Myelodysplastic syndromes and chronic myelomonocytic leukemia (CMML) are characterized by mutations in epigenetic modifiers and aberrant DNA methylation. DNA methyltransferase inhibitors (DMTis) are used to treat these disorders, but response is highly variable with few means to predict which patients will benefit. To develop a molecular means of predicting response at diagnosis, we examined baseline differences in mutations, DNA methylation, and gene expression in 40 CMML patients responsive and resistant to decitabine (DAC). While somatic mutations did not differentiate responders and non-responders, we were able to identify for the first time 158 differentially methylated regions (DMRs) at baseline between responders and non-responders using next-generation sequencing. These DMRs were primarily localized to non-promoter regions and overlapped with distal regulatory enhancers. Using the methylation profiles, we developed an epigenetic classifier that accurately predicted DAC response at the time of diagnosis. We also found 53 differentially expressed genes between responders and non-responders. Genes up-regulated in responders were enriched in the cell cycle, potentially contributing to effective DAC incorporation. Two chemokines overexpressed in non-responders -- CXCL4 and CXCL7 -- were able to block the effect of DAC on normal CD34+ and primary CMML cells in vitro, suggesting their up-regulation contributes to primary DAC resistance. Overall design: mRNA profiling in bone marrow mononuclear cells (BM MNC) from 14 CMML patients (8 decitabine responders vs. 6 non-responders).
Specific molecular signatures predict decitabine response in chronic myelomonocytic leukemia.
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Noncoding RNAs include small transcripts, such as microRNAs and piwi-interacting RNAs, and a wide range of long noncoding RNAs (lncRNAs). Although many lncRNAs have been identified, only a small number of lncRNAs have been characterized functionally. Here, we sought to identify lncRNAs differentially expressed during replicative senescence. We compared lncRNAs expressed in proliferating, early-passage, 'young' human diploid WI-38 fibroblasts [population doubling (PDL) 20] with those expressed in senescent, late-passage, 'old' fibroblasts (PDL 52) by RNA sequencing (RNA-Seq). Numerous transcripts in all lncRNA groups (antisense lncRNAs, pseudogene-encoded lncRNAs, previously described lncRNAs and novel lncRNAs) were validated using reverse transcription (RT) and real-time, quantitative (q)PCR. Among the novel senescence-associated lncRNAs (SAL-RNAs) showing lower abundance in senescent cells, SAL-RNA1 (XLOC_023166) was found to delay senescence, because reducing SAL-RNA1 levels enhanced the appearance of phenotypic traits of senescence, including an enlarged morphology, positive ÃŸ-galactosidase activity, and heightened p53 levels. Our results reveal that the expression of known and novel lncRNAs changes with senescence and suggests that SAL-RNAs play direct regulatory roles in this important cellular process. Overall design: RNA was extracted from both young and senescent WI-38 cells and used for total RNA-Seq.
Senescence-associated lncRNAs: senescence-associated long noncoding RNAs.
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Purpose: Gene expression signatures developed to measure the activity of oncogenic signaling pathways have been used to dissect the heterogeneity of tumor samples and to predict sensitivity to various cancer drugs that target components of the relevant pathways, thus potentially identifying therapeutic options for subgroups of patients. To facilitate broad use, including in a clinical setting, the ability to generate data from formalin-fixed, paraffin-embedded (FFPE) tissues is essential. Experimental Design: Patterns of pathway activity in matched fresh-frozen and FFPE xenograft tumor samples were generated using the MessageAmp Premier methodology in combination with assays using Affymetrix arrays. Results generated were compared with those obtained from fresh-frozen samples using a standard Affymetrix assay. In addition, gene expression data from patient matched fresh-frozen and FFPE melanomas were also utilized to evaluate the consistency of predictions of oncogenic signaling pathway status. Results: Significant correlation of pathway activity predictions was observed between paired fresh-frozen and FFPE xenograft tumor samples. In addition, significant concordance of pathway activity predictions was also observed between patient matched fresh-frozen and FFPE melanomas. Conclusion: Reliable and consistent predictions of oncogenic pathway activities can be obtained from FFPE tumor tissue samples. The ability to reliably utilize FFPE patient tumor tissue samples for genomic analyses will lead to a better understanding of the biology of disease progression and, in the clinical setting, will provide tools to guide the choice of therapeutics to those most likely to be effective in treating a patients disease.
A methodology for utilization of predictive genomic signatures in FFPE samples.
Specimen partView Samples
Male Sprague-Dawley rats 8 weeks old, were adrenalectomized, treated with 300ug/kg corticosterone or vehicle 3 days after surgery then sacrificed 1 hour later. Hippocampi were removed and RNA extracted and processed for sequencing at the Massachusetts General Hospital Nex-Generation Sequening Core. Overall design: Includes 6 cort treated and 6 control biological replicates
Stress and corticosteroids regulate rat hippocampal mitochondrial DNA gene expression via the glucocorticoid receptor.
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Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-loaded macrophages in the arterial wall. Intimal macrophages internalize modified lipoproteins such as oxidized LDL (oxLDL) through scavenger receptors, leading to storage of excess cholesteryl esters in lipid bodies and a "foam cell" phenotype. In addition, stimulation of macrophage Toll-like receptors (TLRs) has been shown to promote lipid body proliferation. We investigated the possibility that there are transcriptional regulators that are common to both pathways for stimulating foam cell formation (modified lipoproteins and TLR stimulation), and identified the transcription factor ATF3 as a candidate regulator.
ATF3 protects against atherosclerosis by suppressing 25-hydroxycholesterol-induced lipid body formation.
Sex, Specimen partView Samples
M21 or M21L cells were grown either in a 2-dimensional culture (on plastic) or in a 3-dimensional-collagen model.
Protein kinase Cα (PKCα) regulates p53 localization and melanoma cell survival downstream of integrin αv in three-dimensional collagen and in vivo.
Cell lineView Samples
We performed RNAseq analysis to determine the effect of MFN1 deletion on oocyte global gene expression profile. RNAseq revealed a total of 982 genes were significantly differentially expressed (p<0.05) in Mfn1-/- oocytes compared to WT (654 up-regulated and 337 down-regulated). Pathway analysis indicated significant over-representation of elements involved in regulation of ceramide biosynthesis, death receptor signaling and adherens junction signaling. Differential expression of these genes (Bad, G2e3, Cdh17 and Myh2) was also confirmed by qRT-PCR.Our findings provide new insight into the role of MFN1 in the oocytes, and may help understand the potential mechanism of infertility and reproductive aging associated with MFN1-deficiency. Overall design: Secondary follicle-enclosed oocytes were collected from 8-week-old Mfn1-/- and WT mice (n=3 for each group) and 5 oocytes from each group were pooled for RNA sequencing analysis.
Mitofusin 1 is required for female fertility and to maintain ovarian follicular reserve.
Cell line, SubjectView Samples
The tumor suppressor p53 can induce various biological responses. Yet it is not clear whether it is p53 in vivo promoter selectivity that triggers different transcription programs leading to different outcomes. Our analysis of genome-wide chromatin occupancy by p53 using ChIP-seq (deposited in Sequence Read Archive database as SRP007261) revealed p53 default program, i.e. the pattern of major p53-bound sites that is similar upon p53 activation by nutlin3a, RITA or 5-FU in breast cancer cells, despite different biological outcomes triggered by these compounds. Parallel analysis of gene expression allowed identification of 280 previously unknown p53 target genes, including p53-repressed AURKA. The consensus p53 binding motif was present more frequently in p53-induced, than in repressed targets, indicating different mechanisms of gene activation versus repression. We identified several possible cofactors of p53, and found that STAT3 antagonised p53-mediated repression of a subset of genes, including AURKA. Finally, we showed that the expression of the novel p53 targets correlates with p53 status and survival in breast cancer patients.
Insights into p53 transcriptional function via genome-wide chromatin occupancy and gene expression analysis.
Cell line, TreatmentView Samples
Since its discovery as a tumour suppressor some fifteen years ago, the transcription factor p53 has attracted paramount attention for its role as the guardian of the genome. TP53 mutations occur so frequently in cancer, regardless of patient age or tumour type, that they appear to be part of the life history of at least 50% of human tumours. In most tumours that retain wild-type p53, its function is inactivated due to deregulated HDM2, a protein which binds to p53 and which can inhibit the transcriptional activity of p53 and induce its degradation.
Ablation of key oncogenic pathways by RITA-reactivated p53 is required for efficient apoptosis.
Specimen part, DiseaseView Samples
RCC cells (786-O) were transfected with VHL. The parental cell line should be compared to the transfectant (+VHL) under nomoxia as well as under hypoxia conditions.
Distinct von Hippel-Lindau gene and hypoxia-regulated alterations in gene and protein expression patterns of renal cell carcinoma and their effects on metabolism.
Cell lineView Samples