mTORC1 Hyperactivation Research Articles

The Bi-Steric Inhibitor RMC-5552 Reduces mTORC1 Signaling and Growth in Lymphangioleiomyomatosis.

Mutations in the Tuberous Sclerosis Complex (TSC) genes result in the hyperactivation of the mechanistic/mammalian target of rapamycin 1 (mTORC1) growth pathway in mesenchymal pulmonary cells. Rapamycin (SirolimusTM), a naturally occurring macrolide, is the only therapeutic approved for women with lymphangioleiomyomatosis (LAM), a progressive, destructive lung disease caused by TSC gene mutations and mTORC1 hyperactivation. However, on cessation of the drug, lung function decline continues. We demonstrated here that pulmonary LAM cancer stem-like cells (SLS) most highly expressed the eukaryotic translation initiation factor 4E (eIF4E)-dependent translation initiation genes. We also showed that the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) gene has the lowest expression in these cells, indicating that the 4E-BP1/eIF4E ratio in LAM SLS cells favors unrestrained eIF4E oncogenic mRNA translation. The bi-steric mTORC1-selective compound RMC-5552 prevented growth of LAM-associated fibroblasts (LAFs) and phosphorylation of proteins in the ribosomal protein S6K1/ribosomal protein S6 (S6K1/S6) and 4E-BP1/eIF4E translation mTORC1-driven pathways, whereas rapamycin only blocked the S6K/S6 axis. Rapamycin inhibition of LAF growth was rapidly reversed, but RMC-5552 inhibition was more durable. RMC-5552, through its potential to eradicate LAM cancer SLS cells, may have therapeutic benefit in LAM and other diseases with mTORC1 hyperactivity.

Regulatory-Associated Protein of mTOR-Mediated Signaling: A Nexus Between Tumorigenesis and Disease

RAPTOR (regulatory-associated protein of mTOR) is a pivotal component of the mammalian target of rapamycin complex 1 (mTORC1), playing a central role in regulating cell growth, metabolism and stress responses. As a scaffold protein, RAPTOR recruits key substrates such as eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and ribosomal protein S6 kinase (S6K), facilitating their phosphorylation by mTORC1, which in turn drives protein synthesis, lipid metabolism and cellular proliferation. Its regulatory function becomes especially crucial under conditions of nutrient deprivation or stress, where it enhances the stability of the mTORC1 complex, allowing cells to adapt to fluctuating environmental cues. The hyperactivation of mTORC1, largely mediated by RAPTOR, is frequently observed in various cancers, contributing to uncontrolled cell proliferation and tumorigenesis. Moreover, RAPTOR’s modulation of immune responses and metabolic pathways extends its influence beyond oncogenesis, impacting inflammatory diseases and metabolic disorders. This review meticulously elucidates RAPTOR’s structure, post-translational modifications as well as its indispensable role within the mTORC1 complex, emphasizing its regulatory functions in cellular growth, metabolic adaptation, immune response and disease pathology including oncogenesis. Furthermore, it explores emergent therapeutic avenues targeting RAPTOR-mediated mTORC1 signaling, underscoring their potential to revolutionize cancer treatment and the management of related pathophysiological conditions.

Somatostatin-expressing inhibitory neurons with mTORC1 activation in cortical layers 4/5 are involved in the epileptogenesis of mice

Aim: Patients with tuberous sclerosis complex (TSC) which is caused by hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) often show giant cells in the brain. These giant cells are thought to be involved in epileptogenesis, but the underlying mechanisms are unknown. In this study, we focused on mTORC1 activation and γ-amino butyric acid (GABA)ergic signaling in somatostatin-expressing inhibitory neurons (SST-INs) using TSC-related epilepsy model mice. Methods: We analyzed the 8-week-old Tsc2 conditional knockout (Tsc2 cKO) mice, which have epileptic seizures that are cured by sirolimus, an mTORC1 inhibitor. After the occurrence of epileptic seizures was confirmed, Tsc2 cKO mice were treated with vehicle or sirolimus. Then, their brains were investigated by hematoxylin and eosin staining, immunohistochemical staining and immunoblotting assay. Results: As in TSC patients, giant cells with hyperactivation of mTORC1 were found in the cerebral cortex of Tsc2 cKO mice. These giant cells were mainly SST-INs in the cortical layers 4/5. Giant cells showed decreased expression of GABA type A receptor subunit α1 (GABAAR-α1) compared with normal size cells in control mice and Tsc2 cKO mice. In addition, decreased GABAAR-α1 expression was also confirmed by immunoblotting assay of the whole cerebral cortex. In the cerebral cortex of sirolimus-treated Tsc2 cKO mice, whose epileptic seizures were cured, decreased GABAAR-α1 expression was recovered to the same level as in control mice. Conclusions: These results suggest that the epileptic seizures in Tsc2 cKO mice are caused by the deregulation of GABAergic signaling through mTORC1 activation of SST-INs localized in cortical layers 4/5.

Tip60-mediated Rheb acetylation links palmitic acid with mTORC1 activation and insulin resistance.

Excess dietary intake of saturated fatty acids (SFAs) induces glucose intolerance and metabolic disorders. In contrast, unsaturated fatty acids (UFAs) elicit beneficial effects on insulin sensitivity. However, it remains elusive how SFAs and UFAs signal differentially toward insulin signaling to influence glucose homeostasis. Here, using a croaker model, we report that dietary palmitic acid (PA), but not oleic acid or linoleic acid, leads to dysregulation of mTORC1, which provokes systemic insulin resistance. Mechanistically, we show that PA profoundly elevates acetyl-CoA derived from mitochondrial fatty acid β oxidation to intensify Tip60-mediated Rheb acetylation, which triggers mTORC1 activation by promoting the interaction between Rheb and FKBPs. Subsequently, hyperactivation of mTORC1 enhances IRS1 serine phosphorylation and inhibits TFEB-mediated IRS1 transcription, inducing impairment of insulin signaling. Collectively, our results reveal a conserved molecular insight into the mechanism by which Tip60-mediated Rheb acetylation induces mTORC1 activation and insulin resistance under the PA condition, which may provide therapeutic avenues to intervene in the development of T2D.

Unraveling CASTOR1's tumor suppressive role: Genetic ablation of CASTOR1 and KRAS-driven mouse model of non-small cell lung cancer.

8528 Background: Cytosolic Arginine Sensor for mTORC1 Subunit 1 (CASTOR1) has emerged as a potent suppressor of mTORC1 activity, playing a pivotal role in various cancer contexts. Previously implicated as a tumor suppressor in multiple cancers, CASTOR1 is tightly regulated in cancer, including inhibition by viral miRNAs in KSHV-induced cancer and degradation via AKT-phosphorylation and RNF167-mediated ubiquitination. Notably, CASTOR1's role extends beyond normal cellular contexts, as evidenced by its tumor-suppressive function in breast cancer xenograft models and its prognostic value in lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). This study aims to elucidate CASTOR1’s role in a transgenic Kras-driven LUAD mouse model, shedding light on its tumor-suppressive potential. Methods: Utilizing a novel CASTOR1 knockout (KO) mouse model crossed with the Kras (LSL-KrasG12D/+) NSCLC model, we established a new mouse model with simultaneous Kras activation and homozygous CASTOR1 ablation. Tumor incidence and growth were monitored in Kras mice harboring wild-type (WT) and KO CASTOR1. Further investigations examined the proliferative dynamics and activation status of various oncogenic pathways, and assessed mTORC1 inhibitor sensitivities in human LUAD cell lines with altered CASTOR1 expression levels. Results: CASTOR1 deficiency markedly heightened lung tumor incidence and size, concomitant with an elevated proliferative index. Intriguingly, a novel antibody identified robust pCASTOR1 levels in CASTOR1 WT mouse LUAD tumors, positively correlated with tumor stage and inversely related to total CASTOR1 abundance, hinting at an active degradation mechanism promoting tumor progression. Elevated mTORC1 signaling, evidenced by increased levels of p4EBP and pS6, underscored the pathway's activation consequent to CASTOR1 loss, likely driving tumor development. Furthermore, heightened pERK levels in CASTOR1 KO tumors unveiled a novel feedback loop intertwining CASTOR1/mTORC1 and the Kras/ERK pathway. Notably, treatment with mTORC1 inhibitors sensitized organoids of Kras tumors with mTORC1 hyperactivation to Kras G12D inhibitor. Conclusions: Our findings elucidate CASTOR1's tumor-suppressive role in Kras-dependent mouse LUAD tumors, suggesting the potential efficacy of combined Kras and mTORC1 inhibition in LUAD subsets characterized by hyperactivated Kras and diminished CASTOR1 levels or mTORC1 hyperactivity.

Dicer-Mediated mTORC1 Signaling and Parathyroid Gland Integrity and Function.

Secondary hyperparathyroidism of CKD contributes significantly to patient morbidity and mortality. The underlining mechanisms of CKD-induced secondary hyperparathyroidism remain elusive. We previously demonstrated that PT-Dicer-/- mice, with parathyroid specific deletion of the miRNA processing enzyme Dicer and consequently miRNA maintain normal basal serum PTH levels but do not develop secondary hyperparathyroidism induced by CKD. Additionally, we showed that the parathyroid mTORC1 pathway is activated in CKD. We now explored the roles of Dicer/miRNA and mTORC1 in parathyroid development and function. We generated mice with parathyroid specific Dicer (PT-Dicer-/-), mTOR (PT-mTOR-/-) or tuberous sclerosis complex 1 (PT-Tsc1-/-) deficiency combined with YFP or tdTomato expression, to identify the parathyroids by fluorescent microscopy. CKD was induced by an adenine-rich high phosphate diet. Despite normal basal serum PTH levels, PT-Dicer-/- mice displayed apoptotic loss of intact parathyroid glands postnatally, and reduced mTOR activity. PT-mTOR-/- mice lacked intact parathyroid glands, yet maintained normal serum PTH levels, mirroring the phenotype of PT-Dicer-/- mice. Conversely, PT-Tsc1-/- mice with hyperactivated mTORC1, exhibited enlarged glands along with elevated basal serum PTH and calcium levels. Significantly, PT-Dicer-/-;Tsc1-/- double knockout mice, preserved intact parathyroid glands and reinstated CKD-induced secondary hyperparathyroidism. mTORC1 operates downstream of Dicer and miRNA in the parathyroid and is essential for maintaining postnatal parathyroid gland integrity throughout life and for the pathogenesis of CKD-induced secondary hyperparathyroidism.

Multi-omic Analysis of Human B-cell Activation Reveals a Key Lysosomal BCAT1 Role in mTOR Hyperactivation by B-cell receptor and TLR9.

B-lymphocytes play major adaptive immune roles, producing antibody and driving T-cell responses. However, how immunometabolism networks support B-cell activation and differentiation in response to distinct receptor stimuli remains incompletely understood. To gain insights, we systematically investigated acute primary human B-cell transcriptional, translational and metabolomic responses to B-cell receptor (BCR), Toll-like receptor 9 (TLR9), CD40-ligand (CD40L), interleukin-4 (IL4) or combinations thereof. T-independent BCR/TLR9 co-stimulation, which drives malignant and autoimmune B-cell states, jointly induced PD-L1 plasma membrane expression, supported by NAD metabolism and oxidative phosphorylation. BCR/TLR9 also highly induced the transaminase BCAT1, which localized to lysosomal membranes to support branched chain amino acid synthesis and mTORC1 hyperactivation. BCAT1 inhibition blunted BCR/TLR9, but not CD40L/IL4-triggered B-cell proliferation, IL10 expression and BCR/TLR pathway-driven lymphoma xenograft outgrowth. These results provide a valuable resource, reveal receptor-mediated immunometabolism remodeling to support key B-cell phenotypes including PD-L1 checkpoint signaling, and identify BCAT1 as a novel B-cell therapeutic target.

Hyperactive mTORC1 disrupts habenula function and light preference in zebrafish model of Tuberous sclerosis complex

Mechanistic target of rapamycin complex 1 (mTORC1) is an integration hub for extracellular and intracellular signals necessary for brain development. Hyperactive mTORC1 is found in autism spectrum disorder (ASD) characterized by atypical reactivity to sensory stimuli, among other symptoms. In Tuberous sclerosis complex (TSC) inactivating mutations in the TSC1 or TSC2 genes result in hyperactivation of the mTORC1 pathway and ASD. Here, we show that lack of light preference of the TSC zebrafish model, tsc2 vu242/vu242 is caused by aberrant processing of light stimuli in the left dorsal habenula and tsc2 vu242/vu242 fish have impaired function of the left dorsal habenula, in which neurons exhibited higher activity and lacked habituation to the light stimuli. These characteristics were rescued by rapamycin. We thus discovered that hyperactive mTorC1 caused aberrant habenula function resulting in lack of light preference. Our results suggest that mTORC1 hyperactivity contributes to atypical reactivity to sensory stimuli in ASD.

Synergistic hyperactivation of both mTORC1 and mTORC2 underlies the neural abnormalities of PTEN-deficient human neurons and cortical organoids

Mutations in the phosphatase and tensin homolog (PTEN) gene are associated with severe neurodevelopmental disorders. Loss of PTEN leads to hyperactivation of the mechanistic target of rapamycin (mTOR), which functions in two distinct protein complexes, mTORC1 and mTORC2. The downstream signaling mechanisms that contribute to PTEN mutant phenotypes are not well delineated. Here, we show that pluripotent stem cell-derived PTEN mutant human neurons, neural precursors, and cortical organoids recapitulate disease-relevant phenotypes, including hypertrophy, electrical hyperactivity, enhanced proliferation, and structural overgrowth. PTEN loss leads to simultaneous hyperactivation of mTORC1 and mTORC2. We dissect the contribution of mTORC1 and mTORC2 by generating double mutants of PTEN and RPTOR or RICTOR, respectively. Our results reveal that the synergistic hyperactivation of both mTORC1 and mTORC2 is essential for the PTEN mutant human neural phenotypes. Together, our findings provide insights into the molecular mechanisms that underlie PTEN-related neural disorders and highlight novel therapeutic targets.

Abstract 3055: Cholesterol And Amino Acids Derived From Dietary Sources Drive Atherosclerosis Via Synergistic Activation Of Macrophage Mtorc1 Signaling.

The pathogenic role for lipids, especially cholesterol, in atherosclerosis is well established although relevant mechanisms of action continue to be uncovered. Recent studies linking cholesterol, cholesterol sensing, and the induction of proatherogenic mammalian target of rapamycin (mTOR) signaling is one area with significant therapeutic potential. Our previous studies demonstrated a critical role for amino acids, especially leucine, derived from dietary protein in promoting plaque progression through activation of macrophage mTORC1. We thus aimed to understand the role of cholesterol in macrophage mTOR signaling, its relation to amino acid-mediated effects, and cooperation between dietary lipids and protein in plaque mTOR activation and atherosclerosis. Here, we show LDL cholesterol as well as atherogenic lipids containing modified cholesterol (7-Ketocholesterol and oxLDL) are potent inducers of macrophage mTORC1 especially inconcert with leucine. Importantly, cholesterol and leucine synergistically dampen autophagy/mitophagy, resulting in mitochondrial dysfunction and apoptosis, a process which is completely mTORC1-dependent. In vivo, we find lesional macrophage mTORC1 is stimulated by elevation of plasma cholesterol in ApoE-null mice (n=8-10 per group) fed a Western Diet, while increases in circulating amino acids and cholesterol as a result of diets enriched in protein and lipids lead to mTORC1 hyperactivation and concomitant reduction in autophagy. In conclusion, our study identifies a previously unrecognized signaling role for cholesterol in macrophages and highlights a unique and therapeutically relevant mechanism by which dietary nutrients such as lipids and amino acids can cooperate to drive atherosclerosis.

Pathogenic hyperactivation of mTORC1 by cytoplasmic EP300 in Hutchinson-Gilford progeria syndrome.

In a recent issue in Nature Cell Biology, Sung Min Son et al. unveil a novel layer in the regulation of the mTORC1/autophagy axis by EP300 which can undergo nucleocytoplasmic shuttling in response to alterations in nutrient availability. The study highlights that, in Hutchinson-Gilford progeria syndrome, overabundant cytoplasmic EP300 results in mTORC1 hyperactivation and impaired autophagy, potentially contributing to premature and accelerated aging.

MTORC1 hyperactivation and resultant suppression of macroautophagy contribute to the induction of cardiomyocyte necroptosis by catecholamine surges.

Previous studies revealed a controversial role of mechanistic target of rapamycin complex 1 (mTORC1) and mTORC1-regulated macroautophagy in isoproterenol (ISO)-induced cardiac injury. Here we investigated the role of mTORC1 and potential underlying mechanisms in ISO-induced cardiomyocyte necrosis. Two consecutive daily injections of ISO (85 mg/kg, s.c.) or vehicle control (CTL) were administered to C57BL/6J mice with or without rapamycin (RAP, 5 mg/kg, i.p.) pretreatment. Western blot analyses showed that myocardial mTORC1 signaling and the RIPK1-RIPK3-MLKL necroptotic pathway were activated, mRNA expression analyses revealed downregulation of representative TFEB target genes, and Evan's blue dye uptake assays detected increased cardiomyocyte necrosis in ISO-treated mice. However, RAP pretreatment prevented or significantly attenuated the ISO-induced cardiomyocyte necrosis, myocardial inflammation, downregulation of TFEB target genes, and activation of the RIPK1-RIPK3-MLKL pathway. LC3-II flux assays confirmed the impairment of myocardial autophagic flux in the ISO-treated mice. In cultured neonatal rat cardiomyocytes, mTORC1 signaling was also activated by ISO, and inhibition of mTORC1 by RAP attenuated ISO-induced cytotoxicity. These findings suggest that mTORC1 hyperactivation and resultant suppression of macroautophagy play a major role in the induction of cardiomyocyte necroptosis by catecholamine surges, identifying mTORC1 inhibition as a potential strategy to treat heart diseases with catecholamine surges.

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.

TFEB drives mTORC1 hyperactivation and kidney disease in Tuberous Sclerosis Complex

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, leading to hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and lesions in multiple organs including lung (lymphangioleiomyomatosis) and kidney (angiomyolipoma and renal cell carcinoma). Previously, we found that TFEB is constitutively active in TSC. Here, we generated two mouse models of TSC in which kidney pathology is the primary phenotype. Knockout of TFEB rescues kidney pathology and overall survival, indicating that TFEB is the primary driver of renal disease in TSC. Importantly, increased mTORC1 activity in the TSC2 knockout kidneys is normalized by TFEB knockout. In TSC2-deficient cells, Rheb knockdown or Rapamycin treatment paradoxically increases TFEB phosphorylation at the mTORC1-sites and relocalizes TFEB from nucleus to cytoplasm. In mice, Rapamycin treatment normalizes lysosomal gene expression, similar to TFEB knockout, suggesting that Rapamycin’s benefit in TSC is TFEB-dependent. These results change the view of the mechanisms of mTORC1 hyperactivation in TSC and may lead to therapeutic avenues.

IMPACT OF MITOCHONDRIAL MALFUNCTION IN AGING IS MEDIATED BY IMPAIRED LYSOSOMES AND ENDOPLASMIC RETICULUM

Abstract The hallmarks of aging are well established, and include mitochondrial malfunction, deregulated nutrient sensing, loss of proteostasis, and senescence, among others. While these hallmarks are all part of the aging process, they have typically been studied independently. We have found that mitochondrial malfunction can affect lysosomal activity, impacting nutrient sensing, and our that the crosstalk between mitochondria and other organelles such as endoplasmic reticulum and lysosomes affects cellular senescence, proteostasis and intercellular communication. Chronic mitochondrial malfunction causes perturbations in the lysosomal membrane which affect the function of this organelle as well as the recruitment of folliculin (FLCN) to the lysosomes. FLCN regulates the balance between anabolism (stimulating mTORC1) and catabolism (repressing AMPK). Increased recruitment of FLCN to the lysosomal membrane in cells subjected to chronic mitochondrial malfunction results in hyperactive mTORC1 and inhibition of AMPK signaling, thus aberrantly promoting anabolism under conditions that are not optimal. Furthermore, peroxisomal β-oxidation increases, and expression of proteasome subunits decreases, with consequent perturbation of proteostasis. Overall, the impact of chronic mitochondrial malfunction over lysosomes triggers multiple perturbations in organelles and organelle crosstalk that culminate in aberrant nutrient sensing, metabolic rewiring, proteostasis impairment, senescence and release of sterile inflammation-related proteins to the extracellular medium that denote alterations in intercellular communication. We will build on these findings to explore an overall picture of organelle homeostasis in aging and in age-related diseases.

Hyperactive mTORC1 in lung mesenchyme induces endothelial cell dysfunction and pulmonary vascular remodeling.

Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease caused by tuberous sclerosis complex 1/2 (TSC1/2) gene mutations in pulmonary mesenchymal cells, resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). A subset of patients with LAM develop pulmonary vascular remodeling and pulmonary hypertension. Little, however, is known regarding how LAM cells communicate with endothelial cells (ECs) to trigger vascular remodeling. In end-stage LAM lung explants, we identified EC dysfunction characterized by increased EC proliferation and migration, defective angiogenesis, and dysmorphic endothelial tube network formation. To model LAM disease, we used an mTORC1 gain-of-function mouse model with a Tsc2 KO (Tsc2KO) specific to lung mesenchyme (Tbx4LME-Cre Tsc2fl/fl), similar to the mesenchyme-specific genetic alterations seen in human disease. As early as 8 weeks of age, ECs from mice exhibited marked transcriptomic changes despite an absence of morphological changes to the distal lung microvasculature. In contrast, 1-year-old Tbx4LME-Cre Tsc2fl/fl mice spontaneously developed pulmonary vascular remodeling with increased medial thickness. Single-cell RNA-Seq of 1-year-old mouse lung cells identified paracrine ligands originating from Tsc2KO mesenchyme, which can signal through receptors in arterial ECs. These ECs had transcriptionally altered genes including those in pathways associated with blood vessel remodeling. The proposed pathophysiologic mesenchymal ligand-EC receptor crosstalk highlights the importance of an altered mesenchymal cell/EC axis in LAM and other hyperactive mTORC1-driven diseases. Since ECs in patients with LAM and in Tbx4LME-Cre Tsc2fl/fl mice did not harbor TSC2 mutations, our study demonstrates that constitutively active mTORC1 lung mesenchymal cells orchestrated dysfunctional EC responses that contributed to pulmonary vascular remodeling.

Abstract A116: TSC1 mutant bladder cancer is characterized by a TSC-associated gene expression signature due to TFE3 transcriptional activity

Abstract Background: Mutations and inactivation of the tumor suppressor gene TSC1 are recurrent (6-10%) events in invasive bladder cancer, but the mechanism by which TSC1 loss promotes bladder cancer development beyond mTORC1 activation is uncertain. The purpose of this study was to assess the functional effects of TSC1 loss in bladder cancer and uncover new therapeutic targets for patients with TSC1 mutations. Materials and methods: We performed differential gene expression and pathway analyses using The Cancer Genome Atlas (TCGA) expression data for TSC1-mutated bladder cancer (TSC1mt-BLCA, n=26) and TSC1 wild-type tumors (TSC1wt-BLCA, n=382) and compared them to an internal cohort of TSC1/TSC2-mutant tumors (n=48). Transcriptomic, epigenetic, and functional analyses, including RNA-seq, H3K27ac ChIP-seq, TFE3 CUT&RUN, ATAC-seq and TFE3 knock-out using CRISPR/Cas9 were conducted on TSC1-mt/wt BLCA cell lines. Results: Comparison of TSC1mt-BLCA and TSC1wt-BLCA TCGA tumors identified 26 genes upregulated in TSC1mt-BLCA out of 60 genes commonly upregulated in Tuberous Sclerosis Complex (TSC) syndromic tumors. Among them, GPNMB showed the greatest increase in expression (median fold change 9.8). GSEA and DESeq2 analyses implicated both mTORC1 hyperactivation and increased lysosomal gene expression in TSC1mt-BLCA. Expression differences in several genes, including GPNMB, and in pS6 were confirmed by immunohistochemistry (IHC) of 5 independent BWH TSC1mt-BLCA and TSC1wt-BLCA specimens each. TFE3, a key regulator of lysosomal gene expression, was elevated in expression and localized to the nucleus in TSC1mt-BLCA by IHC. Molecular studies of TSC1-mt/wt BLCA isogenic cell lines (HCV29, RT4) recapitulated the phenotype found in human tumors. TFE3 was both post-translationally modified (assessed by immunoblot) and predominantly nuclear in TSC1-null cell lines compared to TSC1wt cells, and the localization was partially reversed by rapamycin treatment leading to reduced expression of lysosomal genes. TSC1mt HCV29 cells showed higher expression of multiple genes increased in TSC1mt-BLCA tumors (GPNMB, SQSTM1, GNPDA1). TFE3 knock-out HCV29 clones showed markedly reduced growth, which was not seen with TFE3 KO in TSC1-addback HCV29 cells. GSEA of RNA-seq data indicated that TFE3 KO led to significant reduction in expression of lysosome genes, as well as those involved in ribosome synthesis, DNA replication and DNA excision repair processes. TFE3 CUT&RUN confirmed that TFE3 binds to promoter regions of those genes, thereby directly regulating these pathways. In contrast, TFE3 KO led to an increase in inflammatory and cellular stress genes that could impede TSC1mt cell growth. Conclusions: Our findings show that TSC1mt-BLCA tumors and cell lines have a transcriptional signature similar to that of TSC syndromic tumors and show nuclear localization and transcriptional activation of TFE3. Aberrant TFE3 activation likely contributes to TSC1mt-BLCA development and may therefore be amenable to targeted therapy. Citation Format: Magdalena Losko, Clemens K Probst, Elio Adib, Heng Du, Michelle Hirsch, Krinio Giannikou, David J Kwiatkowski. TSC1 mutant bladder cancer is characterized by a TSC-associated gene expression signature due to TFE3 transcriptional activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A116.

Influence of Insulin Resistance and Metabolic Dysregulation on Synaptic Protein Profile in SH‐SY5Y Neuroblastoma Cells

AbstractBackgroundBrain insulin resistance and metabolic dysregulation are significantly associated with Alzheimer’s disease development[1,2]. Metformin is the most prescribed type‐2‐diabetic drug and has been used to treat peripheral insulin resistance (IR) driven metabolic dysfunction through the activation of AMP‐kinase (AMPK)[3,4], but whether metformin is effective in improving brain metabolism is unknown. We have previously shown that chronic AMPK activation negatively impacts neuronal morphology and impairs synaptic protein content in healthy neurons[5]. However, whether AMPK activation under an IR state will exacerbate neuronal impairments is not known. Thus, we examined the effect of metformin‐driven AMPK activation on SH‐SY5Y cells with and without high‐insulin induced IR.MethodsRetinoic acid‐differentiated SH‐SY5Y neuroblastoma cells were treated for 48h with either 1) vehicle (Ctl), 2) 100 M insulin (INS, mimicking hyperinsulinemia), 3) 2 mM metformin (MET), or 4) a combination of INS/MET.ResultsINS‐treated cells demonstrated higher Akt S473, mTOR S2448, and p70s6k T389 phosphorylation, indicative of hyperactive mTORC1 signaling associated with brain IR. INS cells had lower insulin receptor‐ (IR‐) content and higher IRS‐1 S636 phosphorylation, further demonstrating insulin signaling abnormalities. MET increased AMPK T172 phosphorylation, and increased phosphorylation of its downstream effectors ULK S555 and raptor S792, which, collectively, is indicative of mTORC1 inhibition. These phosphorylation increases occurred alongside higher IRS‐1 and insulin receptor‐ content and reduced IR‐ content. INS/MET‐treated cells produced no change in AMPK T172 or raptor S792 phosphorylation but produced increased ULK S555 phosphorylation. INS/MET did not recover increased Akt S473, mTOR S2448, p70s6k T389, or IRS‐1 S636 phosphorlyation nor did it recover IR‐ content. No differences were seen between groups for pre‐/post‐synaptic protein content. INS and MET treatments showed reduced NeuN and synaptophysin content. INS/MET treatment showed reductions in synaptophysin. Morphologically, cell size & area, and neurite length & area all increased with INS and MET treatments, however, these increases were absent with the INS/MET treatment.ConclusionThese findings indicate that IR and MET impact neuronal morphology and reduce the development of mature neurons. These impairments are likely driven through Akt and AMPK signaling seperately and suggest that MET is not effective in treating IR‐driven impairments in the brian.

Bi-steric mTORC1 inhibitors induce apoptotic cell death in tumor models with hyperactivated mTORC1.

The PI3K/AKT/mTOR pathway is commonly dysregulated in cancer. Rapalogs exhibit modest clinical benefit, likely owing to their lack of effects on 4EBP1. We hypothesized that bi-steric mTORC1-selective inhibitors would have greater potential for clinical benefit than rapalogs in tumors with mTORC1 dysfunction. We assessed this hypothesis in tumor models with high mTORC1 activity both in vitro and in vivo. Bi-steric inhibitors had strong growth inhibition, eliminated phosphorylated 4EBP1, and induced more apoptosis than rapamycin or MLN0128. Multiomics analysis showed extensive effects of the bi-steric inhibitors in comparison with rapamycin. De novo purine synthesis was selectively inhibited by bi-sterics through reduction in JUN and its downstream target PRPS1 and appeared to be the cause of apoptosis. Hence, bi-steric mTORC1-selective inhibitors are a therapeutic strategy to treat tumors driven by mTORC1 hyperactivation.

Metabolomic and Lipidomic Signature of Skeletal Muscle with Constitutively Active Mechanistic Target of Rapamycin Complex 1

BackgroundRegulation of mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in aging and nutrition. For example, caloric restriction reduces mTORC1 signaling and extends lifespan, whereas nutrient abundance and obesity increase mTORC1 signaling and reduce lifespan. Skeletal muscle-specific knockout (KO) of DEP domain-containing 5 protein (DEPDC5) results in constitutively active mTORC1 signaling, muscle hypertrophy and an increase in mitochondrial respiratory capacity. The metabolic profile of skeletal muscle, in the setting of hyperactive mTORC1 signaling, is not well known. ObjectivesTo determine the metabolomic and lipidomic signature in skeletal muscle from female and male wild-type (WT) and DEPDC5 KO mice. MethodsTibialis anterior (TA) muscles from WT and transgenic (conditional skeletal muscle-specific DEPDC5 KO) were obtained from female and male adult mice. Polar metabolites and lipids were extracted using a Bligh–Dyer extraction from 5 samples per group and identified and quantified by LC-MS/MS. Resulting analyte peak areas were analyzed with t-test, analysis of variance, and Volcano plots for group comparisons (e.g., WT compared with KO) and multivariate statistical analysis for genotype and sex comparisons. ResultsA total of 162 polar metabolites (organic acids, amino acids, and amines and acyl carnitines) and 1141 lipid metabolites were detected in TA samples by LC-MS/MS. Few polar metabolites showed significant differences in KO muscles compared with WT within the same sex group. P-aminobenzoic acid, β-alanine, and dopamine were significantly higher in KO male muscle whereas erythrose-4-phosphate and oxoglutaric acid were significantly reduced in KO females. The lipidomic profile of the KO groups revealed an increase of muscle phospholipids and reduced triacylglycerol and diacylglycerol compared with the WT groups. ConclusionsSex differences were detected in polar metabolome and lipids were dependent on genotype. The metabolomic profile of mice with hyperactive skeletal muscle mTORC1 is consistent with an upregulation of mitochondrial function and amino acid utilization for protein synthesis.