Mapping of DDX11 genetic interactions defines sister chromatid cohesion as the major dependency.

DDX11/Chl1R is a conserved DNA helicase with roles in genome maintenance, DNA replication, and chromatid cohesion. Loss of DDX11 in humans leads to the rare cohesinopathy Warsaw breakage syndrome. DDX11 has also been implicated in human cancer where it has been proposed to have an oncogenic role and possibly to constitute a therapeutic target. Given the multiple roles of DDX11 in genome stability and its potential as an anticancer target, we set out to define a complete genetic interaction profile of DDX11 loss in human cell lines. Screening the human genome with clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA drop out screens in DDX11-wildtype (WT) or DDX11-deficient cells revealed a strong enrichment of genes with functions related to sister chromatid cohesion. We confirm synthetic lethal relationships between DDX11 and the tumor suppressor cohesin subunit STAG2, which is frequently mutated in several cancer types and the kinase HASPIN. This screen highlights the importance of cohesion in cells lacking DDX11 and suggests DDX11 may be a therapeutic target for tumors with mutations in STAG2.

Mapping Synthetic Dosage Lethal Genetic Interactions in Saccharomyces cerevisiae.

Synthetic dosage lethality (SDL) is a type of genetic interaction that occurs when increasing the expression of a gene causes a fitness defect, such as lethality, in a specific mutant background but has little effect on fitness in a wild-type background. SDL genetic interactions discovered in model organisms such as the budding yeast, Saccharomyces cerevisiae , represent candidate genetic interactions that may be conserved in human cells. In some cases, SDL genetic interactions can be applied to study the biological implications of genes overexpressed in cancer and to discover potential anticancer therapeutic drug targets. Here, we provide a protocol for screening a query overexpression gene against ordered arrays of yeast mutant strains to identify mutations that sensitize yeast to increased dosage of a specific gene product. We outline applications and procedures for screening with an inducibly overexpressed wild-type gene, a common feature of cancer cells, or with an inducibly overexpressed gene carrying a dominant-negative missense mutation as a model of protein-inhibitor interactions. This high-throughput screening platform is adapted from synthetic genetic array (SGA) technology and enables the generation of large-scale SDL genetic interaction networks that can be applied to study gene/pathway function and to identify cross-species cancer-relevant processes.

Paralogous synthetic lethality underlies genetic dependencies of the cancer-mutated gene STAG2.

STAG2, a component of the mitotically essential cohesin complex, is highly mutated in several different tumour types, including glioblastoma and bladder cancer. Whereas cohesin has roles in many cancer-related pathways, such as chromosome instability, DNA repair and gene expression, the complex nature of cohesin function has made it difficult to determine how STAG2 loss might either promote tumorigenesis or be leveraged therapeutically across divergent cancer types. Here, we have performed whole-genome CRISPR-Cas9 screens for STAG2-dependent genetic interactions in three distinct cellular backgrounds. Surprisingly, STAG1, the paralog of STAG2, was the only negative genetic interaction that was shared across all three backgrounds. We also uncovered a paralogous synthetic lethal mechanism behind a genetic interaction between STAG2 and the iron regulatory gene IREB2 Finally, investigation of an unusually strong context-dependent genetic interaction in HAP1 cells revealed factors that could be important for alleviating cohesin loading stress. Together, our results reveal new facets of STAG2 and cohesin function across a variety of genetic contexts.

Modeling DNA trapping of anticancer therapeutic targets using missense mutations identifies dominant synthetic lethal interactions.

Genetic screens can identify synthetic lethal (SL) interactions and uncover potential anticancer therapeutic targets. However, most SL screens have utilized knockout or knockdown approaches that do not accurately mimic chemical inhibition of a target protein. Here, we test whether missense mutations can be utilized as a model for a type of protein inhibition that creates a dominant gain-of-function cytotoxicity. We expressed missense mutations in the FEN1 endonuclease and the replication-associated helicase, CHL1, that inhibited enzymatic activity but retained substrate binding, and found that these mutations elicited a dominant SL phenotype consistent with the generation of cytotoxic protein-DNA or protein-protein intermediates. Genetic screens with nuclease-defective hFEN1 and helicase-deficient yCHL1 captured dominant SL interactions, in which ectopic expression of the mutant form, in the presence of the wild-type form, caused SL in specific mutant backgrounds. Expression of nuclease-defective hFEN1 in yeast elicited DNA binding-dependent dominant SL with homologous recombination mutants. In contrast, dominant SL interactions with helicase-deficient yCHL1 were observed in spindle-associated, Ctf18-alternative replication factor C (Ctf18-RFC) clamp loader complex, and cohesin mutant backgrounds. These results highlight the different mechanisms underlying SL interactions that occur in the presence of an inhibited form of the target protein and point to the utility of modeling trapping mutations in pursuit of more clinically relevant SL interactions.

A Multimodal Genotoxic Anticancer Drug Characterized by Pharmacogenetic Analysis in Caenorhabditis elegans.

New anticancer therapeutics require extensive in vivo characterization to identify endogenous and exogenous factors affecting efficacy, to measure toxicity and mutagenicity, and to determine genotypes that result in therapeutic sensitivity or resistance. We used Caenorhabditis elegans as a platform with which to characterize properties of the anticancer therapeutic CX-5461. To understand the processes that respond to CX-5461-induced damage, we generated pharmacogenetic profiles for a panel of C. elegans DNA replication and repair mutants with common DNA-damaging agents for comparison with the profile of CX-5461. We found that multiple repair pathways, including homology-directed repair, microhomology-mediated end joining, nucleotide excision repair, and translesion synthesis, were needed for CX-5461 tolerance. To determine the frequency and spectrum of CX-5461-induced mutations, we used a genetic balancer to capture CX-5461-induced mutations. We found that CX-5461 is mutagenic, resulting in both large copy number variations and a high frequency of single-nucleotide variations (SNVs), which are consistent with the pharmacogenetic profile for CX-5461. Whole-genome sequencing of CX-5461-exposed animals found that CX-5461-induced SNVs exhibited a distinct mutational signature. We also phenocopied the CX-5461 photoreactivity observed in clinical trials and demonstrated that CX-5461 generates reactive oxygen species when exposed to UVA radiation. Together, the data from C. elegans demonstrate that CX-5461 is a multimodal DNA-damaging anticancer agent.

Cross-Species Complementation of Nonessential Yeast Genes Establishes Platforms for Testing Inhibitors of Human Proteins.

Cross-species complementation can be used to generate humanized yeast, which is a valuable resource with which to model and study human biology. Humanized yeast can be used as an in vivo platform to screen for chemical inhibition of human protein drug targets. To this end, we report the systematic complementation of nonessential yeast genes implicated in chromosome instability (CIN) with their human homologs. We identified 20 human-yeast complementation pairs that are replaceable in 44 assays that test rescue of chemical sensitivity and/or CIN defects. We selected a human-yeast pair (hFEN1/yRAD27), which is frequently overexpressed in cancer and is an anticancer therapeutic target, to perform in vivo inhibitor assays using a humanized yeast cell-based platform. In agreement with published in vitro assays, we demonstrate that HU-based PTPD is a species-specific hFEN1 inhibitor. In contrast, another reported hFEN1 inhibitor, the arylstibonic acid derivative NSC-13755, was determined to have off-target effects resulting in a synthetic lethal phenotype with yRAD27-deficient strains. Our study expands the list of human-yeast complementation pairs to nonessential genes by defining novel cell-based assays that can be utilized as a broad resource to study human drug targets.

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome.

Advances in long-read single molecule sequencing have opened new possibilities for 'benchtop' whole-genome sequencing. The Oxford Nanopore Technologies MinION is a portable device that uses nanopore technology that can directly sequence DNA molecules. MinION single molecule long sequence reads are well suited for de novo assembly of complex genomes as they facilitate the construction of highly contiguous physical genome maps obviating the need for labor-intensive physical genome mapping. Long sequence reads can also be used to delineate complex chromosomal rearrangements, such as those that occur in tumor cells, that can confound analysis using short reads. Here, we assessed MinION long-read-derived sequences for feasibility concerning: (1) the de novo assembly of a large complex genome, and (2) the elucidation of complex rearrangements. The genomes of two Caenorhabditis elegans strains, a wild-type strain and a strain containing two complex rearrangements, were sequenced with MinION. Up to 42-fold coverage was obtained from a single flow cell, and the best pooled data assembly produced a highly contiguous wild-type C. elegans genome containing 48 contigs (N50 contig length = 3.99 Mb) covering >99% of the 100,286,401-base reference genome. Further, the MinION-derived genome assembly expanded the C. elegans reference genome by >2 Mb due to a more accurate determination of repetitive sequence elements and assembled the complete genomes of two co-extracted bacteria. MinION long-read sequence data also facilitated the elucidation of complex rearrangements in a mutagenized strain. The sequence accuracy of the MinION long-read contigs (∼98%) was improved using Illumina-derived sequence data to polish the final genome assembly to 99.8% nucleotide accuracy when compared to the reference assembly.

The Chromosome Transmission Fidelity Assay for Measuring Chromosome Loss in Yeast.

The budding yeast Saccharomyces cerevisiae has served as an excellent model system for studying highly conserved biological pathways including pathways involved in genome transmission and maintenance. The Chromosome Transmission Fidelity (CTF) colony color assay was developed to assess chromosome instability (CIN) in yeast, by monitoring the loss or gain during cell division of an artificial chromosome fragment carrying a visual marker. The CTF assay monitors changes in chromosome number, allowing the detection of mutants that exhibit increased rates of chromosome nondisjunction or chromosome loss. In this article, we describe the SUP11-marker-based CTF assay system, and the methodologies for both qualitative analysis of mutants affecting chromosome transmission, and quantitative analysis for determining the types and rates of errors in chromosome transmission using half-sector analysis.

Synthetic lethality and cancer.

A synthetic lethal interaction occurs between two genes when the perturbation of either gene alone is viable but the perturbation of both genes simultaneously results in the loss of viability. Key to exploiting synthetic lethality in cancer treatment are the identification and the mechanistic characterization of robust synthetic lethal genetic interactions. Advances in next-generation sequencing technologies are enabling the identification of hundreds of tumour-specific mutations and alterations in gene expression that could be targeted by a synthetic lethality approach. The translation of synthetic lethality to therapy will be assisted by the synthesis of genetic interaction data from model organisms, tumour genomes and human cell lines.

Canonical DNA Repair Pathways Influence R-Loop-Driven Genome Instability.

DNA repair defects create cancer predisposition in humans by fostering a higher rate of mutations. While DNA repair is quite well characterized, recent studies have identified previously unrecognized relationships between DNA repair and R-loop-mediated genome instability. R-loops are three-stranded nucleic acid structures in which RNA binds to genomic DNA to displace a loop of single-stranded DNA. Mutations in homologous recombination, nucleotide excision repair, crosslink repair, and DNA damage checkpoints have all now been linked to formation and function of transcription-coupled R-loops. This perspective will summarize recent literature linking DNA repair to R-loop-mediated genomic instability and discuss how R-loops may contribute to mutagenesis in DNA-repair-deficient cancers.

Dosage Mutator Genes in Saccharomyces cerevisiae: A Novel Mutator Mode-of-Action of the Mph1 DNA Helicase.

Mutations that cause genome instability are considered important predisposing events that contribute to initiation and progression of cancer. Genome instability arises either due to defects in genes that cause an increased mutation rate (mutator phenotype), or defects in genes that cause chromosome instability (CIN). To extend the catalog of genome instability genes, we systematically explored the effects of gene overexpression on mutation rate, using a forward-mutation screen in budding yeast. We screened ∼5100 plasmids, each overexpressing a unique single gene, and characterized the five strongest mutators, MPH1 (mutator phenotype 1), RRM3, UBP12, PIF1, and DNA2 We show that, for MPH1, the yeast homolog of Fanconi Anemia complementation group M (FANCM), the overexpression mutator phenotype is distinct from that of mph1Δ. Moreover, while four of our top hits encode DNA helicases, the overexpression of 48 other DNA helicases did not cause a mutator phenotype, suggesting this is not a general property of helicases. For Mph1 overexpression, helicase activity was not required for the mutator phenotype; in contrast Mph1 DEAH-box function was required for hypermutation. Mutagenesis by MPH1 overexpression was independent of translesion synthesis (TLS), but was suppressed by overexpression of RAD27, a conserved flap endonuclease. We propose that binding of DNA flap structures by excess Mph1 may block Rad27 action, creating a mutator phenotype that phenocopies rad27Δ. We believe this represents a novel mutator mode-of-action and opens up new prospects to understand how upregulation of DNA repair proteins may contribute to mutagenesis.

Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer.

Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1 Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors.

Dependence of Human Colorectal Cells Lacking the FBW7 Tumor Suppressor on the Spindle Assembly Checkpoint.

FBW7 (F-box and WD repeat domain containing 7), also known as FBXW7 or hCDC4, is a tumor suppressor gene mutated in a broad spectrum of cancer cell types. As a component of the SCF E3 ubiquitin ligase, FBW7 is responsible for specifically recognizing phosphorylated substrates, many important for tumor progression, and targeting them for ubiquitin-mediated degradation. Although the role of FBW7 as a tumor suppressor is well established, less well studied is how FBW7-mutated cancer cells might be targeted for selective killing. To explore this further, we undertook a genome-wide RNAi screen using WT and FBW7 knockout colorectal cell lines and identified the spindle assembly checkpoint (SAC) protein BUBR1, as a candidate synthetic lethal target. We show here that asynchronous FBW7 knockout cells have increased levels of mitotic APC/C substrates and are sensitive to knockdown of not just BUBR1 but BUB1 and MPS1, other known SAC components, suggesting a dependence of these cells on the mitotic checkpoint. Consistent with this dependence, knockdown of BUBR1 in cells lacking FBW7 results in significant cell aneuploidy and increases in p53 levels. The FBW7 substrate cyclin E was necessary for the genetic interaction with BUBR1. In contrast, the establishment of this dependence on the SAC requires the deregulation of multiple substrates of FBW7. Our work suggests that FBW7 knockout cells are vulnerable in their dependence on the mitotic checkpoint and that this may be a good potential target to exploit in FBW7-mutated cancer cells.

Complementation of Yeast Genes with Human Genes as an Experimental Platform for Functional Testing of Human Genetic Variants.

While the pace of discovery of human genetic variants in tumors, patients, and diverse populations has rapidly accelerated, deciphering their functional consequence has become rate-limiting. Using cross-species complementation, model organisms like the budding yeast, Saccharomyces cerevisiae, can be utilized to fill this gap and serve as a platform for testing human genetic variants. To this end, we performed two parallel screens, a one-to-one complementation screen for essential yeast genes implicated in chromosome instability and a pool-to-pool screen that queried all possible essential yeast genes for rescue of lethality by all possible human homologs. Our work identified 65 human cDNAs that can replace the null allele of essential yeast genes, including the nonorthologous pair yRFT1/hSEC61A1. We chose four human cDNAs (hLIG1, hSSRP1, hPPP1CA, and hPPP1CC) for which their yeast gene counterparts function in chromosome stability and assayed in yeast 35 tumor-specific missense mutations for growth defects and sensitivity to DNA-damaging agents. This resulted in a set of human-yeast gene complementation pairs that allow human genetic variants to be readily characterized in yeast, and a prioritized list of somatic mutations that could contribute to chromosome instability in human tumors. These data establish the utility of this cross-species experimental approach.

Mechanisms of genome instability induced by RNA-processing defects.

The role of normal transcription and RNA processing in maintaining genome integrity is becoming increasingly appreciated in organisms ranging from bacteria to humans. Several mutations in RNA biogenesis factors have been implicated in human cancers, but the mechanisms and potential connections to tumor genome instability are not clear. Here, we discuss how RNA-processing defects could destabilize genomes through mutagenic R-loop structures and by altering expression of genes required for genome stability. A compelling body of evidence now suggests that researchers should be directly testing these mechanisms in models of human cancer.

Synthetic cytotoxicity: digenic interactions with TEL1/ATM mutations reveal sensitivity to low doses of camptothecin.

Many tumors contain mutations that confer defects in the DNA-damage response and genome stability. DNA-damaging agents are powerful therapeutic tools that can differentially kill cells with an impaired DNA-damage response. The response to DNA damage is complex and composed of a network of coordinated pathways, often with a degree of redundancy. Tumor-specific somatic mutations in DNA-damage response genes could be exploited by inhibiting the function of a second gene product to increase the sensitivity of tumor cells to a sublethal concentration of a DNA-damaging therapeutic agent, resulting in a class of conditional synthetic lethality we call synthetic cytotoxicity. We used the Saccharomyces cerevisiae nonessential gene-deletion collection to screen for synthetic cytotoxic interactions with camptothecin, a topoisomerase I inhibitor, and a null mutation in TEL1, the S. cerevisiae ortholog of the mammalian tumor-suppressor gene, ATM. We found and validated 14 synthetic cytotoxic interactions that define at least five epistasis groups. One class of synthetic cytotoxic interaction was due to telomere defects. We also found that at least one synthetic cytotoxic interaction was conserved in Caenorhabditis elegans. We have demonstrated that synthetic cytotoxicity could be a useful strategy for expanding the sensitivity of certain tumors to DNA-damaging therapeutics.

Genome destabilizing mutator alleles drive specific mutational trajectories in Saccharomyces cerevisiae.

In addition to environmental factors and intrinsic variations in base substitution rates, specific genome-destabilizing mutations can shape the mutational trajectory of genomes. How specific alleles influence the nature and position of accumulated mutations in a genomic context is largely unknown. Understanding the impact of genome-destabilizing alleles is particularly relevant to cancer genomes where biased mutational signatures are identifiable. We first created a more complete picture of cellular pathways that impact mutation rate using a primary screen to identify essential Saccharomyces cerevisiae gene mutations that cause mutator phenotypes. Drawing primarily on new alleles identified in this resource, we measure the impact of diverse mutator alleles on mutation patterns directly by whole-genome sequencing of 68 mutation-accumulation strains derived from wild-type and 11 parental mutator genotypes. The accumulated mutations differ across mutator strains, displaying base-substitution biases, allele-specific mutation hotspots, and break-associated mutation clustering. For example, in mutants of POLα and the Cdc13-Stn1-Ten1 complex, we find a distinct subtelomeric bias for mutations that we show is independent of the target sequence. Together our data suggest that specific genome-instability mutations are sufficient to drive discrete mutational signatures, some of which share properties with mutation patterns seen in tumors. Thus, in a population of cells, genome-instability mutations could influence clonal evolution by establishing discrete mutational trajectories for genomes.

Glioblastoma cells containing mutations in the cohesin component STAG2 are sensitive to PARP inhibition.

Recent data have identified STAG2, a core subunit of the multifunctional cohesin complex, as a highly recurrently mutated gene in several types of cancer. We sought to identify a therapeutic strategy to selectively target cancer cells harboring inactivating mutations of STAG2 using two independent pairs of isogenic glioblastoma cell lines containing either an endogenous mutant STAG2 allele or a wild-type STAG2 allele restored by homologous recombination. We find that mutations in STAG2 are associated with significantly increased sensitivity to inhibitors of the DNA repair enzyme PARP. STAG2-mutated, PARP-inhibited cells accumulated in G2 phase and had a higher percentage of micronuclei, fragmented nuclei, and chromatin bridges compared with wild-type STAG2 cells. We also observed more 53BP1 foci in STAG2-mutated glioblastoma cells, suggesting that these cells have defects in DNA repair. Furthermore, cells with mutations in STAG2 were more sensitive than cells with wild-type STAG2 when PARP inhibitors were used in combination with DNA-damaging agents. These data suggest that PARP is a potential target for tumors harboring inactivating mutations in STAG2, and strongly recommend that STAG2 status be determined and correlated with therapeutic response to PARP inhibitors, both prospectively and retrospectively, in clinical trials.

Sequencing of candidate chromosome instability genes in endometrial cancers reveals somatic mutations in ESCO1, CHTF18, and MRE11A.

Most endometrial cancers can be classified histologically as endometrioid, serous, or clear cell. Non-endometrioid endometrial cancers (NEECs; serous and clear cell) are the most clinically aggressive of the three major histotypes and are characterized by aneuploidy, a feature of chromosome instability. The genetic alterations that underlie chromosome instability in endometrial cancer are poorly understood. In the present study, we used Sanger sequencing to search for nucleotide variants in the coding exons and splice junctions of 21 candidate chromosome instability genes, including 19 genes implicated in sister chromatid cohesion, from 24 primary, microsatellite-stable NEECs. Somatic mutations were verified by sequencing matched normal DNAs. We subsequently resequenced mutated genes from 41 additional NEECs as well as 42 endometrioid ECs (EECs). We uncovered nonsynonymous somatic mutations in ESCO1, CHTF18, and MRE11A in, respectively, 3.7% (4 of 107), 1.9% (2 of 107), and 1.9% (2 of 107) of endometrial tumors. Overall, 7.7% (5 of 65) of NEECs and 2.4% (1 of 42) of EECs had somatically mutated one or more of the three genes. A subset of mutations are predicted to impact protein function. The co-occurrence of somatic mutations in ESCO1 and CHTF18 was statistically significant (P = 0.0011, two-tailed Fisher's exact test). This is the first report of somatic mutations within ESCO1 and CHTF18 in endometrial tumors and of MRE11A mutations in microsatellite-stable endometrial tumors. Our findings warrant future studies to determine whether these mutations are driver events that contribute to the pathogenesis of endometrial cancer.

Synthetic lethal targeting of superoxide dismutase 1 selectively kills RAD54B-deficient colorectal cancer cells.

Synthetic lethality is a rational approach to identify candidate drug targets for selective killing of cancer cells harboring somatic mutations that cause chromosome instability (CIN). To identify a set of the most highly connected synthetic lethal partner genes in yeast for subsequent testing in mammalian cells, we used the entire set of 692 yeast CIN genes to query the genome-wide synthetic lethal datasets. Hierarchical clustering revealed a highly connected set of synthetic lethal partners of yeast genes whose human orthologs are somatically mutated in colorectal cancer. Testing of a small matrix of synthetic lethal gene pairs in mammalian cells suggested that members of a pathway that remove reactive oxygen species that cause DNA damage would be excellent candidates for further testing. We show that the synthetic lethal interaction between budding yeast rad54 and sod1 is conserved within a human colorectal cancer context. Specifically, we demonstrate RAD54B-deficient cells are selectively killed relative to controls via siRNA-based silencing and chemical inhibition and further demonstrate that this interaction is conserved in an unrelated cell type. We further show that the DNA double strand breaks, resulting from increased reactive oxygen species following SOD1 inhibition, persist within the RAD54B-deficient cells and result in apoptosis. Collectively, these data identify SOD1 as a novel candidate cancer drug target and suggest that SOD1 inhibition may have broad-spectrum applicability in a variety of tumor types exhibiting RAD54B deficiencies.