ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition.

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (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 (ataxia telangiectasia and Rad3-related)/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 signaling 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-d ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-d combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify an ability of ATR inhibition to promote NB 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.

Arginine reprograms metabolism in liver cancer via RBM39.

Metabolic reprogramming is a hallmark of cancer. However, mechanisms underlying metabolic reprogramming and how altered metabolism in turn enhances tumorigenicity are poorly understood. Here, we report that arginine levels are elevated in murine and patient hepatocellular carcinoma (HCC), despite reduced expression of arginine synthesis genes. Tumor cells accumulate high levels of arginine due to increased uptake and reduced arginine-to-polyamine conversion. Importantly, the high levels of arginine promote tumor formation via further metabolic reprogramming, including changes in glucose, amino acid, nucleotide, and fatty acid metabolism. Mechanistically, arginine binds RNA-binding motif protein 39 (RBM39) to control expression of metabolic genes. RBM39-mediated upregulation of asparagine synthesis leads to enhanced arginine uptake, creating a positive feedback loop to sustain high arginine levels and oncogenic metabolism. Thus, arginine is a second messenger-like molecule that reprograms metabolism to promote tumor growth.

mTORC2 regulates auditory hair cell structure and function.

mTOR broadly controls cell growth, but little is known about the role of mTOR complex 2 (mTORC2) in the inner ear. To investigate the role of mTORC2 in sensory hair cells (HCs), we generated HC-specific Rictor knockout (HC-RicKO) mice. HC-RicKO mice exhibited early-onset, progressive, and profound hearing loss. Increased DPOAE thresholds indicated outer HC dysfunction. HCs are lost, but this occurs after hearing loss. Ultrastructural analysis revealed stunted and absent stereocilia in outer HCs. In inner HCs, the number of synapses was significantly decreased and the remaining synapses displayed a disrupted actin cytoskeleton and disorganized Ca2+ channels. Thus, the mTORC2 signaling pathway plays an important role in regulating auditory HC structure and function via regulation of the actin cytoskeleton. These results provide molecular insights on a central regulator of cochlear HCs and thus hearing.

Colitis Is Associated with Loss of the Histidine Phosphatase LHPP and Upregulation 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. Using novel monoclonal antibodies to detect pHis, we previously reported that the loss of the histidine phosphatase LHPP (phospholysine phosphohistidine inorganic pyrophosphate phosphatase) results in elevated pHis levels in hepatocellular carcinoma. Here, we show that intestinal inflammation correlates with the loss of LHPP in dextran sulfate sodium (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, the ablation of Lhpp did not cause increased pHis or promote intestinal inflammation under physiological conditions or after DSS treatment. Our observations suggest that increased histidine phosphorylation plays a role in colitis, but the loss of LHPP is not sufficient to increase pHis or to cause inflammation in the intestine.

Raptor levels are critical for ß-cell adaptation to a high-fat diet in male mice.

OBJECTIVE: The essential role of raptor/mTORC1 signaling in ß-cell survival and insulin processing has been recently demonstrated using raptor knock-out models. Our aim was to evaluate the role of mTORC1 function in adaptation of ß-cells to insulin resistant state. METHOD: Here, we use mice with heterozygous deletion of raptor in ß-cells (ßraHet) to assess whether reduced mTORC1 function is critical for ß-cell function in normal conditions or during ß-cell adaptation to high-fat diet (HFD). RESULTS: Deletion of a raptor allele in ß-cells showed no differences at the metabolic level, islets morphology, or ß-cell function in mice fed regular chow. Surprisingly, deletion of only one allele of raptor increases apoptosis without altering proliferation rate and is sufficient to impair insulin secretion when fed a HFD. This is accompanied by reduced levels of critical ß-cell genes like Ins1, MafA, Ucn3, Glut2, Glp1r, and specially PDX1 suggesting an improper ß-cell adaptation to HFD. CONCLUSION: This study identifies that raptor levels play a key role in maintaining PDX1 levels and ß-cell function during the adaptation of ß-cell to HFD. Finally, we identified that Raptor levels regulate PDX1 levels and ß-cell function during ß-cell adaptation to HFD by reduction of the mTORC1-mediated negative feedback and activation of the AKT/FOXA2/PDX1 axis. We suggest that Raptor levels are critical to maintaining PDX1 levels and ß-cell function in conditions of insulin resistance in male mice.

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 display 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 the 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.NEW & NOTEWORTHY Loss of adipose and hepatic mTORC2 causes diabetes.

Diet-induced loss of adipose hexokinase 2 correlates with hyperglycemia.

Chronically high blood glucose (hyperglycemia) leads to diabetes and fatty liver 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 reduced glucose disposal and lipogenesis and enhanced fatty acid release in adipose tissue. In a non-cell-autonomous manner, Hk2 knockout also promoted glucose production in liver. 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 local and systemic glucose homeostasis, and suggest that obesity-induced loss of adipose HK2 is an evolutionarily conserved mechanism for the development of selective insulin resistance and thereby hyperglycemia.

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

Target of rapamycin complex 1 (TORC1) promotes biogenesis and inhibits the degradation of ribosomes in response to nutrient availability. To ensure a basal supply of ribosomes, cells are known to preserve a small pool of dormant ribosomes under nutrient-limited conditions. However, the regulation of these dormant ribosomes is poorly characterized. Here, we show that upon inhibition of yeast TORC1 by rapamycin or nitrogen starvation, the ribosome preservation factor Stm1 mediates the formation of nontranslating, dormant 80S ribosomes. Furthermore, Stm1-bound 80S ribosomes are protected from proteasomal degradation. Upon nutrient replenishment, TORC1 directly phosphorylates and inhibits Stm1 to reactivate translation. Finally, we find that SERBP1, a mammalian ortholog of Stm1, is likewise required for the formation of dormant 80S ribosomes upon mTORC1 inhibition in mammalian cells. These data suggest that TORC1 regulates ribosomal dormancy in an evolutionarily conserved manner by directly targeting a ribosome preservation factor.

Combining metformin with lactate transport inhibitors as a treatment modality for cancer - recommendation proposal.

Highly glycolytic cancer cells excrete lactate to maintain cellular homeostasis. Inhibiting lactate export by pharmacological targeting of plasma membrane lactate transporters is being pursued as an anti-cancer therapy. Work from many laboratories show that the simultaneous inhibition of lactate export and mitochondrial respiration elicits strong synthetic lethality. The mitochondrial inhibitor, metformin, has been the subject of numerous clinical trials as an anti-cancer agent. We propose that, in future clinical trials, metformin be combined with lactate transport inhibitors to exploit this synergistic interaction.

Transcription factors TEAD2 and E2A globally repress acetyl-CoA synthesis to promote tumorigenesis.

Acetyl-coenzyme A (acetyl-CoA) plays an important role in metabolism, gene expression, signaling, and other cellular processes via transfer of its acetyl group to proteins and metabolites. However, the synthesis and usage of acetyl-CoA in disease states such as cancer are poorly characterized. Here, we investigated global acetyl-CoA synthesis and protein acetylation in a mouse model and patient samples of hepatocellular carcinoma (HCC). Unexpectedly, we found that acetyl-CoA levels are decreased in HCC due to transcriptional downregulation of all six acetyl-CoA biosynthesis pathways. This led to hypo-acetylation specifically of non-histone proteins, including many enzymes in metabolic pathways. Importantly, repression of acetyl-CoA synthesis promoted oncogenic dedifferentiation and proliferation. Mechanistically, acetyl-CoA synthesis was repressed by the transcription factors TEAD2 and E2A, previously unknown to control acetyl-CoA synthesis. Knockdown of TEAD2 and E2A restored acetyl-CoA levels and inhibited tumor growth. Our findings causally link transcriptional reprogramming of acetyl-CoA metabolism, dedifferentiation, and cancer.

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.

Adipose mTORC2 is essential for sensory innervation in white adipose tissue and whole-body energy homeostasis.

OBJECTIVE: Adipose tissue, via sympathetic and possibly 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 are poorly characterized. METHODS AND RESULTS: Taking advantage of recent progress in whole-mount three-dimensional imaging, we identified a network of calcitonin gene-related protein (CGRP)-positive sensory neurons in murine white adipose tissue (WAT). We found that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, is required for arborization of sensory neurons, but not of sympathetic neurons. Time course experiments revealed that adipose mTORC2 is required for maintenance of sensory neurons. Furthermore, loss of sensory innervation in WAT coincided with systemic insulin resistance. Finally, we established that neuronal protein growth-associated protein 43 (GAP43) is a marker for sensory neurons in adipose tissue. CONCLUSION: Our findings indicate that adipose mTORC2 is necessary for sensory innervation in WAT. In addition, our results suggest that WAT may affect whole-body energy homeostasis via sensory neurons.

Novel roles of mTORC2 in regulation of insulin secretion by actin filament remodeling.

Mammalian target of rapamycin (mTOR) kinase is an essential hub where nutrients and growth factors converge to control cellular metabolism. mTOR interacts with different accessory proteins to form complexes 1 and 2 (mTORC), and each complex has different intracellular targets. Although mTORC1's role in ß-cells has been extensively studied, less is known about mTORC2's function in ß-cells. Here, we show that mice with constitutive and inducible ß-cell-specific deletion of RICTOR (ßRicKO and ißRicKO mice, respectively) are glucose intolerant due to impaired insulin secretion when glucose is injected intraperitoneally. Decreased insulin secretion in ßRicKO islets was caused by abnormal actin polymerization. Interestingly, when glucose was administered orally, no difference in glucose homeostasis and insulin secretion were observed, suggesting that incretins are counteracting the mTORC2 deficiency. Mechanistically, glucagon-like peptide-1 (GLP-1), but not gastric inhibitory polypeptide (GIP), rescued insulin secretion in vivo and in vitro by improving actin polymerization in ßRicKO islets. In conclusion, mTORC2 regulates glucose-stimulated insulin secretion by promoting actin filament remodeling.NEW & NOTEWORTHY The current studies uncover a novel mechanism linking mTORC2 signaling to glucose-stimulated insulin secretion by modulation of the actin filaments. This work also underscores the important role of GLP-1 in rescuing defects in insulin secretion by modulating actin polymerization and suggests that this effect is independent of mTORC2 signaling.

mTOR substrate phosphorylation in growth control.

The target of rapamycin (TOR), discovered 30 years ago, is a highly conserved serine/threonine protein kinase that plays a central role in regulating cell growth and metabolism. It is activated by nutrients, growth factors, and cellular energy. TOR forms two structurally and functionally distinct complexes, TORC1 and TORC2. TOR signaling activates cell growth, defined as an increase in biomass, by stimulating anabolic metabolism while inhibiting catabolic processes. With emphasis on mammalian TOR (mTOR), we comprehensively reviewed the literature and identified all reported direct substrates. In the context of recent structural information, we discuss how mTORC1 and mTORC2, despite having a common catalytic subunit, phosphorylate distinct substrates. We conclude that the two complexes recruit different substrates to phosphorylate a common, minimal motif.

Integrative proteogenomic characterization of hepatocellular carcinoma across etiologies and stages.

Proteogenomic analyses of hepatocellular carcinomas (HCC) have focused on early-stage, HBV-associated HCCs. Here we present an integrated proteogenomic analysis of HCCs across clinical stages and etiologies. Pathways related to cell cycle, transcriptional and translational control, signaling transduction, and metabolism are dysregulated and differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. We describe candidate copy number-driven driver genes involved in epithelial-to-mesenchymal transition, the Wnt-ß-catenin, AKT/mTOR and Notch pathways, cell cycle and DNA damage regulation. The targetable aurora kinase A and CDKs are upregulated. CTNNB1 and TP53 mutations are associated with altered protein phosphorylation related to actin filament organization and lipid metabolism, respectively. Integrative proteogenomic clusters show that HCC constitutes heterogeneous subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our study provides a comprehensive overview of the proteomic and phophoproteomic landscapes of HCCs, revealing the major pathways altered in the (phospho)proteome.

Regulation of human mTOR complexes by DEPTOR.

The vertebrate-specific DEP domain-containing mTOR interacting protein (DEPTOR), an oncoprotein or tumor suppressor, has important roles in metabolism, immunity, and cancer. It is the only protein that binds and regulates both complexes of mammalian target of rapamycin (mTOR), a central regulator of cell growth. Biochemical analysis and cryo-EM reconstructions of DEPTOR bound to human mTOR complex 1 (mTORC1) and mTORC2 reveal that both structured regions of DEPTOR, the PDZ domain and the DEP domain tandem (DEPt), are involved in mTOR interaction. The PDZ domain binds tightly with mildly activating effect, but then acts as an anchor for DEPt association that allosterically suppresses mTOR activation. The binding interfaces of the PDZ domain and DEPt also support further regulation by other signaling pathways. A separate, substrate-like mode of interaction for DEPTOR phosphorylation by mTOR complexes rationalizes inhibition of non-stimulated mTOR activity at higher DEPTOR concentrations. The multifaceted interplay between DEPTOR and mTOR provides a basis for understanding the divergent roles of DEPTOR in physiology and opens new routes for targeting the mTOR-DEPTOR interaction in disease.

Multi-omics data integration reveals novel drug targets in hepatocellular carcinoma.

BACKGROUND: Genetic aberrations in hepatocellular carcinoma (HCC) are well known, but the functional consequences of such aberrations remain poorly understood. RESULTS: Here, we explored the effect of defined genetic changes on the transcriptome, proteome and phosphoproteome in twelve tumors from an mTOR-driven hepatocellular carcinoma mouse model. Using Network-based Integration of multi-omiCS data (NetICS), we detected 74 'mediators' that relay via molecular interactions the effects of genetic and miRNA expression changes. The detected mediators account for the effects of oncogenic mTOR signaling on the transcriptome, proteome and phosphoproteome. We confirmed the dysregulation of the mediators YAP1, GRB2, SIRT1, HDAC4 and LIS1 in human HCC. CONCLUSIONS: This study suggests that targeting pathways such as YAP1 or GRB2 signaling and pathways regulating global histone acetylation could be beneficial in treating HCC with hyperactive mTOR signaling.

mTOR signaling mediates ILC3-driven immunopathology.

Innate lymphoid cells (ILCs) have a protective immune function at mucosal tissues but can also contribute to immunopathology. Previous work has shown that the serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1) is involved in generating protective ILC3 cytokine responses during bacterial infection. However, whether mTORC1 also regulates IFN-γ-mediated immunopathology has not been investigated. In addition, the role of mTORC2 in ILC3s is unknown. Using mice specifically defective for either mTORC1 or mTORC2 in ILC3s, we show that both mTOR complexes regulate the maintenance of ILC3s at steady state and pathological immune response during colitis. mTORC1 and to a lesser extend mTORC2 promote the proliferation of ILC3s in the small intestine. Upon activation, intestinal ILC3s produce less IFN-γ in the absence of mTOR signaling. During colitis, loss of both mTOR complexes in colonic ILC3s results in the reduced production of inflammatory mediators, recruitment of neutrophils and immunopathology. Similarly, treatment with rapamycin after colitis induction ameliorates the disease. Collectively, our data show a critical role for both mTOR complexes in controlling ILC3 cell numbers and ILC3-driven inflammation in the intestine.

USP29-mediated HIF1α stabilization is associated with Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis.

Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients.

A reference map of sphingolipids in murine tissues.

Sphingolipids (SPs) have both a structural role in the cell membranes and a signaling function that regulates many cellular processes. The enormous structural diversity and low abundance of many SPs pose a challenge for their identification and quantification. Recent advances in lipidomics, in particular liquid chromatography (LC) coupled with mass spectrometry (MS), provide methods to detect and quantify many low-abundant SP species reliably. Here we use LC-MS to compile a "murine sphingolipid atlas," containing the qualitative and quantitative distribution of 114 SPs in 21 tissues of a widely utilized wild-type laboratory mouse strain (C57BL/6). We report tissue-specific SP fingerprints, as well as sex-specific differences in the same tissue. This is a comprehensive, quantitative sphingolipidomic map of mammalian tissues collected in a systematic fashion. It will complement other tissue compendia for interrogation into the role of SP in mammalian health and disease.