Ankur Sharma

Bio: Ankur Sharma is an academic researcher from Genome Institute of Singapore. The author has contributed to research in topics: Notch signaling pathway & Population. The author has an hindex of 13, co-authored 26 publications receiving 685 citations. Previous affiliations of Ankur Sharma include Harry Perkins Institute of Medical Research & Curtin University.

Longitudinal single-cell RNA sequencing of patient-derived primary cells reveals drug-induced infidelity in stem cell hierarchy

Abstract: Chemo-resistance is one of the major causes of cancer-related deaths. Here we used single-cell transcriptomics to investigate divergent modes of chemo-resistance in tumor cells. We observed that higher degree of phenotypic intra-tumor heterogeneity (ITH) favors selection of pre-existing drug-resistant cells, whereas phenotypically homogeneous cells engage covert epigenetic mechanisms to trans-differentiate under drug-selection. This adaptation was driven by selection-induced gain of H3K27ac marks on bivalently poised resistance-associated chromatin, and therefore not expressed in the treatment-naive setting. Mechanistic interrogation of this phenomenon revealed that drug-induced adaptation was acquired upon the loss of stem factor SOX2, and a concomitant gain of SOX9. Strikingly we observed an enrichment of SOX9 at drug-induced H3K27ac sites, suggesting that tumor evolution could be driven by stem cell-switch-mediated epigenetic plasticity. Importantly, JQ1 mediated inhibition of BRD4 could reverse drug-induced adaptation. These results provide mechanistic insights into the modes of therapy-induced cellular plasticity and underscore the use of epigenetic inhibitors in targeting tumor evolution.

A monoclonal antibody against human Notch1 ligand-binding domain depletes subpopulation of putative breast cancer stem-like cells.

Abstract: Overexpression of Notch receptors and ligands has been associated with various cancers and developmental disorders, making Notch a potential therapeutic target. Here, we report characterization of Notch1 monoclonal antibodies (mAb) with therapeutic potential. The mAbs generated against epidermal growth factor (EGF) repeats 11 to 15 inhibited binding of Jagged1 and Delta-like4 and consequently, signaling in a dose-dependent manner, the antibodies against EGF repeats 11 to 12 being more effective than those against repeats 13 to 15. These data emphasize the role of EGF repeats 11 to 12 in ligand binding. One of the mAbs, 602.101, which specifically recognizes Notch1, inhibited ligand-dependent expression of downstream target genes of Notch such as HES-1, HES-5, and HEY-L in the breast cancer cell line MDA-MB-231. The mAb also decreased cell proliferation and induced apoptotic cell death. Furthermore, exposure to this antibody reduced CD44(Hi)/CD24(Low) subpopulation in MDA-MB-231 cells, suggesting a decrease in the cancer stem-like cell subpopulation. This was confirmed by showing that exposure to the antibody decreased the primary, secondary, and tertiary mammosphere formation efficiency of the cells. Interestingly, effect of the antibody on the putative stem-like cells appeared to be irreversible, because the mammosphere-forming efficiency could not be salvaged even after antibody removal during the secondary sphere formation. The antibody also modulated expression of genes associated with stemness and epithelial-mesenchymal transition. Thus, targeting individual Notch receptors by specific mAbs is a potential therapeutic strategy to reduce the potential breast cancer stem-like cell subpopulation. Mol Cancer Ther; 11(1); 77-86. (C) 2011 AACR.

Targeting the BAF57 SWI/SNF Subunit in Prostate Cancer: A Novel Platform to Control Androgen Receptor Activity

Abstract: The androgen receptor (AR) is critical for disseminated prostate cancer proliferation and survival. AR activity is targeted either through prevention of ligand synthesis or through the use of antagonists that bind the COOH-terminal ligand-binding domain. Although initially effective, treatment fails due to restored AR activity in the presence of therapeutics. Thus, new means must be developed to target AR activity. The SWI/SNF chromatin remodeling complex is critical for AR transcriptional activity, and the BAF57 SWI/SNF subunit facilitates direct interaction with the receptor. Although selected SWI/SNF subunit expression is reduced in prostate cancer, we show that BAF57 is retained in human disease and is elevated in a subset of tumors. Functional analyses showed that BAF57 contributes uniquely to androgen-mediated stimulation of transcription without compromising the effectiveness of AR antagonists. Subsequent studies revealed that BAF57 is recruited to the AR DNA-binding domain/hinge region, which occurs concomitant with receptor activation. These data provided the basis for a novel inhibitor derived from BAF57 [BAF57 inhibitory peptide (BIPep)], which blocked AR residence on chromatin and resultant AR-dependent gene activation. Importantly, BIPep expression was sufficient to inhibit androgen-dependent prostate cancer cell proliferation in AR-positive cells. In summary, these data identify blockade of AR-BAF57 interaction as a novel means to target agonist-induced AR function in prostate cancer, and provide the first evidence that abrogation of SWI/SNF function can be developed as a point of therapeutic intervention in prostate cancer. [Cancer Res 2008;68(12):4551–8]

An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites

Abstract: The DNA in a person’s skin cell will contain the same genes as the DNA in their muscle or brain cells. However, these cells have different identities because different genes are active in skin, muscle and brain cells. Proteins called transcription factors dictate the patterns of gene activation in the different kinds of cells by binding to DNA and switching nearby genes on or off. Transcription factors interact with other proteins such as histones that help to package DNA into a structure known as chromatin. Together, transcription factors, histones and other chromatin-associated proteins determine whether or not nearby genes are active. Sometimes transcription factors and other chromatin-associated proteins bind to the wrong sites on DNA; this situation can lead to diseases in humans, such as cancer. This is one of the many reasons why researchers are interested in working out where specific DNA-binding proteins are located in different situations. A technique called chromatin immunoprecipitation (or ChIP for short) can be used to achieve this goal, yet despite being one of the most widely used techniques in molecular biology, ChIP is hampered by numerous problems. As such, many researchers are keen to find alternative approaches. Skene and Henikoff have now developed a new method, called CUTR this means that protein-DNA interactions are more likely to be maintained in their natural state. With CUT&RUN, as in ChIP, a specific antibody identifies the protein of interest. But in CUT&RUN, this antibody binds to the target protein in intact cells and cuts out the DNA that the protein is bound to, releasing the DNA fragment from the cell. This new strategy allows the DNA fragments to be sequenced and identified more efficiently than is currently possible with ChIP. Skene and Henikoff showed that their new method could more accurately identify where transcription factors bind to DNA from yeast and human cells. CUT&RUN also identified a specific histone that is rarely found in yeast chromatin and the technique can be used with a small number of starting cells. Given the advantages that CUT&RUN offers over ChIP, Skene and Henikoff anticipate that the method will be viewed as a cost-effective and versatile alternative to ChIP. In future, the method could be automated so that multiple analyses can be performed at once.

Notch signalling in context

Abstract: The highly conserved Notch signalling pathway functions in many different developmental and homeostatic processes, which raises the question of how this pathway can achieve such diverse outcomes. With a direct route from the membrane to the nucleus, the Notch pathway has fewer opportunities for regulation than do many other signalling pathways, yet it generates exquisitely patterned structures, including sensory hair cells and branched arterial networks. More confusingly, its activity promotes tissue growth and cancers in some circumstances but cell death and tumour suppression in others. Many different regulatory mechanisms help to shape the activity of the Notch pathway, generating functional outputs that are appropriate for each context. These mechanisms include the receptor-ligand landscape, the tissue topology, the nuclear environment and the connectivity of the regulatory networks.

Control of Cell Identity Genes Occurs in Insulated Neighborhoods in Mammalian Chromosomes

Abstract: The pluripotent state of embryonic stem cells (ESCs) is produced by active transcription of genes that control cell identity and repression of genes encoding lineage-specifying developmental regulators. Here, we use ESC cohesin ChIA-PET data to identify the local chromosomal structures at both active and repressed genes across the genome. The results produce a map of enhancer-promoter interactions and reveal that super-enhancer-driven genes generally occur within chromosome structures that are formed by the looping of two interacting CTCF sites co-occupied by cohesin. These looped structures form insulated neighborhoods whose integrity is important for proper expression of local genes. We also find that repressed genes encoding lineage-specifying developmental regulators occur within insulated neighborhoods. These results provide insights into the relationship between transcriptional control of cell identity genes and control of local chromosome structure.

Notch Signaling in Development, Tissue Homeostasis, and Disease

Abstract: Notch signaling is an evolutionarily highly conserved signaling mechanism, but in contrast to signaling pathways such as Wnt, Sonic Hedgehog, and BMP/TGF-β, Notch signaling occurs via cell-cell com...