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Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)(1). Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.

The cyclin-dependent kinase inhibitor CR8 acts as a molecular glue compound by inducing the formation of a complex between CDK12-cyclin K and DDB1, which results in the ubiquitination and degradation of cyclin K.

Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation(1). Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets(2). They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines(3-5), we identify CR8-a cyclin-dependent kinase (CDK) inhibitor(6)-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.

The cryo-electron microscopy structure of the 16-subunit yeast SWI/SNF complex RSC in complex with a nucleosome substrate provides insights into the chromatin-remodelling function of this family of protein complexes.

Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs)(1,2). In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC3 contains 16 subunits, including the ATP-dependent DNA translocase Sth1(4,5). RSC removes nucleosomes from promoter regions(6,7) and positions the specialized +1 and -1 nucleosomes that flank NDRs(8,9). Here we present the cryo-electron microscopy structure of RSC in complex with a nucleosome substrate. The structure reveals that RSC forms five protein modules and suggests key features of the remodelling mechanism. The body module serves as a scaffold for the four flexible modules that we call DNA-interacting, ATPase, arm and actin-related protein (ARP) modules. The DNA-interacting module binds extra-nucleosomal DNA and is involved in the recognition of promoter DNA elements(8,10,11) that influence RSC functionality(12). The ATPase and arm modules sandwich the nucleosome disc with the Snf2 ATP-coupling (SnAC) domain and the finger helix, respectively. The translocase motor of the ATPase module engages with the edge of the nucleosome at superhelical location +2. The mobile ARP module may modulate translocase-nucleosome interactions to regulate RSC activity(5). The RSC-nucleosome structure provides a basis for understanding NDR formation and the structure and function of human SWI/SNF complexes that are frequently mutated in cancer(13).

An in vivo approach to identify proteins whose enrichment near cardiac Ca(V)1.2 channels changes upon beta-adrenergic stimulation finds the G protein Rad, which is phosphorylated by protein kinase A, thereby relieving channel inhibition by Rad and causing an increased Ca2+ current.

Increased cardiac contractility during the fight-or-flight response is caused by beta-adrenergic augmentation of Ca(V)1.2 voltage-gated calcium channels(1-4). However, this augmentation persists in transgenic murine hearts expressing mutant Ca(V)1.2 alpha(1C) and beta subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of beta-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which beta-adrenergic agonists stimulate voltage-gated calcium channels. We express alpha(1C) or beta(2B) subunits conjugated to ascorbate peroxidase(5) in mouse hearts, and use multiplexed quantitative proteomics(6,7) to track hundreds of proteins in the proximity of Ca(V)1.2. We observe that the calcium-channel inhibitor Rad(8,9), a monomeric G protein, is enriched in the Ca(V)1.2 microenvironment but is depleted during beta-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for beta subunits and relieves constitutive inhibition of Ca(V)1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of Ca(V)1.3 and Ca(V)2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.