Biology subjects

Rai, K.

Publications and source records attributed to Rai, K..

40 records · Page 3Linked to original sources

bioRxiv2019

Genomes of intracellular Leishmania parasites directly sequenced from patients

Whole genome sequencing (WGS) is increasingly used for molecular diagnosis and epidemiology of infectious diseases. Current Leishmania genomic studies rely on DNA extracted from cultured parasites, which might introduce sampling and biological biases into the subsequent analyses. Up to now, direct analysis of Leishmania genome in clinical samples is hampered by high levels of human DNA and large variation in parasite load in patient samples. Here, we present a method, based on target enrichment of Leishmania donovani DNA with Agilent SureSelect technology, that allows the analysis of Leishmania genomes directly in clinical samples. We validated our protocol with a set of artificially mixed samples, followed by the analysis of 63 clinical samples (bone marrow or spleen aspirates) from visceral leishmaniasis patients in Nepal. We were able to identify genotypes using a set of diagnostic SNPs in almost all of these samples (97%) and access comprehensive genome-wide information in most (83%). This allowed us to perform phylogenomic analysis, assess chromosome copy number and identify large copy number variants (CNVs). Pairwise comparisons between the parasite genomes in clinical samples and derived in vitro cultured promastigotes showed a lower aneuploidy in amastigotes as well as genomic differences, suggesting polyclonal infections in patients. Altogether our results underline the need for sequencing parasite genomes directly in the host samples.\n\nAuthor summaryVisceral leishmaniasis (VL) is caused by parasitic protozoa of the Leishmania donovani complex and is lethal in the absence of treatment. Whole Genome Sequencing (WGS) of L. donovani clinical isolates revealed hitherto cryptic population structure in the Indian Sub-Continent and provided insights into the epidemiology and potential mechanisms of drug resistance. However, several biases are likely introduced during the culture step. We report here the development of a method that allows determination of parasite genomes directly in clinical samples, and validate it on bone marrow and splenic aspirates of VL patients in Nepal. Our study sheds a new light on the biology of Leishmania in the human host: we found that intracellular parasites of the patients had very low levels of aneuploidy, in sharp contrast to the situation in cultivated isolates. Moreover, the observed differences in genomes between intracellular amastigotes of the patient and the derived cultured parasites suggests polyclonality of infections, with different clones dominating in clinical samples and in culture, likely due to fitness differences. We believe this method is most suitable for clinical studies and for molecular tracking in the context of elimination programs.

microbiology↗

bioRxiv2019

Gene-specific methylation profiles for integrative methylation-expression analysis in cancer research

DNA methylation is a key epigenetic factor regulating gene expression. While promoter-associated methylation has been extensively studied, recent publications have revealed that functionally important methylation also occurs in intergenic and distal regions, and varies across genes and tissue types. Given the growing importance of inter-platform integrative genomic analyses, there is an urgent need to develop methods to construct gene-level methylation summaries that account for the potentially complex relationships between methylation and expression. We introduce a novel sequential penalized regression approach to construct gene-specific methylation profiles (GSMPs) which find for each gene and tissue type a sparse set of CpGs best explaining gene expression and weights indicating direction and strength of association. Using TCGA and MD Anderson colorectal cohorts to build and validate our models, we demonstrate our strategy better explains expression variability than standard approaches and produces gene-level scores showing key methylation differences across recently discovered colorectal cancer subtypes. We share an R Shiny app that presents GSMP results for colorectal, breast, and pancreatic cancer with plans to extend it to all TCGA cancer types. Our approach yields tissue-specific, gene-specific sparse lists of functionally important CpGs that can be used to construct gene-level methylation scores that are maximally correlated with gene expression for use in integrative models, and produce a tissue-specific summary of which genes appear to be strongly regulated by methylation. Our results introduce an important resource to the biomedical community for integrative genomics analyses involving DNA methylation.

genomics↗

bioRxiv2018

Super-enhancer impairment is a link between MLL4 inactivated lung tumors and their vulnerability to glycolysis pathway inhibition

Epigenetic modifiers often harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Here we show that lung-specific loss of the gene encoding the histone methyltransferase MLL4 (alias KMT2D; a COMPASS-like enzyme), which is ranked the most highly inactivated epigenetic modifier in lung cancer, strongly promotes lung adenocarcinoma in mice. Mll4 loss upregulated tumor-promoting programs, including glycolysis. The pharmacological inhibition of glycolysis preferentially impeded tumorigenic growth of human lung cancer cells bearing MLL4-inactivating mutations. Mll4 loss widely impaired epigenomic signals for super-enhancers and enhancers, including the super-enhancer for the circadian rhythm repressor gene Per2, and decreased Per2 expression. Per2 downregulated several glycolytic pathway genes. These findings uncover a distinct tumor-suppressive epigenetic mechanism in which MLL4 enhances Per2-mediated repression of pro-tumorigenic glycolytic genes via super-enhancer activation to suppress lung adenocarcinoma tumorigenesis and also implicate a glycolysis-targeting strategy as a therapeutic intervention for the treatment of MLL4- mutant lung cancer.

cancer biology↗

bioRxiv2018

Enhancer Reprogramming Confers Dependence on Glycolysis and IGF signaling in KMT2D Mutant Melanoma

Epigenetic modifiers have emerged as important regulators of tumor progression. We identified histone methyltransferase KMT2D as a potent tumor-suppressor through an in vivo epigenome-focused pooled RNAi screen in melanoma. KMT2D harbors frequent somatic point mutations in multiple tumor types. How these events contribute to tumorigenesis and whether they impart therapeutic vulnerability are poorly understood. To address these questions, we generated a genetically engineered mouse model of melanoma based on conditional and melanocyte-specific deletion of KMT2D. We demonstrate KMT2D as a bona fide tumor suppressor which cooperates with activated BRAF. KMT2D-deficient tumors showed substantial reprogramming of key metabolic pathways including glycolysis. Glycolysis enzymes, intermediate metabolites and glucose consumption rate were aberrantly upregulated in KMT2D mutant cells. The pharmacological inhibition of glycolysis reduced proliferation and tumorigenesis preferentially in KMT2D mutant cells. Mechanistically, KMT2D loss caused drastic reduction of H3K4me1-marked active enhancer states. Loss of distal enhancer and subsequent reduction in expression of IGFBP5 activated IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating increased usage of glycolysis pathway for enhanced biomass needs via enhancer reprogramming. Our data imply that inhibition of glycolysis or IGFR pathway could be a potential therapeutic strategy in KMT2D mutant tumors.

cancer biology↗