Project description:Here we show that the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance and the other via an epigenetic RNA interference (RNAi)-mediated pathway that results in unstable drug resistance. The peptidyl-prolyl isomerase FKBP12 interacts with FK506 to form a complex that inhibits the protein phosphatase calcineurin. In M. circinelloides, inhibition of calcineurin by FK506 blocks the transition to hyphae and enforces yeast growth. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance, restoring hyphal growth in the presence of this drug. In parallel, RNAi is spontaneously triggered to silence the FKBP12 fkbA gene, giving rise to drug-resistant epimutants. FK506-resistant epimutants readily reverted to the drug-sensitive wild type (WT) phenotype when selection was released by growth without drug. High-throughput small RNA (sRNA) sequencing showed abundant sRNA antisense to fkbA only in the epimutants and not in the WT or revertant strains. Analysis of RNAi mutants revealed components required for the establishment of drug resistant epimutants as well as other factors that constrain or reverse the epimutation pathway. Silencing involves generation of a double stranded RNA trigger intermediate using the fkbA mature mRNA as template. These results uncover a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi regulatory mechanisms in fungi and other eukaryotes.
Project description:Our aim is to analyze the genome of human melanoma cell lines and short term culture from human melanoma samples in order to identify genes that confer drug resistance to clinically relevant targeted therapies. We will perform whole-exome sequencing, copy number variation analysis and methylome analysis in a collection of human melanoma cell lines and short term culture that will be then screened for drug sensitivity/resistance through a library of clinically relevant drugs and drug combinations. By the combined analysis of the genomic lesion and the drug sensitivity/resistance profile of different cell lines, we will look for genes whose mutation is associated to the sensitivity or resistance to a specific drug in different samples.
Project description:Oxamniquine resistance evolved in the human blood fluke (Schistosoma mansoni) in Brazil in the 1970s. We crossed parental parasites differing ~500-fold in drug response, determined drug sensitivity and marker segregation in clonally-derived F2s, and identified a single QTL (LOD=31) on chromosome 6. A sulfotransferase was identified as the causative gene using RNAi knockdown and biochemical complementation assays and we subsequently demonstrated independent origins of loss-of-function mutations in field-derived and laboratory-selected resistant parasites. These results demonstrate the utility of linkage mapping in a human helminth parasite, while crystallographic analyses of protein-drug interactions illuminate the mode of drug action and provide a framework for rational design of oxamniquine derivatives that kill both S. mansoni and S. haematobium, the two species responsible for >99% of schistosomiasis cases worldwide. mRNA profiles from adult worms resistant (HR, 3 replicates) and susceptible (LE, 2 replicates) to the oxamniquine drug were compared to identify differential expression in genes related to drug resistance
Project description:Background
Development of drug resistance is often dynamic and requires time series experiments to understand these processes. The most abundant source of information regarding such progressive activity is tracking single cell gene expression data. Our understanding of the imminent role of phenotype switching in modifying therapeutic response has considerably increased.The analysis of single cell transcriptomic data along with machine learning models offers a constructive tool to describe dynamic cellular processes to elucidate cell-to-cell variability within the population after drug exposure. Here we studied how genomic and non-genomic basis for transcriptome differences can influence the developing drug resistance and clonal fitness.
Results: This study describes the scRNA-seq analysis of untreated, treated and drug holiday PDX transplants of TNBC exposed to platinum. This builds from the materials and findings of our paper by Salehi, Kabeer et al, Nature 2021 (PMID: 3416307), in which we established the measurement of fitness in this time series. In the new work, we have mapped single cell transcription in conjunction, allowing a dissection of copy number driven (in-cis) clonal transcription and non-clonal, ie non-genomic transcription. We developed an experimental and computational platform consisting of four major steps: (1) timeseries sampling of scRNA-seq 126,556 TNBC PDX cells >2.5 years; (2) clustering the tumor cells into groups reflecting their drug sensitivity, resistance and other characteristics; (3) performing trajectory inference to identify lineages of transitional processes along with drug and tumor progression; (4) estimating the longitudinal gene expression of each lineage; and identifying significant genes between-lineage differential expression modules. Interestingly, we identified a small group of genes (18.9%) that display a new cell state that is not towards the untreated (Un-Rx) nor towards treated Rx which are distant from untreated cell states. The pathway results are shared between two different quantitative measurements of clone-aware differential expression and pseudotime analysis.
Project description:Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). We optimized and leveraged this property to identify drug resistance mutations, a technique we term LentiMutate. After validating this technique by identifying clinically relevant EGFR resistance mutations, we applied this technique to two additional anti-cancer drugs, imatinib and AMG 510. We find novel deletions in BCR-ABL1 that confer resistance to BCR-ABL1 inhibitors and point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations, in KRAS-G12C or wild-type KRAS, respectively, that confer resistance to AMG 510. LentiMutate may prove highly valuable to clinical and preclinical cancer drug development.
Project description:Analysis of the mechanism of drug resistance regulated by Twist 1 at gene expression level. The hypothesis tested in the present study was that Twist 1 regulated the expression of drug resistance related genes. Results provide important information of the response of drug resistance related genes to Twist 1, either up- or down-regulated.