Arabidopsis thaliana FANCD2 Promotes Meiotic Crossover Formation.

Fanconi anemia (FA) is a human autosomal recessive disorder characterized by chromosomal instability, developmental pathologies, predisposition to cancer, and reduced fertility. So far, 19 genes have been implicated in FA, most of them involved in DNA repair. Some are conserved across higher eukaryotes, including plants. The Arabidopsis thaliana genome encodes a homolog of the Fanconi anemia D2 gene (FANCD2) whose function in DNA repair is not yet fully understood. Here, we provide evidence that AtFANCD2 is required for meiotic homologous recombination. Meiosis is a specialized cell division that ensures reduction of genomic content by half and DNA exchange between homologous chromosomes via crossovers (COs) prior to gamete formation. In plants, a mutation in AtFANCD2 results in a 14% reduction of CO numbers. Genetic analysis demonstrated that AtFANCD2 acts in parallel to both MUTS HOMOLOG4 (AtMSH4), known for its role in promoting interfering COs and MMS AND UV SENSITIVE81 (AtMUS81), known for its role in the formation of noninterfering COs. AtFANCD2 promotes noninterfering COs in a MUS81-independent manner and is therefore part of an uncharted meiotic CO-promoting mechanism, in addition to those described previously.

MEK inhibitors block growth of lung tumours with mutations in ataxia-telangiectasia mutated.

Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.

Gain- and Loss-of-Function Mutations in the Breast Cancer Gene GATA3 Result in Differential Drug Sensitivity.

Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.

A thesaurus of genetic variation for interrogation of repetitive genomic regions.

Detecting genetic variation is one of the main applications of high-throughput sequencing, but is still challenging wherever aligning short reads poses ambiguities. Current state-of-the-art variant calling approaches avoid such regions, arguing that it is necessary to sacrifice detection sensitivity to limit false discovery. We developed a method that links candidate variant positions within repetitive genomic regions into clusters. The technique relies on a resource, a thesaurus of genetic variation, that enumerates genomic regions with similar sequence. The resource is computationally intensive to generate, but once compiled can be applied efficiently to annotate and prioritize variants in repetitive regions. We show that thesaurus annotation can reduce the rate of false variant calls due to mappability by up to three orders of magnitude. We apply the technique to whole genome datasets and establish that called variants in low mappability regions annotated using the thesaurus can be experimentally validated. We then extend the analysis to a large panel of exomes to show that the annotation technique opens possibilities to study variation in hereto hidden and under-studied parts of the genome.

Targeting a cell state common to triple-negative breast cancers.

Some mutations in cancer cells can be exploited for therapeutic intervention. However, for many cancer subtypes, including triple-negative breast cancer (TNBC), no frequently recurring aberrations could be identified to make such an approach clinically feasible. Characterized by a highly heterogeneous mutational landscape with few common features, many TNBCs cluster together based on their 'basal-like' transcriptional profiles. We therefore hypothesized that targeting TNBC cells on a systems level by exploiting the transcriptional cell state might be a viable strategy to find novel therapies for this highly aggressive disease. We performed a large-scale chemical genetic screen and identified a group of compounds related to the drug PKC412 (midostaurin). PKC412 induced apoptosis in a subset of TNBC cells enriched for the basal-like subtype and inhibited tumor growth in vivo. We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC. Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells. This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies.

TOPS: a versatile software tool for statistical analysis and visualization of combinatorial gene-gene and gene-drug interaction screens.

BACKGROUND: Measuring the impact of combinations of genetic or chemical perturbations on cellular fitness, sometimes referred to as synthetic lethal screening, is a powerful method for obtaining novel insights into gene function and drug action. Especially when performed at large scales, gene-gene or gene-drug interaction screens can reveal complex genetic interactions or drug mechanism of action or even identify novel therapeutics for the treatment of diseases.The result of such large-scale screen results can be represented as a matrix with a numeric score indicating the cellular fitness (e.g. viability or doubling time) for each double perturbation. In a typical screen, the majority of combinations do not impact the cellular fitness. Thus, it is critical to first discern true "hits" from noise. Subsequent data exploration and visualization methods can assist to extract meaningful biological information from the data. However, despite the increasing interest in combination perturbation screens, no user friendly open-source program exists that combines statistical analysis, data exploration tools and visualization. RESULTS: We developed TOPS (Tool for Combination Perturbation Screen Analysis), a Java and R-based software tool with a simple graphical user interface that allows the user to import, analyze, filter and plot data from double perturbation screens as well as other compatible data. TOPS was designed in a modular fashion to allow the user to add alternative importers for data formats or custom analysis scripts not covered by the original release.We demonstrate the utility of TOPS on two datasets derived from functional genetic screens using different methods. Dataset 1 is a gene-drug interaction screen and is based on Luminex xMAP technology. Dataset 2 is a gene-gene short hairpin (sh)RNAi screen exploring the interactions between deubiquitinating enzymes and a number of prominent oncogenes using massive parallel sequencing (MPS). CONCLUSIONS: TOPS provides the benchtop scientist with a free toolset to analyze, filter and visualize data from functional genomic gene-gene and gene-drug interaction screens with a flexible interface to accommodate different technologies and analysis algorithms in addition to those already provided here. TOPS is freely available for academic and non-academic users and is released as open source.

A reversible gene trap collection empowers haploid genetics in human cells.

Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-ß, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.

Meier-Gorlin syndrome and Wolf-Hirschhorn syndrome: two developmental disorders highlighting the importance of efficient DNA replication for normal development and neurogenesis.

Microcephaly represents one of the most obvious clinical manifestations of impaired neurogenesis. Defects in the DNA damage response, in DNA repair, and structural abnormalities in centrosomes, centrioles and the spindle microtubule network have all been demonstrated to cause microcephaly in humans. Work describing novel functional defects in cell lines from individuals with either Meier-Gorlin syndrome or Wolf-Hirschhorn syndrome highlight the significance of optimal DNA replication and S phase progression for normal human development, including neurogenesis. These findings illustrate how different primary defects in processes impacting upon DNA replication potentially influence similar phenotypic outcomes, including growth retardation and microcephaly. Herein, we will describe the nature of the S phase defects uncovered for each of these conditions and highlight some of the overlapping cellular features.

Characterizing the functional consequences of haploinsufficiency of NELF-A (WHSC2) and SLBP identifies novel cellular phenotypes in Wolf-Hirschhorn syndrome.

Wolf-Hirschhorn syndrome (WHS) is a contiguous gene deletion disorder associated with the distal part of the short arm of chromosome 4 (4p16.3). Employing a unique panel of patient-derived cell lines with differing-sized 4p deletions, we provide evidence that haploinsufficiency of SLBP and/or WHSC2 (NELF-A) contributes to several novel cellular phenotypes of WHS, including delayed progression from S-phase into M-phase, reduced DNA replication in asynchronous culture and altered higher order chromatin assembly. The latter is evidenced by reduced histone-chromatin association, elevated levels of soluble chaperone-bound histone H3 and increased sensitivity to micrococcal nuclease digestion in WHS patient-derived cells. We also observed increased camptothecin-induced inhibition of DNA replication and hypersensitivity to killing. Our work provides a novel pathogenomic insight into the aetiology of WHS by describing it, for the first time, as a disorder of impaired chromatin reorganization. Delayed cell-cycle progression and impaired DNA replication likely underlie or contribute to microcephaly, pre- and postnatal growth retardation, which constitute the core clinical features of WHS.

A chemical-genetic screen reveals a mechanism of resistance to PI3K inhibitors in cancer.

Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. However, there has been no systematic approach for analyzing gene-drug interactions in human cells. Here we establish a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, enabling the systematic analysis of thousands of gene-drug interactions. Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug-resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to phosphoinositide 3-kinase (PI3K) inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC over-rode the dependency of cells on the PI3K-mTOR pathway for proliferation. These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.

CUL4B-deficiency in humans: understanding the clinical consequences of impaired Cullin 4-RING E3 ubiquitin ligase function.

Cullin's encode the structural components for one of the most abundant E3 ubiquitin ligase families in eukaryotes accounting for as many as 400 distinct E3 ubiquitin ligases. Because of their modular assembly involving combinations of multiple distinct adaptor and substrate receptor proteins, it comes as no surprise that these E3's are implicated in a plethora of fundamental biochemical processes ranging from DNA replication and repair to transcription and development. Herein, we focus on one member of the cullin family, namely the Cullin 4-RING E3 ligases (CRL4's). More specifically, we overview what has been learned about some of the functions of CRL4's from various model systems. We discuss the unexpected association of defective CUL4B with syndromal X-linked mental retardation in humans and speculate on the biochemical consequences and clinical implications of defective CRL4 function. In particular, mutations in CUL4B highlight a previously unappreciated role for CRL4's in neuronal function and cognition in humans.

Mutations in ORC1, encoding the largest subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome.

Studies into disorders of extreme growth failure (for example, Seckel syndrome and Majewski osteodysplastic primordial dwarfism type II) have implicated fundamental cellular processes of DNA damage response signaling and centrosome function in the regulation of human growth. Here we report that mutations in ORC1, encoding a subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome. We establish that these mutations disrupt known ORC1 functions including pre-replicative complex formation and origin activation. ORC1 deficiency perturbs S-phase entry and S-phase progression. Additionally, we show that Orc1 depletion in zebrafish is sufficient to markedly reduce body size during rapid embryonic growth. Our data suggest a model in which ORC1 mutations impair replication licensing, slowing cell cycle progression and consequently impeding growth during development, particularly at times of rapid proliferation. These findings establish a novel mechanism for the pathogenesis of microcephalic dwarfism and show a surprising but important developmental impact of impaired origin licensing.

Mutations in Cullin 4B result in a human syndrome associated with increased camptothecin-induced topoisomerase I-dependent DNA breaks.

CUL4A and B encode subunits of E3-ubiquitin ligases implicated in diverse processes including nucleotide excision repair, regulating gene expression and controlling DNA replication fork licensing. But, the functional distinction between CUL4A and CUL4B, if any, is unclear. Recently, mutations in CUL4B were identified in humans associated with mental retardation, relative macrocephaly, tremor and a peripheral neuropathy. Cells from these patients offer a unique system to help define at the molecular level the consequences of defective CUL4B specifically. We show that these patient-derived cells exhibit sensitivity to camptothecin (CPT), impaired CPT-induced topoisomerase I (Topo I) degradation and ubiquitination, thereby suggesting Topo I to be a novel Cul4-dependent substrate. Consistent with this, we also find that these cells exhibit increased levels of CPT-induced DNA breaks. Furthermore, over-expression of known CUL4-dependent substrates including Cdt1 and p21 appear to be a feature of these patient-derived cells. Collectively, our findings highlight the interplay between CUL4A and CUL4B and provide insight into the pathogenesis of CUL4B-deficiency in humans.

UVB and caffeine: inhibiting the DNA damage response to protect against the adverse effects of UVB.

The incidence of sunlight-induced skin cancer is increasing. Mouse studies indicate that caffeine, administered orally or topically, promotes apoptosis of UVB-irradiated keratinocytes. In this issue, Heffernan and colleagues identify the pathway targeted by caffeine and suggest that inhibition of this DNA damage response may offer a viable therapeutic option for nonmelanoma skin cancer. This potentially represents an important protective or therapeutic option from the most unlikely of sources: your daily coffee.

Human DNA damage response and repair deficiency syndromes: linking genomic instability and cell cycle checkpoint proficiency.

A plethora of clinically distinct human disorders exist whose underlying cause is a defect in the response to or repair of DNA damage. The clinical spectrum of these conditions provides evidence for the role of the DNA damage response (DDR) in mediating diverse processes such as genomic stability, immune system function and normal human development. Cell lines from these disorders provide a valuable resource to help dissect the consequences of compromised DDR at the molecular level. Here we will discuss some well known, less well known and 'novel' DDR defective disorders with particular reference to the functional interplay between the DNA damage response and cell cycle checkpoints. We will describe recent advances in further delineating the genetic basis of Seckel syndrome and microcephalic osteodysplastic primordial dwarfism type II, which have shed more light on the interplay between the DDR, cycle progression and centrosomes. We will also overview recent developments concerning haploinsufficiency of DDR components and their association with certain genomic disorders such as Miller-Dieker lissencephaly syndrome and Williams-Beuren syndrome. Finally, we will discuss how defects in the DDR result in some unexpected clinical features before describing how the nature of a DDR defect impacts on the management and treatment of individuals with these conditions.

A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene.

Obligatory homologous recombination (HR) is required for chiasma formation and chromosome segregation in meiosis I. Meiotic HR is initiated by DNA double-strand breaks (DSBs), generated by Spo11, a homologue of the archaebacterial topoisomerase subunit Top6A. In Saccharomyces cerevisiae, Rad50, Mre11 and Com1/Sae2 are essential to process an intermediate of the cleavage reaction consisting of Spo11 covalently linked to the 5' termini of DNA. While Rad50 and Mre11 also confer genome stability to vegetative cells and are well conserved in evolution, Com1/Sae2 was believed to be fungal-specific. Here, we identify COM1/SAE2 homologues in all eukaryotic kingdoms. Arabidopsis thaliana Com1/Sae2 mutants are sterile, accumulate AtSPO11-1 during meiotic prophase and fail to form AtRAd51 foci despite the presence of unrepaired DSBs. Furthermore, DNA fragmentation in AtCom1 is suppressed by eliminating AtSPO11-1. In addition, AtCOM1 is specifically required for mitomycin C resistance. Interestingly, we identified CtIP, an essential protein interacting with the DNA repair machinery, as the mammalian homologue of Com1/Sae2, with important implications for the molecular role of CtIP.

The Arabidopsis thaliana MND1 homologue plays a key role in meiotic homologous pairing, synapsis and recombination.

Mnd1 has recently been identified in yeast as a key player in meiotic recombination. Here we describe the identification and functional characterisation of the Arabidopsis homologue, AtMND1, which is essential for male and female meiosis and thus for plant fertility. Although axial elements are formed normally, sister chromatid cohesion is established and recombination initiation appears to be unaffected in mutant plants, chromosomes do not synapse. During meiotic progression, a mass of entangled chromosomes, interconnected by chromatin bridges, and severe chromosome fragmentation are observed. These defects depend on the presence of SPO11-1, a protein that initiates recombination by catalysing DNA double-strand break (DSB) formation. Furthermore, we demonstrate that the AtMND1 protein interacts with AHP2, the Arabidopsis protein closely related to budding yeast Hop2. These data demonstrate that AtMND1 plays a key role in homologous synapsis and in DSB repair during meiotic recombination.

Expression of the ubiquitin variant ubR48 decreases proteolytic activity in Arabidopsis and induces cell death.

The ubiquitin-proteasome pathway is the major route for protein degradation in eukaryotes. We show here that this pathway can be inhibited in Arabidopsis thaliana by expression of a ubiquitin variant that contains Arg instead of Lys at position 48 (ubR48). A major consequence of ubR48 expression is the induction of cell death. Cell death induction coincides with the appearance of reactive oxygen intermediates, but is independent of salicylic acid. We found changes in expression of some defense-related genes, but these changes are apparently insufficient to cause alterations in the response to a bacterial pathogen. Expression of ubR48 from an inducible gene allowed investigation of kinetic parameters of cell death induction. In the absence of additional stress factors, slow death processes dominate if the transgene is induced in seedlings older than 2 weeks. The inducible gene also allowed isolation of suppressor mutants. Expression of ubR48 may cause changes similar to inhibition of the proteasome, which also induces various forms of cell death. Thus, ubR48 is a tool to manipulate protein turnover and to probe cell death programs in plants.