Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, while vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression1–4. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression5–10. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the Bromodomain and Extra Terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by “mimicking” acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages and confers protection against LPS-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fung et al., 2015), that described how we intended to replicate selected experiments from the paper "Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia" (Dawson et al.

Two members of the NF-kappaB (nuclear factor kappaB)/Rel transcription factor family, NF-kappaB1 and NF-kappaB2, are produced as precursor proteins, NF-kappaB1 p105 and NF-kappaB2 p100 respectively. These are proteolytically processed by the proteasome to produce the mature transcription factors NF-kappaB1 p50 and NF-kappaB2 p52. p105 and p100 are known to function additionally as IkappaBs (inhibitors of NF-kappaB), which retain associated NF-kappaB subunits in the cytoplasm of unstimulated cells. The present review focuses on the latest advances in research on the function of NF-kappaB1 and NF-kappaB2 in immune cells. NF-kappaB2 p100 processing has recently been shown to be stimulated by a subset of NF-kappaB inducers, including lymphotoxin-beta, B-cell activating factor and CD40 ligand, via a novel signalling pathway. This promotes the nuclear translocation of p52-containing NF-kappaB dimers, which regulate peripheral lymphoid organogenesis and B-lymphocyte differentiation. Increased p100 processing also contributes to the malignant phenotype of certain T- and B-cell lymphomas. NF-kappaB1 has a distinct function from NF-kappaB2, and is important in controlling lymphocyte and macrophage function in immune and inflammatory responses. In contrast with p100, p105 is constitutively processed to p50. However, after stimulation with agonists, such as tumour necrosis factor-alpha and lipopolysaccharide, p105 is completely degraded by the proteasome. This releases associated p50, which translocates into the nucleus to modulate target gene expression. p105 degradation also liberates the p105-associated MAP kinase (mitogen-activated protein kinase) kinase kinase TPL-2 (tumour progression locus-2), which can then activate the ERK (extracellular-signal-regulated kinase)/MAP kinase cascade. Thus, in addition to its role in NF-kappaB activation, p105 functions as a regulator of MAP kinase signalling.

55 Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which confer a poor prognosis and are often refractory to conventional therapies. Recent efforts have begun to unravel the molecular pathogenesis of these malignancies. Several groups have demonstrated that MLL-fusions associate with two macromolecular chromatin complexes; the polymerase associated factor (PAFc) complex, which interacts with the N-terminal domain of MLL, a portion of the protein that is retained in all the described fusions, or the super elongation complex (SEC), via interaction with the C-terminal fusion partner. These complexes play an integral role in regulating transcriptional elongation and this function appears to be aberrantly co-opted by the MLL-fusions to initiate and perpetuate transcriptional programmes that culminate in leukaemia. In this study we used a systematic global proteomic survey incorporating quantitative mass spectrometry to demonstrate that MLL-fusions, as part of SEC and PAFc complexes, are associated with the BET family of acetyl lysine recognition chromatin “adaptor” proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. Targeting the BET proteins to alter aberrant transcriptional elongation has recently been demonstrated to be possible using small molecule inhibitors that selectively bind the tandem bromodomain at the amino-terminus of the ubiquitously expressed BET proteins (BRD2/BRD3/BRD4). We developed a novel class of potent small molecule inhibitors to the BET family, which is chemically distinct to previously published BET-inhibitors. We then used this new compound (I-BET151) to demonstrate its profound and selective efficacy against human MLL-fusion leukaemic cell lines in liquid culture as well as clonogenic assays in methylcellulose. We also establish that primary murine progenitors retrovirally transformed with MLL-ENL and MLL-AF9 are equally susceptible to treatment with I-BET151. We show that the main phenotypic consequence of BET inhibition in MLL fusion leukaemia is a dramatic early induction of cell cycle arrest and apoptosis. Global gene-expression profiling, following I-BET151 treatment in two different human MLL-fusion leukaemia cell lines (expressing MLL-AF4 and MLL-AF9), highlights a common differentially expressed gene signature that accounts for this phenotype. Importantly, chromatin immunoprecipitation analyses at direct MLL target genes including BCL2, C-MYC and CDK6, indicate that I-BET151 selectively inhibits the recruitment of BET family members BRD3/BRD4, and SEC and PAFc components. These events result in the inefficient phosphorylation and release of paused POL-II from the TSS of these genes providing mechanistic insight into the mode of action of I-BET151 in MLL-fusion leukaemia. We subsequently established the therapeutic efficacy of I-BET151 in vivo by demonstrating dramatic disease control in murine models of MLL-AF4 and MLL-AF9 leukaemia. Finally, we also demonstrate that I-BET151 accelerates apoptosis in primary leukaemic cells from a large number of patients with various MLL-fusion leukaemias, by affecting a similar transcription programme to that identified in the human leukaemic cell lines. Importantly, we also demonstrate that I-BET151 significantly reduces the clonogenic potential of isolated primary leukaemic stem cells, suggesting that disease eradication may be possible. These data highlight a new paradigm for drug discovery targeting the protein-protein interactions of chromatin-associated proteins. We demonstrate that small molecules that perturb the interaction of BRD3/4 with chromatin have therapeutic potential in MLL fusion leukaemias and moreover, we provide the molecular mechanism to account for this therapeutic efficacy. Finally, our results emphasize an emerging role for targeting aberrant transcriptional elongation in oncogenesis. Disclosures: Prinjha: GSK: Employment. Chung:GSK: Employment. Lugo:GSK: Employment. Beinke:GSK: Employment. Soden:GSK: Employment. Mirguet:GSK: Employment. Jeffrey:GSK: Employment. Lee:GSK: Employment. Kouzarides:GSK: Consultancy.

Despite the prevalence and clinical importance of influenza, its long-term effect on lung immunity is unclear. Here we describe that following viral clearance and clinical recovery, at one month post-influenza, mice are better protected from Streptococcus pneumoniae infection due to a population of monocyte-derived alveolar macrophages (AMs) which produce increased IL-6. Influenza-induced monocyte-derived AMs have a surface phenotype similar to resident AMs but display a unique functional, transcriptional and epigenetic profile which is distinct from resident AMs. In contrast, influenza-experienced resident AMs remain largely similar to naive AMs. Thus, influenza changes the composition of the AM population to provide prolonged antibacterial protection. Monocyte-derived AMs persist over time but lose their protective profile. Our results help to understand how transient respiratory infections, a common occurrence in human life, can constantly alter lung immunity by contributing monocyte-derived, recruited cells to the AM population.