Low mTORC1 Activity Research Articles

Dynamic control of mTORC1 facilitates bone healing in mice.

Bone healing requires well-orchestrated sequential actions of osteoblasts and osteoclasts. Previous studies have demonstrated that the mechanistic target of rapamycin complex 1 (mTORC1) plays a critical role in the metabolism of osteoblasts and osteoclasts. However, the role of mTORC1 in bone healing remains unclear. Here, we showed that a dynamic change in mTORC1 activity during the process was essential for proper healing and can be harnessed therapeutically for treatment of bone fractures. Low mTORC1 activity induced by osteoblastic Raptor knockout or rapamycin treatment promoted osteoblast-mediated osteogenesis, thus leading to better bone formation and shorter bone union time. Rapamycin treatment in vitro also revealed that low mTORC1 activity enhanced osteoblast differentiation and maturation. However, rapamycin treatment affected the recruitment of osteoclasts to new bone sites, thus resulting in delayed callus absorption in bone marrow cavity. Mechanistically, decreased mTORC1 activity inhibited the recruitment of osteoclast progenitor cells to healing sites through a decrease in osteoblastic expression of monocyte chemoattractant protein-1, thus inhibiting osteoclast-mediated remodeling. Therefore, normal mTORC1 activity was necessary for bone remodeling stage. Furthermore, through the use of sustained-release materials at the bone defect, we confirmed that localized application of rapamycin in early stages accelerated bone healing without affecting bone remodeling. Together, these findings revealed that the activity of mTORC1 continually changed during bone healing, and staged rapamycin treatment could be used to promote bone healing.

MTORC1 activity oscillates throughout the cell cycle, promoting mitotic entry and differentially influencing autophagy induction

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a master metabolic regulator that is active in nearly all proliferating eukaryotic cells; however, it is unclear whether mTORC1 activity changes throughout the cell cycle. We find that mTORC1 activity oscillates from lowest in mitosis/G1 to highest in S/G2. The interphase oscillation is mediated through the TSC complex but is independent of major known regulatory inputs, including Akt and Mek/Erk signaling. By contrast, suppression of mTORC1 activity in mitosis does not require the TSC complex. mTORC1 has long been known to promote progression through G1. We find that mTORC1 also promotes progression through S and G2 and is important for satisfying the Chk1/Wee1-dependent G2/M checkpoint to allow entry into mitosis. We also find that low mTORC1 activity in G1 sensitizes cells to autophagy induction in response to partial mTORC1 inhibition or reduced nutrient levels. Together, these findings demonstrate that mTORC1 is differentially regulated throughout the cell cycle, with important phase-specific consequences for proliferating cells.

Neuronal mTORC1 inhibition promotes longevity without suppressing anabolic growth and reproduction in C. elegans.

mTORC1 (mechanistic target of rapamycin complex 1) is a metabolic sensor that promotes growth when nutrients are abundant. Ubiquitous inhibition of mTORC1 extends lifespan in multiple organisms but also disrupts several anabolic processes resulting in stunted growth, slowed development, reduced fertility, and disrupted metabolism. However, it is unclear if these pleiotropic effects of mTORC1 inhibition can be uncoupled from longevity. Here, we utilize the auxin-inducible degradation (AID) system to restrict mTORC1 inhibition to C. elegans neurons. We find that neuron-specific degradation of RAGA-1, an upstream activator of mTORC1, or LET-363, the ortholog of mammalian mTOR, is sufficient to extend lifespan in C. elegans. Unlike raga-1 loss of function genetic mutations or somatic AID of RAGA-1, neuronal AID of RAGA-1 robustly extends lifespan without impairing body size, developmental rate, brood size, or neuronal function. Moreover, while degradation of RAGA-1 in all somatic tissues alters the expression of thousands of genes, demonstrating the widespread effects of mTORC1 inhibition, degradation of RAGA-1 in neurons only results in around 200 differentially expressed genes with a specific enrichment in metabolism and stress response. Notably, our work demonstrates that targeting mTORC1 specifically in the nervous system in C. elegans uncouples longevity from growth and reproductive impairments, and that many canonical effects of low mTORC1 activity are not required to promote healthy aging. These data challenge previously held ideas about the mechanisms of mTORC1 lifespan extension and underscore the potential of promoting longevity by neuron-specific mTORC1 modulation.

HDAC6-G3BP2 promotes lysosomal-TSC2 and suppresses mTORC1 under ETV4 targeting-induced low-lactate stress in non-small cell lung cancer.

TSC-mTORC1 inhibition-mediated translational reprogramming is a major adaptation mechanism upon many stresses, such as low-oxygen, -ATP, and -amino acids. But how cancer cells hijack the adaptive pathway to survive under low-lactate stress when targeting glycolysis-related signaling remains uncertain. ETV4 is an oncogenic transcription factor frequently dysregulated in human cancer. We previously found that ETV4 is associated with tumor progression and poor prognosis in non-small cell lung cancer (NSCLC). In this study, we report that ETV4 controls HK1 expression and glycolysis-lactate production to activate mTORC1 by relieving TSC2 repression of Rheb in NSCLC cells. Targeting ETV4-induced low-lactate stress is an important input for TSC2 to inhibit mTORC1 and global protein synthesis, while the core stress granule components G3BP2 and HDAC6 are selectively translated. Mechanistically, G3BP2 recruits lysosomal-TSC2 to suppress mTORC1. HDAC6 deacetylates TSC2 to sustain protein stability and associates with G3BP2 to facilitate more recruiting of TSC2 to inactivate mTORC1. In addition, the microtubule retrograde transport activity of HDAC6 drives the aggregate-like perinuclear-mTOR distribution paralleled by lower mTORC1 activity under stress. Thus, HDAC6-G3BP2 is the key complex that promotes lysosomal-TSC2 and suppresses mTORC1 when targeting ETV4, which might represent a critical adaptive mechanism for cell survival under low-lactate challenges.

Elevated V-ATPase Activity Following PTEN Loss Is Required for Enhanced Oncogenic Signaling in Breast Cancer.

PTEN loss-of-function contributes to hyperactivation of the PI3K pathway and to drug resistance in breast cancer. Unchecked PI3K pathway signaling increases activation of the mechanistic target of rapamycin complex 1 (mTORC1), which promotes tumorigenicity. Several studies have suggested that vacuolar (H+)-ATPase (V-ATPase) complex activity is regulated by PI3K signaling. In this study, we showed that loss of PTEN elevated V-ATPase activity. Enhanced V-ATPase activity was mediated by increased expression of the ATPase H+ transporting accessory protein 2 (ATP6AP2), also known as the prorenin receptor (PRR). PRR is cleaved into a secreted extracellular fragment (sPRR) and an intracellular fragment (M8.9) that remains associated with the V-ATPase complex. Reduced PTEN expression increased V-ATPase complex activity in a PRR-dependent manner. Breast cancer cell lines with reduced PTEN expression demonstrated increased PRR expression. Similarly, PRR expression became elevated upon PTEN deletion in a mouse model of breast cancer. Interestingly, concentration of sPRR was elevated in the plasma of patients with breast cancer and correlated with tumor burden in HER2-enriched cancers. Moreover, PRR was essential for proper HER2 receptor expression, localization, and signaling. PRR knockdown attenuated HER2 signaling and resulted in reduced Akt and ERK 1/2 phosphorylation, and in lower mTORC1 activity. Overall, our study demonstrates a mechanism by which PTEN loss in breast cancer can potentiate multiple signaling pathways through upregulation of the V-ATPase complex. IMPLICATIONS: Our study contributed to the understanding of the role of the V-ATPase complex in breast cancer cell tumorigenesis and provided a potential biomarker in breast cancer.

Glutamine Transporter as a Target of mTOR Signaling Modulating Longevity

Mice that express reduced levels of the c‐Myc gene (Myc+/− heterozygotes) are long‐lived. Myc hypomorphic mice display reduced rates of protein translation and decreased activity of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Given the prominent effect of mTOR on aging, lower mTORC1 activity could contribute to the exceptional longevity and enhanced healthspan of Myc+/− animals. However, given the downstream position of MYC in these signaling cascades, the mechanism through which mTORC1 activity is downregulated in Myc+/− mice is not understood. We have found that the high‐affinity glutamine transporter SLC1A5, which is critical for activation of mTORC1 activity by amino acids, is a transcriptional target of MYC. Myc+/− cells display decreased Slc1a5 gene expression that leads to lower glutamine uptake and consequently reduced mTORC1 activity. Decreased mTORC1 activity in turn mediates an elevation of fatty acid oxidation (FAO) by indirectly upregulating the expression of carnitine palmitoyltransferase 1a (Cpt1a) that mediates the rate‐limiting step of β‐oxidation. Increased FAO has been noted in a number of long‐lived mouse models. Taken together, our results show that transcriptional feedback loops regulated by MYC modulate upstream signaling pathways such as mTOR and impact FAO on an organismal level.Support or Funding InformationSupported by NIH grant R37 AG016694 to J.M.S.

Rapamycin and chloroquine insensitivity reveal mTORC1‐independent regulation of epithelial permeability in differentiated intestinal Caco‐2 cells

Intestinal barrier integrity dictates the intestinal homeostasis by stringently regulating the permeability of the luminal contents into the intestinal tissue of the human body. The protective role of a well‐formed epithelial barrier is substantiated by incidences of increased mucosal inflammation upon epithelial barrier disruption. The pathophysiology of many serious intestinal disorders such as inflammatory bowel disease, necrotizing enterocolitis, and celiac disease is associated with increased permeability of the intestinal wall to luminal contents. In order to identify new therapeutic strategies against these disorders, it is critical to better understand the underpinning molecular mechanisms regulating epithelial barrier permeability. The role of the mTORC1 (mechanistic target of rapamycin complex 1) signaling in the regulation of intestinal barrier function and permeability remains to be fully elucidated. To that end, we genetically manipulated human epithelial intestinal Caco‐2 cells to generate three stable cell lines with baseline (shScramble), low (shRaptor) or high (shTSC2) mTORC1 activity. The three cell lines maintained their intended mTORC1 activity upon differentiation on Transwell® inserts, i.e., shRaptor cells exhibited 50% lower mTORC1 activity vs. shScramble cells, while shTSC2 cells exhibited 80% higher mTORC1 activity vs. shScramble cells. Transepithelial electrical resistance (TEER), a measure of epithelial permeability was inversely proportional to mTORC1 activity (r2= 0.98, P<0.05). shRaptor cells had higher expression of p‐(Thr172)AMPK (+46%, P<0.05), lower abundance of p‐(Ser757)ULK‐1 (−50%, P>0.05), higher autophagic flux (+180%, P<0.05), and lower expression of pore‐forming tight junction protein Claudin‐2 (−65%, P<0.05) compared with shScramble cells. In contrast, shTSC2 cells had higher levels of p‐(Ser757)ULK‐1 (+100%, P<0.05) and Claudin‐2 (+250%, P<0.05) compared with shScramble cells. We treated shScramble cells with rapamycin in an attempt to pharmacologically reproduce the effects of Raptor knockdown on epithelial permeability and found that rapamycin modestly but statistically increased TEER (+34%, P<0.05) and decreased Claudin2 (−36%, P<0.05). The role of autophagy in rapamycin‐mediated increase of TEER was dismissed on the basis that chloroquine (inhibitor of autophagosome‐lysosome fusion) did not significantly reduce TEER in shScramble cells. Our results suggest that although mTORC1 regulates epithelial permeability during the early stages of Caco‐2 cell differentiation, TEER is not substantially affected by rapamycin and chloroquine in fully differentiated cells.Support or Funding InformationSupported by USDA‐NIFA

Cap-independent mRNA translation is upregulated in long-lived endocrine mutant mice.

It has been hypothesized that transcriptional changes associated with lower mTORC1 activity in mice with reduced levels of growth hormone and insulin-like growth factor 1 are responsible for the longer healthy lifespan of these mutant mice. Cell lines and tissues from these mice show alterations in the levels of many proteins that cannot be explained by corresponding changes in mRNAs. Such post-transcriptional modulation may be the result of preferential mRNA translation by the cap-independent translation of mRNA bearing the N6-methyl-adenosine (m6A) modification. The long-lived endocrine mutants - Snell dwarf, growth hormone receptor deletion and pregnancy-associated plasma protein-A knockout - all show increases in the N6-adenosine-methyltransferases (METTL3/14) that catalyze 6-methylation of adenosine (m6A) in the 5' UTR region of select mRNAs. In addition, these mice have elevated levels of YTH domain-containing protein 1 (YTHDF1), which recognizes m6A and promotes translation by a cap-independent mechanism. Consistently, multiple proteins that can be translated by the cap-independent mechanism are found to increase in these mice, including DNA repair and mitochondrial stress response proteins, without changes in corresponding mRNA levels. Lastly, a drug that augments cap-independent translation by inhibition of cap-dependent pathways (4EGI-1) was found to elevate levels of the same set of proteins and able to render cells resistant to several forms of in vitro stress. Augmented translation by cap-independent pathways facilitated by m6A modifications may contribute to the stress resistance and increased healthy longevity of mice with diminished GH and IGF-1 signals.

Inducibility and role of mTORC1 signaling in intestinal epithelial cells as a result of cell differentiation

Intestinal epithelium undergoes rapid turnover owing to its multiple functions and active metabolism. Stem cells (SE) residing at the base of the crypts give rise to the distinct lineages of specialized intestinal epithelial cells (IEC). In doing so, SE ensure epithelial renewal and maintain intestinal homeostasis. However, SE‐based tissue renewal is not the only means of maintaining intestinal homeostasis. Specialized IEC that become damaged by the repeated physical, chemical or microbial insults attempt to self‐repair by deploying a dedifferentiation program supported by mediators of growth and proliferation such as mechanistic target of rapamycin complex 1 (mTORC1); also contributing in tissue regeneration and repair. In an event of insult, physiological stimuli may reactivate mechanistic target of rapamycin complex 1 (mTORC1) signaling in differentiated IEC thereby, contributing to epithelial dysfunction, return to stemness or tissue damage. We investigated the inducibility of mTORC1 signaling in differentiated vs. undifferentiated Caco‐2 cells in response to growth factor (IGF‐1) or pro‐inflammatory cytokines. To ascertain the role of mTORC1 in the cell response to the mucosal inflammation, Caco‐2 cells were genetically altered resulting in high (Caco‐2shTSC2), low (Caco‐2shRaptor) or baseline (Caco‐2shScramble) mTORC1 activity. Results showed that, mTORC1 activity was several fold lower in differentiated vs. undifferentiated Caco‐2 cells. While IGF‐1 induced mTORC1 signaling in undifferentiated Caco‐2 cells (IGF‐1: +95%, P<0.05), the stimuli failed to do so in differentiated Caco‐2 cells. The cocktail of cytokines (IFNγ, TNFα, IL‐1β, LPS) prominently induced IL‐1β and IL‐8 mRNA abundance in either undifferentiated or differentiated Caco‐2 cells (IL‐1b, range +374–1213%, P<0.05; IL‐8, range +4180–3350%, P<0.05). Despite the overall low mTORC1 activity levels in differentiated Caco‐2 cells, the transepithelial electrical resistance (TEER) was markedly affected by the mTORC1 status of these cells. Caco‐2shRaptor cells developed higher TEER (+180%, P<0.05) than Caco‐2shScramble cells, while Caco‐2shTSC2 cells had a lower TEER (−60%, P<0.05) than Caco‐2shScramble cells. Media transfer studies, wherein differentiated stable Caco‐2 cells received the conditioned media from activated macrophages to model mucosal inflammation, revealed that cellular TEER dropped significantly irrespective of mTORC1 status. However, unlike Caco‐2shRaptor and Caco‐2shScramble cells, in which TEER recovered marginally, Caco‐2shTSC2 cells exhibited 100% TEER recovery within 3 days. In conclusion, the overall inducibility of mTORC1 in response to the tested exogenous stimuli in Caco‐2 was dependent upon the state of differentiation and mTORC1 activity in IEC. Henceforth, mTORC1 activity is downregulated and not readily inducible (by IGF‐1 or the cytokine cocktail) as Caco‐2 cells differentiate in culture. However, development of the highest TEER by Caco‐2shRaptor among the three cell lines; and highest rate of TEER recovery in Caco‐2shTSC2 suggests that mTORC1 may have a crucial role in maintaining barrier function and promoting epithelial recovery in response to inflammation in IEC.Support or Funding InformationUSDA‐NIFAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SLC1A5 glutamine transporter is a target of MYC and mediates reduced mTORC1 signaling and increased fatty acid oxidation in long-lived Myc hypomorphic mice.

Mice that express reduced levels of the c‐Myc gene (Myc +/− heterozygotes) are long‐lived. Myc hypomorphic mice display reduced rates of protein translation and decreased activity of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Given the prominent effect of mTOR on aging, lower mTORC1 activity could contribute to the exceptional longevity and enhanced healthspan of Myc +/− animals. However, given the downstream position of MYC in these signaling cascades, the mechanism through which mTORC1 activity is downregulated in Myc +/− mice is not understood. We report that the high‐affinity glutamine transporter SLC1A5, which is critical for activation of mTORC1 activity by amino acids, is a transcriptional target of MYC. Myc +/− cells display decreased Slc1a5 gene expression that leads to lower glutamine uptake and consequently reduced mTORC1 activity. Decreased mTORC1 activity in turn mediates an elevation of fatty acid oxidation (FAO) by indirectly upregulating the expression of carnitine palmitoyltransferase 1a (Cpt1a) that mediates the rate‐limiting step of β‐oxidation. Increased FAO has been noted in a number of long‐lived mouse models. Taken together, our results show that transcriptional feedback loops regulated by MYC modulate upstream signaling pathways such as mTOR and impact FAO on an organismal level.

MTORC1 Is a Local, Postsynaptic Voltage Sensor Regulated by Positive and Negative Feedback Pathways.

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) serves as a regulator of mRNA translation. Recent studies suggest that mTORC1 may also serve as a local, voltage sensor in the postsynaptic region of neurons. Considering biochemical, bioinformatics and imaging data, we hypothesize that the activity state of mTORC1 dynamically regulates local membrane potential by promoting and repressing protein synthesis of select mRNAs. Our hypothesis suggests that mTORC1 uses positive and negative feedback pathways, in a branch-specific manner, to maintain neuronal excitability within an optimal range. In some dendritic branches, mTORC1 activity oscillates between the “On” and “Off” states. We define this as negative feedback. In contrast, positive feedback is defined as the pathway that leads to a prolonged depolarized or hyperpolarized resting membrane potential, whereby mTORC1 activity is constitutively on or off, respectively. We propose that inactivation of mTORC1 increases the expression of voltage-gated potassium alpha (Kv1.1 and 1.2) and beta (Kvβ2) subunits, ensuring that the membrane resets to its resting membrane potential after experiencing increased synaptic activity. In turn, reduced mTORC1 activity increases the protein expression of syntaxin-1A and promotes the surface expression of the ionotropic glutamate receptor N-methyl-D-aspartate (NMDA)-type subunit 1 (GluN1) that facilitates increased calcium entry to turn mTORC1 back on. Under conditions such as learning and memory, mTORC1 activity is required to be high for longer periods of time. Thus, the arm of the pathway that promotes syntaxin-1A and Kv1 protein synthesis will be repressed. Moreover, dendritic branches that have low mTORC1 activity with increased Kv expression would balance dendrites with constitutively high mTORC1 activity, allowing for the neuron to maintain its overall activity level within an ideal operating range. Finally, such a model suggests that recruitment of more positive feedback dendritic branches within a neuron is likely to lead to neurodegenerative disorders.

Significance of low mTORC1 activity in defining the characteristics of brain tumor stem cells.

The significance of mammalian target of rapamycin complex 1 (mTORC1) activity in the maintenance of cancer stem cells (CSCs) remains controversial. Previous findings showed that mTORC1 activation depleted the population of leukemia stem cells in leukemia, while maintaining the stemness in pancreatic CSCs. The purpose of this study was to examine the currently unknown role and significance of mTORC1 activity in brain tumor stem cells (BTSCs). Basal mTORC1 activity and its kinetics were investigated in BTSC clones isolated from patients with glioblastoma and their differentiated progenies (DIFFs). The effects of nutrient deprivation and the mTORC1 inhibitors on cell proliferation were compared between the BTSCs and DIFFs. Tissue sections from patients with brain gliomas were examined for expression of BTSC markers and mTORC1 activity by immunohistochemistry. BTSCs presented lower basal mTORC1 activity under each culture condition tested and a more rapid decline of mTORC1 activity after nutrient deprivation than observed in DIFFs. The self-renewal capacity of BTSCs was unaffected by mTORC1 inhibition, whereas it effectively suppressed DIFF proliferation. In agreement, immunohistochemical staining of glioma tissues revealed low mTORC1 activity in tumor cells positive for BTSC markers. In in vitro culture, BTSCs exhibited resistance to the antitumor agent temozolomide. Our findings indicated the importance of low mTORC1 activity in maintaining the undifferentiated state of BTSCs, implicating the relevance of manipulating mTORC1 activity when developing future strategies that target BTSCs.

Asymmetric inheritance of mTORC1 kinase activity during division dictates CD8(+) T cell differentiation.

The asymmetric partitioning of fate determining proteins has been shown to contribute to the generation of effector and memory CD8+ T cell precursors. Here, we demonstrate the asymmetric partitioning of mTORC1 activity upon activation of naïve CD8+ T cells. This results in the generation of one daughter T cell with increased mTORC1 activity, increased glycolytic activity and increased expression of effector molecules. The other daughter T cell inherits relatively low levels of mTORC1 activity, possesses increased lipid metabolism, expresses increased anti-apoptotic molecules and subsequently displays enhanced long-term survival. Mechanistically, we demonstrate a link between TCR-induced asymmetric expression of amino acid transporters and RagC-mediated translocation of mTOR to the lysosomes. Overall, our data provide important insight into how mTORC1-mediated metabolic reprogramming affects the fate decisions of T cells.

Clinical impact of myocardial mTORC1 activation in nonischemic dilated cardiomyopathy

BackgroundActivity of mTOR complex 1 (mTORC1) has been shown to be up-regulated in animal models of heart failure. Here, we investigated the change and role of mTORC1 in human nonischemic dilated cardiomyopathy (NICM). MethodsEndomyocardial biopsy specimens were obtained from patients with NICM (n=52) and from Brugada syndrome patients with normal LVEF as controls (n=10). The specimens were stained for phospho-ribosomal protein S6 (p-Rps6) and phospho-p70S6K (p-p70S6K), and the area with p-Rps6 signal was used as an index of mTORC1 activity. Using median mTORC1 activity, patients were divided into a high mTORC1 activity (H-mTOR) group and a low mTORC1 activity (L-mTOR) group. ResultsThe ratio of p-Rps6-positive area in biopsy samples was 10-fold larger in patients with NICM than in controls (2.0±2.2% vs. 0.2±0.2%, p<0.01). p-p70S6K signal level was higher in the H-mTOR group than in the L-mTOR group. The proportion of patients with a family history of cardiomyopathy was higher and the proportion of patients on ACE inhibitors or angiotensin receptor blockers was lower in the H-mTOR group than in the L-mTOR group. The p-Rps6-positive area was correlated with extent of myocardial fibrosis (r=0.46, p<0.01). The cardiac event-free survival rate during a 5-year follow-up period tended to be lower in the H-mTOR group than in the L-mTOR group (52.9% vs. 81.6%, P=0.10). ConclusionAberrant activation of mTORC1 in cardiomyocytes was associated with myocardial fibrosis and a trend for worse prognosis in patients with NICM, indicating that persistently activated mTORC1 contributes to progression of human heart failure.

UCH-L1 Is a Novel Regulator of mTOR Signaling That May Predict a Poor Response to mTOR Inhibition in Patients with B-Cell Lymphoma,

Abstract 3691The mTOR kinase is the core component a master regulatory network that controls nutrient-sensitive mRNA translation and the activity of the Akt growth and survival pathway. The importance of this kinase in cancer has made it the target of several new agents for a variety of cancers. Recently, prospective trials have shown strong responses in patients with refractory B-cell non-Hodgkin’s lymphomas treated with mTOR inhibitors. We recently demonstrated that the de-ubiquitiinase UCH-L1 is a potent oncogene that drives the development of B-cell lymphomas in mice and is frequently over-expressed in human B-cell lymphomas of a variety of histologies. UCH-L1 promotes signaling through the Akt pathway, though the molecular basis for this is unknown. Here we describe a novel mechanism by which UCH-L1 regulates the mTOR/Akt signaling pathway and provide data suggesting that UCH-L1 expression predicts a poor response to mTOR inhibition in patients with B-cell lymphoma. Depletion of UCH-L1 in malignant B-cells leads to a dramatic decline in Akt phosphorylation that is not due to changes in the levels of the kinases PDK1, mTOR, or any mTOR complex component. We observe, however, a striking UCH-L1 driven increase in the association of mTOR with the mTORC2 component rictor, and a reciprocal decline in the mTORC1 subunit raptor, suggesting that UCH-L1 promotes mTORC2 assembly. This is verified when we introduce UCH-L1 into HeLa cells and observe an increase in the co-precipitation of rictor with mTOR, with a corresponding decrease in the recovery of raptor. Consistent with these results, we find that mTORC1 activity is greatly reduced in cells with high levels of UCH-L1, and conversely is enhanced in tissues from Uchl1 null mice. This effect is entirely dependent on DUB activity, as expression of a catalytic mutant UCH-L1 construct has no effect on complex assembly. We find that UCH-L1 forms a novel association with the ubiquitin-ligase DDB1 and that the formation of this complex leads to the displacement of DDB1 from raptor, which then leads to the destabilization of mTORC1. As expected, depletion of DDB1 itself produces a phenocopy of UCH-L1 over-expression. We hypothesize that the suppressive effect of UCH-L1 effect on mTORC1 signaling would render malignant cells resistant to mTOR inhibition, as they likely have adapted to low mTORC1 activity. To test this hypothesis, we analyzed data from a recent prospective trial in which patients with refractory mantle cell lymphoma received the mTOR inhibitor temsirolimus along with rituximab. Limiting our analysis to those cases staining positive for phospho-Akt (pAkt), we found a strong trend towards UCH-L1 expression associated with a worse prognosis, despite the small numbers of patients (n = 10 pAkt+/UCH-L1– v. 5 pAkt+/UCH-L1+, median overall survival 913 v. 201 days, respectively; p=0.08). We conclude that UCH-L1 is a physiological regulator of mTOR/Akt signaling and that this function is co-opted in the pathogenesis of B-cell malignancies. Our data suggest that UCH-L1 expression is a negative predictor for responsiveness to mTOR inhibition in B-cell lymphoma, a finding that warrants further evaluation in larger cohorts. Disclosures:Off Label Use: Results from a clinical trial using the drug temsirolimus will be discussed.

FoxOs Inhibit mTORC1 and Activate Akt by Inducing the Expression of Sestrin3 and Rictor

FoxO transcription factors and TORC1 are conserved downstream effectors of Akt. Here, we unraveled regulatory circuits underlying the interplay between Akt, FoxO, and mTOR. Activated FoxO1 inhibits mTORC1 by TSC2-dependent and TSC2-independent mechanisms. First, FoxO1 induces Sestrin3 (Sesn3) gene expression. Sesn3, in turn, inhibits mTORC1 activity in Tsc2-proficient cells. Second, FoxO1 elevates the expression of Rictor, leading to increased mTORC2 activity that consequently activates Akt. In Tsc2-deficient cells, the elevation of Rictor by FoxO increases mTORC2 assembly and activity at the expense of mTORC1, thereby activating Akt while inhibiting mTORC1. FoxO may act as a rheostat that maintains homeostatic balance between Akt and mTOR complexes' activities. In response to physiological stresses, FoxO maintains high Akt activity and low mTORC1 activity. Thus, under stress conditions, FoxO inhibits the anabolic activity of mTORC1, a major consumer of cellular energy, while activating Akt, which increases cellular energy metabolism, thereby maintaining cellular energy homeostasis.