Energy Stress-Mediated Cytotoxicity in Tuberous Sclerosis Complex 2-Deficient Cells with Nelfinavir and Mefloquine Treatment.
ABSTRACT: To find new anti-cancer drug therapies, we wanted to exploit homeostatic vulnerabilities within Tuberous Sclerosis Complex 2 (TSC2)-deficient cells with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivity. We show that nelfinavir and mefloquine synergize to selectively evoke a cytotoxic response in TSC2-deficient cell lines with mTORC1 hyperactivity. We optimize the concentrations of nelfinavir and mefloquine to a clinically viable range that kill cells that lack TSC2, while wild-type cells tolerate treatment. This new clinically viable drug combination causes a significant level of cell death in TSC2-deficient tumor spheroids. Furthermore, no cell recovery was apparent after drug withdrawal, revealing potent cytotoxicity. Transcriptional profiling by RNA sequencing of drug treated TSC2-deficient cells compared to wild-type cells suggested the cytotoxic mechanism of action, involving initial ER stress and an imbalance in energy homeostatic pathways. Further characterization revealed that supplementation with methyl pyruvate alleviated energy stress and reduced the cytotoxic effect, implicating energy deprivation as the trigger of cell death. This work underpins a critical vulnerability with cancer cells with aberrant signaling through the TSC2-mTORC1 pathway that lack flexibility in homeostatic pathways, which could be exploited with combined nelfinavir and mefloquine treatment.
Project description:Uncontrolled cell growth in Tuberous Sclerosis Complex occurs due to inappropriate activation of mechanistic (mammalian) target of rapamycin complex 1 (mTORC1). The current therapy, rapamycin, produced promising clinical trial results, but patient tumours regrow if treatment is discontinued, revealing rapamycin has cytostatic properties rather than a cytotoxic effect. Taking advantage of the enhanced levels of endoplasmic reticulum (ER) stress present in TSC2-null cells, we investigated drug combinations producing a cytotoxic response. We found a nelfinavir and salinomycin combination specifically killed TSC2-deficient, mTORC1 hyperactive cells. Cytotoxicity was rescued by reducing protein synthesis, either through mTORC1 inhibition or cycloheximide treatment. This indicates that the drug combination targets the cells by tipping the protein homeostasis balance of the already metabolically stressed TSC2-deficient cells in favour of cell death. Furthermore, this drug combination also inhibited tumour formation in TSC2-deficient cell models and caused tumour spheroid death in 3D culture. Importantly, the 3D assay could differentiate the cytostatic agent, rapamycin, from the cytotoxic nelfinavir/salinomycin combination. Sporadic cancer cell lines with hyperactive mTORC1 signalling were also susceptible to this nelfinavir/salinomycin drug combination. This work indicates that the protein homeostasis pathway is an attractive therapeutic target in both Tuberous Sclerosis Complex and mTORC1-driven sporadic cancers.
Project description:Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome associated with tumors of the brain, heart, kidney, and lung. The TSC protein complex inhibits the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Inhibitors of mTORC1, including rapamycin, induce a cytostatic response in TSC tumors, resulting in temporary disease stabilization and prompt regrowth when treatment is stopped. The lack of TSC-specific cytotoxic therapies represents an important unmet clinical need. Using a high-throughput chemical screen in TSC2-deficient, patient-derived cells, we identified a series of molecules antagonized by rapamycin and therefore selective for cells with mTORC1 hyperactivity. In particular, the cell-permeable alkaloid chelerythrine induced reactive oxygen species (ROS) and depleted glutathione (GSH) selectively in TSC2-null cells based on metabolic profiling. N-acetylcysteine or GSH cotreatment protected TSC2-null cells from chelerythrine's effects, indicating that chelerythrine-induced cell death is ROS dependent. Induction of heme-oxygenase-1 (HMOX1/HO-1) with hemin also blocked chelerythrine-induced cell death. In vivo, chelerythrine inhibited the growth of TSC2-null xenograft tumors with no evidence of systemic toxicity with daily treatment over an extended period of time. This study reports the results of a bioactive compound screen and the identification of a potential lead candidate that acts via a novel oxidative stress-dependent mechanism to selectively induce necroptosis in TSC2-deficient tumors.This study demonstrates that TSC2-deficient tumor cells are hypersensitive to oxidative stress-dependent cell death, and provide critical proof of concept that TSC2-deficient cells can be therapeutically targeted without the use of a rapalog to induce a cell death response.
Project description:Increased mTORC1 signaling from <i>TSC1/TSC2</i> inactivation is found in cancer and causes tuberous sclerosis complex (TSC). The role of mesenchymal-derived cells in TSC tumorigenesis was investigated through disruption of <i>Tsc2</i> in craniofacial and limb bud mesenchymal progenitors. Tsc2cKO<sup>Prrx1-cre</sup> mice had shortened lifespans and extensive hamartomas containing abnormal tortuous, dilated vessels prominent in the forelimbs. Abnormalities were blocked by the mTORC1 inhibitor sirolimus. A Tsc2/mTORC1 expression signature identified in Tsc2-deficient fibroblasts was also increased in bladder cancers with <i>TSC1</i>/<i>TSC2</i> mutations in the TCGA database. Signature component <i>Lgals3</i> encoding galectin-3 was increased in Tsc2-deficient cells and serum of Tsc2cKO<sup>Prrx1</sup>-cre mice. Galectin-3 was increased in TSC-related skin tumors, angiomyolipomas, and lymphangioleiomyomatosis with serum levels in patients with lymphangioleiomyomatosis correlating with impaired lung function and angiomyolipoma presence. Our results demonstrate Tsc2-deficient mesenchymal progenitors cause aberrant morphogenic signals, and identify an expression signature including <i>Lgals3</i> relevant for human disease of <i>TSC1/TSC2</i> inactivation and mTORC1 hyperactivity.
Project description:FoxO transcription factors and TORC1 are conserved downstream effectors of Akt. Here, we unraveled regulatory circuits underlying the interplay between Akt, FoxO, and mTOR. Activated FoxO1 inhibits mTORC1 by TSC2-dependent and TSC2-independent mechanisms. First, FoxO1 induces Sestrin3 (Sesn3) gene expression. Sesn3, in turn, inhibits mTORC1 activity in Tsc2-proficient cells. Second, FoxO1 elevates the expression of Rictor, leading to increased mTORC2 activity that consequently activates Akt. In Tsc2-deficient cells, the elevation of Rictor by FoxO increases mTORC2 assembly and activity at the expense of mTORC1, thereby activating Akt while inhibiting mTORC1. FoxO may act as a rheostat that maintains homeostatic balance between Akt and mTOR complexes' activities. In response to physiological stresses, FoxO maintains high Akt activity and low mTORC1 activity. Thus, under stress conditions, FoxO inhibits the anabolic activity of mTORC1, a major consumer of cellular energy, while activating Akt, which increases cellular energy metabolism, thereby maintaining cellular energy homeostasis.
Project description:The mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis and autophagy1. Its hyperactivation contributes to disease in numerous organs, including the heart1,2, although broad inhibition of mTORC1 risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 that acts through modulation of RHEB (Ras homologue enriched in brain). TSC2 constitutively inhibits mTORC1; however, this activity is modified by phosphorylation from multiple signalling kinases that in turn inhibits (AMPK and GSK-3?) or stimulates (AKT, ERK and RSK-1) mTORC1 activity3-9. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here we show that phosphorylation or gain- or loss-of-function mutations at either of two adjacent serine residues in TSC2 (S1365 and S1366 in mice; S1364 and S1365 in humans) can bidirectionally control mTORC1 activity stimulated by growth factors or haemodynamic stress, and consequently modulate cell growth and autophagy. However, basal mTORC1 activity remains unchanged. In the heart, or in isolated cardiomyocytes or fibroblasts, protein kinase G1 (PKG1) phosphorylates these TSC2 sites. PKG1 is a primary effector of nitric oxide and natriuretic peptide signalling, and protects against heart disease10-13. Suppression of hypertrophy and stimulation of autophagy in cardiomyocytes by PKG1 requires TSC2 phosphorylation. Homozygous knock-in mice that express a phosphorylation-silencing mutation in TSC2 (TSC2(S1365A)) develop worse heart disease and have higher mortality after sustained pressure overload of the heart, owing to mTORC1 hyperactivity that cannot be rescued by PKG1 stimulation. However, cardiac disease is reduced and survival of heterozygote Tsc2S1365A knock-in mice subjected to the same stress is improved by PKG1 activation or expression of a phosphorylation-mimicking mutation (TSC2(S1365E)). Resting mTORC1 activity is not altered in either knock-in model. Therefore, TSC2 phosphorylation is both required and sufficient for PKG1-mediated cardiac protection against pressure overload. The serine residues identified here provide a genetic tool for bidirectional regulation of the amplitude of stress-stimulated mTORC1 activity.
Project description: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.
Project description: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<sup>+/-</sup> 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.
Project description:Inappropriate activation of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is common in cancer and has many cellular consequences including elevated endoplasmic reticulum (ER) stress. Cells employ autophagy as a critical compensatory survival mechanism during ER stress. This study utilised drug-induced ER stress through nelfinavir in order to examine ER stress tolerance in cell lines with hyper-active mTORC1 signalling. Our initial findings in wild type cells showed nelfinavir inhibited mTORC1 signalling and upregulated autophagy, as determined by decreased rpS6 and S6K1 phosphorylation, and SQTSM1 protein expression, respectively. Contrastingly, cells with hyper-active mTORC1 displayed basally elevated levels of ER stress which was greatly exaggerated following nelfinavir treatment, seen through increased CHOP mRNA and XBP1 splicing. To further enhance the effects of nelfinavir, we introduced chloroquine as an autophagy inhibitor. Combination of nelfinavir and chloroquine significantly increased ER stress and caused selective cell death in multiple cell line models with hyper-active mTORC1, whilst control cells with normalised mTORC1 signalling tolerated treatment. By comparing chloroquine to other autophagy inhibitors, we uncovered that selective toxicity invoked by chloroquine was independent of autophagy inhibition yet entrapment of chloroquine to acidified lysosomal/endosomal compartments was necessary for cytotoxicity. Our research demonstrates that combination of nelfinavir and chloroquine has therapeutic potential for treatment of mTORC1-driven tumours.
Project description:Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by germline inactivating mutations of TSC1 or TSC2. In TSC-associated tumors of the brain, heart, skin, kidney and lung, inactivation of both alleles of TSC1 or TSC2 leads to hyperactivation of the mTORC1 pathway. The TSC/mTORC1 pathway is a key regulator of cellular processes related to growth, proliferation and autophagy. We and others have previously found that mTORC1 regulates microRNA biogenesis, but the mechanisms are not fully understood. Microprocessor, a multi-protein complex including the nuclease Drosha, processes the primary miR transcript. Using a dual-luciferase reporter, we found that inhibition of mTORC1 or downregulation of Raptor decreased Microprocessor activity, while loss of TSC2 led to a striking increase (?5-fold) in Microprocessor activity. To determine the global impact of TSC2 on microRNAs we quantitatively analyzed 752 microRNAs in Tsc2-expressing and Tsc2-deficient cells. Out of 259 microRNAs expressed in both cell lines, 137 were significantly upregulated and 24 were significantly downregulated in Tsc2-deficient cells, consistent with the increased Microprocessor activity. Microprocessor activity is known to be regulated in part by GSK3?. We found that total GSK3? levels were higher in Tsc2-deficient cells, and the increase in Microprocessor activity associated with Tsc2 loss was reversed by three different GSK3? inhibitors. Furthermore, mTOR inhibition increased the levels of phospho-GSK3? (S9), which negatively affects Microprocessor activity. Taken together these data reveal that TSC2 regulates microRNA biogenesis and Microprocessor activity via GSK3?.
Project description:mTORC1 hyperactivation drives the multi-organ hamartomatous disease tuberous sclerosis complex (TSC). Rapamycin inhibits mTORC1, inducing partial tumor responses; however, the tumors regrow following treatment cessation. We discovered that the oncogenic miRNA, miR-21, is increased in Tsc2-deficient cells and, surprisingly, further increased by rapamycin. To determine the impact of miR-21 in TSC, we inhibited miR-21 in vitro. miR-21 inhibition significantly repressed the tumorigenic potential of Tsc2-deficient cells and increased apoptosis sensitivity. Tsc2-deficient cells' clonogenic and anchorage independent growth were reduced by ?50% (p<0.01) and ?75% (p<0.0001), respectively, and combined rapamycin treatment decreased soft agar growth by ?90% (p<0.0001). miR-21 inhibition also increased sensitivity to apoptosis. Through a network biology-driven integration of RNAseq data, we discovered that miR-21 promotes mitochondrial adaptation and homeostasis in Tsc2-deficient cells. miR-21 inhibition reduced mitochondrial polarization and function in Tsc2-deficient cells, with and without co-treatment with rapamycin. Importantly, miR-21 inhibition limited Tsc2-deficient tumor growth in vivo, reducing tumor size by approximately 3-fold (p<0.0001). When combined with rapamcyin, miR-21 inhibition showed even more striking efficacy, both during treatment and after treatment cessation, with a 4-fold increase in median survival following rapamycin cessation (p=0.0008). We conclude that miR-21 promotes mTORC1-driven tumorigenesis via a mechanism that involves the mitochondria, and that miR-21 is a potential therapeutic target for TSC-associated hamartomas and other mTORC1-driven tumors, with the potential for synergistic efficacy when combined with rapalogs.