R-loop-mediated genome instability in mRNA cleavage and polyadenylation mutants.

Genome instability via RNA:DNA hybrid-mediated R loops has been observed in mutants involved in various aspects of transcription and RNA processing. The prevalence of this mechanism among essential chromosome instability (CIN) genes remains unclear. In a secondary screen for increased Rad52 foci in CIN mutants, representing ∼25% of essential genes, we identified seven essential subunits of the mRNA cleavage and polyadenylation (mCP) machinery. Genome-wide analysis of fragile sites by chromatin immunoprecipitation (ChIP) and microarray (ChIP-chip) of phosphorylated H2A in these mutants supported a transcription-dependent mechanism of DNA damage characteristic of R loops. In parallel, we directly detected increased RNA:DNA hybrid formation in mCP mutants and demonstrated that CIN is suppressed by expression of the R-loop-degrading enzyme RNaseH. To investigate the conservation of CIN in mCP mutants, we focused on FIP1L1, the human ortholog of yeast FIP1, a conserved mCP component that is part of an oncogenic fusion in eosinophilic leukemia. We found that truncation fusions of yeast FIP1 analogous to those in cancer cause loss of function and that siRNA knockdown of FIP1L1 in human cells increases DNA damage and chromosome breakage. Our findings illuminate how mCP maintains genome integrity by suppressing R-loop formation and suggest that this function may be relevant to certain human cancers.

Synthetic lethality of cohesins with PARPs and replication fork mediators.

Synthetic lethality has been proposed as a way to leverage the genetic differences found in tumor cells to affect their selective killing. Cohesins, which tether sister chromatids together until anaphase onset, are mutated in a variety of tumor types. The elucidation of synthetic lethal interactions with cohesin mutants therefore identifies potential therapeutic targets. We used a cross-species approach to identify robust negative genetic interactions with cohesin mutants. Utilizing essential and non-essential mutant synthetic genetic arrays in Saccharomyces cerevisiae, we screened genome-wide for genetic interactions with hypomorphic mutations in cohesin genes. A somatic cell proliferation assay in Caenorhabditis elegans demonstrated that the majority of interactions were conserved. Analysis of the interactions found that cohesin mutants require the function of genes that mediate replication fork progression. Conservation of these interactions between replication fork mediators and cohesin in both yeast and C. elegans prompted us to test whether other replication fork mediators not found in the yeast were required for viability in cohesin mutants. PARP1 has roles in the DNA damage response but also in the restart of stalled replication forks. We found that a hypomorphic allele of the C. elegans SMC1 orthologue, him-1(e879), genetically interacted with mutations in the orthologues of PAR metabolism genes resulting in a reduced brood size and somatic cell defects. We then demonstrated that this interaction is conserved in human cells by showing that PARP inhibitors reduce the viability of cultured human cells depleted for cohesin components. This work demonstrates that large-scale genetic interaction screening in yeast can identify clinically relevant genetic interactions and suggests that PARP inhibitors, which are currently undergoing clinical trials as a treatment of homologous recombination-deficient cancers, may be effective in treating cancers that harbor cohesin mutations.

The complete spectrum of yeast chromosome instability genes identifies candidate CIN cancer genes and functional roles for ASTRA complex components.

Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ∼ 2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease.

Predisposition to cancer caused by genetic and functional defects of mammalian Atad5.

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5(+/m)) mice that were haploinsuffficient for Atad5. Atad5(+/m) mice displayed high levels of genomic instability in vivo, and Atad5(+/m) mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5(+/m) mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5(+/m) mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis.

Metagenomics of hydrocarbon resource environments indicates aerobic taxa and genes to be unexpectedly common.

Oil in subsurface reservoirs is biodegraded by resident microbial communities. Water-mediated, anaerobic conversion of hydrocarbons to methane and CO2, catalyzed by syntrophic bacteria and methanogenic archaea, is thought to be one of the dominant processes. We compared 160 microbial community compositions in ten hydrocarbon resource environments (HREs) and sequenced twelve metagenomes to characterize their metabolic potential. Although anaerobic communities were common, cores from oil sands and coal beds had unexpectedly high proportions of aerobic hydrocarbon-degrading bacteria. Likewise, most metagenomes had high proportions of genes for enzymes involved in aerobic hydrocarbon metabolism. Hence, although HREs may have been strictly anaerobic and typically methanogenic for much of their history, this may not hold today for coal beds and for the Alberta oil sands, one of the largest remaining oil reservoirs in the world. This finding may influence strategies to recover energy or chemicals from these HREs by in situ microbial processes.

Interaction between organochlorines and the AHR gene, and risk of non-Hodgkin lymphoma.

BACKGROUND: Plasma organochlorines have been implicated to increase the risk of non-Hodgkin lymphoma (NHL), and interaction with the aryl hydrocarbon receptor gene (AHR) may modify this risk. METHODS: In this case-control study conducted in British Columbia, Canada, five single nucleotide polymorphisms (SNPs) of AHR were genotyped in 422 NHL cases and 459 controls to measure the association between individual SNPs, haplotypes, and risk of NHL. Pre-chemotherapy organochlorine levels were measured and gene-environment interaction analysis was performed. RESULTS: The IVS1 + 4640G/A SNP was significantly associated with NHL risk, with an odds ratio of 1.32 (95% CI = 1.05-1.65) for G/A or A/A genotypes compared to the G/G genotype. Interactions were observed with PCB 118, a known inducer of AHR, and chlordane-related analytes oxychlordane and trans-nonachlor, although no interactions were statistically significant after controlling for multiple comparisons. The observed interactions were consistent across NHL subtypes. CONCLUSION: Results suggest that the AHR gene may play a role in determining the risk of NHL with exposure to organochlorines, and highlight the importance of understanding gene-environment interactions.

Genetic variation in H2AFX contributes to risk of non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) comprises a group of lymphoid tumors that have in common somatic translocations. H2AFX encodes a key histone involved in the detection of the DNA double-stranded breaks that can lead to translocations. H2afx is a dosage-dependent gene that protects against B-cell lymphomas in mice, making its human orthologue an ideal candidate gene for susceptibility to lymphoma. We did a population-based genetic association study of H2AFX variants in 487 NHL cases and 531 controls. Complete resequencing of the human H2AFX gene in 95 NHL cases was done to establish the spectrum of variation in affected individuals; this was followed by both direct and indirect tests for association at the level of individual single nucleotide polymorphisms (SNP) and as haplotypes. Homozygosity for the AA genotype of a SNP 417 bp upstream of the translational start of H2AFX is strongly associated [odds ratio (OR), 0.54; P = 0.001] with protection from NHL. We find a strong association of this SNP with the follicular lymphoma subtype of NHL (AA genotype: OR, 0.40; P = 0.004) and with mantle cell lymphoma (AA genotype: OR, 0.20; P = 0.01) that remains significant after adjustment for the false discovery rate, but not with diffuse large B-cell lymphoma. These data support the hypothesis that genetic variation in the H2AFX gene influences genetic susceptibility or resistance to some subtypes of NHL by contributing to the maintenance of genome stability.

miR-509-3p is clinically significant and strongly attenuates cellular migration and multi-cellular spheroids in ovarian cancer.

Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E-3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E-3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3p's most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers.

Saccharomyces cerevisiae genetics predicts candidate therapeutic genetic interactions at the mammalian replication fork.

The concept of synthetic lethality has gained popularity as a rational guide for predicting chemotherapeutic targets based on negative genetic interactions between tumor-specific somatic mutations and a second-site target gene. One hallmark of most cancers that can be exploited by chemotherapies is chromosome instability (CIN). Because chromosome replication, maintenance, and segregation represent conserved and cell-essential processes, they can be modeled effectively in simpler eukaryotes such as Saccharomyces cerevisiae. Here we analyze and extend genetic networks of CIN cancer gene orthologs in yeast, focusing on essential genes. This identifies hub genes and processes that are candidate targets for synthetic lethal killing of cancer cells with defined somatic mutations. One hub process in these networks is DNA replication. A nonessential, fork-associated scaffold, CTF4, is among the most highly connected genes. As Ctf4 lacks enzymatic activity, potentially limiting its development as a therapeutic target, we exploited its function as a physical interaction hub to rationally predict synthetic lethal interactions between essential Ctf4-binding proteins and CIN cancer gene orthologs. We then validated a subset of predicted genetic interactions in a human colorectal cancer cell line, showing that siRNA-mediated knockdown of MRE11A sensitizes cells to depletion of various replication fork-associated proteins. Overall, this work describes methods to identify, predict, and validate in cancer cells candidate therapeutic targets for tumors with known somatic mutations in CIN genes using data from yeast. We affirm not only replication stress but also the targeting of DNA replication fork proteins themselves as potential targets for anticancer therapeutic development.

A histology-based model for predicting microsatellite instability in colorectal cancers.

Identifying colorectal cancers (CRCs) with high levels of microsatellite instability (MSI-H) is clinically important. MSI-H is a positive prognostic marker for CRC, a predictive marker for resistance to standard 5-fluorouracil-based adjuvant chemotherapy, and an important feature for identifying individuals and families with Lynch syndrome. Our aim was to compare and improve upon the existing predictive pathology models for MSI-H CRCs. We tested 2 existing models used to predict MSI-H tumors, (1) Revised Bethesda Guidelines and (2) MsPath, in our population-based cohort of CRCs diagnosed less than 75 years from Newfoundland (N=710). We also scored additional histologic features not described in the other models. From this analysis, we developed a model for the prediction of MSI-H CRCs; Pathologic Role in Determination of Instability in Colorectal Tumors (PREDICT). An independent pathologist validated this model in a second cohort of all CRCs (N=276). Tumor histology was a better predictor of MSI status than was personal and family history of cancer. MsPath identified MSI-H CRCs with a sensitivity of 92.1% and a specificity of 37.8%, whereas the Revised Bethesda Guidelines had a sensitivity of 81.3% and a specificity of 39.5%. PREDICT included some new histology features, including peritumoral lymphocytic reaction and increased proportion of plasma cells in the tumor stroma. PREDICT was superior to both existing models in the development cohort with a sensitivity of 97.4% and a specificity of 53.9%. In the validation cohort, sensitivity was 96.9% and specificity 76.6%. We conclude that PREDICT is a good predictor of MSI-H CRC.

A systematic evaluation of the ataxia telangiectasia mutated gene does not show an association with non-Hodgkin lymphoma.

The ataxia telangiectasia mutated (ATM) gene is critical for the detection and repair of DNA double-stranded breaks. Mutations in this gene cause the autosomal recessive syndrome ataxia telangiectasia (AT), an attribute of which is an increased risk of cancer, particularly lymphoma. We have undertaken a population-based case/control study to assess the influence of genetic variation in ATM on the risk of non-Hodgkin lymphoma (NHL). A number of the subtypes that constitute NHL have in common the occurrence of specific somatic translocations that contribute to lymphomagenesis. We hypothesize that ATM function is slightly attenuated by some variants, which could reduce double-stranded break repair capacity, contributing to the occurrence of translocations and subsequent lymphomas. We sequenced the promoter and all exons of ATM in the germline DNA of 86 NHL patients and identified 79 variants. Eighteen of these variants correspond to nonsynonymous amino acid differences, 6 of which were predicted to be deleterious to protein function; these variants were all rare. Eleven common variants make up 10 haplotypes that are specified by 7 tagSNPs. Linkage disequilibrium across the ATM gene is high but incomplete. TagSNPs and the 6 putatively deleterious variants were genotyped in 798 NHL cases and 793 controls. Our results indicate that common variants of ATM do not significantly contribute to the risk of NHL in the general population. However, some rare, functionally deleterious variants may contribute to an increased risk of development of rare subtypes of the disease.