Genotyping panel for assessing response to cancer chemotherapy

Background

Variants in numerous genes are thought to affect the success or failure of cancer chemotherapy. Interindividual variability can result from genes involved in drug metabolism and transport, drug targets (receptors, enzymes, etc), and proteins relevant to cell survival (e.g., cell cycle, DNA repair, and apoptosis). The purpose of the current study is to establish a flexible, cost-effective, high-throughput genotyping platform for candidate genes involved in chemoresistance and -sensitivity, and treatment outcomes.

Methods

We have adopted SNPlex for genotyping 432 single nucleotide polymorphisms (SNPs) in 160 candidate genes implicated in response to anticancer chemotherapy.

Results

The genotyping panels were applied to 39 patients with chronic lymphocytic leukemia undergoing flavopiridol chemotherapy, and 90 patients with colorectal cancer. 408 SNPs (94%) produced successful genotyping results. Additional genotyping methods were established for polymorphisms undetectable by SNPlex, including multiplexed SNaPshot for CYP2D6 SNPs, and PCR amplification with fluorescently labeled primers for the UGT1A1 promoter (TA)nTAA repeat polymorphism.

Conclusion

This genotyping panel is useful for supporting clinical anticancer drug trials to identify polymorphisms that contribute to interindividual variability in drug response. Availability of population genetic data across multiple studies has the potential to yield genetic biomarkers for optimizing anticancer therapy.     

Genome‐scale screening in a rat haploid system identifies Thop1 as a modulator of pluripotency exit

ObjectivesThe rats are crucial animal models for the basic medical researches. Rat embryonic stem cells (ESCs), which are widely studied, can self‐renew and exhibit pluripotency in long‐term culture, but the mechanism underlying how they exit pluripotency remains obscure. To investigate the key modulators on pluripotency exiting in rat ESCs, we perform genome‐wide screening using a unique rat haploid system.Materials and MethodsRat haploid ESCs (haESCs) enable advances in the discovery of unknown functional genes owing to their homozygous and pluripotent characteristics. REX1 is a sensitive marker for the naïve pluripotency that is often utilized to monitor pluripotency exit, thus rat haESCs carrying a Rex1‐GFP reporter are used for genetic screening. Genome‐wide mutations are introduced into the genomes of rat Rex1‐GFP haESCs via piggyBac transposon, and differentiation‐retarded mutants are obtained after random differentiation selection. The exact mutations are elucidated by high‐throughput sequencing and bioinformatic analysis. The role of candidate mutation is validated in rat ESCs by knockout and overexpression experiments, and the phosphorylation of ERK1/2 (p‐ERK1/2) is determined by western blotting.ResultsHigh‐throughput sequencing analysis reveals numerous insertions related to various pathways affecting random differentiation. Thereafter, deletion of Thop1 (one candidate gene in the screened list) arrests the differentiation of rat ESCs by inhibiting the p‐ERK1/2, whereas overexpression of Thop1 promotes rat ESCs to exit from pluripotency.ConclusionsOur findings provide an ideal tool to study functional genomics in rats: a homozygous haploid system carrying a pluripotency reporter that facilitates robust discovery of the mechanisms involved in the self‐renewal or pluripotency of rat ESCs.

Differentiation of pluripotent rat embryonic stem cells (ESCs) in vitro is difficult to achieve for unknown mechanisms. Rat haploid ESCs (haESCs) have been validated as a powerful tool to target unknown functional genes and pathways based on homozygous genetic screening. Xu et al. utilized Rex1‐GFP labelled‐rat haESCs to conduct genome‐scale screening of genes modulating pluripotency exiting. Validation experiments showed that Thop1 (one of the screened out genes) played very important roles in the random differentiation of rat ESCs in vitro via modulating phosphorylation of ERK.     

Ionizing radiation-induced DNA injury and damage detection in patients with breast cancer

Breast cancer is the most common malignancy in women. Radiotherapy is frequently used in patients with breast cancer, but some patients may be more susceptible to ionizing radiation, and increased exposure to radiation sources may be associated to radiation adverse events. This susceptibility may be related to deficiencies in DNA repair mechanisms that are activated after cell-radiation, which causes DNA damage, particularly DNA double strand breaks. Some of these genetic susceptibilities in DNA-repair mechanisms are implicated in the etiology of hereditary breast/ovarian cancer (pathologic mutations in the BRCA 1 and 2 genes), but other less penetrant variants in genes involved in sporadic breast cancer have been described. These same genetic susceptibilities may be involved in negative radiotherapeutic outcomes. For these reasons, it is necessary to implement methods for detecting patients who are susceptible to radiotherapy-related adverse events. This review discusses mechanisms of DNA damage and repair, genes related to these functions, and the diagnosis methods designed and under research for detection of breast cancer patients with increased radiosensitivity.     

Genome-Wide Association Analysis of Radiation Resistance in Drosophila melanogaster

Background

Ionizing radiation is genotoxic to cells. Healthy tissue toxicity in patients and radiation resistance in tumors present common clinical challenges in delivering effective radiation therapies. Radiation response is a complex, polygenic trait with unknown genetic determinants. The Drosophila Genetic Reference Panel (DGRP) provides a model to investigate the genetics of natural variation for sensitivity to radiation.

Methods and Findings

Radiation response was quantified in 154 inbred DGRP lines, among which 92 radiosensitive lines and 62 radioresistant lines were classified as controls and cases, respectively. A case-control genome-wide association screen for radioresistance was performed. There are 32 single nucleotide polymorphisms (SNPs) associated with radio resistance at a nominal p<10⁻⁵; all had modest effect sizes and were common variants with the minor allele frequency >5%. All the genes implicated by those SNP hits were novel, many without a known role in radiation resistance and some with unknown function. Variants in known DNA damage and repair genes associated with radiation response were below the significance threshold of p<10⁻⁵ and were not present among the significant hits. No SNP met the genome-wide significance threshold (p = 1.49×10⁻⁷), indicating a necessity for a larger sample size.

Conclusions

Several genes not previously associated with variation in radiation resistance were identified. These genes, especially the ones with human homologs, form the basis for exploring new pathways involved in radiation resistance in novel functional studies. An improved DGRP model with a sample size of at least 265 lines and ideally up to 793 lines is recommended for future studies of complex traits.     

Upregulation of DNA repair genes and cell extrusion underpin the remarkable radiation resistance of Trichoplax adhaerens

Trichoplax adhaerens is the simplest multicellular animal with tissue differentiation and somatic cell turnover. Like all other multicellular organisms, it should be vulnerable to cancer, yet there have been no reports of cancer in T. adhaerens or any other placozoan. We investigated the cancer resistance of T. adhaerens, discovering that they are able to tolerate high levels of radiation damage (218.6 Gy). To investigate how T. adhaerens survive levels of radiation that are lethal to other animals, we examined gene expression after the X-ray exposure, finding overexpression of genes involved in DNA repair and apoptosis including the MDM2 gene. We also discovered that T. adhaerens extrudes clusters of inviable cells after X-ray exposure. T. adhaerens is a valuable model organism for studying the molecular, genetic, and tissue-level mechanisms underlying cancer suppression.

The placozoan Trichoplax adhaerens is able to tolerate high levels of radiation and is resilient to DNA damage; this study reveals that exposure to X-rays triggers the extrusion of cell clusters which subsequently die, and that radiation exposure induces the overexpression of genes involved in DNA repair.     

Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes

The reactivation of X‐linked genes is observed in some primary breast tumors. Two active X chromosomes are also observed in female embryonic stem cells (ESCs), but whether double doses of X‐linked genes affect DNA repair efficiency remains unclear. Here, we establish isogenic female/male ESCs and show that the female ESCs are more sensitive to camptothecin and have lower gene targeting efficiency than male ESCs, suggesting that homologous recombination (HR) efficiency is reduced in female ESCs. We also generate Xist‐inducible female ESCs and show that the lower HR efficiency is restored when X chromosome inactivation is induced. Finally, we assess the X‐linked genes with a role in DNA repair and find that Brcc3 is one of the genes involved in a network promoting proper HR. Our findings link the double doses of X‐linked genes with lower DNA repair activity, and this may have relevance for common diseases in female patients, such as breast cancer.     

Identification of Druggable Cancer Driver Genes Amplified across TCGA Datasets

The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 14 cancer subtypes and identified 461 genes that were amplified in two or more datasets. The list was narrowed to 73 cancer-associated genes with potential “druggable” properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 40 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 40 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapter GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts.     

Genome-wide screening in the haploid system reveals Slc25a43 as a target gene of oxidative toxicity

Reactive oxygen species (ROS) are extensively assessed in physiological and pathological studies; however, the genes and mechanisms involved in antioxidant reactions are elusive. To address this knowledge gap, we used a forward genetic approach with mouse haploid embryonic stem cells (haESCs) to generate high-throughput mutant libraries, from which numerous oxidative stress-targeting genes were screened out. We performed proof-of-concept experiments to validate the potential inserted genes. Slc25a43 (one of the candidates) knockout (KO) ESCs presented reduced damage caused by ROS and higher cell viability when exposed to H₂O₂. Subsequently, ROS production and mitochondrial function analysis also confirmed that Slc25a43 was a main target gene of oxidative toxicity. In addition, we identified that KO of Slc25a43 activated mitochondria-related genes including Nlrx1 to protect ESCs from oxidative damage. Overall, our findings facilitated revealing target genes of oxidative stress and shed lights on the mechanism underlying oxidative death.Subject terms: Cell death, Stem cells, Screening, Target identification     

The potential of effector-target genes in breeding for plant innate immunity

Increasing numbers of infectious crop diseases that are caused by fungi and oomycetes urge the need to develop alternative strategies for resistance breeding. As an alternative for the use of resistance (R) genes, the application of mutant susceptibility (S) genes has been proposed as a potentially more durable type of resistance. Identification of S genes is hampered by their recessive nature. Here we explore the use of pathogen-derived effectors as molecular probes to identify S genes. Effectors manipulate specific host processes thereby contributing to disease. Effector targets might therefore represent S genes. Indeed, the Pseudomonas syringae effector HopZ2 was found to target MLO2, an Arabidopsis thaliana homologue of the barley S gene Mlo. Unfortunately, most effector targets identified so far are not applicable as S genes due to detrimental effects they have on other traits. However, some effector targets such as Mlo are successfully used, and with the increase in numbers of effector targets being identified, the numbers of S genes that can be used in resistance breeding will rise as well.     

High-Content,Image-Based Screening for Drug Targets in Yeast

Background

Drug discovery and development are predicated on elucidation of the potential mechanisms of action and cellular targets of candidate chemical compounds. Recent advances in high-content imaging techniques allow simultaneous analysis of a range of cellular events. In this study, we propose a novel strategy to identify drug targets by combining genetic screening and high-content imaging in yeast.

Methodology

In this approach, we infer the cellular functions affected by candidate drugs by comparing morphologic changes induced by the compounds with the phenotypes of yeast mutants.

Conclusions

Using this method and four well-characterized reagents, we successfully identified previously known target genes of the compounds as well as other genes involved with functionally related cellular pathways. This is the first demonstration of a genetic high-content assay that can be used to identify drug targets based on morphologic phenotypes of a reference mutant panel.     

Integrative Analysis of Epigenetic Modulation in Melanoma Cell Response to Decitabine: Clinical Implications

Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21^Cip1 in a p53-independent manner, identified the TGFβ pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated β-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patient''s selection and monitoring response, as well as targets for improved combination therapy.     

Exome Sequencing Identifies Three Novel Candidate Genes Implicated in Intellectual Disability

Intellectual disability (ID) is a major health problem mostly with an unknown etiology. Recently exome sequencing of individuals with ID identified novel genes implicated in the disease. Therefore the purpose of the present study was to identify the genetic cause of ID in one syndromic and two non-syndromic Pakistani families. Whole exome of three ID probands was sequenced. Missense variations in two plausible novel genes implicated in autosomal recessive ID were identified: lysine (K)-specific methyltransferase 2B (KMT2B), zinc finger protein 589 (ZNF589), as well as hedgehog acyltransferase (HHAT) with a de novo mutation with autosomal dominant mode of inheritance. The KMT2B recessive variant is the first report of recessive Kleefstra syndrome-like phenotype. Identification of plausible causative mutations for two recessive and a dominant type of ID, in genes not previously implicated in disease, underscores the large genetic heterogeneity of ID. These results also support the viewpoint that large number of ID genes converge on limited number of common networks i.e. ZNF589 belongs to KRAB-domain zinc-finger proteins previously implicated in ID, HHAT is predicted to affect sonic hedgehog, which is involved in several disorders with ID, KMT2B associated with syndromic ID fits the epigenetic module underlying the Kleefstra syndromic spectrum. The association of these novel genes in three different Pakistani ID families highlights the importance of screening these genes in more families with similar phenotypes from different populations to confirm the involvement of these genes in pathogenesis of ID.     

Negative Regulation of Systemic Acquired Resistance by Replication Factor C Subunit3 in Arabidopsis

Systemic acquired resistance (SAR) is a plant immune response induced by local necrotizing pathogen infections. Expression of SAR in Arabidopsis (Arabidopsis thaliana) plants correlates with accumulation of salicylic acid (SA) and up-regulation of Pathogenesis-Related (PR) genes. SA is an essential and sufficient signal for SAR. In a genetic screen to search for negative regulators of PR gene expression and SAR, we found a new mutant that is hypersensitive to SA and exhibits enhanced induction of PR genes and resistance against the virulent oomycete Hyaloperonospora arabidopsidis Noco2. The enhanced pathogen resistance in the mutant is Nonexpressor of PR genes1 independent. The mutant gene was identified by map-based cloning, and it encodes a protein with high homology to Replication Factor C Subunit3 (RFC3) of yeast and other eukaryotes; thus, the mutant was named rfc3-1. rfc3-1 mutant plants are smaller than wild-type plants and have narrower leaves and petals. On the epidermis of true leaves, there are fewer cells in rfc3-1 compared with the wild type. Cell production rate is reduced in rfc3-1 mutant roots, indicating that the mutated RFC3 slows down cell proliferation. As Replication Factor C is involved in replication-coupled chromatin assembly, our data suggest that chromatin assembly and remodeling may play important roles in the negative control of PR gene expression and SAR.