Enhancer-activated RET confers protection against oxidative stress to KMT2A-rearranged acute myeloid leukemia.

Ectopic activation of rearranged during transfection (RET) has been reported to facilitate lineage differentiation and cell proliferation in different cytogenetic subtypes of acute myeloid leukemia (AML). Herein, we demonstrate that RET is significantly (p < 0.01) upregulated in AML subtypes containing rearrangements of the lysine methyltransferase 2A gene (KMT2A), commonly referred to as KMT2A-rearranged (KMT2A-r) AML. Integrating multi-epigenomics data, we show that the KMT2A-MLLT3 fusion induces the development of CCCTC-binding (CTCF)-guided de novo extrusion enhancer loop to upregulate RET expression in KMT2A-r AML. Based on the finding that RET expression is tightly correlated with the selective chromatin remodeler and mediator (MED) proteins, we used a small-molecule inhibitor having dual inhibition against RET and MED12-associated cyclin-dependent kinase 8 (CDK8) in KMT2A-r AML cells. Dual inhibition of RET and CDK8 restricted cell proliferation by producing multimodal oxidative stress responses in treated cells. Our data suggest that epigenetically enhanced RET protects KMT2A-r AML cells from oxidative stresses, which could be exploited as a potential therapeutic strategy.

CRISPR/dCas9-KRAB-Mediated Suppression of S100b Restores p53-Mediated Apoptosis in Melanoma Cells.

Overexpression of S100B is routinely used for disease-staging and for determining prognostic outcomes in patients with malignant melanoma. Intracellular interactions between S100B and wild-type (WT)-p53 have been demonstrated to limit the availability of free WT-p53 in tumor cells, inhibiting the apoptotic signaling cascade. Herein, we demonstrate that, while oncogenic overexpression of S100B is poorly correlated (R < 0.3; p > 0.05) to alterations in S100B copy number or DNA methylation in primary patient samples, the transcriptional start site and upstream promoter of the gene are epigenetically primed in melanoma cells with predicted enrichment of activating transcription factors. Considering the regulatory role of activating transcription factors in S100B upregulation in melanoma, we stably suppressed S100b (murine ortholog) by using a catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor, Krüppel-associated box (KRAB). Selective combination of S100b-specific single-guide RNAs and the dCas9-KRAB fusion significantly suppressed expression of S100b in murine B16 melanoma cells without noticeable off-target effects. S100b suppression resulted in recovery of intracellular WT-p53 and p21 levels and concomitant induction of apoptotic signaling. Expression levels of apoptogenic factors (i.e., apoptosis-inducing factor, caspase-3, and poly-ADP ribose polymerase) were altered in response to S100b suppression. S100b-suppressed cells also showed reduced cell viability and increased susceptibility to the chemotherapeutic agents, cisplatin and tunicamycin. Targeted suppression of S100b therefore offers a therapeutic vulnerability to overcome drug resistance in melanoma.

Expression of integrin ß-7 is epigenetically enhanced in multiple myeloma subgroups with high-risk cytogenetics.

BACKGROUND: Oncogenic overexpression of integrin-ß7 (ITGB7) in cases of high-risk multiple myeloma (MM) was reported to promote enhanced interactions between neoplastic plasma-B cells and stromal cells to develop cell-adhesion mediated drug resistance. METHODS: Expression profiles of adhesion related genes were analyzed in a cohort of MM patients containing major IgH translocations or hyperdiploidies (HY), diagnosed at the premalignant monoclonal gammopathy of undetermined significance (MGUS; n = 103), smoldering multiple myeloma; (SMM; n = 190) or MM (MM; n = 53) stage. Differential expression was integrated with loci-specific alterations in DNA-methylation and chromatin marks in MM patients. A CRISPR-based targeted induction of DNA-methylation at the ITGB7 super-enhancer (SE) in MM.1S cells was employed to intersect the impact of cis-regulatory elements on ITGB7 expression. RESULTS: ITGB7 was significantly (p < 0.05) upregulated in patients with t(14;16) and t(14;20) subgroups in all MGUS, SMM and MM stages, but sporadically upregulated in t(4;14) subgroup at the MM stage. We demonstrate a predetermined enhancer state on ITGB7 in primary-B cells that is maintained under bivalent chromatin, which undergoes a process of chromatin-state alterations and develops into an active enhancer in cases of the t(4;14) subgroup or SE in cases of the t(14;16) subgroup. We also demonstrate that while targeted induction of DNA-methylation at the ITGB7-SE further upregulated the gene, inhibition of ITGB7-SE-associated transcription factor bromodomain-4 downregulated expression of the gene. CONCLUSIONS: Our findings suggest an epigenetic regulation of oncogenic overexpression of ITGB7 in MM cells, which could be critical in MM progression and an attractive therapeutic target.

Epigenetic Deregulation of Telomere-Related Genes in Newly Diagnosed Multiple Myeloma Patients.

High-risk Multiple Myeloma (MM) patients were found to maintain telomere length (TL), below the margin of short critical length, consistent with proactive overexpression of telomerase. Previously, DNA methylation has been shown as a determinant of telomere-related gene (TRG) expression and TL to assess risk in different types of cancer. We mapped genome-wide DNA methylation in a cohort of newly diagnosed MM (NDMM; n = 53) patients of major molecular subgroups, compared to age-matched healthy donors (n = 4). Differential methylation and expression at TRG-loci were analyzed in combination with overlapping chromatin marks and underlying DNA-sequences. We observed a strong correlation (R2 ≥ 0.5) between DNA methylation and expression amongst selective TRGs, such that demethylation at the promoters of DDX1 and TERF1 were associated to their oncogenic upregulation, while demethylation at the bodies of two key tumor suppressors ZNF208 and RAP1A led to downregulation of the genes. We demonstrated that TRG expression may be controlled by DNA methylation alone or in cooperation with chromatin modifications or CCCTC-binding factor at the regulatory regions. Additionally, we showed that hypomethylated DMRs of TRGs in NDMM are stabilized with G-quadruplex forming sequences, suggesting a crucial role of these epigenetically vulnerable loci in MM pathogenesis. We have identified a panel of five TRGs, which are epigenetically deregulated in NDMM patients and may serve as early detection biomarkers or therapeutic targets in the disease.

Investigation of antimicrobial physiology of orthorhombic and monoclinic nanoallotropes of sulfur at the interface of transcriptome and metabolome.

Nanosized elemental sulfur (ES) is already reported to exert superior antimicrobial efficacy than micron-sized ES, which encourages their use in drugs and therapeutics. The aim of the present study is to explore the possible route and mode of antimicrobial action of orthorhombic (α-SNPs) and monoclinic (ß-SNPs) allotropes of sulfur, respectively, at their nano-dimensions. The antimicrobial efficacy of α- and ß-SNPs was determined against both the conventionally ES-resistant and ES-susceptible fungi and bacteria. Both the SNPs inhibited the microbial growth, irrespective of their resistance profile to ES and caused significant deformities on the microbial cell surfaces. However, the extent of antimicrobial efficacy was found to be optimum for α-SNPs, which can be attributed to their size, shape, and surface modification. Subsequent transcript profiling, metabolite profiling, and enzymatic analyses revealed that α- and ß-SNPs impaired a cluster of mitochondrial enzymes involved in cellular respiration and oxidative phosphorylation. ES and SNPs stress were found to elicit the NADPH-dependent glutathione reductase mediated ES-detoxification response in fungi and caused them to undertake the glyoxylate shunt in favor of energy conservation. A simultaneous study was also undertaken to assess the biocompatible or bio-adverse properties of SNPs in terms of their cytotoxic and genotoxic effects against the human derived lung fibroblast cell line (MRC-5). The present study hence explores the antimicrobial physiology of two novel functional materials and demonstrates their compatibility as a future putative antimicrobial drug.

Inhibitory effects of sulfur nanoparticles on membrane lipids of Aspergillus niger: a novel route of fungistasis.

Orthorhombic (spherical; ~10 nm) and monoclinic (cylindrical; ~50 nm) sulfur nanoparticles (SNPs) were synthesized and examined for their effects on the total lipid content and desaturase enzymes of Aspergillus niger. Synthesized SNPs were characterized for size with transmission electron microscopy, elemental composition with energy dispersive X-ray spectroscopy and allotropic nature with X-ray diffraction pattern. Both the SNPs considerably reduced total lipid content of the treated fungal isolates with significant down regulation of the expression of various desaturase enzymes (linoleoyl-CoA desaturase, stearoyl-CoA 9-desaturase and phosphatidylcholine desaturase). Unusual high accumulation of saturated fatty acids with depleted lipid layer can be inferred as one of the major reasons of SNPs mediated fungistasis.