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Biology subjects

Hall, M. N.

Publications and source records attributed to Hall, M. N..

15 recordsLinked to original sources

Generation of functional adipoids from human induced pluripotent stem cells

Human adipose tissue models are lacking although essential for metabolic research. Traditional primary cell cultures are poorly scalable and unsuitable for drug screening or modeling of genetic disease. Conventional two-dimensional adipocyte cultures cannot withstand the long-term treatment required to mimic chronic conditions such as obesity and insulin resistance. To overcome these limitations, we developed an accessible protocol to produce three-dimensional human induced pluripotent stem cell (hiPSC)-derived adipocyte microtissues, termed adipoids. hiPSC-derived adipoids display long-term stability and viability. Single-nucleus RNA sequencing revealed that adipoids contain mature adipocytes and adipocyte stem/progenitor cells, similar to human adipose tissue. Furthermore, hiPSC-derived adipoids contain unilocular adipocytes, exhibit stimulated lipolysis, and secrete adipokines. Thus, hiPSC-derived adipoids provide a functional, scalable and genetically tractable model to perform large-scale pharmacological screens and to investigate human metabolic disease.

molecular biology↗

Antizyme regulates polyamine uptake via ATP13A3

Intracellular polyamine levels are tightly regulated and frequently elevated in cancer. While the regulation of polyamine synthesis is well characterized, the regulation of polyamine uptake is poorly understood. Here we identify ATP13A3 as a plasma membrane polyamine transporter. An increase in intracellular polyamine levels, due to polyamine supplementation or induction of polyamine synthesis, causes rapid internalization of the transporter and inhibition of polyamine uptake. Mechanistically, increased polyamine concentrations lead to expression of antizyme (AZ) which binds ATP13A3 and triggers its internalization. Mutations in the AZ binding site of ATP13A3 prevent AZ binding and lead to polyamine toxicity through uncontrolled polyamine influx. These findings establish ATP13A3 as a key polyamine transporter and provide a framework for targeting polyamine metabolism in cancer.

cell biology↗

Proteomics of Transglutaminase 2 polyaminated proteins

Post-translational modification of proteins by polyamines (putrescine, spermine and spermidine) is poorly studied due to technical challenges in identification. Polyamination is mainly performed by transglutaminases via transamidation of a polyamine to an acceptor glutamine-residue on the target protein. We performed polyamination reactions on whole cell lysates using 2 different polyamine probes (Biotin-pentylamine and DNP-pentylamine) and identified modified peptides by mass spectrometry. 51 protein targets modified on 66 different sites were identified, with an overlap between hits tagged with the different probes. Many of the targets are involved in translation, cytoskeletal organization or have roles in cancer.

molecular biology↗

ALK signalling primes the DNA damage response sensitizing ALK-driven neuroblastoma to ATR inhibition

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and ALK, which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signalling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-day ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-day combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumour cells to a neuronal/Schwann cell lineage identity. Our results identify a unique ability of ATR inhibition to trigger neuroblastoma differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.

cancer biology↗

Hepatic mTORC2 compensates for loss of adipose mTORC2 in mediating energy storage and glucose homeostasis

Mammalian target of rapamycin complex 2 (mTORC2) is a protein kinase complex that plays an important role in energy homeostasis. Loss of adipose mTORC2 reduces lipogenic enzyme expression and de novo lipogenesis in adipose tissue. Adipose-specific mTORC2 knockout mice also displays triglyceride accumulation in the liver. However, the mechanism and physiological role of hepatic triglyceride accumulation upon loss of adipose mTORC2 are unknown. Here, we show that loss of adipose mTORC2 increases expression of de novo lipogenic enzymes in the liver, thereby causing accumulation of hepatic triglyceride and hypertriglyceridemia. Simultaneous inhibition of lipogenic enzymes in adipose tissue and liver by ablating mTORC2 in both tissues prevented accumulation of hepatic triglycerides and hypertriglyceridemia. However, loss of adipose and hepatic mTORC2 caused severe insulin resistance and glucose intolerance. Thus, our findings suggest that increased hepatic lipogenesis is a compensatory mechanism to cope with loss of lipogenesis in adipose tissue, and further suggest that mTORC2 in adipose tissue and liver plays a crucial role in maintaining whole-body energy homeostasis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/521172v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@904812org.highwire.dtl.DTLVardef@6b9f43org.highwire.dtl.DTLVardef@1a507b9org.highwire.dtl.DTLVardef@19e08a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

TORC1 phosphorylates and inhibits the ribosome preservation factor Stm1 to activate dormant ribosomes

Target of rapamycin complex 1 (TORC1) promotes biogenesis and inhibits degradation of ribosomes in response to nutrient availability. To ensure a basal supply of ribosomes, cells preserve a small pool of dormant ribosomes under nutrient-limited conditions. The regulation of dormant ribosomes is poorly characterized. Here, we show that upon inhibition of TORC1 by rapamycin or nitrogen starvation, Stm1 (suppressor of target of Myb protein 1) forms non-translating, dormant 80S ribosomes. Furthermore, Stm1-bound 80S ribosomes are protected from proteasomal degradation. Upon re-feeding, TORC1 directly phosphorylates and inhibits Stm1, thereby reactivating translation. Finally, SERBP1 (SERPINE1 mRNA binding protein), a mammalian ortholog of Stm1, forms dormant 80S ribosomes upon mTORC1 inhibition in mammalian cells. Thus, TORC1 regulates ribosomal dormancy in an evolutionarily conserved manner via a ribosome preservation factor.

molecular biology↗

Elevated arginine levels in liver tumors promote metabolic reprogramming and tumor growth

Arginine auxotropy, due to reduced expression of urea cycle genes, is common in cancer. However, little is known about the levels of arginine in these cancers. Here, we report that arginine levels are elevated in hepatocellular carcinoma (HCC) despite reduced expression of urea cycle enzymes. Liver tumors accumulate high levels specifically of arginine via increased uptake and, more importantly, via suppression of arginine-to-polyamine conversion due to reduced arginase 1 (ARG1) and agmatinase (AGMAT) expression. Furthermore, the high levels of arginine are required for tumor growth. Mechanistically, high levels of arginine promote tumorigenesis via transcriptional regulation of metabolic genes, including upregulation of asparagine synthetase (ASNS). ASNS-derived asparagine further enhances arginine uptake, creating a positive feedback loop to sustain high arginine levels and oncogenic metabolism. Thus, arginine is a novel second messenger-like molecule that reprograms metabolism to promote tumor growth.

cancer biology↗

Adipose mTORC2 is essential for arborization of sensory neurons in white adipose tissue and whole-body energy homeostasis

Adipose tissue, via sympathetic and sensory neurons, communicates with the central nervous system (CNS) to mediate energy homeostasis. In contrast to the sympathetic nervous system, the morphology, role and regulation of the sensory nervous system in adipose tissue is poorly characterized. Taking advantage of recent progress in whole-mount three-dimensional imaging of adipose tissue, we identified a neuronal network of calcitonin gene-related protein (CGRP)-positive sensory neurons in white adipose tissue (WAT). Furthermore, we show that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, mediates sensory innervation in WAT. Based on visualization of neuronal networks, mTORC2-deficient WAT displayed reduced arborization of (CGRP)-positive sensory neurons, while sympathetic neurons were unaffected. This selective loss of sensory innervation followed reduced expression of growth-associated protein 43 (GAP43) in CGRP-positive sensory neurons. Finally, we found that loss of sensory innervation in WAT correlated with systemic insulin resistance. Our findings suggest that adipose mTORC2 is necessary for sensory innervation in WAT which likely contributes to WAT-to-CNS communication.

physiology↗

Multi-omics subtyping of hepatocellular carcinoma patients using a Bayesian network mixture model

Comprehensive molecular characterization of cancer subtypes is essential for predicting clinical outcomes and searching for personalized treatments. We present bnClustOmics, a statistical model and computational tool for multi-omics unsupervised clustering, which serves a dual purpose: Clustering patient samples based on a Bayesian network mixture model and learning the networks of omics variables representing these clusters. The discovered networks encode interactions among all omics variables and provide a molecular characterization of each patient subgroup. We conducted simulation studies that demonstrated the advantages of our approach compared to other clustering methods in the case where the generative model is a mixture of Bayesian networks. We applied bnClustOmics to a hepatocellular carcinoma (HCC) dataset comprising genome (mutation and copy number), transcriptome, proteome, and phosphoproteome data. We identified three main HCC subtypes together with molecular characteristics, some of which are associated with survival even when adjusting for the clinical stage. Cluster-specific networks shed light on the links between genotypes and molecular phenotypes of samples within their respective clusters and suggest targets for personalized treatments. Author summaryMulti-omics approaches to cancer subtyping can provide more insights into molecular changes in tumors compared to single-omics approaches. However, most multi-omics clustering methods do not take into account that gene products interact, for example, as parts of protein complexes or signaling networks. Here we present bnClustOmics, a Bayesian network mixture model for unsupervised clustering of multi-omics data, which can represent dependencies among molecular changes of various omics types explicitly. Unlike other approaches that use data from public interaction databases as ground truth, bnClustOmics learns the dependencies between genes from the analyzed multi-omics dataset. At the same time, our approach can also account for prior knowledge from public interaction databases and use it to guide network learning without losing the ability to learn new dependencies. We applied bnClustOmics to a multi-omics HCC dataset and identified three subtypes similar to those identified in other HCC studies. The cluster-specific networks learned by bnClustOmics revealed additional insights into the molecular characterization of the discovered subgroups and highlighted the changes in signaling networks leading to distinct HCC phenotypes.

systems biology↗

Proteogenomic characterization of hepatocellular carcinoma

We performed a proteogenomic analysis of hepatocellular carcinomas (HCCs) across clinical stages and etiologies. We identified pathways differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. These pathways are involved in the organization of cellular components, cell cycle control, signaling pathways, transcriptional and translational control and metabolism. Analyses of CNA-mRNA and mRNA-protein correlations identified candidate driver genes involved in epithelial-to-mesenchymal transition, the Wnt-{beta}-catenin pathway, transcriptional control, cholesterol biosynthesis and sphingolipid metabolism. The activity of targetable kinases aurora kinase A and CDKs was upregulated. We found that CTNNB1 mutations are associated with altered phosphorylation of proteins involved in actin filament organization, whereas TP53 mutations are associated with elevated CDK1/2/5 activity and altered phosphorylation of proteins involved in lipid and mRNA metabolism. Integrative clustering identified HCC subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our analysis provides insights into the molecular processes underlying HCCs.

genomics↗

Colitis is associated with loss of LHPP and up-regulation of histidine phosphorylation in intestinal epithelial cells

Protein histidine phosphorylation (pHis) is a posttranslational modification involved in cell cycle regulation, ion channel activity and phagocytosis (1). Using novel monoclonal antibodies to detect pHis (2), we recently reported that loss of the histidine phosphatase LHPP results in elevated pHis levels in hepatocellular carcinoma (3). Here, we show that intestinal inflammation correlates with loss of LHPP, in DSS-treated mice and in inflammatory bowel disease (IBD) patients. Increased histidine phosphorylation was observed in intestinal epithelial cells (IECs), as determined by pHis immunofluorescence staining of colon samples from a colitis mouse model. However, ablation of Lhpp did not cause increased pHis or promote intestinal inflammation in physiological conditions or after DSS treatment. Our observations suggest that increased histidine phosphorylation plays a role in colitis, but loss of LHPP is not sufficient to increase pHis or to cause inflammation in the intestine.

biochemistry↗

The 3.2A resolution structure of human mTORC2

The protein kinase mammalian target of rapamycin (mTOR) is the central regulator of cell growth. Aberrant mTOR signaling is linked to cancer, diabetes and neurological disorders. mTOR exerts its functions in two distinct multiprotein complexes, mTORC1 and mTORC2. Here we report a 3.2 [A] resolution cryo-EM reconstruction of mTORC2. It reveals entangled folds of the defining Rictor and the substrate-binding SIN1 subunits, identifies the C-terminal domain of Rictor as the source of the rapamycin insensitivity of mTORC2, and resolves mechanisms for mTORC2 regulation by complex destabilization. Two novel small molecule binding sites are visualized, an inositol hexakisphosphate (InsP6) pocket in mTOR and an mTORC2-specific nucleotide binding site in Rictor which also forms a zinc finger. Structural and biochemical analyses suggest that InsP6 and nucleotide binding do not control mTORC2 activity directly but rather have roles in folding or ternary interactions. These insights provide a firm basis for studying mTORC2 signaling and for developing mTORC2-specific inhibitors.

biochemistry↗

Diet-induced loss of adipose Hexokinase 2 triggers hyperglycemia

Chronically high blood glucose (hyperglycemia) leads to diabetes, fatty liver disease, and cardiovascular disease. Obesity is a major risk factor for hyperglycemia, but the underlying mechanism is unknown. Here we show that a high fat diet (HFD) in mice causes early loss of expression of the glycolytic enzyme Hexokinase 2 (HK2) specifically in adipose tissue. Adipose-specific knockout of Hk2 caused enhanced gluconeogenesis and lipogenesis in liver, a condition known as selective insulin resistance, leading to glucose intolerance. Furthermore, we observed reduced hexokinase activity in adipose tissue of obese and diabetic patients, and identified a loss-of-function mutation in the hk2 gene of naturally hyperglycemic Mexican cavefish. Mechanistically, HFD in mice led to loss of HK2 by inhibiting translation of Hk2 mRNA. Our findings identify adipose HK2 as a critical mediator of systemic glucose homeostasis, and suggest that obesity-induced loss of adipose HK2 is an evolutionarily conserved, non-cell-autonomous mechanism for the development of hyperglycemia. One Sentence SummaryLoss of the glycolytic enzyme Hexokinase 2 in adipose tissue is a mechanism underlying high blood glucose levels.

physiology↗

mTOR dependent transformed human cells have a distinct set of essential genes from bcr-abl transformed cells

Constitutively active intracellular signaling drives and sustains cancer growth. The mTOR kinase integrates multiple inputs sensing nutrient, energy and growth factor levels to promote protein synthesis and anabolic metabolism, and is hyperactivated in a broad range of cancers. The bcr-abl kinase is a fusion protein generated by chromosomal translocation and gives rise to chronic myeloid leukemia and a small sub-set of leukemias. Using an in vitro transformed murine cell model, we performed shRNA knockdown of 25 genes previously shown to be essential for mTOR dependent oncogenic growth and survival. None of the genes were essential in the bcr-abl transformed line. Interrogation of this gene set in human cancer cell lines revealed that many of these genes were essential in cells dependent on mTOR signaling (defined by sensitivity to pharmacological mTOR inhibition). However, none of the genes were essential in bcr-abl transformed K562 cells that are insensitive to mTOR inhibition. Thus there is a clear divide between cells transformed via bcr-abl directed oncogenesis and other modes of transformation where mTOR, due to its central role in regulating cell growth and metabolism, is recruited as part of an oncogenic program. These validated hits represent a set of genes essential for executing critical functions downstream of mTOR and may be novel therapeutic targets for cancer.

cell biology↗

Patterns of selection reveal shared molecular targets over short and long evolutionary timescales

Standing and de novo genetic variants can both drive adaptation to environmental changes, but their relative contributions and interplay remain poorly understood. Here we investigated the dynamics of drug adaptation in yeast populations with different levels of standing variation by experimental evolution coupled with time-resolved sequencing and phenotyping. We found a doubling of standing variation alone boost the adaptation by 64.1% and 51.5% in hydroxyuea and rapamycin respectively. The causative standing and de novo variants were selected on shared targets of RNR4 in hydroxyurea and TOR1, TOR2 in rapamycin. The standing and de novo TOR variants map to different functional domains and act via distinct mechanisms. Interestingly, standing TOR variants from two domesticated strains exhibited opposite resistance effects, reflecting lineage-specific functional divergence. This study provides a dynamic view on how standing and de novo variants interactively drive adaptation and deepens our understanding of clonally evolving diseases.

genetics↗