Effect of BET inhibitors (JQ1 and RVX-208) on gene expression in HepG2 cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE51143 | GEO | 2013/09/24
SECONDARY ACCESSION(S): PRJNA221333
REPOSITORIES: GEO
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Project description:Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo-and-extra-terminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here we report that RVX-208, a compound currently in phase II clinical trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2). Co-crystal structures revealed binding modes of RVX-208 and its synthetic precursor and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation HepG2 Cells were treated with eitther DMSO or 0.5uM JQ1 or 5uM RVX-208. Three samples per condition, total of nine samples. Inhbitor treatment was carried out for 4h before RNA was extracted. HepG2 cells (ATCC: HB-8065) were maintained in M-NM-1-MEM (Cat.#BE12-169F; BioWhittaker) supplemented with 10 % heat-inactivated foetal calf serum (PAA #A15-152), non-essential amino acids (Cat. #M7145; Sigma), glutamine (Cat.#M11-004; PAA), and vitamins (Cat.#M6895; Sigma). Cells were grown at 37 M-BM-0C in a humidified cabinet at 5 % CO2 (Heraeus Function Line). For experiments, cells were seeded the day prior to treatment at 2x105/ml. Treatments were performed for 4 h so that a final concentration of 0.1 % DMSO (Cat.#D1435; Sigma) was achieved. At harvest, cells were washed once with PBS (Cat.#H15-002; PAA), and lysed in situ using RLT buffer supplemented with 10 M-NM-<l/ml M-NM-2-mercaptoethanol (Cat.#M7522; Sigma). Total RNA was extracted and prepared using RNeasy columns (Cat.#74106 plus; Qiagen) including a Qia shredding step (Cat.#79656; Qiagen) and an on-column DNAse digestion (Cat.#EN0521; Fermentas), according to the manufacturerM-bM-^@M-^Ys instructions. The resulting RNA was quantified and quality controlled using a Nanodrop spectrophotometer (model ND1000; Thermo Fisher). RNA integrity was assessed on a BioAnalyzer (model G2938C; Agilent Laboratories, USA) and all samples had a RNA Integrity Number (RIN) M-bM-^IM-% 9. Labelled sense ssDNA for hybridization was generated from 200 ng starting RNA with the Ambion WT expression kit (Cat.#4411973; Ambion) and the Affymetrix GeneChip WT Terminal Labelling and Controls Kit (Cat.#901525; Affymetrix) according to the manufacturerM-bM-^@M-^Ys instructions. The distribution of fragmented sense ssDNA lengths was measured on the BioAnalyser. The fragmented ssDNA was labelled and hybridized for 17 hours at 45 M-BM-0C on the Affymetrix GeneChip Human Gene 1.0 ST Array (Affymetrix). Chips were processed on an Affymetrix GeneChip Fluidics Station 450 and Scanner 3000 and the affymetrix Command Console (v.3.2.4; Affymetrix) was used to generate CEL files.
2013-09-24 | E-GEOD-51143 | biostudies-arrayexpress
Project description:The evolutionarily conserved BET bromodomain family contain a distinctive tandem bromodomain structure that enables their chromatin binding to facilitate transcription. Whilst first-in-class drugsthat equally inhibit both bromodomains have shown pre-clinicaland clinical efficacy in a range of malignant and inflammatory pathologies, the functional contribution of the first (BD1) and second (BD2) bromodomains to these pathologies remains largely unknown. Here we haveexploited detailedstructuralinsights and our extensivemedicinal chemistry capabilities to develop novel compounds that are exquisitely selectivefor either BD1 or BD2 of the BET proteins. Using thesewell characterised domain-specific inhibitors we show that BD1 is required by all BET proteins to associate with chromatin and maintain established malignant transcriptional programs.Therefore, targeting BD1 alone phenocopies the therapeutic effects of current non-selective BET inhibitorsin models of cancer. Whilst steady state gene expression primarily requires BD1,we find that the rapid increase of gene expression effectedby a wide range of inflammatory stimuli requiresboth BD1 and BD2. Moreover, although BRD4 is the principal member required for the maintenance of established gene expression, efficient gene induction also requires BRD2 and BRD3. Selective inhibition of BD2 is effective in perturbing the recruitment of all the BET proteins for stimulated gene expression and consequently small molecule inhibitors of BD2 amelioratethe immunoinflammatory end organ damage seen in models of inflammatory arthritis, psoriasis and hepatitis. Together,these data provide novel biological and functional insights into this family of key transcriptional regulators. Importantly, they also help refine the future therapeutic approach when targeting the BET proteinsin distinct pathologies such as cancer and immuno-inflammation.
2020-06-17 | GSE138084 | GEO
Project description:The evolutionarily conserved BET bromodomain family contain a distinctive tandem bromodomain structure that enables their chromatin binding to facilitate transcription. Whilst first-in-class drugsthat equally inhibit both bromodomains have shown pre-clinicaland clinical efficacy in a range of malignant and inflammatory pathologies, the functional contribution of the first (BD1) and second (BD2) bromodomains to these pathologies remains largely unknown. Here we haveexploited detailedstructuralinsights and our extensivemedicinal chemistry capabilities to develop novel compounds that are exquisitely selectivefor either BD1 or BD2 of the BET proteins. Using thesewell characterised domain-specific inhibitors we show that BD1 is required by all BET proteins to associate with chromatin and maintain established malignant transcriptional programs.Therefore, targeting BD1 alone phenocopies the therapeutic effects of current non-selective BET inhibitorsin models of cancer. Whilst steady state gene expression primarily requires BD1,we find that the rapid increase of gene expression effectedby a wide range of inflammatory stimuli requiresboth BD1 and BD2. Moreover, although BRD4 is the principal member required for the maintenance of established gene expression, efficient gene induction also requires BRD2 and BRD3. Selective inhibition of BD2 is effective in perturbing the recruitment of all the BET proteins for stimulated gene expression and consequently small molecule inhibitors of BD2 amelioratethe immunoinflammatory end organ damage seen in models of inflammatory arthritis, psoriasis and hepatitis. Together,these data provide novel biological and functional insights into this family of key transcriptional regulators. Importantly, they also help refine the future therapeutic approach when targeting the BET proteinsin distinct pathologies such as cancer and immuno-inflammation.
2020-06-17 | GSE138209 | GEO
Project description:The evolutionarily conserved BET bromodomain family contain a distinctive tandem bromodomain structure that enables their chromatin binding to facilitate transcription. Whilst first-in-class drugsthat equally inhibit both bromodomains have shown pre-clinicaland clinical efficacy in a range of malignant and inflammatory pathologies, the functional contribution of the first (BD1) and second (BD2) bromodomains to these pathologies remains largely unknown. Here we haveexploited detailedstructuralinsights and our extensivemedicinal chemistry capabilities to develop novel compounds that are exquisitely selectivefor either BD1 or BD2 of the BET proteins. Using thesewell characterised domain-specific inhibitors we show that BD1 is required by all BET proteins to associate with chromatin and maintain established malignant transcriptional programs.Therefore, targeting BD1 alone phenocopies the therapeutic effects of current non-selective BET inhibitorsin models of cancer. Whilst steady state gene expression primarily requires BD1,we find that the rapid increase of gene expression effectedby a wide range of inflammatory stimuli requiresboth BD1 and BD2. Moreover, although BRD4 is the principal member required for the maintenance of established gene expression, efficient gene induction also requires BRD2 and BRD3. Selective inhibition of BD2 is effective in perturbing the recruitment of all the BET proteins for stimulated gene expression and consequently small molecule inhibitors of BD2 amelioratethe immunoinflammatory end organ damage seen in models of inflammatory arthritis, psoriasis and hepatitis. Together,these data provide novel biological and functional insights into this family of key transcriptional regulators. Importantly, they also help refine the future therapeutic approach when targeting the BET proteinsin distinct pathologies such as cancer and immuno-inflammation.
2020-06-17 | GSE138089 | GEO
Project description:Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically-defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis demonstrated downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knock-down phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in three in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma. Significance: Biomarkers of response to small-molecule inhibitors of BET bromodomains, a new compound class with promising anti-cancer activity, have been lacking. Here, we reveal MYCN amplification as a strong genetic predictor of sensitivity to BET bromodomain inhibitors, demonstrate a mechanistic rationale for this finding, and provide a translational framework for clinical trial development of BET bromodomain inhibitors for pediatric patients with MYCN-amplified neuroblastoma. JQ1 is a novel thieno-triazolo-1,4-diazepine, which displaces BET bromodomains from chromatin by competitively binding to the acetyl lysine recognition pocket. BE(2)-C and Kelly cells were treated in triplicate with 1 µM JQ1 or DMSO for 24 hours. RNA was extracted and a decrease in MYCN transcript was confirmed by real time RT-PCR as described above. The samples were profiled using the Affymetrix PrimeView Human Gene Expression Array (Affymetrix) at Beth Israel Deaconess Medical Center (Boston, MA, USA).
2013-02-25 | E-GEOD-43392 | biostudies-arrayexpress
Project description:We comprehensively analyzed the differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) underlying the antitumor functions of BET inhibitors of human HCC cells using RNA sequencing (RNA-Seq). The pan-BET inhibitor ABBV-075 and BD2 specific inhibitor ABBV-744 attenuates the IFNγ-induced inflammatory response. In addition, BD2 inhibitors were predominantly effective in inflammatory response.
2022-05-04 | GSE189376 | GEO
Project description:One critical task in pluripotent reprogramming is to erase the somatic transcriptional program of starting cells. No strategy or theory exists for achieving erasure of somatic gene expression memory. Here, we present a proof-of-principle strategy in which reprogramming to pluripotency is facilitated by small molecules that erase somatic cell transcription memory. We show that mild chemical targeting of the acetyllysine-binding pockets of the BET bromodomains, the transcriptional bookmarking domains, robustly enhances reprogramming. Furthermore, we show that chemical targeting of the transcriptional bookmarking BET bromodomains dramatically downregulates specific somatic gene expression programs in both naïve and reprogramming fibroblasts. Chemical blocking of the BET bromodomains also resulted in loss of fibroblast morphology early in reprograming. In this study, we experimentally demonstrate a concept for cell fate conversion: facilitating the conversion by chemically targeting the transcriptional bookmarking BET bromodomains responsible for transcriptional memory. human BJ cells were treated with JQ1 at 50 nM for 48 hours. Differential expression was compared with DMSO treatment. The same treatments and comparsion were conducted for reprogramming BJ cells, which were transduced with OCT4, SOX2, and KLF4. JQ1iPSC5 is a iPSC (induced pluripotent stem cell) line generated in this study using small molecules JQ1.
2016-08-22 | E-GEOD-75364 | biostudies-arrayexpress
Project description:One critical task in pluripotent reprogramming is to erase the somatic transcriptional program of starting cells. No strategy or theory exists for achieving erasure of somatic gene expression memory. Here, we present a proof-of-principle strategy in which reprogramming to pluripotency is facilitated by small molecules that erase somatic cell transcription memory. We show that mild chemical targeting of the acetyllysine-binding pockets of the BET bromodomains, the transcriptional bookmarking domains, robustly enhances reprogramming. Furthermore, we show that chemical targeting of the transcriptional bookmarking BET bromodomains dramatically downregulates specific somatic gene expression programs in both naïve and reprogramming fibroblasts. Chemical blocking of the BET bromodomains also resulted in loss of fibroblast morphology early in reprograming. In this study, we experimentally demonstrate a concept for cell fate conversion: facilitating the conversion by chemically targeting the transcriptional bookmarking BET bromodomains responsible for transcriptional memory.
2016-08-22 | GSE75364 | GEO
Project description:The epigenome is often deregulated in cancer and treatment with inhibitors of bromodomain and extra-terminal proteins, the readers of epigenetic acetylation marks, represents a novel therapeutic approach. Here, we have characterized the anti-tumour activity of the novel bromodomain and extra-terminal (BET) inhibitor BAY 1238097 in preclinical lymphoma mod- els. BAY 1238097 showed anti-proliferative activity in a large panel of lym- phoma-derived cell lines, with a median 50% inhibitory concentration between 70 and 208 nmol/l. The compound showed strong anti-tumour efficacy in vivo as a single agent in two diffuse large B cell lymphoma mod- els. Gene expression profiling showed BAY 1238097 targeted the NFKB/ TLR/JAK/STAT signalling pathways, MYC and E2F1-regulated genes, cell cycle regulation and chromatin structure. The gene expression profiling sig- natures also highly overlapped with the signatures obtained with other BET Bromodomain inhibitors and partially overlapped with HDAC-inhibitors, mTOR inhibitors and demethylating agents. Notably, BAY 1238097 presented in vitro synergism with EZH2, mTOR and BTK inhibitors. In conclusion, the BET inhibitor BAY 1238097 presented promising anti-lym- phoma preclinical activity in vitro and in vivo, mediated by the interference with biological processes driving the lymphoma cells. Our data also indicate the use of combination schemes targeting EZH2, mTOR and BTK along- side BET bromodomains.
2017-09-05 | GSE93032 | GEO
Project description:The MYC transcription factor is a master regulator of diverse cancer pathways and somatic cell reprogramming. MYC is a compelling therapeutic target that exhibits cancer-specific cellular effects. Pharmacologic inhibition of MYC function has proven challenging due to its numerous modes of forced expression and the difficulty of disrupting protein-DNA interactions. Here we demonstrate the rapid and potent abrogation of MYC gene transcription by representative small molecule bromodomain inhibitors of the BET family of chromatin adaptors. This transcriptional suppression of MYC was observed in the context of the natural, chromosomally translocated, and amplified gene locus. Inhibition of BET bromodomain-promoter interactions and subsequent reduction of MYC transcript and protein levels resulted in G1 arrest and extensive apoptosis in a variety of leukemia and lymphoma cell lines. Exogenous expression of MYC from an artificial promoter that is resistant to BET regulation significantly protected cells from growth suppression by BET inhibitors and revealed that MYC exerts a direct and tight control of key pro-growth and anti-apoptotic target genes. Transcriptional profiling of two cells after 4 and 8 hours of treatment with BET inhibitor shows that both MYC and its targets are strongly down-regulated. We thus demonstrate that pharmacologic inhibition of MYC is achievable through targeting BET bromodomains, and suggest that such inhibitors may have broad clinical applicability given the widespread pathogenetic role of MYC in cancer. LP-1, a multiple myeloma cell line, and Raji, a Burkitt lymphoma cell line, were treated with BET inhibitor and an inactive enantiomer for 4 or 8 hours. Two biological replicates of each sample were profiled on exon arrays.
2011-09-18 | E-GEOD-29449 | biostudies-arrayexpress