Genetic and functional correction of argininosuccinate lyase deficiency using CRISPR adenine base editors.

Argininosuccinate lyase deficiency (ASLD) is a recessive metabolic disorder caused by variants in ASL. In an essential step in urea synthesis, ASL breaks down argininosuccinate (ASA), a pathognomonic ASLD biomarker. The severe disease forms lead to hyperammonemia, neurological injury, and even early death. The current treatments are unsatisfactory, involving a strict low-protein diet, arginine supplementation, nitrogen scavenging, and in some cases, liver transplantation. An unmet need exists for improved, efficient therapies. Here, we show the potential of a lipid nanoparticle-mediated CRISPR approach using adenine base editors (ABEs) for ASLD treatment. To model ASLD, we first generated human-induced pluripotent stem cells (hiPSCs) from biopsies of individuals homozygous for the Finnish founder variant (c.1153C>T [p.Arg385Cys]) and edited this variant using the ABE. We then differentiated the hiPSCs into hepatocyte-like cells that showed a 1,000-fold decrease in ASA levels compared to those of isogenic non-edited cells. Lastly, we tested three different FDA-approved lipid nanoparticle formulations to deliver the ABE-encoding RNA and the sgRNA targeting the ASL variant. This approach efficiently edited the ASL variant in fibroblasts with no apparent cell toxicity and minimal off-target effects. Further, the treatment resulted in a significant decrease in ASA, to levels of healthy donors, indicating restoration of the urea cycle. Our work describes a highly efficient approach to editing the disease-causing ASL variant and restoring the function of the urea cycle. This method relies on RNA delivered by lipid nanoparticles, which is compatible with clinical applications, improves its safety profile, and allows for scalable production.

Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells.

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.

Metabolic determination of cell fate through selective inheritance of mitochondria.

Metabolic characteristics of adult stem cells are distinct from their differentiated progeny, and cellular metabolism is emerging as a potential driver of cell fate conversions1-4. How these metabolic features are established remains unclear. Here we identified inherited metabolism imposed by functionally distinct mitochondrial age-classes as a fate determinant in asymmetric division of epithelial stem-like cells. While chronologically old mitochondria support oxidative respiration, the electron transport chain of new organelles is proteomically immature and they respire less. After cell division, selectively segregated mitochondrial age-classes elicit a metabolic bias in progeny cells, with oxidative energy metabolism promoting differentiation in cells that inherit old mitochondria. Cells that inherit newly synthesized mitochondria with low levels of Rieske iron-sulfur polypeptide 1 have a higher pentose phosphate pathway activity, which promotes de novo purine biosynthesis and redox balance, and is required to maintain stemness during early fate determination after division. Our results demonstrate that fate decisions are susceptible to intrinsic metabolic bias imposed by selectively inherited mitochondria.

NOTUM from Apc-mutant cells biases clonal competition to initiate cancer.

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling1, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)2. Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.

UBR5 Is Coamplified with MYC in Breast Tumors and Encodes an Ubiquitin Ligase That Limits MYC-Dependent Apoptosis.

For maximal oncogenic activity, cellular MYC protein levels need to be tightly controlled so that they do not induce apoptosis. Here, we show how ubiquitin ligase UBR5 functions as a molecular rheostat to prevent excess accumulation of MYC protein. UBR5 ubiquitinates MYC and its effects on MYC protein stability are independent of FBXW7. Silencing of endogenous UBR5 induced MYC protein expression and regulated MYC target genes. Consistent with the tumor suppressor function of UBR5 (HYD) in Drosophila, HYD suppressed dMYC-dependent overgrowth of wing imaginal discs. In contrast, in cancer cells, UBR5 suppressed MYC-dependent priming to therapy-induced apoptosis. Of direct cancer relevance, MYC and UBR5 genes were coamplified in MYC-driven human cancers. Functionally, UBR5 suppressed MYC-mediated apoptosis in p53-mutant breast cancer cells with UBR5/MYC coamplification. Furthermore, single-cell immunofluorescence analysis demonstrated reciprocal expression of UBR5 and MYC in human basal-type breast cancer tissues. In summary, UBR5 is a novel MYC ubiquitin ligase and an endogenous rheostat for MYC activity. In MYC-amplified, and p53-mutant breast cancer cells, UBR5 has an important role in suppressing MYC-mediated apoptosis priming and in protection from drug-induced apoptosis. SIGNIFICANCE: These findings identify UBR5 as a novel MYC regulator, the inactivation of which could be very important for understanding of MYC dysregulation on cancer cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/7/1414/F1.large.jpg.

Stem Cell Intrinsic Hexosamine Metabolism Regulates Intestinal Adaptation to Nutrient Content.

The intestine is an organ with an exceptionally high rate of cell turnover, and perturbations in this process can lead to severe diseases such as cancer or intestinal atrophy. Nutrition has a profound impact on intestinal volume and cellular architecture. However, how intestinal homeostasis is maintained in fluctuating dietary conditions remains insufficiently understood. By utilizing the Drosophila midgut model, we reveal a novel stem cell intrinsic mechanism coupling cellular metabolism with stem cell extrinsic growth signal. Our results show that intestinal stem cells (ISCs) employ the hexosamine biosynthesis pathway (HBP) to monitor nutritional status. Elevated activity of HBP promotes Warburg effect-like metabolic reprogramming required for adjusting the ISC division rate according to nutrient content. Furthermore, HBP activity is an essential facilitator for insulin signaling-induced ISC proliferation. In conclusion, ISC intrinsic hexosamine synthesis regulates metabolic pathway activities and defines the stem cell responsiveness to niche-derived growth signals.

Regulation of Carbohydrate Energy Metabolism in Drosophila melanogaster.

Carbohydrate metabolism is essential for cellular energy balance as well as for the biosynthesis of new cellular building blocks. As animal nutrient intake displays temporal fluctuations and each cell type within the animal possesses specific metabolic needs, elaborate regulatory systems are needed to coordinate carbohydrate metabolism in time and space. Carbohydrate metabolism is regulated locally through gene regulatory networks and signaling pathways, which receive inputs from nutrient sensors as well as other pathways, such as developmental signals. Superimposed on cell-intrinsic control, hormonal signaling mediates intertissue information to maintain organismal homeostasis. Misregulation of carbohydrate metabolism is causative for many human diseases, such as diabetes and cancer. Recent work in Drosophila melanogaster has uncovered new regulators of carbohydrate metabolism and introduced novel physiological roles for previously known pathways. Moreover, genetically tractable Drosophila models to study carbohydrate metabolism-related human diseases have provided new insight into the mechanisms of pathogenesis. Due to the high degree of conservation of relevant regulatory pathways, as well as vast possibilities for the analysis of gene-nutrient interactions and tissue-specific gene function, Drosophila is emerging as an important model system for research on carbohydrate metabolism.

PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

Ribosome biogenesis regulates animal growth and is controlled by nutrient-responsive mTOR signaling. How ribosome biogenesis is regulated during the developmental growth of animals and how nutrient-responsive signaling adjusts ribosome biogenesis in this setting have remained insufficiently understood. We uncover PWP1 as a chromatin-associated regulator of developmental growth with a conserved role in RNA polymerase I (Pol I)-mediated rRNA transcription. We further observed that PWP1 epigenetically maintains the rDNA loci in a transcription-competent state. PWP1 responds to nutrition in Drosophila larvae via mTOR signaling through gene expression and phosphorylation, which controls the nucleolar localization of dPWP1. Our data further imply that dPWP1 acts synergistically with mTOR signaling to regulate the nucleolar localization of TFIIH, a known elongation factor of Pol I. Ribosome biogenesis is often deregulated in cancer, and we demonstrate that high PWP1 levels in human head and neck squamous cell carcinoma tumors are associated with poor prognosis.

Salt-Inducible Kinase 3 Provides Sugar Tolerance by Regulating NADPH/NADP+ Redox Balance.

Nutrient-sensing pathways respond to changes in the levels of macronutrients, such as sugars, lipids, or amino acids, and regulate metabolic pathways to maintain organismal homeostasis [1, 2]. Consequently, nutrient sensing provides animals with the metabolic flexibility necessary for enduring temporal fluctuations in nutrient intake. Recent studies have shown that an animal's ability to survive on a high-sugar diet is determined by sugar-responsive gene regulation [3-8]. It remains to be elucidated whether other levels of metabolic control, such as post-translational regulation of metabolic enzymes, also contribute to organismal sugar tolerance. Furthermore, the sugar-regulated metabolic pathways contributing to sugar tolerance remain insufficiently characterized. Here, we identify Salt-inducible kinase 3 (SIK3), a member of the AMP-activated protein kinase (AMPK)-related kinase family, as a key determinant of Drosophila sugar tolerance. SIK3 allows sugar-feeding animals to increase the reductive capacity of nicotinamide adenine dinucleotide phosphate (NADPH/NADP+). NADPH mediates the reduction of the intracellular antioxidant glutathione, which is essential for survival on a high-sugar diet. SIK3 controls NADP+ reduction by phosphorylating and activating Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. SIK3 gene expression is regulated by the sugar-regulated transcription factor complex Mondo-Mlx, which was previously identified as a key determinant of sugar tolerance. SIK3 converges with Mondo-Mlx in sugar-induced activation of G6PD, and simultaneous inhibition of SIK3 and Mondo-Mlx leads to strong synergistic lethality on a sugar-containing diet. In conclusion, SIK3 cooperates with Mondo-Mlx to maintain organismal sugar tolerance through the regulation of NADPH/NADP+ redox balance.

p53- and ERK7-dependent ribosome surveillance response regulates Drosophila insulin-like peptide secretion.

Insulin-like signalling is a conserved mechanism that coordinates animal growth and metabolism with nutrient status. In Drosophila, insulin-producing median neurosecretory cells (IPCs) regulate larval growth by secreting insulin-like peptides (dILPs) in a diet-dependent manner. Previous studies have shown that nutrition affects dILP secretion through humoral signals derived from the fat body. Here we uncover a novel mechanism that operates cell autonomously in the IPCs to regulate dILP secretion. We observed that impairment of ribosome biogenesis specifically in the IPCs strongly inhibits dILP secretion, which consequently leads to reduced body size and a delay in larval development. This response is dependent on p53, a known surveillance factor for ribosome biogenesis. A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation. We show that ERK7 is sufficient and essential to inhibit dILP secretion upon impaired ribosome biogenesis, and it acts epistatically to p53. Moreover, we provide evidence that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation. Thus, we conclude that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions.

ER stress potentiates insulin resistance through PERK-mediated FOXO phosphorylation.

Endoplasmic reticulum (ER) stress is emerging as a potential contributor to the onset of type 2 diabetes by making cells insulin-resistant. However, our understanding of the mechanisms by which ER stress affects insulin response remains fragmentary. Here we present evidence that the ER stress pathway acts via a conserved signaling mechanism involving the protein kinase PERK to modulate cellular insulin responsiveness. Insulin signaling via AKT reduces activity of FOXO transcription factors. In some cells, PERK can promote insulin responsiveness. However, we found that PERK also acts oppositely via phosphorylation of FOXO to promote FOXO activity. Inhibition of PERK improves cellular insulin responsiveness at the level of FOXO activity. We suggest that the protein kinase PERK may be a promising pharmacological target for ameliorating insulin resistance.

Glucose sensing by ChREBP/MondoA-Mlx transcription factors.

The paralogous transcription factors ChREBP and MondoA, together with their common binding partner Mlx, have emerged as key mediators of intracellular glucose sensing. By regulating target genes involved in glycolysis and lipogenesis, they mediate metabolic adaptation to changing glucose levels. As disturbed glucose homeostasis plays a central role in human metabolic diseases and as cancer cells often display altered glucose metabolism, better understanding of cellular glucose sensing will likely uncover new therapeutic opportunities. Here we review the regulation, function and evolutionary conservation of the ChREBP/MondoA-Mlx glucose sensing system and discuss possible directions for future research.

Characterization of Drosophila GDNF receptor-like and evidence for its evolutionarily conserved interaction with neural cell adhesion molecule (NCAM)/FasII.

BACKGROUND: Glial cell line-derived neurotrophic factor (GDNF) family ligands are secreted growth factors distantly related to the TGF-ß superfamily. In mammals, they bind to the GDNF family receptor α (Gfrα) and signal through the Ret receptor tyrosine kinase. In order to gain insight into the evolution of the Ret-Gfr-Gdnf signaling system, we have cloned and characterized the first invertebrate Gfr-like cDNA (DmGfrl) from Drosophila melanogaster and generated a DmGfrl mutant allele. RESULTS: We found that DmGfrl encodes a large GPI-anchored membrane protein with four GFR-like domains. In line with the fact that insects lack GDNF ligands, DmGfrl mediated neither Drosophila Ret phosphorylation nor mammalian RET phosphorylation. In situ hybridization analysis revealed that DmGfrl is expressed in the central and peripheral nervous systems throughout Drosophila development, but, surprisingly, DmGfrl and DmRet expression patterns were largely non-overlapping. We generated a DmGfrl null allele by genomic FLP deletion and found that both DmGfrl null females and males are viable but display fertility defects. The female fertility defect manifested as dorsal appendage malformation, small size and reduced viability of eggs laid by mutant females. In male flies DmGfrl interacted genetically with the Drosophila Ncam (neural cell adhesion molecule) homolog FasII to regulate fertility. CONCLUSION: Our results suggest that Ret and Gfrl did not function as an in cis receptor-coreceptor pair before the emergence of GDNF family ligands, and that the Ncam-Gfr interaction predated the in cis Ret-Gfr interaction in evolution. The fertility defects that we describe in DmGfrl null flies suggest that GDNF receptor-like has an evolutionarily ancient role in regulating male fertility and a previously unrecognized role in regulating oogenesis. SIGNIFICANCE: These results shed light on the evolutionary aspects of the structure, expression and function of Ret-Gfrα and Ncam-Gfrα signaling complexes.

MAPK/ERK signaling regulates insulin sensitivity to control glucose metabolism in Drosophila.

The insulin/IGF-activated AKT signaling pathway plays a crucial role in regulating tissue growth and metabolism in multicellular animals. Although core components of the pathway are well defined, less is known about mechanisms that adjust the sensitivity of the pathway to extracellular stimuli. In humans, disturbance in insulin sensitivity leads to impaired clearance of glucose from the blood stream, which is a hallmark of diabetes. Here we present the results of a genetic screen in Drosophila designed to identify regulators of insulin sensitivity in vivo. Components of the MAPK/ERK pathway were identified as modifiers of cellular insulin responsiveness. Insulin resistance was due to downregulation of insulin-like receptor gene expression following persistent MAPK/ERK inhibition. The MAPK/ERK pathway acts via the ETS-1 transcription factor Pointed. This mechanism permits physiological adjustment of insulin sensitivity and subsequent maintenance of circulating glucose at appropriate levels.

TOR complex 2 is needed for cell cycle progression and anchorage-independent growth of MCF7 and PC3 tumor cells.

BACKGROUND: AKT signaling promotes cell growth, proliferation and survival and is hyperactivated in many cancers. TOR complex 2 (TORC2) activates AKT by phosphorylating it on the 'hydrophobic motif' site. Hydrophobic motif site phosphorylation is needed only for a subset of AKT functions. Whether proliferation of tumor cells depends on TORC2 activity has not been thoroughly explored. METHODS: We used RNAi-mediated knockdown of rictor to inhibit TORC2 activity in MCF7 and PC3 tumor cells to analyze the importance of TORC2 on proliferation of tumor cells. RESULTS: TORC2 inhibition reduced proliferation and anchorage-independent growth of both cell lines. Rictor depleted cells accumulated G1 phase, and showed prominent downregulation of Cyclin D1. CONCLUSION: This study provides further evidence that inhibition of TORC2 activity might be a useful strategy to inhibit proliferation of tumor cells and subsequent tumor growth.

Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila.

Animals use the insulin/TOR signaling pathway to mediate their response to fluctuations in nutrient availability. Energy and amino acids are monitored at the single-cell level via the TOR branch of the pathway and systemically via insulin signaling to regulate cellular growth and metabolism. Using a combination of genetics, expression profiling, and chromatin immunoprecipitation, we examine nutritional control of gene expression and identify the transcription factor Myc as an important mediator of TOR-dependent regulation of ribosome biogenesis. We also identify myc as a direct target of FOXO and provide genetic evidence that Myc has a key role in mediating the effects of TOR and FOXO on growth and metabolism. FOXO and TOR also converge to regulate protein synthesis, acting via 4E-BP and Lk6, regulators of the translation factor eIF4E. This study uncovers a network of convergent regulation of protein biosynthesis by the FOXO and TOR branches of the nutrient-sensing pathway.

Re-evaluating AKT regulation: role of TOR complex 2 in tissue growth.

Phosphatidylinositol-3-kinase (PI3K)/AKT signaling is essential for growth and metabolism and is elevated in many cancers. Enzymatic activity of AKT has been shown to depend on phosphorylation of two conserved sites by PDK1 and TOR (target of rapamycin) complex 2 (TORC2) in a PI3K-dependent manner. Here we analyze the role of TORC2-mediated AKT phosphorylation in Drosophila. Mutants removing critical TORC2 components, rictor and sin1, strongly reduced AKT hydrophobic motif (HM) phosphorylation and AKT activity, but showed only minor growth impairment. A mutant form of AKT lacking the HM phosphorylation site displayed comparable activity. In contrast to the mild effects of removing HM site phosphorylation at normal levels of PI3K activity, loss of TORC2 activity strongly inhibited hyperplasia caused by elevated pathway activity, as in mutants of the tumor suppressor PTEN. Thus, TORC2 acts as a rheostat to broaden the range of AKT signaling at the high end of its range.

Rapid turnover of c-FLIPshort is determined by its unique C-terminal tail.

The caspase-8 inhibitor c-FLIP exists as two splice variants, c-FLIP(L) and c-FLIP(S), with distinct roles in death receptor signaling. The mechanisms determining their turnover have not been established. We found that in differentiating K562 erythroleukemia cells both c-FLIP isoforms were inducibly degraded by the proteasome, but c-FLIP(S) was more prone to ubiquitylation and had a considerably shorter half-life. Analysis of the c-FLIP(S)-specific ubiquitylation revealed two lysines, 192 and 195, C-terminal to the death effector domains, as principal ubiquitin acceptors in c-FLIP(S) but not in c-FLIP(L). Furthermore the c-FLIP(S)-specific tail of 19 amino acids, adjacent to the two target lysines, was demonstrated to be the key element determining the isoform-specific instability of c-FLIP(S). Molecular modeling in combination with site-directed mutagenesis demonstrated that the C-terminal tail is required for correct positioning and subsequent ubiquitylation of the target lysines. Because the antiapoptotic operation of c-FLIP(S) was not affected by the tail deletion, the antiapoptotic activity and ubiquitin-mediated degradation of c-FLIP(S) are functionally and structurally independent processes. The presence of a small destabilizing sequence in c-FLIP(S) constitutes an important determinant of c-FLIP(S)/c-FLIP(L) ratios by allowing differential degradation of c-FLIP isoforms. The conformation-based predisposition of c-FLIP(S) to ubiquitin-mediated degradation introduces a novel concept to the regulation of the death-inducing signaling complex.

Erythroid differentiation sensitizes K562 leukemia cells to TRAIL-induced apoptosis by downregulation of c-FLIP.

Regulation of the apoptotic threshold is of great importance in the homeostasis of both differentiating and fully developed organ systems. Triggering differentiation has been employed as a strategy to inhibit cell proliferation and accelerate apoptosis in malignant cells, in which the apoptotic threshold is often characteristically elevated. To better understand the mechanisms underlying differentiation-mediated regulation of apoptosis, we have studied death receptor responses during erythroid differentiation of K562 erythroleukemia cells, which normally are highly resistant to tumor necrosis factor (TNF) alpha-, FasL-, and TRAIL-induced apoptosis. However, upon hemin-mediated erythroid differentiation, K562 cells specifically lost their resistance to TNF-related apoptosis-inducing ligand (TRAIL), which efficiently killed the differentiating cells independently of mitochondrial apoptotic signaling. Concomitantly with the increased sensitivity, the expression of both c-FLIP splicing variants, c-FLIP(L) and c-FLIP(S), was downregulated, resulting in an altered caspase 8 recruitment and cleavage in the death-inducing signaling complex (DISC). Stable overexpression of both c-FLIP(L) and c-FLIP(S) rescued the cells from TRAIL-mediated apoptosis with isoform-specific effects on DISC-recruited caspase 8. Our results show that c-FLIP(L) and c-FLIP(S) potently control TRAIL responses, both by distinct regulatory features, and further imply that the differentiation state of malignant cells determines their sensitivity to death receptor signals.