Constitutive Activation Of mTORC1 Research Articles

Abstract P3066: TSC2-mTOR Axis Exerts Biased Control Over Macrophage Infiltration Following Myocardial Infarction

Introduction: The mechanistic target of rapamycin complex 1 (mTORC1) is an evolutionarily conserved serine/threonine kinase, which regulates pivotal physiological processes. mTORC1 activity is tightly regulated upstream by the tuberous sclerosis complex (TSC) which consists of TSC1 and TSC2. In macrophages as throughout the immune system, mTORC1 is a potent regulator of cell activation and function. Myocardial infarction (MI) is associated with stimulation of mTORC1; however, studies vary as to whether this activation is beneficial or detrimental, and its influence on macrophage differentiation, and infiltration after MI is unknown. Hypothesis: We tested the hypothesis that cardiac infiltration of macrophages following MI is regulated by their expression of TSC2. Methods/Results: Macrophage-specific TSC2 knock-out mice (TSC2-LyzM) were made by crossing mice with floxed-TSC2 and LysM-Cre that display a significant reduction of macrophage TSC2 protein. TSC2-LyzM bone marrow-derived macrophages (BMDMs) have constitutive mTORC1 activation reflected by phosphorylation of downstream targets S6K1 and 4E-BP1 and were correspondingly larger. To assess macrophage polarization, cells were stimulated with LPS to promote an M1-like phenotype or IL-4 to induce an M2-like phenotype. LPS- treated TSC2-LyzM BMDMs secreted more of the proinflammatory cytokine TNF-a, while IL-4 stimulation resulted in less of an M2 program, with significantly less Arg1, Mrc1, and Rentla expression. These findings show TSC2 is required for normal BMDM polarization. We next tested if TSC2 impacts macrophage myocardial infiltration after MI. Macrophages were identified as CD45+CD11b+CD64+ and further parsed by CCR2 and MHC-II. In concordance with prior studies, we found substantial infiltration of CCR2+ (BMDMs) macrophages in both genotype groups after MI. However, more TSC2-LyzM had increased CCR2+ MHC-II+ phenotype (infiltrating macrophages) versus wild-type. Conclusion: Overall, our findings highlight a key role for the TSC2-mTORC1 pathway in regulating macrophage fate specification and cardiac infiltration following a MI. This supports novel signaling control over BMDMs by TSC2-phosphorylation mediated mTORC1 activation or inhibition.

Exosome Release Delays Senescence by Disposing of Obsolete Biomolecules.

Accumulation of obsolete biomolecules can accelerate cell senescence and organism aging. The two efficient intracellular systems, namely the ubiquitin-proteasome system and the autophagy-lysosome system, play important roles in dealing with cellular wastes. However, how multicellular organisms orchestrate the processing of obsolete molecules and delay aging remains unclear. Herein, it is shown that prevention of exosome release by GW4869 or Rab27a-/- accelerated senescence in various cells and mice, while stimulating exosome release by nutrient restriction delays aging. Interestingly, exosomes isolate from serum-deprived cells or diet-restricted human plasma, enriched with garbage biomolecules, including misfolded proteins, oxidized lipids, and proteins. These cellular wastes can be englobed by macrophages, eventually, for disintegration in vivo. Inhibition of nutrient-sensing mTORC1 signaling increases exosome release and delays senescence, while constitutive activation of mTORC1 reduces exosome secretion and exacerbates senescence in vitro and in mice. Notably, inhibition of exosome release attenuates nutrient restriction- or rapamycin-delayed senescence, supporting a key role for exosome secretion in this process. This study reveals a potential mechanism by which stimulated exosome release delays aging in multicellular organisms, by orchestrating the harmful biomolecules disposal via exosomes and macrophages.

GPNMB expression identifies TSC1/2/mTOR-associated and MiT family translocation-driven renal neoplasms.

GPNMB (glycoprotein nonmetastatic B) and other TFE3/TFEB transcriptional targets have been proposed as markers for microphthalmia (MiT) translocation renal cell carcinomas (tRCCs). We recently demonstrated that constitutive mTORC1 activation via TSC1/2 loss leads to increased activity of TFE3/TFEB, suggesting that the pathogenesis and molecular markers for tRCCs and TSC1/2‐associated tumors may be overlapping. We examined GPNMB expression in human kidney and angiomyolipoma (AML) cell lines with TSC2 and/or TFE3/TFEB loss produced using CRISPR–Cas9 genome editing as well as in a mouse model of Tsc2 inactivation‐driven renal tumorigenesis. Using an automated immunohistochemistry (IHC) assay for GPNMB, digital image analysis was employed to quantitatively score expression in clear cell RCC (ccRCC, n = 87), papillary RCC (papRCC, n = 53), chromophobe RCC (chRCC, n = 34), oncocytoma (n = 4), TFE3‐ or TFEB‐driven tRCC (n = 56), eosinophilic solid and cystic RCC (ESC, n = 6), eosinophilic vacuolated tumor (EVT, n = 4), and low‐grade oncocytic tumor (LOT, n = 3), as well as AML (n = 29) and perivascular epithelioid cell tumors (PEComas, n = 8). In cell lines, GPNMB was upregulated following TSC2 loss in a MiT/TFE‐ and mTORC1‐dependent fashion. Renal tumors in Tsc2 +/− A/J mice showed upregulation of GPNMB compared with normal kidney. Mean GPNMB expression was significantly higher in tRCC than in ccRCC (p < 0.0001), papRCC (p < 0.0001), and chRCC (p < 0.0001). GPNMB expression in TSC1/2/MTOR alteration‐associated renal tumors (including ESC, LOT, AML, and PEComa) was comparable to that in tRCC. The immunophenotype of tRCC and TSC1/2/MTOR alteration‐associated renal tumors is highly overlapping, likely due to the increased activity of TFE3/TFEB in both, revealing an important caveat regarding the use of TFE3/TFEB‐transcriptional targets as diagnostic markers. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Activation of Liver mTORC1 Protects Against NASH via Dual Regulation of VLDL-TAG Secretion and De Novo Lipogenesis.

Background & AimsDysregulation of liver lipid metabolism is associated with the development and progression of nonalcoholic fatty liver disease, a spectrum of liver diseases including nonalcoholic steatohepatitis (NASH). In the liver, insulin controls lipid homeostasis by increasing triglyceride (TAG) synthesis, suppressing fatty acid oxidation, and enhancing TAG export via very low-density lipoproteins. Downstream of insulin signaling, the mechanistic target of rapamycin complex 1 (mTORC1), is a key regulator of lipid metabolism. Here, we define the role of hepatic mTORC1 activity in mouse models of NASH and investigate the mTORC1-dependent mechanisms responsible for protection against liver damage in NASH.MethodsUtilizing 2 rodent NASH-promoting diets, we demonstrate that hepatic mTORC1 activity was reduced in mice with NASH, whereas under conditions of insulin resistance and benign fatty liver, mTORC1 activity was elevated. To test the beneficial effects of hepatic mTORC1 activation in mouse models of NASH, we employed an acute, liver-specific knockout model of TSC1 (L-TSC-KO), a negative regulator of mTORC1.ResultsL-TSC-KO mice are protected from and have improved markers of NASH including reduced steatosis, decreased circulating transaminases, and reduced expression of inflammation and fibrosis genes. Mechanistically, protection from hepatic inflammation and fibrosis by constitutive mTORC1 activity occurred via promotion of the phosphatidylcholine synthesizing enzyme, CCTα, and enhanced very low-density lipoprotein-triglyceride export. Additionally, activation of mTORC1 protected from hepatic steatosis via negative feedback of the mTORC2-AKT-FOXO-SREBP1c lipogenesis axis.ConclusionsCollectively, this study identifies a protective role for liver mTORC1 signaling in the initiation and progression of NASH in mice via dual control of lipid export and synthesis.

Rheb-mTOR activation rescues Aβ-induced cognitive impairment and memory function by restoring miR-146 activity in glial cells

Deposition of amyloid beta plaques in adult rat or human brain is associated with increased production of proinflammatory cytokines by associated glial cells that are responsible for degeneration of the diseased tissue. The expression of these cytokines is usually under check and is controlled at the post-transcriptional level via several microRNAs. Computational analysis of gene expression profiles of cortical regions of Alzheimer’s disease patients’ brain suggests ineffective target cytokine mRNA suppression by existing micro-ribonucleoproteins (miRNPs) in diseased brain. Exploring the mechanism of amyloid beta-induced cytokine expression, we have identified how the inactivation of the repressive miR-146 miRNPs causes increased production of cytokines in amyloid beta-exposed glial cells. In exploration of the cause of miRNP inactivation, we have noted amyloid beta oligomer-induced sequestration of the mTORC1 complex to early endosomes that results in decreased Ago2 phosphorylation, limited Ago2-miRNA uncoupling, and retarded Ago2-cytokine mRNA interaction in rat astrocytes. Interestingly, constitutive activation of mTORC1 by Rheb activator restricts proinflammatory cytokine production by reactivating miR-146 miRNPs in amyloid beta-exposed glial cells to rescue the disease phenotype in the in vivo rat model of Alzheimer’s disease.

Amino Acid Transporters Are a Vital Focal Point in the Control of mTORC1 Signaling and Cancer.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates signals from growth factors and nutrients to control biosynthetic processes, including protein, lipid, and nucleic acid synthesis. Dysregulation in the mTORC1 network underlies a wide array of pathological states, including metabolic diseases, neurological disorders, and cancer. Tumor cells are characterized by uncontrolled growth and proliferation due to a reduced dependency on exogenous growth factors. The genetic events underlying this property, such as mutations in the PI3K-Akt and Ras-Erk signaling networks, lead to constitutive activation of mTORC1 in nearly all human cancer lineages. Aberrant activation of mTORC1 has been shown to play a key role for both anabolic tumor growth and resistance to targeted therapeutics. While displaying a growth factor-independent mTORC1 activity and proliferation, tumors cells remain dependent on exogenous nutrients such as amino acids (AAs). AAs are an essential class of nutrients that are obligatory for the survival of any cell. Known as the building blocks of proteins, AAs also act as essential metabolites for numerous biosynthetic processes such as fatty acids, membrane lipids and nucleotides synthesis, as well as for maintaining redox homeostasis. In most tumor types, mTORC1 activity is particularly sensitive to intracellular AA levels. This dependency, therefore, creates a targetable vulnerability point as cancer cells become dependent on AA transporters to sustain their homeostasis. The following review will discuss the role of AA transporters for mTORC1 signaling in cancer cells and their potential as therapeutic drug targets.

New Drug Repositioning Candidates for T-ALL Identified Via Human/Murine Gene Signature Comparison.

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive subtype of leukemia for which important progress in treatment efficiency have been made in the past decades to reach a cure rate of 75%–80% nowadays. It is nevertheless mandatory to find new targets and active molecules for innovative therapeutic strategies as relapse is associated with a very dismal outcome. We designed an experimental workflow to highlight the conserved core pathways associated with leukemogenesis by confronting the gene expression profiles (GEPs) of human T-ALL cases to the GEP of a murine T-ALL representative model, generated by the conditional deletion of the PTEN tumor suppressor gene in T cell precursors (tPTEN-/-). We identified 844 differentially expressed genes, common GEPs (cGEP) that were conserved between human T-ALL and murine signatures, and also similarly differentially expressed, compared to normal T cells. Using bioinformatic tools we highlighted in cGEPan upregulation of E2F, MYC and mTORC1. Next, using Connectivity Map (CMAP) and CMAPViz a visualization procedure for CMAP data that we developed, we selected in silico three FDA-approved, bioactive molecule candidates: α-estradiol (α-E), nordihydroguaiaretic acid (NDGA) and prochlorperazine dimaleate (PCZ). At a biological level, we showed that the three drugs triggered an apoptotic cell death in a panel of T-ALL cell lines, activated a DNA damage response and interfered with constitutive mTORC1 activation and c-MYC expression. This analysis shows that the investigation of conserved leukemogenesis pathways could be a strategy to reveal new avenues for pharmacological intervention.

Abstract 6316: High throughput small molecule screening with synthetic 3D lung-mimetic hydrogels in the rare lung cancer lymphangioleiomyomatosis

Abstract Lymphangioleiomyomatosis (LAM) is a rare lung cancer characterized by immature smooth muscle-like cell invasion of the lung parenchyma, leading to cystonodular destruction and respiratory decline. Interestingly, LAM is a monogetic disease caused by loss of TSC2, conferring constitutive mTORC1 activation. Currently, there are no approved therapeutics which exhibit LAM cell-specific cytotoxicity. Here, we describe a novel 3D drug screening platform which couples a rationally designed, synthetic, lung-mimetic hydrogel to high content imaging. We have designed and synthesized a hyaluronic acid-based hydrogel which mimics endogenous lung tissue. Human LAM cell models maintain mTORC1 activation in the hydrogel compared to matched controls, reflective of human LAM lesions. We observe LAM cells to exhibit both protease-dependent and independent modes of invasion: the former imparted by gel crosslinking with an MMP-cleavable peptide, the latter enabled by methylcellulose conjugation. Proliferation is inhibited as LAM cells actively invade through the gel. We coupled our culture system to high content confocal microscopy, permitting measurements of cell viability and invasion depth in response to therapeutic screening. We subsequently tested an 800-drug library of Health Canada-approved small molecule cancer therapeutics on LAM cells and matched controls. Surprisingly, LAM cells exhibited pan-therapeutic resistance as measured by both invasion and viability metrics. Enrichment analysis revealed categories of therapeutics which demonstrated LAM-selective cytotoxicity and/or anti-invasiveness. We performed a refinement screen on select therapeutics and identified AURA inhibition as an effective LAM cell-specific therapeutic avenue. Work is ongoing to establish the most efficacious small molecule inhibitor. In conclusion, we describe a novel drug screening platform which enables high content measurements of viability and invasion in response to drug screening in a lung-mimetic system. We have identified AURA inhibition as a potential therapeutic avenue for LAM patients using this screening platform. Future work will involve target validation, elucidation of the mechanism of action, and testing in a pre-clinical mouse model. As these small molecules are Health Canada-approved, we anticipate rapid clinical translation of our preferred candidate. Citation Format: Adam Pietrobon, Julien Yockell-Lelievre, Carole Doré, Roger Y. Tam, Sean P. Delaney, Molly Shoichet, William L. Stanford. High throughput small molecule screening with synthetic 3D lung-mimetic hydrogels in the rare lung cancer lymphangioleiomyomatosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6316.

Role of long non-coding RNA MALAT1 in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a pathological inflammatory condition of the lungs that is associated with high rates of mortality. Although long non-coding RNAs (lncRNAs) serve a role in lung diseases, their functions in COPD pathogenesis are relatively unknown. The present study aimed to assess the role of differentially expressed lncRNAs in COPD. Expression profile analysis of six lncRNAs in age-matched COPD and non-COPD tissues were conducted. Among the six tested lncRNAs, metastasis-associated in lung adenocarcinoma transcript 1 (MALAT1) was the most consistently overexpressed in COPD lung tissue specimens. To model COPD in vitro, human lung fibroblasts were treated with transforming growth factor-β (TGF-β) and MALAT1 was knocked down by small interfering RNA. This promoted cell viability and concurrently inhibited the expression of mesenchymal proteins, fibronectin and α-smooth muscle actin. In COPD, cell senescence is linked to the activation of mammalian target of rapamycin complex 1 (mTORC1). Upon gene silencing of MALAT1 in non-TGF-β-treated cells, cells demonstrated constitutive activation of mTORC1, which was assessed by the protein expression levels of mTORC1 substrate S6 kinase (S6K1). By contrast, upon MALAT1 silencing in the TGF-β-treated cells, mTORC1 activation was not suppressed, despite the mesenchymal cell markers protein expression levels being downregulated. Thus, lncRNA MALAT1 may represent a potent biomarker in COPD patients and may act as a target for both diagnostic and therapeutic purposes.

Genetic dissection of Ragulator structure and function in amino acid-dependent regulation of mTORC1.

Ragulator is a heteropentameric protein complex consisting of two roadblock heterodimers wrapped by the membrane anchor p18/Lamtor1. The Ragulator complex functions as a lysosomal membrane scaffold for Rag GTPases to recruit and activate mechanistic target of rapamycin complex 1 (mTORC1). However, the roles of Ragulator structure in the regulation of mTORC1 function remain elusive. In this study, we disrupted Ragulator structure by directly anchoring RagC to lysosomes and monitored the effect on amino acid-dependent mTORC1 activation. Expression of lysosome-anchored RagC in p18-deficient cells resulted in constitutive lysosomal localization and amino acid-independent activation of mTORC1. Co-expression of Ragulator in this system restored the amino acid dependency of mTORC1 activation. Furthermore, ablation of Gator1, a suppressor of Rag GTPases, induced amino acid-independent activation of mTORC1 even in the presence of Ragulator. These results demonstrate that Ragulator structure is essential for amino acid-dependent regulation of Rag GTPases via Gator1. In addition, our genetic analyses revealed new roles of amino acids in the regulation of mTORC1 as follows: amino acids could activate a fraction of mTORC1 in a Rheb-independent manner, and could also drive negative-feedback regulation of mTORC1 signalling via protein phosphatases. These intriguing findings contribute to our overall understanding of the regulatory mechanisms of mTORC1 signalling.

Proximal Tubule mTORC1 Is a Central Player in the Pathophysiology of Diabetic Nephropathy and Its Correction by SGLT2 Inhibitors

SummaryDiabetic kidney disease (DKD) increases the risk for mortality and is the leading cause of end-stage renal disease. Treatment with sodium-glucose cotransporter 2 inhibitors (SGLT2i) attenuates the progression of DKD, especially in patients with advanced kidney disease. Herein, we show that in diabetes, mTORC1 activity is increased in renal proximal tubule cells (RPTCs) along with enhanced tubule-interstitial fibrosis; this is prevented by SGLT2i. Constitutive activation of mTORC1 in RPTCs induces renal fibrosis and failure and abolishes the renal-protective effects of SGLT2i in diabetes. On the contrary, partial inhibition of mTORC1 in RPTCs prevents fibrosis and the decline in renal function. Stimulation of mTORC1 in RPTCs turns on a pro-fibrotic program in the renal cortex, whereas its inhibition in diabetes reverses the alterations in gene expression. We suggest that RPTC mTORC1 is a critical node that mediates kidney dysfunction in diabetes and the protective effects of SGLT2i by regulating fibrogenesis.

Tsc1-mTORC1 signaling controls striatal dopamine release and cognitive flexibility

Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in TSC1 or TSC2, which encode proteins that negatively regulate mTOR complex 1 (mTORC1). TSC is associated with significant cognitive, psychiatric, and behavioral problems, collectively termed TSC-Associated Neuropsychiatric Disorders (TAND), and the cell types responsible for these manifestations are largely unknown. Here we use cell type-specific Tsc1 deletion to test whether dopamine neurons, which modulate cognitive, motivational, and affective behaviors, are involved in TAND. We show that loss of Tsc1 and constitutive activation of mTORC1 in dopamine neurons causes somatodendritic hypertrophy, reduces intrinsic excitability, alters axon terminal structure, and impairs striatal dopamine release. These perturbations lead to a selective deficit in cognitive flexibility, preventable by genetic reduction of the mTOR-binding protein Raptor. Our results establish a critical role for Tsc1-mTORC1 signaling in setting the functional properties of dopamine neurons, and indicate that dopaminergic dysfunction may contribute to cognitive inflexibility in TSC.

MTORC1 feedback to AKT modulates lysosomal biogenesis through MiT/TFE regulation.

The microphthalmia family of transcription factors (MiT/TFEs) controls lysosomal biogenesis and is negatively regulated by the nutrient sensor mTORC1. However, the mechanisms by which cells with constitutive mTORC1 signaling maintain lysosomal catabolism remain to be elucidated. Using the murine epidermis as a model system, we found that epidermal Tsc1 deletion resulted in a phenotype characterized by wavy hair and curly whiskers, and was associated with increased EGFR and HER2 degradation. Unexpectedly, constitutive mTORC1 activation with Tsc1 loss increased lysosomal content via upregulated expression and activity of MiT/TFEs, whereas genetic deletion of Rheb or Rptor or prolonged pharmacologic mTORC1 inactivation had the reverse effect. This paradoxical increase in lysosomal biogenesis by mTORC1 was mediated by feedback inhibition of AKT, and a resulting suppression of AKT-induced MiT/TFE downregulation. Thus, inhibiting hyperactive AKT signaling in the context of mTORC1 loss-of-function fully restored MiT/TFE expression and activity. These data suggest that signaling feedback loops work to restrain or maintain cellular lysosomal content during chronically inhibited or constitutively active mTORC1 signaling, respectively, and reveal a mechanism by which mTORC1 regulates upstream receptor tyrosine kinase signaling.

Chromosomal Instability and mTORC1 Activation through PTEN Loss Contribute to Proteotoxic Stress in Ovarian Carcinoma.

High-grade serous ovarian carcinoma commonly arises from fallopian tube secretory epithelium and is characterized by a high level of chromosomal instability. To model the acquisition of aneuploidy during early carcinogenesis, chromosome missegregation was induced in immortalized tubal epithelial cells, which proved acutely detrimental to cellular fitness. The phenotype was characterized by accumulation of misfolded proteins, activation of the unfolded protein response (UPR), decreased protein synthesis, and enhanced vulnerability to proteasome inhibition. However, chromosome missegregation also resulted in heightened transformation potential, assessed by colony formation in soft agar. Ovarian cancer cells retained intrinsic sensitivity to proteasome inhibitors under adherent culture conditions, but acquired resistance as spheroids (recapitulating their native configuration in ascites) by downregulating protein synthesis via mTORC1 suppression. Loss of PTEN drove constitutive mTORC1 activity, enhanced proteotoxic stress, as evidenced by UPR induction, and resensitized tumor spheroids to proteasome inhibition both in vitro and in vivo. In cohorts of primary ovarian carcinomas, mTORC1 and UPR signaling pathways were closely associated. These results implicate attenuation of protein synthesis as a protective mechanism in tumor spheroids, which may explain the overall poor response to bortezomib in clinical trials of patients with advanced ovarian cancer. However, patients with PTEN-deficient tumors may represent a subpopulation potentially amenable to treatment with proteasome inhibitors or other therapeutic agents that disrupt protein homeostasis. SIGNIFICANCE: Chromosome instability and protein synthesis are important factors that determine the efficacy of proteotoxic stress-inducing agents, such as proteasome inhibitors, in the treatment of ovarian cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/21/5536/F1.large.jpg.

ER-lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signalling in Niemann-Pick type C.

Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes via the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signaling are unknown. We show that Oxysterol Binding Protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA/B, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, mTORC1 recruitment by the Rag GTPases is inhibited due to impaired cholesterol transport to lysosomes. Conversely, OSBP-mediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by loss of the lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signaling and restores autophagic function in cellular models of NPC. Thus, ER-lysosome contacts are signaling hubs that enable cholesterol sensing by mTORC1, and targeting their sterol-transfer activity could be beneficial in NPC.

Iron overload inhibits late stage autophagic flux leading to insulin resistance

Iron overload, a common clinical occurrence, is implicated in the metabolic syndrome although the contributing pathophysiological mechanisms are not fully defined. We show that prolonged iron overload results in an autophagy defect associated with accumulation of dysfunctional autolysosomes and loss of free lysosomes in skeletal muscle. These autophagy defects contribute to impaired insulin-stimulated glucose uptake and insulin signaling. Mechanistically, we show that iron overload leads to a decrease in Akt-mediated repression of tuberous sclerosis complex (TSC2) and Rheb-mediated mTORC1 activation on autolysosomes, thereby inhibiting autophagic-lysosome regeneration. Constitutive activation of mTORC1 or iron withdrawal replenishes lysosomal pools via increased mTORC1-UVRAG signaling, which restores insulin sensitivity. Induction of iron overload via intravenous iron-dextran delivery in mice also results in insulin resistance accompanied by abnormal autophagosome accumulation, lysosomal loss, and decreased mTORC1-UVRAG signaling in muscle. Collectively, our results show that chronic iron overload leads to a profound autophagy defect through mTORC1-UVRAG inhibition and provides new mechanistic insight into metabolic syndrome-associated insulin resistance.

Abstract 4267: mTORC1-dependent tumors have innate vulnerability to autophagic cell death by HDAC inhibitors

Abstract mTORC1 plays a significant role in the development and progression of many different types of cancers. Although mTORC1 inhibitors, such as Rapamycin, have anti-tumor effects on some specific cancers, their efficacy is still largely limited to anti-proliferative effects. Histone deacetylase (HDAC) inhibitors have been proven to be effective in cancer treatment including some solid cancers. We have previously established a vascular tumor cell line, Tsc1Δ EC, which was derived from Tsc1 deletion in mouse endothelial cells leading to constitutive activation of mTORC1. Here, we found that a pan-HDAC inhibitor, SAHA, caused Tsc1Δ EC cell death and growth arrest in vitro and in vivo. Our data showed that a selective Class I HDAC inhibitor CI994 treatment also caused Tsc1Δ EC cell death. Unlike mTORC1 inhibitors, the anti-tumor activity of SAHA was dependent on mTORC1 activation but it had little effect on mTORC1 activity in mTORC1 activated tumor cells. We found that Z-VAD-FMK, a pan-caspase inhibitor, had no inhibitory effect on SAHA treatment. Cell death pathway array analysis revealed that SAHA treatment in Tsc1ΔEC upregulated autophagy-related genes. Further experiments revealed that both autophagy gene Fip200 deletion and autophagy inhibition by spautin-1 in Tsc1Δ EC impaired tumor cell death by SAHA treatment. We further found that SAHA could play a role by increasing reactive oxygen species (ROS) via autophagy. Fip200 deletion caused Nrf2 activation through p62 accumulation, which can lead to antioxidation effect through p62-Keap1-Nrf2 pathway. We showed that inhibition of p62 expression re-sensitized Fip200 deleted Tsc1Δ EC to SAHA treatment. Taken together, HDAC inhibitors treatment provides an alternative promising therapeutic strategy for mTORC1-dependent cancers. Note: This abstract was not presented at the meeting. Citation Format: Fuchun Yang, Chenran Wang, Shaogang Sun, Michael Haas, Syn Yeo, Jun-Lin Guan. mTORC1-dependent tumors have innate vulnerability to autophagic cell death by HDAC inhibitors [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 4267.

Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is an autosomal dominant inherited disorders that is characterized by the systemic hamartomas, along with epilepsy, cognitive impairment and hypopigmented macules. It is caused by genetic mutations in either TSC1 or TSC2 gene which encodes hamartin and tuberin, respectively. As the hamartin-tuberin-complex downregulates the mechanistic/mammalian target of the rapamycin complex1 (mTORC1), dysfunction in either hamartin or tuberin induces the constitutive activation of mTORC1. In fact, almost all the symptoms in TSC are derived from the activation of mTORC1. Therefore, mTORC1 inhibitors improves all the symptoms, including skin lesions and neural symptoms. Among the many symptoms, skin lesions appear earlier than renal or pulmonary lesions and are more specific than neuronal symptoms. Therefore, skin lesions are useful for the diagnosis of TSC. This chapter focuses on the features of skin lesions and mechanistic their potential role in differential diagnosis and therapy including the therapeutic use of mTORC1 inhibitors.

Activation of mTORC1 in subchondral bone preosteoblasts promotes osteoarthritis by stimulating bone sclerosis and secretion of CXCL12

Increasing evidences show that aberrant subchondral bone remodeling plays an important role in the development of osteoarthritis (OA). However, how subchondral bone formation is activated and the mechanism by which increased subchondral bone turnover promotes cartilage degeneration during OA remains unclear. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) pathway is activated in subchondral bone preosteoblasts (Osterix+) from OA patients and mice. Constitutive activation of mTORC1 in preosteoblasts by deletion of the mTORC1 upstream inhibitor, tuberous sclerosis 1, induced aberrant subchondral bone formation, and sclerosis with little-to-no effects on articular cartilage integrity, but accelerated post-traumatic OA development in mice. In contrast, inhibition of mTORC1 in preosteoblasts by disruption of Raptor (mTORC1-specific component) reduced subchondral bone formation and cartilage degeneration, and attenuated post-traumatic OA in mice. Mechanistically, mTORC1 activation promoted preosteoblast expansion and Cxcl12 secretion, which induced subchondral bone remodeling and cartilage degeneration during OA. A Cxcl12-neutralizing antibody reduced cartilage degeneration and alleviated OA in mice. Altogether, these findings demonstrate that mTORC1 activation in subchondral preosteoblasts is not sufficient to induce OA, but can induce aberrant subchondral bone formation and secrete of Cxcl12 to accelerate disease progression following surgical destabilization of the joint. Pharmaceutical inhibition of the pathway presents a promising therapeutic approach for OA treatment.

Constitutive Activation of the Nutrient Sensor mTORC1 in Myeloid Cells Induced by Tsc1 Deletion Protects Mice from Diet‐Induced Obesity

To test whether myeloid cells Tsc1 deletion and therefore constitutive activation of the nutrient sensor mTORC1 protects from high-fat diet (HFD)-induced obesity, glucose intolerance, and adipose tissue inflammation. Mice with Tsc1 deletion in myeloid cells (MTsc1KO) and littermate controls (MTsc1WT) were fed with HFD for 8 weeks and evaluated for body weight, glucose homeostasis, and adipose tissue inflammation. MTsc1KO mice were protected from HFD-induced obesity and glucose intolerance. MTsc1KO, however, displayed, independently of the diet, abnormal behavior, episodes of intense movement, and muscle spasms followed by temporary paralysis. To investigate whether obesity protection was due to myeloid cells Tsc1 deletion, bone marrow was transplanted from MTsc1WT and MTsc1KO into irradiated C57BL6/J mice. Mice transplanted with MTsc1KO bone marrow displayed reduced body weight gain, adiposity, and inflammation, and enhanced energy expenditure, glucose tolerance and adipose tissue M2 macrophage content upon HFD feeding, in the absence of abnormal behavior. In vitro, Tsc1 deletion increased in a mTORC1-dependent manner macrophage polarization to M2 profile and mRNA levels of fatty acid binding protein 4 and PPARγ. Constitutive mTORC1 activation in myeloid cells protects mice from HFD-induced obesity, adipose tissue inflammation, and glucose intolerance by promoting macrophage polarization to M2 pro-resolution profile and increasing energy expenditure.