TSC2 regulates lysosome biogenesis via a non-canonical RAGC and TFEB-dependent mechanism.

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2+/- mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells.

Leucyl-tRNA synthetase 1 is required for proliferation of TSC-null cells.

Uncontrolled cell proliferation associated with cancer depends on the functional abrogation of at least one of tumor suppressor. In response to nutrient cue, tuberous sclerosis complex (TSC) works as a tumor suppressor which inhibits cell growth via negative regulation of the mammalian target of rapamycin complex (mTORC1). However, the regulation mechanism of nutrient-dependent cell proliferation in TSC-null cells remains unclear. Here, we demonstrate that leucine is required for cell proliferation through the activation of leucyl-tRNA synthetase (LARS1)-mTORC1 pathway in TSC-null cells. Cell proliferation and survival were attenuated by LARS1 knock-down or inhibitors in TSC-null cells. In addition, either rapamycin or LARS1 inhibitors significantly decreased colony formation ability while their combined treatment drastically attenuated it. Taken together, we suggest that LARS1 inhibitors might considered as novel tools for the regression of tumor growth and proliferation in TSC-null tumor cells which regrow upon discontinuation of the mTORC1 inhibition.

Therapeutic Targeting of DGKA-Mediated Macropinocytosis Leads to Phospholipid Reprogramming in Tuberous Sclerosis Complex.

Lymphangioleiomyomatosis is a rare destructive lung disease affecting primarily women and is the primary lung manifestation of tuberous sclerosis complex (TSC). In lymphangioleiomyomatosis, biallelic loss of TSC1/2 leads to hyperactivation of mTORC1 and inhibition of autophagy. To determine how the metabolic vulnerabilities of TSC2-deficient cells can be targeted, we performed a high-throughput screen utilizing the "Repurposing" library at the Broad Institute of MIT and Harvard (Cambridge, MA), with or without the autophagy inhibitor chloroquine. Ritanserin, an inhibitor of diacylglycerol kinase alpha (DGKA), was identified as a selective inhibitor of proliferation of Tsc2-/- mouse embryonic fibroblasts (MEF), with no impact on Tsc2+/+ MEFs. DGKA is a lipid kinase that metabolizes diacylglycerol to phosphatidic acid, a key component of plasma membranes. Phosphatidic acid levels were increased 5-fold in Tsc2-/- MEFs compared with Tsc2+/+ MEFs, and treatment of Tsc2-/- MEFs with ritanserin led to depletion of phosphatidic acid as well as rewiring of phospholipid metabolism. Macropinocytosis is known to be upregulated in TSC2-deficient cells. Ritanserin decreased macropinocytic uptake of albumin, limited the number of lysosomes, and reduced lysosomal activity in Tsc2-/- MEFs. In a mouse model of TSC, ritanserin treatment decreased cyst frequency and volume, and in a mouse model of lymphangioleiomyomatosis, genetic downregulation of DGKA prevented alveolar destruction and airspace enlargement. Collectively, these data indicate that DGKA supports macropinocytosis in TSC2-deficient cells to maintain phospholipid homeostasis and promote proliferation. Targeting macropinocytosis with ritanserin may represent a novel therapeutic approach for the treatment of TSC and lymphangioleiomyomatosis. SIGNIFICANCE: This study identifies macropinocytosis and phospholipid metabolism as novel mechanisms of metabolic homeostasis in mTORC1-hyperactive cells and suggest ritanserin as a novel therapeutic strategy for use in mTORC1-hyperactive tumors, including pancreatic cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2086/F1.large.jpg.

Dynamic analysis of 4E-BP1 phosphorylation in neurons with Tsc2 or Depdc5 knockout.

TSC1 or TSC2 mutations cause Tuberous Sclerosis Complex (TSC), and lead to mechanistic target of rapamycin (mTOR) hyperactivation evidenced by hyperphosphorylation of ribosomal S6 protein and 4-elongation factor binding protein 1 (4E-BP1). Amino acid (AA) levels modulate mTOR-dependent S6 and 4E-BP1 phosphorylation in non-neural cells, but this has not been comprehensively investigated in neurons. The effects of AA levels on mTOR signaling and S6 and 4E-BP1 phosphorylation were analyzed in Tsc2 and Depdc5 (a distinct mTOR regulatory gene associated with epilepsy) CRISPR-edited Neuro2a (N2a) cells and differentiated neurons. Tsc2 or Depdc5 knockout (KO) led to S6 and 4E-BP1 hyperphosphorylation and cell soma enlargement, but while Tsc2 KO N2a cells exhibited reduced S6 phosphorylation (Ser240/244) and cell soma size after incubation in AA free (AAF) media, Depdc5 KO cells did not. Using a CFP/YFP FRET-biosensor coupled to 4E-BP1, we assayed 4E-BP1 phosphorylation in living N2a cells and differentiated neurons following Tsc2 or Depdc5 KO. AAF conditions reduced 4E-BP1 phosphorylation in Tsc2 KO N2a cells but had no effect in Depdc5 KO cells. Rapamycin blocked S6 protein phosphorylation but had no effect on 4E-BP1 phosphorylation, following either Tsc2 or Depdc5 KO. Confocal imaging demonstrated that AAF media promoted movement of mTOR off the lysosome, functionally inactivating mTOR, in Tsc2 KO but not Depdc5 KO cells, demonstrating that AA levels modulate lysosomal mTOR localization and account, in part, for differential effects of AAF conditions following Tsc2 versus Depdc5 KO. AA levels and rapamycin differentially modulate S6 and 4E-BP1 phosphorylation and mTOR lysosomal localization in neurons following Tsc2 KO versus Depdc5 KO. Neuronal mTOR signaling in mTOR-associated epilepsies may have distinct responses to mTOR inhibitors and to levels of cellular amino acids.

Adoptive T-Cell Transfer to Treat Lymphangioleiomyomatosis.

Patients with lymphangioleiomyomatosis (LAM) develop pulmonary cysts associated with neoplastic, smooth muscle-like cells that feature neuroendocrine cell markers. The disease preferentially affects premenopausal women. Existing therapeutics do not cure LAM. As gp100 is a diagnostic marker expressed by LAM lesions, we proposed to target this immunogenic glycoprotein using TCR transgenic T cells. To reproduce the genetic mutations underlying LAM, we cultured Tsc2-/- kidney tumor cells from aged Tsc2 heterozygous mice and generated a stable gp100-expressing cell line by lentiviral transduction. T cells were isolated from major histocompatibility complex-matched TCR transgenic pmel-1 mice to measure cytotoxicity in vitro, and 80% cytotoxicity was observed within 48 hours. Antigen-specific cytotoxicity was likewise observed using pmel-1 TCR-transduced mouse T cells, suggesting that transgenic T cells may likewise be useful to treat LAM in vivo. On intravenous injection, slow-growing gp100+ LAM-like cells formed lung nodules that were readily detectable in severe combined immunodeficient/beige mice. Adoptive transfer of gp100-reactive but not wild-type T cells into mice significantly shrunk established lung tumors, even in the absence of anti-PD-1 therapy. These results demonstrate the treatment potential of adoptively transferred T cells to eliminate pulmonary lesions in LAM.

Upregulation of 6-phosphofructo-2-kinase (PFKFB3) by hyperactivated mammalian target of rapamycin complex 1 is critical for tumor growth in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by the benign tumor formation in multiple organs. The main etiology of TSC is the loss-of-function mutation of TSC1 or TSC2 gene, which leads to aberrant activation of mammalian target of rapamycin complex 1 (mTORC1). In this research, we found a significant increase of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression in Tsc1-/- and Tsc2-/- mouse embryonic fibroblasts (MEFs) compared with the control cells. Inhibition of mTORC1 led to a dramatic decrease of PFKFB3 expression, indicating PFKFB3 regulation by mTORC1. Moreover, suppression of mTORC1 inhibited the expression of PFKFB3 in rat uterine leiomyoma-derived Tsc2-null ELT3 cells and human tumor cells. Furthermore, we identified hypoxia-inducible factor 1α (HIF-1α) as a mediator transmitting the signal from mTORC1 to PFKFB3. Depletion of PFKFB3 inhibited proliferation and tumorigenicity of Tsc1- or Tsc2-deficient cells. In addition, combination of rapamycin with PFK15, a PFKFB3 inhibitor, exerts a stronger inhibitory effect on cell proliferation of Tsc1- or Tsc2-null MEFs than treatment with single drug. We conclude that loss of TSC1 or TSC2 led to upregulated expression of PFKFB3 through activation of mTORC1/HIF-1α signaling pathway and co-administration of rapamycin and PFK15 may be a promising strategy for the treatment of TSC tumors as well as other hyperactivated mTORC1-related tumors.

Epilepsy in a melanocyte-lineage mTOR hyperactivation mouse model: A novel epilepsy model.

OBJECTIVE: To clarify the complex mechanism underlying epileptogeneis, a novel animal model was generated. METHODS: In our previous research, we have generated a melanocyte-lineage mTOR hyperactivation mouse model (Mitf-M-Cre Tsc2 KO mice; cKO mice) to investigate mTOR pathway in melanogenesis regulation, markedly reduced skin pigmentation was observed. Very unexpectedly, spontaneous recurrent epilepsy was also developed in this mouse model. RESULTS: Compared with control littermates, no change was found in either brain size or brain mass in cKO mice. Hematoxylin staining revealed no obvious aberrant histologic features in the whole brains of cKO mice. Histoimmunofluorescence staining and electron microscopy examination revealed markedly increased mTOR signaling and hyperproliferation of mitochondria in cKO mice, especially in the hippocampus. Furthermore, rapamycin treatment reversed these abnormalities. CONCLUSIONS: This study suggests that our melanocyte-lineage mTOR hyperactivation mouse is a novel animal model of epilepsy, which may promote the progress of both epilepsy and neurophysiology research.

Inhibition of Growth of TSC2-Null Cells by a PI3K/mTOR Inhibitor but Not by a Selective MNK1/2 Inhibitor.

Lymphangioleiomyomatosis (LAM) is a rare metastatic cystic lung disease due to a mutation in a TSC tumor suppressor, resulting in hyperactive mTOR growth pathways. Sirolimus (rapamycin), an allosteric mTORC1 inhibitor, is a therapeutic option for women with LAM but it only maintains lung volume during treatment and does not provide benefit for all LAM patients. The two major mTORC1 protein synthesis pathways are via S6K/S6 or 4E-BP/eIF4E activation. We aimed to investigate rapamycin in combination with compounds that target associated growth pathways, with the potential to be additive to rapamycin. In this study we demonstrated that rapamycin, at a clinically tolerable concentration (10 nM), inhibited the phosphorylation of S6, but not the critical eIF4E releasing Thr 37/46 phosphorylation sites of 4E-BP1 in TSC2-deficient LAM-derived cells. We also characterized the abundant protein expression of peIF4E within LAM lesions. A selective MNK1/2 inhibitor eFT508 inhibited the phosphorylation of eIF4E but did not reduce TSC2-null cell growth. In contrast, a PI3K/mTOR inhibitor omipalisib blocked the phosphorylation of Akt and both S6K/S6 and 4E-BP/eIF4E branches, and additively decreased the growth of TSC2-null cells with rapamycin. Omipalisib, or another inhibitor of both major mTORC1 growth pathways and pAkt, might provide therapeutic options for TSC2-deficient cancers including, but not limited to, LAM.

Alterations in Polyamine Metabolism in Patients With Lymphangioleiomyomatosis and Tuberous Sclerosis Complex 2-Deficient Cells.

BACKGROUND: Lymphangioleiomyomatosis (LAM), a destructive lung disease that affects primarily women, is caused by loss-of-function mutations in TSC1 or TSC2, leading to hyperactivation of mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Rapamycin (sirolimus) treatment suppresses mTORC1 but also induces autophagy, which promotes the survival of TSC2-deficient cells. Based on the hypothesis that simultaneous inhibition of mTORC1 and autophagy would limit the availability of critical nutrients and inhibit LAM cells, we conducted a phase 1 clinical trial of sirolimus and hydroxychloroquine for LAM. Here, we report the analyses of plasma metabolomic profiles from the clinical trial. METHODS: We analyzed the plasma metabolome in samples obtained before, during, and after 6 months of treatment with sirolimus and hydroxychloroquine, using univariate statistical models and machine learning approaches. Metabolites and metabolic pathways were validated in TSC2-deficient cells derived from patients with LAM. Single-cell RNA-Seq was employed to assess metabolic enzymes in an early-passage culture from an LAM lung. RESULTS: Metabolomic profiling revealed changes in polyamine metabolism during treatment, with 5'-methylthioadenosine and arginine among the most highly upregulated metabolites. Similar findings were observed in TSC2-deficient cells derived from patients with LAM. Single-cell transcriptomic profiling of primary LAM cultured cells revealed that mTORC1 inhibition upregulated key enzymes in the polyamine metabolism pathway, including adenosylmethionine decarboxylase 1. CONCLUSIONS: Our data demonstrate that polyamine metabolic pathways are targeted by the combination of rapamycin and hydroxychloroquine, leading to upregulation of 5'-methylthioadenosine and arginine in the plasma of patients with LAM and in TSC2-deficient cells derived from a patient with LAM upon treatment with this drug combination. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01687179; URL: www.clinicaltrials.gov. Partners Human Research Committee, protocol No. 2012P000669.

Allosteric and ATP-Competitive Inhibitors of mTOR Effectively Suppress Tumor Progression-Associated Epithelial-Mesenchymal Transition in the Kidneys of Tsc2+/- Mice.

In tuberous sclerosis (TSC)-associated tumors, mutations in the TSC genes lead to aberrant activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. mTORC1 signaling impacts many biological processes including the epithelial-mesenchymal transition (EMT), which is suggested to promote tumor progression and metastasis in various types of cancer. In this study, we report hybrid cells with epithelial and mesenchymal features in angiomyolipomas and partial EMT in carcinomas from TSC patients and describe a new model of EMT activation during tumor progression from cyst to papillary adenoma to solid carcinoma in the kidneys of Tsc2+/- mice. Features of EMT occurred infrequently in TSC-associated cysts but increased as the lesions progressed through papillary adenoma to solid carcinoma where epithelial-mesenchymal hybrid cells were abundant, indicating partial EMT. We also compared the effects of the novel ATP-competitive mTOR inhibitor AZD2014 with the allosteric mTOR inhibitor rapamycin on EMT and tumor burden. Both AZD2014 and rapamycin potently suppressed EMT of renal tumors and effectively blocked tumor progression in Tsc2+/- mice. These results suggest that partial EMT is a shared feature of TSC-associated renal tumors in humans and mice and occurs during TSC-associated tumor progression. EMT-related signaling pathways may represent therapeutic targets for tumors associated with mutations in the TSC genes.

mTOR-dependent upregulation of xCT blocks melanin synthesis and promotes tumorigenesis.

Loss of either TSC1 or TSC2 causes tuberous sclerosis complex (TSC) via activation of mTOR signaling pathway. The two prominent features of TSC are skin lesions including hypomelanic macules and benign tumors in multiple organs, whose molecular alterations are largely unknown. We report here that Xc- cystine/glutamate antiporter (xCT) was elevated in Tsc2-/- or Pten-/- cells, Tsc1 knockout mouse tissues and TSC2-deficient human kidney tumor. xCT was transcriptionally boosted by mTOR-mediated Oct1 signaling cascade. Augmented xCT led to reduction of eumelanin and elevation of pheomelanin in Tsc1 skin knockout mice through mTOR signaling pathway. Disruption of xCT suppressed the proliferation and tumorigenesis of Pten-null cells and Tsc2-null cells. mTOR hyperactive cells were more sensitive to inhibitors of mTOR or xCT. Combined inhibition of mTOR and xCT synergistically blocked the propagation and oncogenesis of mTOR hyperactive cells. Therefore, oncogenic mTOR activation of xCT is a key connection between aberrant melanin synthesis and tumorigenesis. We suggest that xCT is a novel therapeutic target for TSC and other aberrant mTOR-related diseases.

Hyperactivation of mTORC1 disrupts cellular homeostasis in cerebellar Purkinje cells.

Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolism. The importance of mTORC1 signaling in neuronal development and functions has been highlighted by its strong relationship with many neurological and neuropsychiatric diseases. Previous studies demonstrated that hyperactivation of mTORC1 in forebrain recapitulates tuberous sclerosis and neurodegeneration. In the mouse cerebellum, Purkinje cell-specific knockout of Tsc1/2 has been implicated in autistic-like behaviors. However, since TSC1/2 activity does not always correlate with clinical manifestations as evident in some cases of tuberous sclerosis, the intriguing possibility is raised that phenotypes observed in Tsc1/2 knockout mice cannot be attributable solely to mTORC1 hyperactivation. Here we generated transgenic mice in which mTORC1 signaling is directly hyperactivated in Purkinje cells. The transgenic mice exhibited impaired synapse elimination of climbing fibers and motor discoordination without affecting social behaviors. Furthermore, mTORC1 hyperactivation induced prominent apoptosis of Purkinje cells, accompanied with dysregulated cellular homeostasis including cell enlargement, increased mitochondrial respiratory activity, and activation of pseudohypoxic response. These findings suggest the different contributions between hyperactivated mTORC1 and Tsc1/2 knockout in social behaviors, and reveal the perturbations of cellular homeostasis by hyperactivated mTORC1 as possible underlying mechanisms of neuronal dysfunctions and death in tuberous sclerosis and neurodegenerative diseases.

Vps34-mediated macropinocytosis in Tuberous Sclerosis Complex 2-deficient cells supports tumorigenesis.

Tuberous Sclerosis Complex (TSC), a rare genetic disorder with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivation, is characterized by multi-organ hamartomatous benign tumors including brain, skin, kidney, and lung (Lymphangioleiomyomatosis). mTORC1 hyperactivation drives metabolic reprogramming including glucose and glutamine utilization, protein, nucleic acid and lipid synthesis. To investigate the mechanisms of exogenous nutrients uptake in Tsc2-deficient cells, we measured dextran uptake, a polysaccharide internalized via macropinocytosis. Tsc2-deficient cells showed a striking increase in dextran uptake (3-fold, p < 0.0001) relative to Tsc2-expressing cells, which was decreased (3-fold, p < 0.0001) with mTOR inhibitor, Torin1. Pharmacologic and genetic inhibition of the lipid kinase Vps34 markedly abrogated uptake of Dextran in Tsc2-deficient cells. Macropinocytosis was further increased in Tsc2-deficient cells that lack autophagic mechanisms, suggesting that autophagy inhibition leads to dependence on exogenous nutrient uptake in Tsc2-deficient cells. Treatment with a macropinocytosis inhibitor, ethylisopropylamiloride (EIPA), resulted in selective growth inhibition of Atg5-deficient, Tsc2-deficient cells (50%, p < 0.0001). Genetic inhibition of autophagy (Atg5-/- MEFs) sensitized cells with Tsc2 downregulation to the Vps34 inhibitor, SAR405, resulting in growth inhibition (75%, p < 0.0001). Finally, genetic downregulation of Vps34 inhibited tumor growth and increased tumor latency in an in vivo xenograft model of TSC. Our findings show that macropinocytosis is upregulated with Tsc2-deficiency via a Vps34-dependent mechanism to support their anabolic state. The dependence of Tsc2-deficient cells on exogenous nutrients may provide novel approaches for the treatment of TSC.

Identification of a novel TSC2 c.3610G > A, p.G1204R mutation contribute to aberrant splicing in a patient with classical tuberous sclerosis complex: a case report.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomas in any organ systems. Mutations in the TSC1 or TSC2 gene lead to the dysfunction of hamartin or tuberin proteins, which cause tuberous sclerosis complex. CASE PRESENTATION: We describe the clinical characteristics of patients from a Chinese family with tuberous sclerosis complex and analyze the functional consequences of their causal genetic mutations. A novel heterozygous mutation (c.3610G > A) at the last nucleotide of exon 29 in TSC2 was identified. On the protein level, this variant was presumed to be a missense mutation (p.Gly1204Arg). However, the splicing assay revealed that this mutation also leads to the whole TSC2 exon 29 skipping, besides the wild-type transcript. The mutated transcript results in an in-frame deletion of 71 amino acids (p.Gly1133_Thr1203del) and its ratio with the normal splice product is of about 44:56. CONCLUSIONS: The novel c.3610G > A TSC2 mutation was identified in association with tuberous sclerosis complex. And it was proven to code both for a missense-carrying transcript (56%), and for an isoform lacking exon 29 (44%).

Acid Suspends the Circadian Clock in Hypoxia through Inhibition of mTOR.

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

Deletion of Tsc2 in Nociceptors Reduces Target Innervation, Ion Channel Expression, and Sensitivity to Heat.

The mechanistic target of rapamycin complex 1 (mTORC1) is known to regulate cellular growth pathways, and its genetic activation is sufficient to enhance regenerative axon growth following injury to the central or peripheral nervous systems. However, excess mTORC1 activation may promote innervation defects, and mTORC1 activity mediates injury-induced hypersensitivity, reducing enthusiasm for the pathway as a therapeutic target. While mTORC1 activity is required for full expression of some pain modalities, the effects of pathway activation on nociceptor phenotypes and sensory behaviors are currently unknown. To address this, we genetically activated mTORC1 in mouse peripheral sensory neurons by conditional deletion of its negative regulator Tuberous Sclerosis Complex 2 (Tsc2). Consistent with the well-known role of mTORC1 in regulating cell size, soma size and axon diameter of C-nociceptors were increased in Tsc2-deleted mice. Glabrous skin and spinal cord innervation by C-fiber neurons were also disrupted. Transcriptional profiling of nociceptors enriched by fluorescence-associated cell sorting (FACS) revealed downregulation of multiple classes of ion channels as well as reduced expression of markers for peptidergic nociceptors in Tsc2-deleted mice. In addition to these changes in innervation and gene expression, Tsc2-deleted mice exhibited reduced noxious heat sensitivity and decreased injury-induced cold hypersensitivity, but normal baseline sensitivity to cold and mechanical stimuli. Together, these data show that excess mTORC1 activity in sensory neurons produces changes in gene expression, neuron morphology and sensory behavior.

TSC2-deficient tumors have evidence of T cell exhaustion and respond to anti-PD-1/anti-CTLA-4 immunotherapy.

Tuberous sclerosis complex (TSC) is an incurable multisystem disease characterized by mTORC1-hyperactive tumors. TSC1/2 mutations also occur in other neoplastic disorders, including lymphangioleiomyomatosis (LAM) and bladder cancer. Whether TSC-associated tumors will respond to immunotherapy is unknown. We report here that the programmed death 1 coinhibitory receptor (PD-1) is upregulated on T cells in renal angiomyolipomas (AML) and pulmonary lymphangioleiomyomatosis (LAM). In C57BL/6J mice injected with syngeneic TSC2-deficient cells, anti-PD-1 alone decreased 105K tumor growth by 67% (P < 0.0001); the combination of PD-1 and CTLA-4 blockade was even more effective in suppressing tumor growth. Anti-PD-1 induced complete rejection of TSC2-deficient 105K tumors in 37% of mice (P < 0.05). Double blockade of PD-1 and CTLA-4 induced rejection in 62% of mice (P < 0.01). TSC2 reexpression in TSC2-deficient TMKOC cells enhanced antitumor immunity by increasing T cell infiltration and production of IFN-γ/TNF-α by T cells, suggesting that TSC2 and mTORC1 play specific roles in the induction of antitumor immunity. Finally, 1 month of anti-PD-1 blockade reduced renal tumor burden by 53% (P < 0.01) in genetically engineered Tsc2+/- mice. Taken together, these data demonstrate for the first time to our knowledge that checkpoint blockade may have clinical efficacy for TSC and LAM, and possibly other benign tumor syndromes, potentially yielding complete and durable clinical responses.

Downregulation of PERK activity and eIF2α serine 51 phosphorylation by mTOR complex 1 elicits pro-oxidant and pro-death effects in tuberous sclerosis-deficient cells.

Oxidative stress determines cell fate through several mechanisms, among which regulation of mRNA translation by the phosphorylation of the alpha (α) subunit of the translation initiation factor eIF2α at serine 51 (eIF2αP) plays a prominent role. Increased eIF2αP can contribute to tumor progression as well as tumor suppression. While eIF2αP is increased in most cells to promote survival and adaptation to different forms of stress, we demonstrate that eIF2αP is reduced in tuberous sclerosis complex 2 (TSC2)-deficient cells subjected to oxidative insults. Decreased eIF2αP in TSC2-deficient cells depends on reactive oxygen species (ROS) production and is associated with a reduced activity of the endoplasmic reticulum (ER)-resident kinase PERK owing to the hyper-activation of the mammalian target of rapamycin complex 1 (mTORC1). Downregulation of PERK activity and eIF2αP is accompanied by increased ROS production and enhanced susceptibility of TSC2-deficient cells to extrinsic pro-oxidant stress. The decreased levels of eIF2αP delay tumor formation of TSC2-deficient cells in immune deficient mice, an effect that is significantly alleviated in mice subjected to an anti-oxidant diet. Our findings reveal a previously unidentified connection between mTORC1 and eIF2αP in TSC2-deficient cells with potential implications in tumor suppression in response to oxidative insults.

mGluR5 Modulation of Behavioral and Epileptic Phenotypes in a Mouse Model of Tuberous Sclerosis Complex.

Drugs targeting metabotropic glutamate receptor 5 (mGluR5) have therapeutic potential in autism spectrum disorders (ASD), including tuberous sclerosis complex (TSC). The question whether inhibition or potentiation of mGluR5 could be beneficial depends, among other factors, on the specific indication. To facilitate the development of mGluR5 treatment strategies, we tested the therapeutic utility of mGluR5 negative and positive allosteric modulators (an mGluR5 NAM and PAM) for TSC, using a mutant mouse model with neuronal loss of Tsc2 that demonstrates disease-related phenotypes, including behavioral symptoms of ASD and epilepsy. This model uniquely enables the in vivo characterization and rescue of the electrographic seizures associated with TSC. We demonstrate that inhibition of mGluR5 corrects hyperactivity, seizures, and elevated de novo synaptic protein synthesis. Conversely, positive allosteric modulation of mGluR5 results in the exacerbation of hyperactivity and epileptic phenotypes. The data suggest a meaningful therapeutic potential for mGluR5 NAMs in TSC, which warrants clinical exploration and the continued development of mGluR5 therapies.