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

Osteoclast (OC) development in response to nuclear factor kappa-Β ligand (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 OC-affiliated transcription factors (TFs) during osteoclastogenesis are not known. Here, we demonstrate that in response to RANKL, early OC development entails regulation of two alternative cell fate transcriptional programmes, OC 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 OC development in co-operation with Brd2/4 and Max and by establishing negative and positive regulatory loops with other lineage-affiliated TFs. These insights into the transcriptional regulation of osteoclastogenesis suggest the clinical potential of selective targeting of Brd2/4 to abrogate pathological OC activation

Selective small molecule induced degradation of the BET bromodomain protein BRD4

The Bromo- and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation, epigenetics, and cancer and are the targets of pan-BET selective bromodomain inhibitor JQ1. However, the lack of intra-BET selectivity limits the scope of current inhibitors as probes for target validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We designed Proteolysis Targeted Chimeras (PROTACs) that tether JQ1 to a ligand for the E3 ubiquitin ligase VHL, aimed at triggering the intracellular destruction of BET proteins. Compound MZ1 potently and rapidly induces reversible, long-lasting, and unexpectedly selective removal of BRD4 over BRD2 and BRD3. The activity of MZ1 is dependent on binding to VHL but is achieved at a sufficiently low concentration not to induce stabilization of HIF-1α. Gene expression profiles of selected cancer-related genes responsive to JQ1 reveal distinct and more limited transcriptional responses induced by MZ1, consistent with selective suppression of BRD4. Our discovery opens up new opportunities to elucidate the cellular phenotypes and therapeutic implications associated with selective targeting of BRD4

Genome-wide association analysis of susceptibility and clinical phenotype in multiple sclerosis

Multiple sclerosis (MS), a chronic disorder of the central nervous system and common cause of neurological disability in young adults, is characterized by moderate but complex risk heritability. Here we report the results of a genome-wide association study performed in a 1000 prospective case series of well-characterized individuals with MS and group-matched controls using the Sentrix® HumanHap550 BeadChip platform from Illumina. After stringent quality control data filtering, we compared allele frequencies for 551 642 SNPs in 978 cases and 883 controls and assessed genotypic influences on susceptibility, age of onset, disease severity, as well as brain lesion load and normalized brain volume from magnetic resonance imaging exams. A multi-analytical strategy identified 242 susceptibility SNPs exceeding established thresholds of significance, including 65 within the MHC locus in chromosome 6p21.3. Independent replication confirms a role for GPC5, a heparan sulfate proteoglycan, in disease risk. Gene ontology-based analysis shows a functional dichotomy between genes involved in the susceptibility pathway and those affecting the clinical phenotyp

The discovery of I-BRD9, a selective cell active chemical probe for bromodomain containing protein 9 inhibition

Acetylation of histone lysine residues is one of the most well-studied post-translational modifications of chromatin, selectively recognized by bromodomain “reader” modules. Inhibitors of the bromodomain and extra terminal domain (BET) family of bromodomains have shown profound anticancer and anti-inflammatory properties, generating much interest in targeting other bromodomain-containing proteins for disease treatment. Herein, we report the discovery of I-BRD9, the first selective cellular chemical probe for bromodomain-containing protein 9 (BRD9). I-BRD9 was identified through structure-based design, leading to greater than 700-fold selectivity over the BET family and 200-fold over the highly homologous bromodomain-containing protein 7 (BRD7). I-BRD9 was used to identify genes regulated by BRD9 in Kasumi-1 cells involved in oncology and immune response pathways and to the best of our knowledge, represents the first selective tool compound available to elucidate the cellular phenotype of BRD9 bromodomain inhibition

An Evolutionarily Conserved Function of Polycomb Silences the MHC Class I Antigen Presentation Pathway and Enables Immune Evasion in Cancer.

Loss of MHC class I (MHC-I) antigen presentation in cancer cells can elicit immunotherapy resistance. A genome-wide CRISPR/Cas9 screen identified an evolutionarily conserved function of polycomb repressive complex 2 (PRC2) that mediates coordinated transcriptional silencing of the MHC-I antigen processing pathway (MHC-I APP), promoting evasion of T cell-mediated immunity. MHC-I APP gene promoters in MHC-I low cancers harbor bivalent activating H3K4me3 and repressive H3K27me3 histone modifications, silencing basal MHC-I expression and restricting cytokine-induced upregulation. Bivalent chromatin at MHC-I APP genes is a normal developmental process active in embryonic stem cells and maintained during neural progenitor differentiation. This physiological MHC-I silencing highlights a conserved mechanism by which cancers arising from these primitive tissues exploit PRC2 activity to enable immune evasion.Cancer Research UK Clinician Scientist Fellowship C53779/A20097 (M.L.B), Leukaemia Foundation Australia Senior Fellowship and Howard Hughes Medical Institute International Research Scholarship 55008729 (M.A.D), Peter and Julie Alston Centenary fellowship (K.D.S.), Wellcome Trust Principal Research Fellowship 101835/Z/13/Z (P.J.L), Peter MacCallum Postgraduate Scholarship (C.E.S), NHMRC Postgraduate Scholarship (K.L.C.), Maddie Riewoldt's Vision 064728 (Y-C.C), Victorian Cancer Agency (E.Y.N.L), CSL Centenary fellowship (S-J.D), National Breast Cancer Foundation Fellowship ECF-17-005 (P.A.B.), Addenbrooke’s Charitable Trust and NIHR Cambridge BRC (M.L.B., P.J.L), NHMRC grant 1085015, 1106444 (M.A.D) and 1128984 (M.A.D, S-J.D)

Structure-guided design of a domain-selective bromodomain and extra terminal N-terminal bromodomain chemical probe

Small molecule mediated disruption of the protein-protein interactions between acetylated histone tails and the tandem bromodomains of the bromodomain and extra terminal (BET) family of proteins is an important mechanism of action for the potential modulation of immuno-inflammatory and oncology disease. High quality chemical probes have proven invaluable in elucidating profound BET bromodomain biology, with seminal publications of both pan- and domain-selective BET family bromodomain inhibitors enabling academic and industrial research. To enrich the toolbox of structurally differentiated N-terminal bromodomain (BD1) BET family chemical probes, this work describes an analysis of the GSK BRD4 bromodomain dataset through a lipophilic efficiency lens, which enabled identification of a BD1 domain biased benzimidazole series. Structure guided growth targeting a key Asp/His BD1/BD2 switch enabled delivery of GSK023, a high-quality chemical probe with 300–1000-fold BET BD1 domain selectivity and a phenotypic cellular fingerprint consistent with BET bromodomain inhibition

Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia.

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance

Structure-based design of a bromodomain and extraterminal domain (BET) inhibitor selective for the N-terminal bromodomains that retains an anti-inflammatory and antiproliferative phenotype

The bromodomain and extraterminal domain (BET) family of epigenetic regulators comprises four proteins (BRD2, BRD3, BRD4, BRDT), each containing tandem bromodomains. To date, small molecule inhibitors of these proteins typically bind all eight bromodomains of the family with similar affinity, resulting in a diverse range of biological effects. To enable further understanding of the broad phenotype characteristic of pan-BET inhibition, the development of inhibitors selective for individual, or sets of, bromodomains within the family is required. In this regard, we report the discovery of a potent probe molecule possessing up to 150-fold selectivity for the N-terminal bromodomains (BD1s) over the C-terminal bromodomains (BD2s) of the BETs. Guided by structural information, a specific amino acid difference between BD1 and BD2 domains was targeted for selective interaction with chemical functionality appended to the previously developed I-BET151 scaffold. Data presented herein demonstrate that selective inhibition of BD1 domains is sufficient to drive anti-inflammatory and antiproliferative effects