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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.

Mitochondria take up Ca2+ through the mitochondrial calcium uniporter complex to regulate energy production, cytosolic Ca2+ signalling and cell death(1,2). In mammals, the uniporter complex (uniplex) contains four core components: the pore-forming MCU protein, the gatekeepers MICU1 and MICU2, and an auxiliary subunit, EMRE, essential for Ca2+ transport(3-8). To prevent detrimental Ca2+ overload, the activity of MCU must be tightly regulated by MICUs, which sense changes in cytosolic Ca2+ concentrations to switch MCU on and off(9,10). Here we report cryo-electron microscopic structures of the human mitochondrial calcium uniporter holocomplex in inhibited and Ca2+-activated states. These structures define the architecture of this multicomponent Ca2+-uptake machinery and reveal the gating mechanism by which MICUs control uniporter activity. Our work provides a framework for understanding regulated Ca2+ uptake in mitochondria, and could suggest ways of modulating uniporter activity to treat diseases related to mitochondrial Ca2+ overload.

Cryo-electron microscopy reveals the structures of the mitochondrial calcium uniporter holocomplex in low- and high-calcium conditions, showing the gating mechanism that underlies uniporter activation in response to intracellular calcium signals.

Breakdown of the blood-brain barrier in individuals carrying the epsilon 4 allele of the APOE gene, but not the epsilon 3 allele, increases with and predicts cognitive impairment and is independent of amyloid beta or tau pathology.

The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts(1). Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types(2,3). However, whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis, we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E-2 (PGE(2)). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic, autochthonous tumorigenesis. Mechanistically, single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE(2) drives the expansion omicron f a population of Sca-1(+) reserve-like stem cells. These express a strong regenerative/tumorigenic program, driven by the Hippo pathway effector Yap. In vivo, Yap is indispensable for Sca-1(+) cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments, we identified a molecular mechanism whereby PGE(2) promotes Yap dephosphorylation, nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1(+) cell expansion and sporadic tumour initiation in mutant mice, thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE(2)-Ptger4. Analyses of patient-derived organoids established that PGE(2)-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE(2)-Ptger4-Yap signalling axis.

Single-cell RNA-sequencing analysis of intestinal mesenchyme identified a population of fibroblasts that produce prostaglandin E-2, which, when disrupted, prevented initiation of intestinal tumours.

Phosphorylation of INSIG1 and INSIG2 by PCK1 leads to a reduction in the binding of sterols, the activation of SREBP1 and SREBP2 and the downstream transcription of lipogenesis-associated genes that promote tumour growth.

Cancer cells increase lipogenesis for their proliferation and the activation of sterol regulatory element-binding proteins (SREBPs) has a central role in this process. SREBPs are inhibited by a complex composed of INSIG proteins, SREBP cleavage-activating protein (SCAP) and sterols in the endoplasmic reticulum. Regulation of the interaction between INSIG proteins and SCAP by sterol levels is critical for the dissociation of the SCAP-SREBP complex from the endoplasmic reticulum and the activation of SREBPs(1,2). However, whether this protein interaction is regulated by a mechanism other than the abundance of sterol-and in particular, whether oncogenic signalling has a role-is unclear. Here we show that activated AKT in human hepatocellular carcinoma (HCC) cells phosphorylates cytosolic phosphoenolpyruvate carboxykinase 1 (PCK1), the rate-limiting enzyme in gluconeogenesis, at Ser90. Phosphorylated PCK1 translocates to the endoplasmic reticulum, where it uses GTP as a phosphate donor to phosphorylate INSIG1 at Ser207 and INSIG2 at Ser151. This phosphorylation reduces the binding of sterols to INSIG1 and INSIG2 and disrupts the interaction between INSIG proteins and SCAP, leading to the translocation of the SCAP-SREBP complex to the Golgi apparatus, the activation of SREBP proteins (SREBP1 or SREBP2) and the transcription of downstream lipogenesis-related genes, proliferation of tumour cells, and tumorigenesis in mice. In addition, phosphorylation of PCK1 at Ser90, INSIG1 at Ser207 and INSIG2 at Ser151 is not only positively correlated with the nuclear accumulation of SREBP1 in samples from patients with HCC, but also associated with poor HCC prognosis. Our findings highlight the importance of the protein kinase activity of PCK1 in the activation of SREBPs, lipogenesis and the development of HCC.