Ras Homolog Enriched In Brain Research Articles

Anti-seizure gene therapy for focal cortical dysplasia.

Focal cortical dysplasias are a common subtype of malformation of cortical development, which frequently presents with a spectrum of cognitive and behavioural abnormalities as well as pharmacoresistant epilepsy. Focal cortical dysplasia type II is typically caused by somatic mutations resulting in mammalian target of rapamycin (mTOR) hyperactivity, and is the commonest pathology found in children undergoing epilepsy surgery. However, surgical resection does not always result in seizure freedom, and is often precluded by proximity to eloquent brain regions. Gene therapy is a promising potential alternative treatment and may be appropriate in cases that represent an unacceptable surgical risk. Here, we evaluated a gene therapy based on overexpression of the Kv1.1 potassium channel in a mouse model of frontal lobe focal cortical dysplasia. An engineered potassium channel (EKC) transgene was placed under control of a human promoter that biases expression towards principal neurons (CAMK2A) and packaged in an adeno-associated viral vector (AAV9). We used an established focal cortical dysplasia model generated by in utero electroporation of frontal lobe neural progenitors with a constitutively active human Ras homolog enriched in brain (RHEB) plasmid, an activator of mTOR complex 1. We characterized the model by quantifying electrocorticographic and behavioural abnormalities, both in mice developing spontaneous generalized seizures and in mice only exhibiting interictal discharges. Injection of AAV9-CAMK2A-EKC in the dysplastic region resulted in a robust decrease (∼64%) in the frequency of seizures. Despite the robust anti-epileptic effect of the treatment, there was neither an improvement nor a worsening of performance in behavioural tests sensitive to frontal lobe function. AAV9-CAMK2A-EKC had no effect on interictal discharges or behaviour in mice without generalized seizures. AAV9-CAMK2A-EKC gene therapy is a promising therapy with translational potential to treat the epileptic phenotype of mTOR-related malformations of cortical development. Cognitive and behavioural co-morbidities may, however, resist an intervention aimed at reducing circuit excitability.

RHEB is a potential therapeutic target in T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes. Although various therapeutic approaches have been developed, refractoriness of chemotherapy and relapse cause a poor prognosis of the disease and further therapeutic strategies are required. Here, we report that Ras homolog enriched in brain (RHEB), a critical regulator of mTOR complex 1 activity, is a potential target for T-ALL therapy. In this study, we established an sgRNA library that comprehensively targeted mTOR upstream and downstream pathways, including autophagy. CRISPR/Cas9 dropout screening revealed critical roles of mTOR-related molecules in T-ALL cell survival. Among the regulators, we focused on RHEB because we previously found that it is dispensable for normal hematopoiesis in mice. Transcriptome and metabolic analyses revealed that RHEB deficiency suppressed de novo nucleotide biosynthesis, leading to human T-ALL cell death. Importantly, RHEB deficiency suppressed tumor growth in both mouse and xenograft models. Our data provide a potential strategy for efficient therapy of T-ALL by RHEB-specific inhibition.

HDAC6 Negatively Regulates miR-155-5p Expression to Elicit Proliferation by Targeting RHEB in Microvascular Endothelial Cells under Mechanical Unloading.

Mechanical unloading contributes to significant cardiovascular deconditioning. Endothelial dysfunction in the sites of microcirculation may be one of the causes of the cardiovascular degeneration induced by unloading, but the detailed mechanism is still unclear. Here, we first demonstrated that mechanical unloading inhibited brain microvascular endothelial cell proliferation and downregulated histone deacetylase 6 (HDAC6) expression. Furthermore, HDAC6 promoted microvascular endothelial cell proliferation and attenuated the inhibition of proliferation caused by clinorotation unloading. To comprehensively identify microRNAs (miRNAs) that are regulated by HDAC6, we analyzed differential miRNA expression in microvascular endothelial cells after transfection with HDAC6 siRNA and selected miR-155-5p, which was the miRNA with the most significantly increased expression. The ectopic expression of miR-155-5p inhibited microvascular endothelial cell proliferation and directly downregulated Ras homolog enriched in brain (RHEB) expression. Moreover, RHEB expression was downregulated under mechanical unloading and was essential for the miR-155-5p-mediated promotion of microvascular endothelial cell proliferation. Taken together, these results are the first to elucidate the role of HDAC6 in unloading-induced cell growth inhibition through the miR-155-5p/RHEB axis, suggesting that the HDAC6/miR-155-5p/RHEB pathway is a specific target for the preventative treatment of cardiovascular deconditioning.

Over-expressed RHEB promotes the progression of pancreatic adenocarcinoma

AimsMammalian/mechanistic target of rapamycin (mTOR) is essential in the progression of pancreatic adenocarcinoma (PAAD). But the role of Ras homolog enriched in brain (RHEB), a key activator of mTORC1, is unclear in this disease. This work aims to clarify the function of RHEB in PAAD. Materials and methodsA pan-cancer analysis of RHEB was conducted by using data from several public available databases. Immunohistochemical (IHC) staining on a tissue microarray was used to validate the expression of RHEB in PAAD. In vitro experiments were conducted to explore the function of RHEB in the disease. An integrated bioinformatics tools were used to understand the mechanism of RHEB and construct a RHEB-related prognostic signature. Key findingsRHEB was significantly overexpressed in PAAD and high expression of the gene was associated with poor prognosis. RHEB promoted proliferation, migration and invasion of pancreatic cancer cells. Gene set enrichment analysis (GSEA) showed that RHEB participated in cell cycle progression and WNT signaling pathway. A RHEB-related prognostic signature was developed, and PAAD patients with high risk score had a significantly shorter overall survival. SignificanceRHEB was up-regulated in PAAD and might be a useful therapeutic target.

The Neurodevelopmental Pathogenesis of Tuberous Sclerosis Complex (TSC).

Tuberous sclerosis complex (TSC) is a model disorder for understanding brain development because the genes that cause TSC are known, many downstream molecular pathways have been identified, and the resulting perturbations of cellular events are established. TSC, therefore, provides an intellectual framework to understand the molecular and biochemical pathways that orchestrate normal brain development. The TSC1 and TSC2 genes encode Hamartin and Tuberin which form a GTPase activating protein (GAP) complex. Inactivating mutations in TSC genes (TSC1/TSC2) cause sustained Ras homologue enriched in brain (RHEB) activation of the mammalian isoform of the target of rapamycin complex 1 (mTORC1). TOR is a protein kinase that regulates cell size in many organisms throughout nature. mTORC1 inhibits catabolic processes including autophagy and activates anabolic processes including mRNA translation. mTORC1 regulation is achieved through two main upstream mechanisms. The first mechanism is regulation by growth factor signaling. The second mechanism is regulation by amino acids. Gene mutations that cause too much or too little mTORC1 activity lead to a spectrum of neuroanatomical changes ranging from altered brain size (micro and macrocephaly) to cortical malformations to Type I neoplasias. Because somatic mutations often underlie these changes, the timing, and location of mutation results in focal brain malformations. These mutations, therefore, provide gain-of-function and loss-of-function changes that are a powerful tool to assess the events that have gone awry during development and to determine their functional physiological consequences. Knowledge about the TSC-mTORC1 pathway has allowed scientists to predict which upstream and downstream mutations should cause commensurate neuroanatomical changes. Indeed, many of these predictions have now been clinically validated. A description of clinical imaging and histochemical findings is provided in relation to laboratory models of TSC that will allow the reader to appreciate how human pathology can provide an understanding of the fundamental mechanisms of development.

Silencing of RHEB inhibits cell proliferation and promotes apoptosis in colorectal cancer cells via inhibition of the mTOR signaling pathway.

Colorectal cancer (CRC) is commonly known as one of the most prominent reasons forcancer-related death in China. Ras homolog enriched in brain (RHEB) and the mammalian target activity of rapamycin (mTOR) signaling pathway were found correlated with CRC, but their specific interaction in CRC was still to be investigated. Therefore, we explored whether RHEB gene silencing affected the cell proliferation, differentiation, and apoptosis by directly targeting the mTOR signaling pathway in cells previously harvested from CRC patients. A microarray analysis was subsequently conducted to investigate the relationship between RHEB and mTOR. Eighty-three adjacent normal tissues and CRC tissues were selected. Immunohistochemistry was carried out to detect the positive expression rates of RHEB and Ki-67 in the CRC tissues. Cells were then transfected with different siRNAs to investigate the potential effects RHEB would have on CRC progression. The expressions of RHEB, 4EBP1, ribosomal protein S6 kinase (p70S6K), proliferating cell nuclear antigen (PCNA), B cell lymphoma 2 (bcl-2), and bcl-2-associated X protein (bax) were determined and then the cell cycle, cell proliferation, and apoptotic rate were also measured. We identified RHEB and mTOR as upregulated genes in CRC. Cells treated with RHEB silencing showed a decreased extent of mTOR, p70S6K, 4EBP1 phosphorylationand expression of RHEB, Ki-67, mTOR, p70S6K, 4EBP1, bcl-2, and PCNAas well as decreased activity of cell proliferation and differentiation; although, the expression of bax was evidently higher. Collectively, our data propose the idea that RHEB gene silencing might repress cell proliferation and differentiation while accelerating apoptosis via inactivating the mTOR signaling pathway.

RHEB gene therapy maintains the chondrogenic characteristics and protects cartilage tissue from degenerative damage during experimental murine osteoarthritis

Osteoarthritis (OA) is characterized by cartilage degeneration resulting from hypertrophic changes in chondrocytes caused by altered gene expression. The involvement of Ras homolog enriched in brain (RHEB) in OA regulation is unclear. Human knee articular cartilage samples - were analyzed for structural and biological changes by histology, immunohistochemistry, real time PCR and western blotting. OA-mouse model developed by surgical destabilization of the medial meniscus (DMM) were treated with adenovirus harboring Rheb gene to analyze onset and progression of OA. Histological scoring, immunohistochemistry, and TUNEL assay was performed to assess cartilage damage across the entire joint. Human and mouse OA cartilage is degenerated and has markedly reduced levels of RHEB. Human OA-degenerated chondrocytes (DC) exhibited a fibroblastic phenotype and 80 % of degenerative cartilage were senescent, with higher levels of reactive oxygen species (ROS). Gene expression analysis of DC revealed almost no COL2A1 expression and reduced SOX9 and RHEB expression. Transient transfection of RHEB rescued the DC phenotype and reduced senescence and ROS levels markedly. RHEB overexpression also increased COL2A1 and SOX9 expression. In an OA-mouse model, the Rheb protein level decreased as the severity of OA increased. Ectopic expression of Rheb using adenovirus in mouse-OA cartilage suppressed surgically-induced OA pathogenesis accompanied by modulation of Adamts5, Mmp 13, Col 10, and Col2a1 expression. Rheb induction significantly reduced apoptosis in OA-cartilage. RHEB plays an important role in maintaining the chondrogenic characteristics of chondrocytes, and has potential in preventing progression of OA in the destabilize the medial meniscus (DMM) mouse model of OA.

MTOR Senses Intracellular pH through Lysosome Dispersion from RHEB.

Acidity, generated in hypoxia or hypermetabolic states, perturbs homeostasis and is a feature of solid tumors. That acid peripherally disperses lysosomes is a three-decade-old observation, yet one little understood or appreciated. However, recent work has recognized the inhibitory impact this spatial redistribution has on mechanistic target of rapamycin complex 1 (mTORC1), a key regulator of metabolism. This finding argues for a paradigm shift in localization of mTORC1 activator Ras homolog enriched in brain (RHEB), a conclusion several others have now independently reached. Thus, mTORC1, known to sense amino acids, mitogens, and energy to restrict biosynthesis to times of adequate resources, also senses pH and, via dampened mTOR-governed synthesis of clock proteins, regulates the circadian clock to achieve concerted responses to metabolic stress. While this may allow cancer to endure metabolic deprivation, immune cell mTOR signaling likewise exhibits pH sensitivity, suggesting that suppression of antitumor immune function by solid tumor acidity may additionally fuel cancers, an obstacle potentially reversible through therapeutic pH manipulation.

PKG1-modified TSC2 regulates mTORC1 activity to counter adverse cardiac stress

SUMMARYThe mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis, and autophagy1. Its hyper-activation contributes to disease in many organs including the heart1,2, though broad mTORC1 inhibition risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 by modulating Rheb (Ras homolog enriched in brain). TSC2 constitutively inhibits mTORC1, but this activity is modified by phosphorylation from multiple signaling kinases to in turn inhibit (AMPK, GSK3β) or stimulate (Akt, ERK, RSK-1) mTORC1 activity3–9. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here, we reveal phosphorylation or gain-or-loss of function mutations at either of two adjacent serines in TSC2 (S1365/1366 mouse; 1364/1365 human), with no prior known function, is sufficient to bi-directionally potently control growth-factor and hemodynamic-stress stimulated mTORC1 activity and consequent cell growth and autophagy. Basal mTORC1 activity, however, is unchanged. In heart, myocytes, and fibroblasts, phosphorylation occurs by protein kinase G (PKG), a primary effector of nitric oxide and natriuretic peptide signaling whose activation is protective against heart disease10–13. PKG suppression of hypertrophy and stimulation of autophagy in myocytes requires TSC2 phosphorylation. Homozygous knock-in (KI) mice expressing a phospho-silenced TSC2 (S1365A) mutation develop far worse heart disease and mortality from sustained pressure-overload (PO) due to hyperactive mTORC1 that cannot be rescued by PKG stimulation. Heterozygote SA-KI are rescued, and KI-mice expressing a phospho-mimetic (S1365E) mutation are protected. Neither KI model alters resting mTORC1 activity. Thus, TSC2 phosphorylation is both required and sufficient for PKG-mediated cardiac protection against pressure-overload. These newly identified serines provide a genetic tool to bi-directionally regulate the amplitude of stress-stimulated mTORC1 activity.

Amino Acid- and Insulin-Induced Activation of mTORC1 in Neonatal Piglet Skeletal Muscle Involves Sestrin2-GATOR2, Rag A/C-mTOR, and RHEB-mTOR Complex Formation

Feeding stimulates protein synthesis in skeletal muscle of neonates and this response is regulated through activation of mechanistic target of rapamycin complex 1 (mTORC1). The identity of signaling components that regulate mTORC1 activation in neonatal muscle has not been fully elucidated. We investigated the independent effects of the rise in amino acids (AAs) and insulin after a meal on the abundance and activation of potential regulators of mTORC1 in muscle and whether the responses are modified by development. Overnight-fasted 6- and 26-d-old pigs were infused for 2 h with saline (control group) or with a balanced AA mixture (AA group) or insulin (INS group) to achieve fed levels while insulin or AAs, respectively, and glucose were maintained at fasting levels. Muscles were analyzed for potential mTORC1 regulatory mechanisms and results were analyzed by 2-factor ANOVA followed by Tukey's post hoc test. The abundances of DEP domain-containing mTOR-interacting protein (DEPTOR), growth factor receptor bound protein 10 (GRB10), and regulated in development and DNA damage response 2 (REDD2) were lower (65%, 73%, and 53%, respectively; P<0.05) and late endosomal/lysosomal adaptor, MAPK and mTOR activator 1/2 (LAMTOR1/2), vacuolar H+-ATPase (V-ATPase), and Sestrin2 were higher (94%, 141%, 145%, and 127%, respectively; P<0.05) in 6- than in 26-d-old pigs. Both AA and INS groups increased phosphorylation of GRB10 (P<0.05) compared with control in 26- but not in 6-d-old pigs. Formation of Ras-related GTP-binding protein A (RagA)-mTOR, RagC-mTOR, and Ras homolog enriched in brain (RHEB)-mTOR complexes was increased (P<0.05) and Sestrin2-GTPase activating protein activity towards Rags 2 (GATOR2) complex was decreased (P<0.05) by both AA and INS groups and these responses were greater (P<0.05) in 6- than in 26-d-old pigs. The results suggest that formation of RagA-mTOR, RagC-mTOR, RHEB-mTOR, and Sestrin2-GATOR2 complexes may be involved in the AA- and INS-induced activation of mTORC1 in skeletal muscle of neonates after a meal and that enhanced activation of the mTORC1 signaling pathway in neonatal muscle is in part due to regulation by DEPTOR, GRB10, REDD2, LAMTOR1/2, V-ATPase, and Sestrin2.

Overexpression of RHEB is associated with metastasis and poor prognosis in hepatocellular carcinoma.

Aberrant expression of Ras homolog enriched in brain (RHEB) has been observed in a variety of cancer tissues and is closely associated with clinicopathological features. However, the expression profile of RHEB in patients with hepatocellular carcinoma (HCC) and its clinical signature with underlying mechanisms have not been explored thus far. To analyze the association between RHEB expression and clinicopathological features, the RHEB expression levels were determined in the present study using gene microarrays, immunohistochemistry and western blotting in 60 liver cancer tissues and 35 normal liver tissues. Downregulation of RHEB expression in liver cancer cell lines was achieved by RNA interfering technology to explore its biological function in HCC. RHEB expression was high in liver cancer tissues, with an increase of 2.00±0.19-fold compared with normal tissues and of 2.00±0.27-fold compared with adjacent non-cancer tissues. RHEB expression increased along with the clinical staging of HCC, and the overall survival and mortality of patients were closely correlated to RHEB levels, micro-vascular invasion, hepatitis B virus-DNA titer, tumor differentiation and pathological satellites (P<0.05). After knocking down RHEB in SMMC-7721 cells, the growth of liver cancer cells was significantly reduced. The majority of cells were blocked in S-phase, and their colony-forming and proliferating abilities significantly decreased (P<0.05). In vivo, upon downregulation of RHEB expression, the tumorigenic ability of HCC significantly decreased (P<0.05). These data suggest that RHEB expression is a significant prognostic factor and may be important in HCC cell growth. The present study highlights the importance of RHEB as a novel prognostic marker of HCC.

Mechanisms of mTORC1 activation by RHEB and inhibition by PRAS40.

The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and metabolism in response to nutrients, energy levels and growth factors. It contains the atypical kinase mTOR and the RAPTOR subunit that binds to the TOS motif of substrates and regulators. mTORC1 is activated by the small GTPase RHEB and inhibited by PRAS40. Here we present the 3.0 Å cryo-EM structure of mTORC1 and the 3.4 Å structure of activated RHEB-mTORC1. RHEB binds to mTOR distally from the kinase active site, yet causes a global conformational change that allosterically realigns active-site residues, accelerating catalysis. Cancer-associated hyperactivating mutations map to structural elements that maintain the inactive state, and we provide biochemical evidence that they mimic RHEB relieving auto-inhibition. We also present crystal structures of RAPTOR-TOS motif complexes that define the determinants of TOS recognition, of an mTOR FKBP12-rapamycin-binding (FRB) domain–substrate complex that establishes a second substrate-recruitment mechanism, and of a truncated mTOR-PRAS40 complex that reveals PRAS40 inhibits both substrate-recruitment sites.

MiR-185 Inhibits Fibrogenic Activation of Hepatic Stellate Cells and Prevents Liver Fibrosis.

Recent studies have shown the effect of microRNAs on HSC activation and transformation, which is essential for the pathogenesis of liver fibrosis. In our study, we explored the role of miR-185 in liver fibrosis. Plasma miR-185 was detected in hepatitis B virus-related liver fibrosis patients (S2/3, n = 10) by Illumina HiSeq sequencing, and healthy volunteers were selected (n = 8) as the control group. We found that the plasma miR-185 level in fibrosis patients was significantly downregulated. CCl4-induced fibrosis tissues in mouse livers and TGF-β1-activated HSCs also presented downregulated miR-185 concomitant with an increased expression of RHEB and RICTOR. To explore the correlations, LX-2 cells were transiently transfected with miR-185 mimics. The expression levels of α-SMA, collagen I, and collagen III were decreased as well as RHEB and RICTOR. Inhibition of endogenous miR-185 increased fibrogenic activity. Furthermore, dual-luciferase reporter assays indicated that miR-185 inhibited the expression of RHEB and RICTOR by directly targeting their 3′ UTRs. Moreover, silencing RHEB and RICTOR suppressed α-SMA and collagen expression levels. In conclusion, miR-185 prevents liver fibrogenesis by inhibiting HSC activation via inhibition of RHEB and RICTOR. These results provide new insights into the mechanisms behind the anti-fibrotic effect of miR-185.

Evolutionary Conservation of the Components in the TOR Signaling Pathways.

Target of rapamycin (TOR) is an evolutionarily conserved protein kinase that controls multiple cellular processes upon various intracellular and extracellular stimuli. Since its first discovery, extensive studies have been conducted both in yeast and animal species including humans. Those studies have revealed that TOR forms two structurally and physiologically distinct protein complexes; TOR complex 1 (TORC1) is ubiquitous among eukaryotes including animals, yeast, protozoa, and plants, while TOR complex 2 (TORC2) is conserved in diverse eukaryotic species other than plants. The studies have also identified two crucial regulators of mammalian TORC1 (mTORC1), Ras homolog enriched in brain (RHEB) and RAG GTPases. Of these, RAG regulates TORC1 in yeast as well and is conserved among eukaryotes with the green algae and land plants as apparent exceptions. RHEB is present in various eukaryotes but sporadically missing in multiple taxa. RHEB, in the budding yeast Saccharomyces cerevisiae, appears to be extremely divergent with concomitant loss of its function as a TORC1 regulator. In this review, we summarize the evolutionarily conserved functions of the key regulatory subunits of TORC1 and TORC2, namely RAPTOR, RICTOR, and SIN1. We also delve into the evolutionary conservation of RHEB and RAG and discuss the conserved roles of these GTPases in regulating TORC1.

Abstract 329: Selinexor or KPT-8602 mediated XPO1 inhibition synergizes with dexamethasone to repress convergent pathways in the mTORC1 signaling network and drive cell death in multiple myeloma

Abstract Background: Dexamethasone (DEX), a synthetic glucocorticoid (GC), is administered to nearly all multiple myeloma (MM) patients as a single agent and in combination with other chemotherapies or targeted agents. DEX and other GCs bind to glucocorticoid receptors (GR) in the cytoplasm, induce nuclear translocation and regulate GR-dependent gene expression networks in the nucleus. Selective Inhibitor of Nuclear Export (SINE) compounds (selinexor and KPT-8602) exhibit potent anti-tumor activity in MM especially when combined with DEX. SINE compounds enhance nuclear localization of tumor suppressor proteins (TSPs) through inhibition of the nuclear export protein, XPO1. We discovered that inhibition of the mechanistic Target of Rapamycin Complex 1 (mTORC1) pathway is a primary driver of the combination effect. Here we further elucidate the molecular mechanism of action of the SINE-DEX synergy in MM. Methods: GR+ MM.1S and GRnull MM.1R MM cell lines were treated with SINE compounds and/or DEX for 24 hours. Whole cell lysates were subjected to SDS-PAGE and western blot analysis. Gene expression and GR transcriptional activity was analyzed using qPCR and ELISA, respectively. Cell viability was examined using the Celltiter-Fluor assay. Results: We found that MM cell lines treated with SINE compounds (selinexor or KPT-8602) have increased basal GR protein levels. Consistent with these results, the SINE-DEX combination shows enhanced GR transcriptional activity. Several GR-DEX target genes are known to inhibit the GTPase, Ras homolog enriched in brain (RHEB), which is required for mTORC1 activation. We discovered that the SINE-DEX combination not only reduces RHEB protein but also induces the RHEB inhibitory pathways containing REDD1 and the KLF15-BCAT2 axis. Although SINE compound-mediated inhibition of mTORC1 (i.e. reduced phosphorylation of S6K1 and 4E-BP1) is GR independent, SINE-DEX inhibition is more robust in GR+ MM.1S cell line when compared to the GRnull MM.1R cells. The combination resulted in the selinexor IC50 in MM.1S cells shifting from 40 nM to 11 nM in the presence of low dose DEX. As expected, DEX did not modulate the SINE compounds IC50s in MM.1R cells. Conclusion: We show that SINE compound inhibition of MM cell viability is enhanced with DEX. Our results indicate that this combinatorial effect is due to convergent suppression of mTORC1 signaling by GR targets. The findings provide mechanism of action data around the SINE-DEX combination in MM with suggestive biomarkers (REDD1, KLF15, BCAT2 and GR) that may predict best response to the combination. Therefore, these data may translate directly to the current clinical development of SINE compounds. Citation Format: Christian Argueta, Trinayan Kashyap, Boris Klebanov, Hua Chang, Sharon Friedlander, Erkan Baloglu, Yosef Landesman, Margaret Lee, Humphrey Gardner, Sharon Shacham, William Senapedis. Selinexor or KPT-8602 mediated XPO1 inhibition synergizes with dexamethasone to repress convergent pathways in the mTORC1 signaling network and drive cell death in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 329. doi:10.1158/1538-7445.AM2017-329

The characterization of RHEB gene and its responses to hypoxia and thermal stresses in the small abalone Haliotis diversicolor.

RHEB (Ras Homolog Enriched in Brain) is a GTP-binding protein that is ubiquitously expressed in humans and other mammals. The protein is largely involved in the mechanistic target of rapamycin (mTOR) pathway, and regulates the cell cycle progression and growth. The goal of this study was to characterize the RHEB gene in the small abalone Haliotis diversicolor, and identify the responses of RHEB gene to stresses of hypoxia or/and thermal. The objectives were to: 1) clone the full-length cDNA RHEB gene in the H. diversicolor (HdRHEB); 2) quantify the expression of HdRHEB gene in tissues of haemocytes, mantle, kidney, gill, digestive tract, colleterial gland, and hepatopancreas by using RT-PCR, and 3) evaluate the responses of HdRHEB in gill and haemocyte to stresses of hypoxia (0.2mg/l00ml), thermal (31°C), and combination of hypoxia (0.4mg/l00ml) and thermal (30°C) at exposure time of 0, 4, 24, 96, and 192h. The full length cDNA of HdRHEB was 1044bp encoding a peptide of 182 amino acid residues. Expression of HdRHEB gene was detected in all of the 7 tissues and showed the highest in mantle (P<0.05). Under hypoxia, expression of HdRHEB in gill increased significantly at 4h, 24h and 96h (P<0.05), and that in haemocyte increased significantly at 24h, 96h and 192h (P<0.05). Under thermal stress, expression of HdRHEB gene in gill decreased significantly at 4h and 24h, while expression in haemocyte decreased significantly all the time. Under thermal and hypoxia stresses, expression of HdRHEB gene in gill and haemocyte was up-regulated significantly at 24h and 96h (P<0.05). The results in this study demonstrated for the first time that RHEB gene in abalones is able to response to stress stimuli of hypoxia or/and thermal.

Role of RHEB in Regulating Differentiation Fate of Mesenchymal Stem Cells for Cartilage and Bone Regeneration.

Advances in mesenchymal stem cells (MSCs) and cell replacement therapies are promising approaches to treat cartilage and bone defects since substantial differentiation capacities of MSCs match the demands of tissue regeneration. Our understanding of the dynamic process requiring indispensable differentiation of MSCs remains limited. Herein, we describe the role of RHEB (Ras homolog enriched in brain) regulating gene signature for differentiation of human adipose derived mesenchymal stem cells (ASCs) into chondrogenic, osteogenic, and adipogenic lineages. RHEB-overexpression increases the proliferation of the ASCs. RHEB enhances the chondrogenic differentiation of ASCs in 3D culture via upregulation of SOX9 with concomitant increase in glycosaminoglycans (GAGs), and type II collagen (COL2). RHEB increases the osteogenesis via upregulation of runt related transcription factor 2 (RUNX2) with an increase in the calcium and phosphate contents. RHEB also increases the expression of osteogenic markers, osteonectin and osteopontin. RHEB knockdown ASCs were incapable of expressing sufficient SRY (Sex determining region Y)-box 9 (SOX9) and RUNX2, and therefore had decreased chondrogenic and osteogenic differentiation. RHEB-overexpression impaired ASCs differentiation into adipogenic lineage, through downregulation of CCAAT/enhancer binding protein beta (C/EBPβ). Conversely, RHEB knockdown abolished the negative regulation of adipogenesis. We demonstrate that RHEB is a novel regulator, with a critical role in ASCs lineage determination, and RHEB-modulated ASCs may be useful as a cell therapy for cartilage and bone defect treatments.

RHEB: a potential regulator of chondrocyte phenotype for cartilage tissue regeneration.

As articular cartilage has a limited ability to self-repair, successful cartilage regeneration requires clinical-grade chondrocytes with innate characteristics. However, cartilage regeneration via chondrocyte transplantation is challenging, because chondrocytes lose their innate characteristics during in vitro expansion. Here, we investigated the mechanistic underpinning of the gene Ras homologue enriched in brain (RHEB) in the control of senescence and dedifferentiation through the modulation of oxidative stress in chondrocytes, a hallmark of osteoarthritis. Serial expansion of human chondrocytes led to senescence, dedifferentiation and oxidative stress. RHEB maintained the innate characteristics of chondrocytes by regulating senescence, dedifferentiation and oxidative stress, leading to the upregulation of COL2 expression via SOX9 and the downregulation of p27 expression via MCL1. RHEB also decreased the expression of COL10. RHEB knockdown mimics decreased the expression of SOX9, COL2 and MCL1, while abrogating the suppressive function of RHEB on p27 and COL10 in chondrocytes. RHEB-overexpressing chondrocytes successfully formed cartilage tissue in vitro as well as in vivo, with increased expression of GAG matrix and chondrogenic markers. RHEB induces a distinct gene expression signature that maintained the innate chondrogenic properties over a long period. Therefore, RHEB expression represents a potentially useful mechanism in terms of cartilage tissue regeneration from chondrocytes, by which chondrocyte phenotypic and molecular characteristics can be retained through the modulation of senescence, dedifferentiation and oxidative stress. Copyright © 2016 John Wiley & Sons, Ltd.

Point mutations of the mTOR-RHEB pathway in renal cell carcinoma.

Aberrations in the mTOR (mechanistic target of rapamycin) axis are frequently reported in cancer. Using publicly available tumor genome sequencing data, we identified several point mutations in MTOR and its upstream regulator RHEB (Ras homolog enriched in brain) in patients with clear cell renal cell carcinoma (ccRCC), the most common histology of kidney cancer. Interestingly, we found a prominent cluster of hyperactivating mutations in the FAT (FRAP-ATM-TTRAP) domain of mTOR in renal cell carcinoma that led to an increase in both mTORC1 and mTORC2 activities and led to an increased proliferation of cells. Several of the FAT domain mutants demonstrated a decreased binding of DEPTOR (DEP domain containing mTOR-interacting protein), while a subset of these mutations showed altered binding of the negative regulator PRAS40 (proline rich AKT substrate 40). We also identified a recurrent mutation in RHEB in ccRCC patients that leads to an increase in mTORC1 activity. In vitro characterization of this RHEB mutation revealed that this mutant showed considerable resistance to TSC2 (Tuberous Sclerosis 2) GAP (GTPase activating protein) activity, though its interaction with TSC2 remained unaltered. Mutations in the FAT domain of MTOR and in RHEB remained sensitive to rapamycin, though several of these mutations demonstrated residual mTOR kinase activity after treatment with rapamycin at clinically relevant doses. Overall, our data suggests that point mutations in the mTOR pathway may lead to downstream mTOR hyperactivation through multiple different mechanisms to confer a proliferative advantage to a tumor cell.

Amino Acid-Dependent mTORC1 Regulation by the Lysosomal Membrane Protein SLC38A9.

The serine/threonine kinase mTORC1 regulates cellular homeostasis in response to many cues, such as nutrient status and energy level. Amino acids induce mTORC1 activation on lysosomes via the small Rag GTPases and the Ragulator complex, thereby controlling protein translation and cell growth. Here, we identify the human 11-pass transmembrane protein SLC38A9 as a novel component of the Rag-Ragulator complex. SLC38A9 localizes with Rag-Ragulator complex components on lysosomes and associates with Rag GTPases in an amino acid-sensitive and nucleotide binding state-dependent manner. Depletion of SLC38A9 inhibits mTORC1 activity in the presence of amino acids and in response to amino acid replenishment following starvation. Conversely, SLC38A9 overexpression causes RHEB (Ras homolog enriched in brain) GTPase-dependent hyperactivation of mTORC1 and partly sustains mTORC1 activity upon amino acid deprivation. Intriguingly, during amino acid starvation mTOR is retained at the lysosome upon SLC38A9 depletion but fails to be activated. Together, the findings of our study reveal SLC38A9 as a Rag-Ragulator complex member transducing amino acid availability to mTORC1 activity.