Reactivation Of mTORC1 Research Articles

21 Unconventional mechanisms of action and resistance to rapalogs in renal cancer

Abstract Background Clear cell renal cell carcinoma (ccRCC) is a prevalent cancer type in the United States driven by inactivation of the von Hippel-Lindau (VHL) gene and with frequent hyperactivation of mammalian target of rapamycin complex 1 (mTORC1). Multiple drugs have been developed to target VEGF/VEGFR2 and mTORC1 for advanced RCC treatment. Two specific mTORC1 inhibitors, temsirolimus and everolimus (known as rapalogs), are FDA-approved for the treatment of RCC but their clinical efficacy is hindered by the emergence of resistance. Methods To study the development of rapalog resistance in RCC, we utilized patient-derived tumorgrafts (TGs) implanted in immunocompromised mice and treated the mice with rapamycin for prolonged periods to generate resistance. Resistance was studied by transplanting resistant tumors into additional cohorts of mice and establishing primary cultures. The impact of rapamycin on tumor growth and mTORC1 activity in both tumor and stromal cells was assessed using molecular techniques. To dissect the role of the tumor microenvironment (TME), we generated genetically engineered immunocompromised recipient mice with an mTOR inhibitor resistance mutation (S2035T). Coculture experiments were performed to further explore the interplay. Results Prolonged rapamycin treatment resulted in the development of resistance in TGs. Notably, this occurred despite persistent inhibition of mTORC1 in tumor cells. Resistance was transient and lost with subsequent transplantation and in cell culture. Surprisingly, resistance was accompanied by mTORC1 reactivation in the TME, specifically in cancer associated fibroblasts. Introducing an mTOR resistance mutation (S2035T) into recipient mice was sufficient to cause resistance. Co-culture experiments with fibroblasts further demonstrated that rapamycin-resistant mutant fibroblasts conferred resistance in tumor cells even in the absence of mTORC1 activity. Conclusions This study uncovers a critical role of the TME, in mediating the response and resistance to rapalogs in RCC. Our data show that inhibition of mTORC1 in non-tumor cells is essential for the anti-tumor activity of rapalogs. These findings shed light on why mTOR mutations are rarely observed in resistant RCC patients and highlight the significance of the TME in modulating drug response and resistance. Moreover, our study emphasizes the potential of targeting TME cells as a therapeutic strategy in RCC and possibly other cancers. DOD CDMRP Funding: yes

Unexpected roles for AMPK in the suppression of autophagy and the reactivation of MTORC1 signaling during prolonged amino acid deprivation

ABSTRACT AMPK promotes catabolic and suppresses anabolic cell metabolism to promote cell survival during energetic stress, in part by inhibiting MTORC1, an anabolic kinase requiring sufficient levels of amino acids. We found that cells lacking AMPK displayed increased apoptotic cell death during nutrient stress caused by prolonged amino acid deprivation. We presumed that impaired macroautophagy/autophagy explained this phenotype, as a prevailing view posits that AMPK initiates autophagy (often a pro-survival response) through phosphorylation of ULK1. Unexpectedly, however, autophagy remained unimpaired in cells lacking AMPK, as monitored by several autophagic readouts in several cell lines. More surprisingly, the absence of AMPK increased ULK1 signaling and MAP1LC3B/LC3B lipidation during amino acid deprivation while AMPK-mediated phosphorylation of ULK1 S555 (a site proposed to initiate autophagy) decreased upon amino acid withdrawal or pharmacological MTORC1 inhibition. In addition, activation of AMPK with compound 991, glucose deprivation, or AICAR blunted autophagy induced by amino acid withdrawal. These results demonstrate that AMPK activation and glucose deprivation suppress autophagy. As AMPK controlled autophagy in an unexpected direction, we examined how AMPK controls MTORC1 signaling. Paradoxically, we observed impaired reactivation of MTORC1 in cells lacking AMPK upon prolonged amino acid deprivation. Together these results oppose established views that AMPK promotes autophagy and inhibits MTORC1 universally. Moreover, they reveal unexpected roles for AMPK in the suppression of autophagy and the support of MTORC1 signaling in the context of prolonged amino acid deprivation. These findings prompt a reevaluation of how AMPK and its control of autophagy and MTORC1 affect health and disease.

Inhibition of mitochondrial folate metabolism drives differentiation through mTORC1 mediated purine sensing

Supporting cell proliferation through nucleotide biosynthesis is an essential requirement for cancer cells. Hence, inhibition of folate-mediated one carbon (1C) metabolism, which is required for nucleotide synthesis, has been successfully exploited in anti-cancer therapy. Here, we reveal that mitochondrial folate metabolism is upregulated in patient-derived leukaemic stem cells (LSCs). We demonstrate that inhibition of mitochondrial 1C metabolism through impairment of de novo purine synthesis has a cytostatic effect on chronic myeloid leukaemia (CML) cells. Consequently, changes in purine nucleotide levels lead to activation of AMPK signalling and suppression of mTORC1 activity. Notably, suppression of mitochondrial 1C metabolism increases expression of erythroid differentiation markers. Moreover, we find that increased differentiation occurs independently of AMPK signalling and can be reversed through reconstitution of purine levels and reactivation of mTORC1. Of clinical relevance, we identify that combination of 1C metabolism inhibition with imatinib, a frontline treatment for CML patients, decreases the number of therapy-resistant CML LSCs in a patient-derived xenograft model. Our results highlight a role for folate metabolism and purine sensing in stem cell fate decisions and leukaemogenesis.

P300 nucleocytoplasmic shuttling underlies mTORC1 hyperactivation in Hutchinson-Gilford progeria syndrome.

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism and autophagy. Multiple pathways modulate mTORC1 in response to nutrients. Here we describe that nucleus-cytoplasmic shuttling of p300/EP300 regulates mTORC1 activity in response to amino acid or glucose levels. Depletion of these nutrients causes cytoplasm-to-nucleus relocalization of p300 that decreases acetylation of the mTORC1 component raptor, thereby reducing mTORC1 activity and activating autophagy. This is mediated by AMP-activated protein kinase-dependent phosphorylation of p300 at serine 89. Nutrient addition to starved cells results in protein phosphatase 2A-dependent dephosphorylation of nuclear p300, enabling its CRM1-dependent export to the cytoplasm to mediate mTORC1 reactivation. p300 shuttling regulates mTORC1 in most cell types and occurs in response to altered nutrients in diverse mouse tissues. Interestingly, p300 cytoplasm-nucleus shuttling is altered in cells from patients with Hutchinson-Gilford progeria syndrome. p300 mislocalization by the disease-causing protein, progerin, activates mTORC1 and inhibits autophagy, phenotypes that are normalized by modulating p300 shuttling. These results reveal how nutrients regulate mTORC1, a cytoplasmic complex, by shuttling its positive regulator p300 in and out of the nucleus, and how this pathway is misregulated in Hutchinson-Gilford progeria syndrome, causing mTORC1 hyperactivation and defective autophagy.

Cholesterol regulates insulin-induced mTORC1 signaling.

The rapid activation of the crucial kinase mechanistic target of rapamycin complex-1 (mTORC1) by insulin is key to cell growth in mammals, but the regulatory factors remain unclear. Here, we demonstrate that cholesterol plays a crucial role in the regulation of insulin-stimulated mTORC1 signaling. The rapid progression of insulin-induced mTORC1 signaling declines in sterol-depleted cells and restores in cholesterol-repleted cells. In insulin-stimulated cells, cholesterol promotes recruitment of mTORC1 onto lysosomes without affecting insulin-induced dissociation of the TSC complex from lysosomes, thereby enabling complete activation of mTORC1. We also show that under prolonged starvation conditions, cholesterol coordinates with autophagy to support mTORC1 reactivation on lysosomes thereby restoring insulin-responsive mTORC1 signaling. Furthermore, we identify that fibroblasts from individuals with Smith-Lemli-Opitz Syndrome (SLOS) and model HeLa-SLOS cells, which are deficient in cholesterol biosynthesis, exhibit defects in the insulin-mTORC1 growth axis. These defects are rescued by supplementation of exogenous cholesterol or by expression of constitutively active Rag GTPase, a downstream activator of mTORC1. Overall, our findings propose novel signal integration mechanisms to achieve spatial and temporal control of mTORC1-dependent growth signaling and their aberrations in disease.

Role of Nuclear receptor subfamily 1 group D member 1 in the proliferation, migration of VSMC and vascular intimal hyperplasia.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) causes neointimal hyperplasia after percutaneous vascular interventions. Nuclear receptor subfamily 1 group D member 1 (NR1D1), a crucial member of circadian clock, is involved in regulation on atherosclerosis and cellular proliferation. However, whether NR1D1 affects vascular neointimal hyperplasia remains unclear. In the current study, we found that activating NR1D1 reduced injury-induced vascular neointimal hyperplasia. Overexpression of NR1D1 reduced the number of Ki-67-positive VSMCs and migrated VSMCs after platelet-derived growth factor (PDGF)-BB treatment. Mechanistically, NR1D1 suppressed the phosphorylation of AKT and two main effectors of mammalian target of rapamycin complex 1 (mTORC1), S6 and 4EBP1 in PDGF-BB-challenged VSMCs. Both re-activation of mTORC1 by Tuberous sclerosis 1 siRNA (siTsc1) and re-activation of AKT by SC-79 abolished NR1D1-mediated inhibitory effects on proliferation and migration of VSMCs. Moreover, decreased mTORC1 activity induced by NR1D1 was also reversed by SC-79. Simultaneously, Tsc1 knockdown abolished the vascular protective effects of NR1D1 in vivo. In conclusion, NR1D1 reduces vascular neointimal hyperplasia by suppressing proliferation and migration of VSMCs in an AKT/mTORC1-dependent manner.

Inhibition of VRK1 suppresses proliferation and migration of vascular smooth muscle cells and intima hyperplasia after injury via mTORC1/β-catenin axis

Characterized by abnormal proliferation and migration of vascular smooth muscle cells (VSMCs), neointima hyperplasia is a hallmark of vascular restenosis after percutaneous vascular interventions. Vaccinia-related kinase 1 (VRK1) is a stress adaption-associated ser/thr protein kinase that can induce the proliferation of various types of cells. However, the role of VRK1 in the proliferation and migration of VSMCs and neointima hyperplasia after vascular injury remains unknown. We observed increased expression of VRK1 in VSMCs subjected to platelet-derived growth factor (PDGF)-BB by western blotting. Silencing VRK1 by shVrk1 reduced the number of Ki-67-positive VSMCs and attenuated the migration of VSMCs. Mechanistically, we found that relative expression levels of β-catenin and effectors of mTOR complex 1 (mTORC1) such as phospho (p)-mammalian target of rapamycin (mTOR), p-S6, and p-4EBP1 were decreased after silencing VRK1. Restoration of β-catenin expression by SKL2001 and re-activation of mTORC1 by Tuberous sclerosis 1 siRNA (siTsc1) both abolished shVrk1-mediated inhibitory effect on VSMC proliferation and migration. siTsc1 also rescued the reduced expression of β-catenin caused by VRK1 inhibition. Furthermore, mTORC1 re-activation failed to recover the attenuated proliferation and migration of VSMC resulting from shVrk1 after silencing β-catenin. We also found that the vascular expression of VRK1 was increased after injury. VRK1 inactivation in vivo inhibited vascular injury-induced neointima hyperplasia in a β-catenindependent manner. These results demonstrate that inhibition of VRK1 can suppress the proliferation and migration of VSMC and neointima hyperplasia after vascular injury via mTORC1/β-catenin pathway.

Prolonged deprivation of arginine or leucine induces PI3K/Akt-dependent reactivation of mTORC1

The mechanistic target of rapamycin complex 1 (mTORC1) is a serine/threonine kinase complex that promotes anabolic processes including protein, lipid, and nucleotide synthesis, while suppressing catabolic processes such as macroautophagy. mTORC1 activity is regulated by growth factors and amino acids, which signal through distinct but integrated molecular pathways: growth factors largely signal through the PI3K/Akt-dependent pathway, whereas the availabilities of amino acids leucine and arginine are communicated to mTORC1 by the Rag-GTPase pathway. While it is relatively well described how acute changes in leucine and arginine levels affect mTORC1 signaling, the effects of prolonged amino acid deprivation remain less well understood. Here, we demonstrate that prolonged deprivation of arginine and/or leucine leads to reactivation of mTORC1 activity, which reaches activation levels similar to those observed in nutrient-rich conditions. Surprisingly, we find that this reactivation is independent of the regeneration of amino acids by canonical autophagy or proteasomal degradation but is dependent on PI3K/Akt signaling. Together, our data identify a novel crosstalk between the amino acid and PI3K/Akt signaling pathways upstream of mTORC1. These observations extend our understanding of the role of mTORC1 in growth-related diseases and indicate that dietary intervention by removal of leucine and/or arginine may be an ineffective therapeutic approach.

Elimination of AKT in Cardiomyocytes Causes Complex Reorganization of Signal Transduction leading to AMPK Inhibition and Fatal Cardiac Atrophy

Akt1 and Akt2 are the main protein kinase B (Akt) isoforms expressed in the mammalian heart. Tamoxifen(OHTx) ‐inducible, cardiomyocyte‐specific Akt1/ Akt2 double knockout mice (iCM‐Akt12) show progressive cardiac atrophy and loss of contractile function leading to terminal heart failure 23.9 ± 2 days after first OHTx injection.TUNEL staining of iCM‐Akt12 hearts revealed that cardiomyocyte apoptosis did not contribute substantially to cardiac atrophy. Rather, cellular atrophy caused the loss of cardiac mass. The cellular area (a), width (w), and length (l) declined between day 9 and day 14 [‐18 (a)/‐18 (w)/‐9% (l)] as compared to WT controls. This size reduction was retarded between d14 and d21 (‐20 (a)/‐24 (w)/‐8% (l). In vivo 31P‐imaging identified a progressive energetic deficiency of iCM‐Akt12 hearts on d15 and d20 after OHTx injection.Further analyses showed that in the early phase up to day 14 increased autophagic activity and reduced de novo protein synthesis seemed to cause cellular atrophy. As expected, deletion of Akt led to impaired mTORC1 signaling indicated by reduced phosphorylation of S6K, RPS6, 4E‐BP1, all involved in translation. Concomitantly, we measured reduced protein synthesis. Autophagy was increased in iCM‐Akt12 hearts, as shown by increased LC3‐II/I ratio and higher expression of autophagic genes.From day 14 after KO induction (late phase), protein synthesis appeared to increase and mTORC1 substrates involved in translation (S6K, RPS6 and 4E‐BP1) were higher phosphorylated. Of note, also a higher phosphorylation of the inhibitory mTORC1 target site in the central autophagy kinase Ulk1 (S757) was found. Moreover, autophagy appeared to be disturbed, as p62 accumulated.Despite increasing energetic depletion, the cellular energy sensor AMPK was not activated in iCM‐Akt12 hearts. Rather, AMPK was inhibited by phosphorylation of the α‐subunit on serine 485/491. The S485/491 phosphorylation has been attributed to several kinases (Akt, PKD1, S6K, PKC, PKA). Akt deletion led to a pronounced hyperactivation of Pdk1 signaling upstream of Akt, causing an activation of protein kinases regulated by Pdk1 (S6K, PKA, PKC, PKD). This was demonstrated by increased phosphorylation of specific kinase substrates and consensus motifs in late iCM‐Akt12 hearts and might promote the AMPK inactivation.To test to what extent a non‐inhibitable AMPK might attenuate the fatal phenotype of cardiac Akt loss, AAV‐mediated infection of iCM‐Akt12 mice with a phospho‐defective AMPKα2 S491A mutant prior to KO induction was performed. This led to an increased survival and improved heart function, demonstrating that the AMPK inhibition contributes to the lethal phenotype.In conclusion, loss of Akt signaling leads to a widespread dysregulation of intracellular signal transduction. Whereas enhanced autophagy and inhibition of mTORC1 are direct consequences of Akt deletion, the later reactivation of mTORC1 and inhibition of AMPK causes attenuation of autophagy, increased translation, deceleration of cellular atrophy, worsenes energetic shortage, and aggravates cardiac dysfunction.

The mTOR-Autophagy Axis and the Control of Metabolism.

The mechanistic target of rapamycin (mTOR), master regulator of cellular metabolism, exists in two distinct complexes: mTOR complex 1 and mTOR complex 2 (mTORC1 and 2). MTORC1 is a master switch for most energetically onerous processes in the cell, driving cell growth and building cellular biomass in instances of nutrient sufficiency, and conversely, allowing autophagic recycling of cellular components upon nutrient limitation. The means by which the mTOR kinase blocks autophagy include direct inhibition of the early steps of the process, and the control of the lysosomal degradative capacity of the cell by inhibiting the transactivation of genes encoding structural, regulatory, and catalytic factors. Upon inhibition of mTOR, autophagic recycling of cellular components results in the reactivation of mTORC1; thus, autophagy lies both downstream and upstream of mTOR. The functional relationship between the mTOR pathway and autophagy involves complex regulatory loops that are significantly deciphered at the cellular level, but incompletely understood at the physiological level. Nevertheless, genetic evidence stemming from the use of engineered strains of mice has provided significant insight into the overlapping and complementary metabolic effects that physiological autophagy and the control of mTOR activity exert during fasting and nutrient overload.

MTORC1-dependent reprogramming

DDIT4 and IFRD1 trigger the inhibition and reactivation of MTORC1 during regenerative repair.

DDIT4 Licenses Only Healthy Cells to Proliferate During Injury-induced Metaplasia

In stomach, metaplasia can arise from differentiated chief cells that become mitotic via paligenosis, a stepwise program. In paligenosis, mitosis initiation requires reactivation of the cellular energy hub mTORC1 after initial mTORC1 suppression by DNA damage induced transcript 4 (DDIT4 aka REDD1). Here, we use DDIT4-deficient mice and human cells to study how metaplasia increases tumorigenesis risk. A tissue microarray of human gastric tissue specimens was analyzed by immunohistochemistry for DDIT4. C57BL/6 mice were administered combinations of intraperitoneal injections of high-dose tamoxifen (TAM) to induce spasmolytic polypeptide-expressing metaplasia (SPEM) and rapamycin to block mTORC1 activity, and N-methyl-N-nitrosourea (MNU) in drinking water to induce spontaneous gastric tumors. Stomachs were analyzed for proliferation, DNA damage, and tumor formation. CRISPR/Cas9-generated DDIT4-/- and control human gastric cells were analyzed for growth invitro and in xenografts with and without 5-fluorouracil (5-FU) treatment. DDIT4 was expressed in normal gastric chief cells in mice and humans and decreased as chief cells became metaplastic. Paligenotic Ddit4-/- chief cells maintained constitutively high mTORC1, causing increased mitosis of metaplastic cells despite DNA damage. Lower DDIT4 expression correlated with longer survival of patients with gastric cancer. 5-FU-treated DDIT4-/- human gastric epithelial cells had significantly increased cells entering mitosis despite DNA damage and increased proliferation invitro and in xenografts. MNU-treated Ddit4-/- mice had increased spontaneous tumorigenesis after multiple rounds of paligenosis induced by TAM. During injury-induced metaplastic proliferation, failure of licensing mTORC1 reactivation correlates with increased proliferation of cells harboring DNA damage, as well as increased tumor formation and growth in mice and humans.

A Dedicated Evolutionarily Conserved Molecular Network Licenses Differentiated Cells to Return to the Cell Cycle.

Differentiated cells can re-enter the cell cycle to repair tissue damage via a series of discrete morphological and molecular stages coordinated by the cellular energetics regulator mTORC1. We previously proposed the term "paligenosis" to describe this conserved cellular regeneration program. Here, we detail a molecular network regulating mTORC1 during paligenosis in both mouse pancreatic acinar and gastric chief cells. DDIT4 initially suppresses mTORC1 to induce autodegradation of differentiated cell components and damaged organelles. Later in paligenosis, IFRD1 suppresses p53 accumulation. Ifrd1-/- cells do not complete paligenosis because persistent p53 prevents mTORC1 reactivation and cell proliferation. Ddit4-/- cells never suppress mTORC1 and bypass the IFRD1 checkpoint on proliferation. Previous reports and our current data implicate DDIT4/IFRD1 in governing paligenosis in multiple organs and species. Thus, we propose that an evolutionarily conserved, dedicated molecular network has evolved to allow differentiated cells to re-enter the cell cycle (i.e., undergo paligenosis) after tissue injury. VIDEO ABSTRACT.

CYLD exaggerates pressure overload-induced cardiomyopathy via suppressing autolysosome efflux in cardiomyocytes

Deubiquitinating enzymes (DUBs) appear to be a new class of regulators of cardiac homeostasis and disease. However, DUB-mediated signaling in the heart is not well understood. Herein we report a novel mechanism by which cylindromatosis (CYLD), a DUB mediates cardiac pathological remodeling and dysfunction. Cardiomyocyte-restricted (CR) overexpression of CYLD (CR-CYLD) did not cause gross health issues and hardly affected cardiac function up to age of one year in both female and male mice at physiological conditions. However, CR-CYLD overexpression exacerbated pressure overload (PO)-induced cardiac dysfunction associated with suppressed cardiac hypertrophy and increased myocardial apoptosis in mice independent of the gender. At the molecular level, CR-CYLD overexpression enhanced PO-induced increases in poly-ubiquitinated proteins marked by lysine (K)48-linked ubiquitin chains and autophagic vacuoles containing undegraded contents while suppressing autophagic flux. Augmentation of cardiac autophagy via CR-ATG7 overexpression protected against PO-induced cardiac pathological remodeling and dysfunction in both female and male mice. Intriguingly, CR-CYLD overexpression switched the CR-ATG7 overexpression-dependent cardiac protection into myocardial damage and dysfunction associated with increased accumulation of autophagic vacuoles containing undegraded contents in the heart. Genetic manipulation of Cyld in combination with pharmacological modulation of autophagic functional status revealed that CYLD suppressed autolysosomal degradation and promoted cell death in cardiomyocytes. In addition, Cyld gene gain- and/or loss-of-function approaches in vitro and in vivo demonstrated that CYLD mediated cardiomyocyte death associated with impaired reactivation of mechanistic target of rapamycin complex 1 (mTORC1) and upregulated Ras genes from rat brain 7 (Rab7), two key components for autolysosomal degradation. These results demonstrate that CYLD serves as a novel mediator of cardiac pathological remodeling and dysfunction by suppressing autolysosome efflux in cardiomyocytes. Mechanistically, it is most likely that CYLD suppresses autolysosome efflux via impairing mTORC1 reactivation and interrupting Rab7 release from autolysosomes in cardiomyocytes.

Rheb1-Independent Activation of mTORC1 in Mammary Tumors Occurs through Activating Mutations in mTOR

Mechanistic target of rapamycin complex 1 (mTORC1) is a master modulator of cellular growth, and its aberrant regulation is recurrently documented within breast cancer. While the small GTPase Rheb1 is the canonical activator of mTORC1, Rheb1-independent mechanisms of mTORC1 activation have also been reported but have not been fully understood. Employing multiple transgenic mouse models of breast cancer, we report that ablation of Rheb1 significantly impedes mammary tumorigenesis. In the absence of Rheb1, a block in tumor initiation can be overcome by multiple independent mutations in Mtor to allow Rheb1-independent reactivation of mTORC1. We further demonstrate that the mTOR kinase is indispensable for tumor initiation as the genetic ablation of mTOR abolishes mammary tumorigenesis. Collectively, our findings demonstrate that mTORC1 activation is indispensable for mammary tumor initiation and that tumors acquire alternative mechanisms of mTORC1 activation.

Glycine Enhances Satellite Cell Proliferation, Cell Transplantation, and Oligonucleotide Efficacy in Dystrophic Muscle.

The need to distribute therapy evenly systemically throughout the large muscle volume within the body makes Duchenne muscular dystrophy (DMD) therapy a challenge. Cell and exon-skipping therapies are promising but have limited effects, and thus enhancing their therapeutic potency is of paramount importance to increase the accessibility of these therapies to DMD patients. In this study, we demonstrate that co-administered glycine improves phosphorodiamidate morpholino oligomer (PMO) potency in mdx mice with marked functional improvement and an up to 50-fold increase of dystrophin in abdominal muscles compared to PMO in saline. Glycine boosts satellite cell proliferation and muscle regeneration by increasing activation of mammalian target of rapamycin complex 1 (mTORC1) and replenishing the one-carbon unit pool. The expanded regenerating myofiber population then results in increased PMO uptake. Glycine also augments the transplantation efficiency of exogenous satellite cells and primary myoblasts in mdx mice. Our data provide evidence that glycine enhances satellite cell proliferation, cell transplantation, and oligonucleotide efficacy in mdx mice, and thus it has therapeutic utility for cell therapy and drug delivery in muscle-wasting diseases.

Abstract 3656: The mTOR-activating GATOR2 complex is essential in PAX3-FOXO1-positive rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, and can be genomically classified into fusion-positive and fusion-negative subsets. The former is driven by PAX3-FOXO1 or related chimeric transcription factors and has few other genetic alterations; the latter harbors recurrent mutations within the PI3K-RAS pathway. Despite these fundamental differences in cancer initiating events, current therapeutic approaches are identical between the two groups aside from chemotherapy intensification for some fusion-positive patients given their poorer outcomes. Current pharmacologic strategies do not permit direct targeting of the PAX3-FOXO1 oncoprotein, so we undertook a CRISPRi genetic screen to identify genes whose loss was toxic to PAX3-FOXO1+ RMS cells, but tolerated in cells with knockdown of the fusion protein. We hypothesized that this would identify targetable dependencies created by PAX3-FOXO1 expression that might permit precision therapy for fusion positive RMS. Through this screen, we identified two genes within the mTOR-activating GATOR2 complex, MIOS and WDR24, as essential for the growth of PAX3-FOXO1 positive RMS cells. We find that loss of MIOS is toxic to fusion-positive RMS cells due to a loss of mTORC1 signaling that normally initiates from signals of amino acid sufficiency. Restoration of mTORC1 activation by amino acids could be achieved by genetic suppression of either DEPDC5, a component of the RAG GAP GATOR1 complex that is normally inhibited by GATOR2, or of TSC1, which suppresses mTORC1 activity in the absence of PI3K signaling. Either genetic alteration completely rescued fusion positive RMS cells from the deleterious effects of MIOS loss. Correspondingly, we found that a fusion-negative RMS cell line with an activating NRAS mutation was insensitive to MIOS loss, suggesting that in this genetic context, amino acid signaling through GATOR2 is no longer necessary to activate mTORC1 and promote RMS survival. In contrast, loss of WDR24 was toxic to both fusion positive and fusion negative RMS cells, and could not be rescued by genetic reactivation of mTORC1. Preliminary data demonstrate alterations in lysosome size and quantity after WDR24 suppression, adding to existing data in Drosophila that this gene plays a critical, mTOR-independent role in lysosome biogenesis, and showing that this role supports RMS survival. As mTOR inhibitors are being tested in frontline phase 3 studies in North America, our work lends timely molecular insight into how the genetic basis of distinct RMS subtypes can influence the signals that promote mTORC1 function to support cancer cell survival. Based on our finding that amino acid activation of mTORC1 is a fusion-positive dependency, we are now testing combined mTOR inhibition with amino acid depletion as a precision therapeutic strategy in preclinical models of this aggressive childhood cancer. Citation Format: Amit J. Sabnis, David V. Allegakoen, Trever G. Bivona. The mTOR-activating GATOR2 complex is essential in PAX3-FOXO1-positive rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3656.

Phosphorylated mTORC1 represses autophagic-related mRNA translation in neurons exposed to ischemia-reperfusion injury.

The sequential reactivation of mechanistic target of rapamycin (mTOR) inhibited autophagic flux in neurons exposed to oxygen-glucose deprivation/reperfusion (OGD/R), which was characterized by reduction of autophagosome formation and restriction of autolysosome degradation. However, its detailed molecular mechanism was still unknown. In this study, we further explore the existing form of mTOR and its suppression on the transcriptional levels of related mRNA from neurons exposed to ischemia-reperfusion injury. The OGD/R or middle cerebral artery occlusion/reperfusion (MCAO/R)-treated neurons was used to simulate ischemia/reperfusion injury . Autophagy flux was monitored by means of microtubule-associated protein 1 light chain 3 (LC3) and p62. The reactivation of mTOR was determined by phosphorylation of ribosomal protein S6 kinase 1 (S6K1). Then the inhibitors of mTOR were used to confirm its existence form. Finally, the mRNA transcription levels were analyzed to observe the negative regulation of mTOR. The sequential phosphorylation of mTOR contributed to the neuronal autophagy flux blocking. mTOR was re-phosphorylated and existed as mTOR complex 1 (mTORC1), which was supported by phosphorylation of S6K1 at Thr 389 in neurons. In addition, the phosphorylation of S6K1 was decreased roughly by applying mTORC1 inhibitors, rapamycin and torin 1. However, the administration of mTORC1/2 inhibitor PP242 could recover the phosphorylation of S6K1, which suggested that mTORC2 was involved in the regulation of mTORC1 activity. In paralleling with reactivation of mTORC1, related mRNA transcription was repressed in neurons under ischemia-reperfusion exposure in vivo and in vitro. The mRNA expression levels of LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b were decreased in neurons after reperfusion, comparing with ischemia-treated neurons. The reactivated mTORC1 could suppress the transcription levels of related mRNA, such as LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b. The research will expand the horizons that mTOR would negatively regulate autophagy at transcription and post-translation levels in neurons suffering ischemia-reperfusion injury.

MTORC1 Is Transiently Reactivated in Injured Nerves to Promote c-Jun Elevation and Schwann Cell Dedifferentiation.

Schwann cells (SCs) are endowed with a remarkable plasticity. When peripheral nerves are injured, SCs dedifferentiate and acquire new functions to coordinate nerve repair as so-called repair SCs. Subsequently, SCs redifferentiate to remyelinate regenerated axons. Given the similarities between SC dedifferentiation/redifferentiation in injured nerves and in demyelinating neuropathies, elucidating the signals involved in SC plasticity after nerve injury has potentially wider implications. c-Jun has emerged as a key transcription factor regulating SC dedifferentiation and the acquisition of repair SC features. However, the upstream pathways that control c-Jun activity after nerve injury are largely unknown. We report that the mTORC1 pathway is transiently but robustly reactivated in dedifferentiating SCs. By inducible genetic deletion of the functionally crucial mTORC1-subunit Raptor in mouse SCs (including male and female animals), we found that mTORC1 reactivation is necessary for proper myelin clearance, SC dedifferentiation, and consequently remyelination, without major alterations in the inflammatory response. In the absence of mTORC1 signaling, c-Jun failed to be upregulated correctly. Accordingly, a c-Jun binding motif was found to be enriched in promoters of genes with reduced expression in injured mutants. Furthermore, using cultured SCs, we found that mTORC1 is involved in c-Jun regulation by promoting its translation, possibly via the eIF4F-subunit eIF4A. These results provide evidence that proper c-Jun elevation after nerve injury involves also mTORC1-dependent post-transcriptional regulation to ensure timely dedifferentiation of SCs.SIGNIFICANCE STATEMENT A crucial evolutionary acquisition of vertebrates is the envelopment of axons in myelin sheaths produced by oligodendrocytes in the CNS and Schwann cells (SCs) in the PNS. When myelin is damaged, conduction of action potentials along axons slows down or is blocked, leading to debilitating diseases. Unlike oligodendrocytes, SCs have a high regenerative potential, granted by their remarkable plasticity. Thus, understanding the mechanisms underlying SC plasticity may uncover new therapeutic targets in nerve regeneration and demyelinating diseases. Our work reveals that reactivation of the mTORC1 pathway in SCs is essential for efficient SC dedifferentiation after nerve injury. Accordingly, modulating this signaling pathway might be of therapeutic relevance in peripheral nerve injury and other diseases.

P110α of PI3K is necessary and sufficient for quiescence exit in adult muscle satellite cells.

Adult mouse muscle satellite cells (MuSCs) are quiescent in uninjured muscles. Upon injury, MuSCs exit quiescence invivo to become activated, re-enter the cell cycle to proliferate, and differentiate to repair the damaged muscles. It remains unclear which extrinsic cues and intrinsic signaling pathways regulate quiescence exit during MuSC activation. Here, we demonstrated that inducible MuSC-specific deletion of p110α, a catalytic subunit of phosphatidylinositol 3-kinase (PI3K), rendered MuSCs unable to exit quiescence, resulting in severely impaired MuSC proliferation and muscle regeneration. Genetic reactivation of mTORC1, or knockdown of FoxOs, in p110α-null MuSCs partially rescued the above defects, making them key effectors downstream of PI3K in regulating quiescence exit. c-Jun was found to be a key transcriptional target of the PI3K/mTORC1 signaling axis essential for MuSC quiescence exit. Moreover, induction of a constitutively active PI3K in quiescent MuSCs resulted in spontaneous MuSC activation in uninjured muscles and subsequent depletion of the MuSC pool. Thus, PI3K-p110α is both necessary and sufficient for MuSCs to exit quiescence in response to activating signals.