Drug-induced loss of imprinting revealed using bioluminescent reporters of Cdkn1c.

Genomic imprinting is an epigenetically mediated mechanism that regulates allelic expression of genes based upon parent-of-origin and provides a paradigm for studying epigenetic silencing and release. Here, bioluminescent reporters for the maternally-expressed imprinted gene Cdkn1c are used to examine the capacity of chromatin-modifying drugs to reverse paternal Cdkn1c silencing. Exposure of reporter mouse embryonic stem cells (mESCs) to 5-Azacytidine, HDAC inhibitors, BET inhibitors or GSK-J4 (KDM6A/B inhibitor) relieved repression of paternal Cdkn1c, either selectively or by inducing biallelic effects. Treatment of reporter fibroblasts with HDAC inhibitors or GSK-J4 resulted in similar paternal Cdkn1c activation, whereas BET inhibitor-induced loss of imprinting was specific to mESCs. Changes in allelic expression were generally not sustained in dividing cultures upon drug removal, indicating that the underlying epigenetic memory of silencing was maintained. In contrast, Cdkn1c de-repression by GSK-J4 was retained in both mESCs and fibroblasts following inhibitor removal, although this impact may be linked to cellular stress and DNA damage. Taken together, these data introduce bioluminescent reporter cells as tools for studying epigenetic silencing and disruption, and demonstrate that Cdkn1c imprinting requires distinct and cell-type specific chromatin features and modifying enzymes to enact and propagate a memory of silencing

BET protein inhibition shows efficacy against JAK2V617F-driven neoplasms.

Small molecule inhibition of the BET family of proteins, which bind acetylated lysines within histones, has been shown to have a marked therapeutic benefit in pre-clinical models of mixed lineage leukemia (MLL) fusion protein-driven leukemias. Here, we report that I-BET151, a highly specific BET family bromodomain inhibitor, leads to growth inhibition in a human erythroleukemic (HEL) cell line as well as in erythroid precursors isolated from polycythemia vera patients. One of the genes most highly downregulated by I-BET151 was LMO2, an important oncogenic regulator of hematopoietic stem cell development and erythropoiesis. We previously reported that LMO2 transcription is dependent upon Janus kinase 2 (JAK2) kinase activity in HEL cells. Here, we show that the transcriptional changes induced by a JAK2 inhibitor (TG101209) and I-BET151 in HEL cells are significantly over-lapping, suggesting a common pathway of action. We generated JAK2 inhibitor resistant HEL cells and showed that these retain sensitivity to I-BET151. These data highlight I-BET151 as a potential alternative treatment against myeloproliferative neoplasms driven by constitutively active JAK2 kinase.The Kouzarides laboratory was supported by Cancer Research UK, Leukaemia and Lymphoma Research, GlaxoSmithKline and BBSRC. The Green laboratory was supported by Cancer Research UK and Leukaemia and Lymphoma Research, UK. The Gottgens laboratory was supported by Cancer Research UK and Leukaemia and Lymphoma Research, UK. The Huntly laboratory was supported by Cancer Research UK and Leukaemia and Lymphoma Research, UK. M. A Dawson, E Cannizzaro and M. Wiese are funded by the Wellcome Trust Beit Fellowship.This is the accepted manuscript version of the article. The final version is available from http://www.nature.com/leu/journal/v28/n1/full/leu2013234a.html

Selective inhibition of BET bromodomain epigenetic signalling interferes with the bone-associated tumour vicious cycle

The vicious cycle established between bone-associated tumours and bone resorption is the central problem with therapeutic strategies against primary bone tumours and bone metastasis. Here we report data to support inhibition of BET bromodomain proteins as a promising therapeutic strategy that target simultaneously the three partners of the vicious cycle. Treatment with JQ1, a BET bromodomain inhibitor, reduces cell viability of osteosarcoma cells and inhibits osteoblastic differentiation both in vitro and in vivo. These effects are associated with transcriptional silencing of MYC and RUNX2, resulting from the depletion of BRD4 from their respective loci. Moreover, JQ1 also inhibits osteoclast differentiation by interfering with BRD4-dependent RANKL activation of NFATC1 transcription. Collectively, our data indicate that JQ1 is a potent inhibitor of osteoblast and osteoclast differentiation as well as bone tumour development

Brd2/4 and Myc regulate alternative cell lineage programmes during early osteoclast differentiation in vitro

Osteoclast development in response to RANKL is critical for bone homeostasis in health and in disease. The early and direct chromatin regulatory changes imparted by the BET chromatin readers Brd2-4 and osteoclast-affiliated transcription factors (TF) during osteoclastogenesis are not known. Here, we demonstrate that in response to RANKL, early osteoclast development entails regulation of two alternative cell fate transcriptional programmes, osteoclast vs macrophage, with repression of the latter following activation of the former. Both programmes are regulated in a non-redundant manner by increased chromatin binding of Brd2 at promoters and of Brd4 at enhancers/super-enhancers. Myc, the top RANKL-induced TF, regulates osteoclast development in co-operation with Brd2/4 and Max and by establishing negative and positive regulatory loops with other lineage-affiliated TF. These insights into the transcriptional regulation of osteoclastogenesis suggest the clinical potential of selective targeting of Brd2/4 to abrogate pathological OC activation

Inhibition of histone H3K27 demethylase selectively modulates inflammatory phenotypes of natural killer cells

Natural killer (NK) cells are innate lymphocytes, important in immune surveillance and elimination of stressed, transformed, or virus-infected cells. They critically shape the inflammatory cytokine environment to orchestrate interactions of cells of the innate and adaptive immune systems. Some studies have reported that NK cell activation and cytokine secretion are controlled epigenetically, but have yielded only limited insight into the mechanisms. Using chemical screening with small-molecule inhibitors of chromatin methylation and acetylation, further validated by knockdown approaches, we here identified Jumonji-type histone H3K27 demethylases as key regulators of cytokine production in human NK cell subsets. The prototypic Jumonji domain–containing protein 3 (JMJD3/UTX) H3K27 demethylase inhibitor GSK-J4 increased global levels of the repressive H3K27me3 mark around transcription start sites of effector cytokine genes. Moreover, GSK-J4 reduced IFN-γ, TNFα, granulocyte–macrophage colony-stimulating factor (GM-CSF), and IL- 10 levels in cytokine-stimulated NK cells, while sparing their cytotoxic killing activity against cancer cells. The anti-inflammatory effect of GSK-J4 in NK cell subsets, isolated from peripheral blood or tissue from individuals with rheumatoid arthritis (RA), coupled with an inhibitory effect on formation of bone-resorbing osteoclasts, suggested that histone demethylase inhibition has broad utility for modulating immune and inflammatory responses. Overall, our results indicate that H3K27me3 is a dynamic and important epigenetic modification during NK cell activation and that JMJD3/UTX-driven H3K27 demethylation is critical for NK cell function

Combining BET and HDAC inhibitors synergistically induces apoptosis of melanoma and suppresses AKT and YAP signaling

Histone acetylation marks have an important role in controlling gene expression and are removed by histone deacetylases (HDACs). These marks are read by bromodomain and extra-terminal (BET) proteins and novel inhibitiors of these proteins are currently in clinical development. Inhibitors of HDAC and BET proteins have individually been shown to cause apoptosis and reduce growth of melanoma cells. Here we show that combining the HDAC inhibitor LBH589 and BET inhibitor I-BET151 synergistically induce apoptosis of melanoma cells but not of melanocytes. Induction of apoptosis proceeded through the mitochondrial pathway, was caspase dependent and involved upregulation of the BH3 pro-apoptotic protein BIM. Analysis of signal pathways in melanoma cell lines resistant to BRAF inhibitors revealed that treatment with the combination strongly downregulated anti-apoptotic proteins and proteins in the AKT and Hippo/YAP signaling pathways. Xenograft studies showed that the combination of inhibitors was more effective than single drug treatment and confirmed upregulation of BIM and downregulation of XIAP as seen in vitro. These results support the combination of these two classes of epigenetic regulators in treatment of melanoma including those resistant to BRAF inhibitors

Histone H3K27me3 demethylases regulate human Th17 cell development and effector functions by impacting on metabolism

T helper (Th) cells are CD4+ effector T cells that play an instrumental role in immunity by shaping the inflammatory cytokine environment in a variety of physiological and pathological situations. Using a combined chemico-genetic approach we identify histone H3K27 demethylases KDM6A and KDM6B as central regulators of human Th subsets. The prototypic KDM6 inhibitor GSK-J4 increases genome-wide levels of the repressive H3K27me3 chromatin mark and leads to suppression of the key transcription factor RORγt during Th17 differentiation, whereas in mature Th17 cells an altered transcriptional program leads to a profound metabolic reprogramming with concomitant suppression of IL-17 cytokine levels and reduced proliferation. Single cell analysis reveals a specific shift from highly inflammatory cell subsets towards a resting state upon demethylase inhibition. The root cause of the observed anti-inflammatory phenotype in stimulated Th17 cells is reduced expression of key metabolic transcription factors, such as PPRC1 and c-myc. Overall, this leads to reduced mitochondrial biogenesis resulting in a metabolic switch with concomitant anti-inflammatory effects. These data are consistent with an opposing effect of GSK-J4 on Th17 T-cell differentiation pathways directly related to proliferation and effector cytokine profiles