A cell-based high-throughput screen identifies inhibitors that overcome P-glycoprotein (Pgp)-mediated multidrug resistance.

Multidrug resistance (MDR) to chemotherapeutic drugs remains one of the major impediments to the treatment of cancer. Discovery and development of drugs that can prevent and reverse the acquisition of multidrug resistance constitute a foremost challenge in cancer therapeutics. In this work, we screened a library of 1,127 compounds with known targets for their ability to overcome Pgp-mediated multidrug resistance in cancer cell lines. We identified four compounds (CHIR-124, Elesclomol, Tyrphostin-9 and Brefeldin A) that inhibited the growth of two pairs of parental and Pgp-overexpressing multidrug-resistant cell lines with similar potency irrespective of their Pgp status. Mechanistically, CHIR-124 (a potent inhibitor of Chk1 kinase) inhibited Pgp activity in both multidrug-resistant cell lines (KB-V1 and A2780-Pac-Res) as determined through cell-based Pgp-efflux assays. Other three inhibitors on the contrary, were effective in Pgp-overexpressing resistant cells without increasing the cellular accumulation of a Pgp substrate, indicating that they overcome resistance by avoiding efflux through Pgp. None of these compounds modulated the expression of Pgp in resistant cell lines. PIK-75, a PI3 Kinase inhibitor, was also determined to inhibit Pgp activity, despite being equally potent in only one of the two pairs of resistant and parental cell lines. Strong binding of both CHIR-124 and PIK-75 to Pgp was predicted through docking studies and both compounds inhibited Pgp in a biochemical assay. The inhibition of Pgp causes accumulation of these compounds in the cells where they can modulate the function of their target proteins and thereby inhibit cell proliferation. In conclusion, we have identified compounds with various cellular targets that overcome multidrug resistance in Pgp-overexpressing cell lines through mechanisms that include Pgp inhibition and efflux evasion. These compounds, therefore, can avoid challenges associated with the co-administration of Pgp inhibitors with chemotherapeutic or targeted drugs such as additive toxicities and differing pharmacokinetic properties.

DNA mismatch repair preferentially protects genes from mutation.

Mutation is the source of genetic variation and fuels biological evolution. Many mutations first arise as DNA replication errors. These errors subsequently evade correction by cellular DNA repair, for example, by the well-known DNA mismatch repair (MMR) mechanism. Here, we determine the genome-wide effects of MMR on mutation. We first identify almost 9000 mutations accumulated over five generations in eight MMR-deficient mutation accumulation (MA) lines of the model plant species, Arabidopsis thaliana We then show that MMR deficiency greatly increases the frequency of both smaller-scale insertions and deletions (indels) and of single-nucleotide variant (SNV) mutations. Most indels involve A or T nucleotides and occur preferentially in homopolymeric (poly A or poly T) genomic stretches. In addition, we find that the likelihood of occurrence of indels in homopolymeric stretches is strongly related to stretch length, and that this relationship causes ultrahigh localized mutation rates in specific homopolymeric stretch regions. For SNVs, we show that MMR deficiency both increases their frequency and changes their molecular mutational spectrum, causing further enhancement of the GC to AT bias characteristic of organisms with normal MMR function. Our final genome-wide analyses show that MMR deficiency disproportionately increases the numbers of SNVs in genes, rather than in nongenic regions of the genome. This latter observation indicates that MMR preferentially protects genes from mutation and has important consequences for understanding the evolution of genomes during both natural selection and human tumor growth.

HANDS2: accurate assignment of homoeallelic base-identity in allopolyploids despite missing data.

Characterization of homoeallelic base-identity in allopolyploids is difficult since homeologous subgenomes are closely related and becomes further challenging if diploid-progenitor data is missing. We present HANDS2, a next-generation sequencing-based tool that enables highly accurate (>90%) genome-wide discovery of homeolog-specific base-identity in allopolyploids even in the absence of a diploid-progenitor. We applied HANDS2 to the transcriptomes of various cruciferous plants belonging to genus Brassica. Our results suggest that the three C genomes in Brassica are more similar to each other than the three A genomes, and provide important insights into the relationships between various Brassica tetraploids and their diploid-progenitors at a single-base resolution.

Environmentally responsive genome-wide accumulation of de novo Arabidopsis thaliana mutations and epimutations.

Evolution is fueled by phenotypic diversity, which is in turn due to underlying heritable genetic (and potentially epigenetic) variation. While environmental factors are well known to influence the accumulation of novel variation in microorganisms and human cancer cells, the extent to which the natural environment influences the accumulation of novel variation in plants is relatively unknown. Here we use whole-genome and whole-methylome sequencing to test if a specific environmental stress (high-salinity soil) changes the frequency and molecular profile of accumulated mutations and epimutations (changes in cytosine methylation status) in mutation accumulation (MA) lineages of Arabidopsis thaliana. We first show that stressed lineages accumulate ∼100% more mutations, and that these mutations exhibit a distinctive molecular mutational spectrum (specific increases in relative frequency of transversion and insertion/deletion [indel] mutations). We next show that stressed lineages accumulate ∼45% more differentially methylated cytosine positions (DMPs) at CG sites (CG-DMPs) than controls, and also show that while many (∼75%) of these CG-DMPs are inherited, some can be lost in subsequent generations. Finally, we show that stress-associated CG-DMPs arise more frequently in genic than in nongenic regions of the genome. We suggest that commonly encountered natural environmental stresses can accelerate the accumulation and change the profiles of novel inherited variants in plants. Our findings are significant because stress exposure is common among plants in the wild, and they suggest that environmental factors may significantly alter the rates and patterns of incidence of the inherited novel variants that fuel plant evolution.

Patterns of homoeologous gene expression shown by RNA sequencing in hexaploid bread wheat.

BACKGROUND: Bread wheat (Triticum aestivum) has a large, complex and hexaploid genome consisting of A, B and D homoeologous chromosome sets. Therefore each wheat gene potentially exists as a trio of A, B and D homoeoloci, each of which may contribute differentially to wheat phenotypes. We describe a novel approach combining wheat cytogenetic resources (chromosome substitution 'nullisomic-tetrasomic' lines) with next generation deep sequencing of gene transcripts (RNA-Seq), to directly and accurately identify homoeologue-specific single nucleotide variants and quantify the relative contribution of individual homoeoloci to gene expression. RESULTS: We discover, based on a sample comprising ~5-10% of the total wheat gene content, that at least 45% of wheat genes are expressed from all three distinct homoeoloci. Most of these genes show strikingly biased expression patterns in which expression is dominated by a single homoeolocus. The remaining ~55% of wheat genes are expressed from either one or two homoeoloci only, through a combination of extensive transcriptional silencing and homoeolocus loss. CONCLUSIONS: We conclude that wheat is tending towards functional diploidy, through a variety of mechanisms causing single homoeoloci to become the predominant source of gene transcripts. This discovery has profound consequences for wheat breeding and our understanding of wheat evolution.

HANDS: a tool for genome-wide discovery of subgenome-specific base-identity in polyploids.

BACKGROUND: The analysis of polyploid genomes is problematic because homeologous subgenome sequences are closely related. This relatedness makes it difficult to assign individual sequences to the specific subgenome from which they are derived, and hinders the development of polyploid whole genome assemblies. RESULTS: We here present a next-generation sequencing (NGS)-based approach for assignment of subgenome-specific base-identity at sites containing homeolog-specific polymorphisms (HSPs): 'HSP base Assignment using NGS data through Diploid Similarity' (HANDS). We show that HANDS correctly predicts subgenome-specific base-identity at >90% of assayed HSPs in the hexaploid bread wheat (Triticum aestivum) transcriptome, thus providing a substantial increase in accuracy versus previous methods for homeolog-specific base assignment. CONCLUSION: We conclude that HANDS enables rapid and accurate genome-wide discovery of homeolog-specific base-identity, a capability having multiple applications in polyploid genomics.