionary conservation. We develop a machine-learning classifier based on these features to predict which paralog pairs are most likely to be synthetic lethal and to explain why. We show that our classifier accurately predicts the results of combinatorial CRISPR screens in cancer cell lines and furthermore can distinguish pairs that are synthetic lethal in multiple cell lines from those that are cell-line specific. A record of this paper's transparent peer review process is included in the supplemental information.

Proteomic signatures of radioresistance: Alteration of inflammation, angiogenesis and metabolism-related factors in radioresistant oesophageal adenocarcinoma.

The clinical management of locally advanced oesophageal adenocarcinoma (OAC) involves neoadjuvant chemoradiotherapy (CRT), but as radioresistance remains a major clinical challenge, complete pathological response to CRT only occurs in 20-30% of patients. In this study we used an established isogenic cell line model of radioresistant OAC to detect proteomic signatures of radioresistance to identify novel molecular and cellular targets of radioresistance in OAC. A total of 5785 proteins were identified of which 251 were significantly modulated in OE33R cells, when compared to OE33P. Gene ontology and pathway analysis of these significantly modulated proteins demonstrated altered metabolism in radioresistant cells accompanied by an inhibition of apoptosis. In addition, inflammatory and angiogenic pathways were positively regulated in radioresistant cells compared to the radiosensitive cells. In this study, we demonstrate, for the first time, a comprehensive proteomic profile of the established isogenic cell line model of radioresistant OAC. This analysis provides insights into the molecular and cellular pathways which regulate radioresistance in OAC. Furthermore, it identifies pathway specific signatures of radioresistance that will direct studies on the development of targeted therapies and personalised approaches to radiotherapy.

Cross-species identification of PIP5K1-, splicing- and ubiquitin-related pathways as potential targets for RB1-deficient cells.

The RB1 tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions with RB1 could lead to new approaches to treating cancers with inactivated RB1. We identified 95 SL partners of RB1 based on a Drosophila screen for genetic modifiers of the eye phenotype caused by defects in the RB1 ortholog, Rbf1. We validated 38 mammalian orthologs of Rbf1 modifiers as RB1 SL partners in human cancer cell lines with defective RB1 alleles. We further show that for many of the RB1 SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels of RB1 was associated with improved patient survival. We investigated higher order combinatorial gene interactions by creating a novel Drosophila cancer model with co-occurring Rbf1, Pten and Ras mutations, and found that targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, we found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, we identified several high confidence, evolutionarily conserved, novel targets for RB1-deficient cells that may be further adapted for the treatment of human cancer.

Accurate prediction of kinase-substrate networks using knowledge graphs.

Phosphorylation of specific substrates by protein kinases is a key control mechanism for vital cell-fate decisions and other cellular processes. However, discovering specific kinase-substrate relationships is time-consuming and often rather serendipitous. Computational predictions alleviate these challenges, but the current approaches suffer from limitations like restricted kinome coverage and inaccuracy. They also typically utilise only local features without reflecting broader interaction context. To address these limitations, we have developed an alternative predictive model. It uses statistical relational learning on top of phosphorylation networks interpreted as knowledge graphs, a simple yet robust model for representing networked knowledge. Compared to a representative selection of six existing systems, our model has the highest kinome coverage and produces biologically valid high-confidence predictions not possible with the other tools. Specifically, we have experimentally validated predictions of previously unknown phosphorylations by the LATS1, AKT1, PKA and MST2 kinases in human. Thus, our tool is useful for focusing phosphoproteomic experiments, and facilitates the discovery of new phosphorylation reactions. Our model can be accessed publicly via an easy-to-use web interface (LinkPhinder).

Integrative analysis of large-scale loss-of-function screens identifies robust cancer-associated genetic interactions.

Genetic interactions, including synthetic lethal effects, can now be systematically identified in cancer cell lines using high-throughput genetic perturbation screens. Despite this advance, few genetic interactions have been reproduced across multiple studies and many appear highly context-specific. Here, by developing a new computational approach, we identified 220 robust driver-gene associated genetic interactions that can be reproduced across independent experiments and across non-overlapping cell line panels. Analysis of these interactions demonstrated that: (i) oncogene addiction effects are more robust than oncogene-related synthetic lethal effects; and (ii) robust genetic interactions are enriched among gene pairs whose protein products physically interact. Exploiting the latter observation, we used a protein-protein interaction network to identify robust synthetic lethal effects associated with passenger gene alterations and validated two new synthetic lethal effects. Our results suggest that protein-protein interaction networks can be used to prioritise therapeutic targets that will be more robust to tumour heterogeneity.

Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRASG13D.

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.

Paralog buffering contributes to the variable essentiality of genes in cancer cell lines.

What makes a gene essential for cellular survival? In model organisms, such as budding yeast, systematic gene deletion studies have revealed that paralog genes are less likely to be essential than singleton genes and that this can partially be attributed to the ability of paralogs to buffer each other's loss. However, the essentiality of a gene is not a fixed property and can vary significantly across different genetic backgrounds. It is unclear to what extent paralogs contribute to this variation, as most studies have analyzed genes identified as essential in a single genetic background. Here, using gene essentiality profiles of 558 genetically heterogeneous tumor cell lines, we analyze the contribution of paralogy to variable essentiality. We find that, compared to singleton genes, paralogs are less frequently essential and that this is more evident when considering genes with multiple paralogs or with highly sequence-similar paralogs. In addition, we find that paralogs derived from whole genome duplication exhibit more variable essentiality than those derived from small-scale duplications. We provide evidence that in 13-17% of cases the variable essentiality of paralogs can be attributed to buffering relationships between paralog pairs, as evidenced by synthetic lethality. Paralog pairs derived from whole genome duplication and pairs that function in protein complexes are significantly more likely to display such synthetic lethal relationships. Overall we find that many of the observations made using a single strain of budding yeast can be extended to understand patterns of essentiality in genetically heterogeneous cancer cell lines.

Cells Lacking the RB1 Tumor Suppressor Gene Are Hyperdependent on Aurora B Kinase for Survival.

Small cell lung cancer (SCLC) accounts for 15% of lung cancers and is almost always linked to inactivating RB1 and TP53 mutations. SCLC frequently responds, albeit briefly, to chemotherapy. The canonical function of the RB1 gene product RB1 is to repress the E2F transcription factor family. RB1 also plays both E2F-dependent and E2F-independent mitotic roles. We performed a synthetic lethal CRISPR/Cas9 screen in an RB1 -/- SCLC cell line that conditionally expresses RB1 to identify dependencies that are caused by RB1 loss and discovered that RB1 -/- SCLC cell lines are hyperdependent on multiple proteins linked to chromosomal segregation, including Aurora B kinase. Moreover, we show that an Aurora B kinase inhibitor is efficacious in multiple preclinical SCLC models at concentrations that are well tolerated in mice. These results suggest that RB1 loss is a predictive biomarker for sensitivity to Aurora B kinase inhibitors in SCLC and perhaps other RB1 -/- cancers. SIGNIFICANCE: SCLC is rarely associated with actionable protooncogene mutations. We did a CRISPR/Cas9-based screen that showed that RB1 -/- SCLC are hyperdependent on AURKB, likely because both genes control mitotic fidelity, and confirmed that Aurora B kinase inhibitors are efficacious against RB1 -/- SCLC tumors in mice at nontoxic doses.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.

Synthetic Lethality and Cancer - Penetrance as the Major Barrier.

Synthetic lethality has long been proposed as an approach for targeting genetic defects in tumours. Despite a decade of screening efforts, relatively few robust synthetic lethal targets have been identified. Improved genetic perturbation techniques, including CRISPR/Cas9 gene editing, have resulted in renewed enthusiasm for searching for synthetic lethal effects in cancer. An implicit assumption behind this enthusiasm is that the lack of reproducibly identified targets can be attributed to limitations of RNAi technologies. We argue here that a bigger hurdle is that most synthetic lethal interactions (SLIs) are not highly penetrant, in other words they are not robust to the extensive molecular heterogeneity seen in tumours. We outline strategies for identifying and prioritising SLIs that are most likely to be highly penetrant.

Systems biology: old news or new stimulus for biochemistry.

In this issue of Essays in Biochemistry, biochemistry meets systems biology-a blind date that may hold all the promises, pitfalls and failures of a relationship where a new discipline has been sprung upon a well-established one. As the articles in this issue show, the blind date in this case has great potential to develop into a long-term relationship, where both partners share common values but can benefit from different complementary approaches. Together this partnership is well poised to address and solve some of the major challenges in modern biology.

Author Correction: Chemosensitivity profiling of osteosarcoma tumour cell lines identifies a model of BRCAness.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Chemosensitivity profiling of osteosarcoma tumour cell lines identifies a model of BRCAness.

Osteosarcoma (OS) is an aggressive sarcoma, where novel treatment approaches are required. Genomic studies suggest that a subset of OS, including OS tumour cell lines (TCLs), exhibit genomic loss of heterozygosity (LOH) patterns reminiscent of BRCA1 or BRCA2 mutant tumours. This raises the possibility that PARP inhibitors (PARPi), used to treat BRCA1/2 mutant cancers, could be used to target OS. Using high-throughput drug sensitivity screening we generated chemosensitivity profiles for 79 small molecule inhibitors, including three clinical PARPi. Drug screening was performed in 88 tumour cell lines, including 18 OS TCLs. This identified known sensitivity effects in OS TCLs, such as sensitivity to FGFR inhibitors. When compared to BRCA1/2 mutant TCLs, OS TCLs, with the exception of LM7, were PARPi resistant, including those with previously determined BRCAness LoH profiles. Post-screen validation experiments confirmed PARPi sensitivity in LM7 cells as well as a defect in the ability to form nuclear RAD51 foci in response to DNA damage. LM7 provides one OS model for the study of PARPi sensitivity through a potential defect in RAD51-mediated DNA repair. The drug sensitivity dataset we generated in 88 TCLs could also serve as a resource for the study of drug sensitivity effects in OS.