Metabolic regulation of the hallmarks of stem cell biology.

Stem cells perform many different functions, each of which requires specific metabolic adaptations. Over the past decades, studies of pluripotent and tissue stem cells have uncovered a range of metabolic preferences and strategies that correlate with or exert control over specific cell states. This review aims to describe the common themes that emerge from the study of stem cell metabolism: (1) metabolic pathways supporting stem cell proliferation, (2) metabolic pathways maintaining stem cell quiescence, (3) metabolic control of cellular stress responses and cell death, (4) metabolic regulation of stem cell identity, and (5) metabolic requirements of the stem cell niche.

Amino acid intake strategies define pluripotent cell states.

Mammalian preimplantation development is associated with marked metabolic robustness, and embryos can develop under a wide variety of nutrient conditions, including even the complete absence of soluble amino acids. Here we show that mouse embryonic stem cells (ESCs) capture the unique metabolic state of preimplantation embryos and proliferate in the absence of several essential amino acids. Amino acid independence is enabled by constitutive uptake of exogenous protein through macropinocytosis, alongside a robust lysosomal digestive system. Following transition to more committed states, ESCs reduce digestion of extracellular protein and instead become reliant on exogenous amino acids. Accordingly, amino acid withdrawal selects for ESCs that mimic the preimplantation epiblast. More broadly, we find that all lineages of preimplantation blastocysts exhibit constitutive macropinocytic protein uptake and digestion. Taken together, these results highlight exogenous protein uptake and digestion as an intrinsic feature of preimplantation development and provide insight into the catabolic strategies that enable embryos to sustain viability before implantation.

A non-canonical tricarboxylic acid cycle underlies cellular identity.

The tricarboxylic acid (TCA) cycle is a central hub of cellular metabolism, oxidizing nutrients to generate reducing equivalents for energy production and critical metabolites for biosynthetic reactions. Despite the importance of the products of the TCA cycle for cell viability and proliferation, mammalian cells display diversity in TCA-cycle activity1,2. How this diversity is achieved, and whether it is critical for establishing cell fate, remains poorly understood. Here we identify a non-canonical TCA cycle that is required for changes in cell state. Genetic co-essentiality mapping revealed a cluster of genes that is sufficient to compose a biochemical alternative to the canonical TCA cycle, wherein mitochondrially derived citrate exported to the cytoplasm is metabolized by ATP citrate lyase, ultimately regenerating mitochondrial oxaloacetate to complete this non-canonical TCA cycle. Manipulating the expression of ATP citrate lyase or the canonical TCA-cycle enzyme aconitase 2 in mouse myoblasts and embryonic stem cells revealed that changes in the configuration of the TCA cycle accompany cell fate transitions. During exit from pluripotency, embryonic stem cells switch from canonical to non-canonical TCA-cycle metabolism. Accordingly, blocking the non-canonical TCA cycle prevents cells from exiting pluripotency. These results establish a context-dependent alternative to the traditional TCA cycle and reveal that appropriate TCA-cycle engagement is required for changes in cell state.

Repurposing an adenine riboswitch into a fluorogenic imaging and sensing tag.

Fluorogenic RNA aptamers are used to genetically encode fluorescent RNA and to construct RNA-based metabolite sensors. Unlike naturally occurring aptamers that efficiently fold and undergo metabolite-induced conformational changes, fluorogenic aptamers can exhibit poor folding, which limits their cellular fluorescence. To overcome this, we evolved a naturally occurring well-folded adenine riboswitch into a fluorogenic aptamer. We generated a library of roughly 1015 adenine aptamer-like RNAs in which the adenine-binding pocket was randomized for both size and sequence, and selected Squash, which binds and activates the fluorescence of green fluorescent protein-like fluorophores. Squash exhibits markedly improved in-cell folding and highly efficient metabolite-dependent folding when fused to a S-adenosylmethionine (SAM)-binding aptamer. A Squash-based ratiometric sensor achieved quantitative SAM measurements, revealed cell-to-cell heterogeneity in SAM levels and revealed metabolic origins of SAM. These studies show that the efficient folding of naturally occurring aptamers can be exploited to engineer well-folded cell-compatible fluorogenic aptamers and devices.

Cytomegalovirus Infection Drives Avidity Selection of Natural Killer Cells.

The process of affinity maturation, whereby T and B cells bearing antigen receptors with optimal affinity to the relevant antigen undergo preferential expansion, is a key feature of adaptive immunity. Natural killer (NK) cells are innate lymphocytes capable of "adaptive" responses after cytomegalovirus (CMV) infection. However, whether NK cells are similarly selected on the basis of their avidity for cognate ligand is unknown. Here, we showed that NK cells with the highest avidity for the mouse CMV glycoprotein m157 were preferentially selected to expand and comprise the memory NK cell pool, whereas low-avidity NK cells possessed greater capacity for interferon-γ (IFN-γ) production. Moreover, we provide evidence for avidity selection occurring in human NK cells during human CMV infection. These results delineate how heterogeneity in NK cell avidity diversifies NK cell effector function during antiviral immunity, and how avidity selection might serve to produce the most potent memory NK cells.

lncRNA Spehd Regulates Hematopoietic Stem and Progenitor Cells and Is Required for Multilineage Differentiation.

Long non-coding RNAs (lncRNAs) show patterns of tissue- and cell type-specific expression that are very similar to those of protein coding genes and consequently have the potential to control stem and progenitor cell fate decisions along a differentiation trajectory. To understand the roles that lncRNAs may play in hematopoiesis, we selected a subset of mouse lncRNAs with potentially relevant expression patterns and refined our candidate list using evidence of conserved expression in human blood lineages. For each candidate, we assessed its possible role in hematopoietic differentiation in vivo using competitive transplantation. Our studies identified two lncRNAs that were required for hematopoiesis. One of these, Spehd, showed defective multilineage differentiation, and its silencing yielded common myeloid progenitors that are deficient in their oxidative phosphorylation pathway. This effort not only suggests that lncRNAs can contribute to differentiation decisions during hematopoiesis but also provides a path toward the identification of functional lncRNAs in other differentiation hierarchies.

Tumor-derived CSF-1 induces the NKG2D ligand RAE-1δ on tumor-infiltrating macrophages.

NKG2D is an important immunoreceptor expressed on the surface of NK cells and some T cells. NKG2D recognizes a set of ligands typically expressed on infected or transformed cells, but recent studies have also documented NKG2D ligands on subsets of host non-tumor cells in tumor-bearing animals and humans. Here we show that in transplanted tumors and genetically engineered mouse cancer models, tumor-associated macrophages are induced to express the NKG2D ligand RAE-1δ. We find that a soluble factor produced by tumor cells is responsible for macrophage RAE-1δ induction, and we identify tumor-derived colony-stimulating factor-1 (CSF-1) as necessary and sufficient for macrophage RAE-1δ induction in vitro and in vivo. Furthermore, we show that induction of RAE-1δ on macrophages by CSF-1 requires PI3K p110α kinase signaling. Thus, production of CSF-1 by tumor cells leading to activation of PI3K p110α represents a novel cellular and molecular pathway mediating NKG2D ligand expression on tumor-associated macrophages.

NF-κB inducing kinase is a therapeutic target for systemic lupus erythematosus.

NF-κB-inducing kinase (NIK) mediates non-canonical NF-κB signaling downstream of multiple TNF family members, including BAFF, TWEAK, CD40, and OX40, which are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we show that experimental lupus in NZB/W F1 mice can be treated with a highly selective and potent NIK small molecule inhibitor. Both in vitro as well as in vivo, NIK inhibition recapitulates the pharmacological effects of BAFF blockade, which is clinically efficacious in SLE. Furthermore, NIK inhibition also affects T cell parameters in the spleen and proinflammatory gene expression in the kidney, which may be attributable to inhibition of OX40 and TWEAK signaling, respectively. As a consequence, NIK inhibition results in improved survival, reduced renal pathology, and lower proteinuria scores. Collectively, our data suggest that NIK inhibition is a potential therapeutic approach for SLE.

Endothelial cells express NKG2D ligands and desensitize antitumor NK responses.

Natural Killer (NK) cells confer protection from tumors and infections by releasing cytotoxic granules and pro-inflammatory cytokines upon recognition of diseased cells. The responsiveness of NK cells to acute stimulation is dynamically tuned by steady-state receptor-ligand interactions of an NK cell with its cellular environment. Here, we demonstrate that in healthy WT mice the NK activating receptor NKG2D is engaged in vivo by one of its ligands, RAE-1ε, which is expressed constitutively by lymph node endothelial cells and highly induced on tumor-associated endothelium. This interaction causes internalization of NKG2D from the NK cell surface and transmits an NK-intrinsic signal that desensitizes NK cell responses globally to acute stimulation, resulting in impaired NK antitumor responses in vivo.

lncRNA requirements for mouse acute myeloid leukemia and normal differentiation.

A substantial fraction of the genome is transcribed in a cell-type-specific manner, producing long non-coding RNAs (lncRNAs), rather than protein-coding transcripts. Here, we systematically characterize transcriptional dynamics during hematopoiesis and in hematological malignancies. Our analysis of annotated and de novo assembled lncRNAs showed many are regulated during differentiation and mis-regulated in disease. We assessed lncRNA function via an in vivo RNAi screen in a model of acute myeloid leukemia. This identified several lncRNAs essential for leukemia maintenance, and found that a number act by promoting leukemia stem cell signatures. Leukemia blasts show a myeloid differentiation phenotype when these lncRNAs were depleted, and our data indicates that this effect is mediated via effects on the MYC oncogene. Bone marrow reconstitutions showed that a lncRNA expressed across all progenitors was required for the myeloid lineage, whereas the other leukemia-induced lncRNAs were dispensable in the normal setting.