Critical Review Tuberous Sclerosis Complex: From Molecular Biology to Novel Therapeutic Approaches

Katarzyna Switon1 Katarzyna Kotulska2 Aleksandra JanuszKaminska1 Justyna Zmorzynska1 Jacek Jaworski1*

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Laboratory of Molecular and Cellular Neurobiology, International Institute of Molecular and Cell Biology, Warsaw, Poland 2 Department of Neurology and Epileptology, Children’s Memorial Health Institute, Warsaw, Poland

Abstract Tuberous sclerosis complex (TSC) is a rare multi-system disorder, primary manifestations of which are benign tumors and lesions in various organs of the body, including the brain. TSC patients often suffer from epilepsy, mental retardation, and autism spectrum disorder (ASD). Therefore, TSC serves as a model of epilepsy, ASD, and tumorigenesis. TSC is caused by the lack of functional Tsc1-Tsc2 complex, which serves as a major cellular inhibitor of mammalian Target of Rapamycin Complex 1 (mTORC1). mTORC1 is a kinase controlling most of anabolic processes in eukaryotic cells. Consequently, mTORC1 inhibitors, such as rapamycin, serve as experimental or already approved drugs for several TSC symptoms. However, rapalogs, although quite effective, need to be administered

chronically and likely for a lifetime, since therapy discontinuation results in tumor regrowth and epilepsy recurrence. Recent studies revealed that metabolism and excitability (in the case of neurons) of cells lacking Tsc1-Tsc2 complex are changed, and these features may potentially be used to treat some of TSC symptoms. In this review, we first provide basic facts about TSC and its molecular background, to next discuss the newest findings in TSC cell biology that can be used to improve existing therapies of TSC and other diseases linked to C 2016 The Authors IUBMB Life mTORC1 hyperactivation. V published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 00(0):000–000, 2016

Keywords: tuberous sclerosis complex; mTOR; epilepsy; tumors; rapamycin; therapy; ROS

Introduction Tuberous Sclerosis Complex (TSC), also known as Tuberous Sclerosis or Bourneville-Pringle disease, is an autosomal

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Katarzyna Switon and Katarzyna Kotulska contributed equally to this work. C 2016 The Authors IUBMB Life published by Wiley Periodicals, Inc. on V behalf of International Union of Biochemistry and Molecular Biology Volume 00, Number 00, Month 2016, Pages 00–00 *Address correspondence to: Jacek Jaworski, International Institute of Molecular and Cell Biology, 4 Ks. Trojdena St., 02-109 Warsaw, Poland. E-mail: [email protected] Received 28 September 2016; Accepted 9 October 2016 DOI 10.1002/iub.1579 Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com)

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dominant genetic disorder, which belongs to a group of neurocutaneous syndromes (phakomatoses) together with SturgeWeber syndrome, von Hippel-Lindau disease, and neurofibromatosis (1,2). Although TSC is classified as a rare disease (1 in 6000 births; (3)), it has gained substantial attention as a model of epilepsy, autism spectrum disorder (ASD) and tumorigenesis, with a clearly defined genetic trigger. Thus far, mutations in TSC1 or TSC2 genes were found in 85% of TSC cases (4,5). However, additional mutations in these genes are expected to be identified in the remaining cases, as improvement of sequencing technologies progresses (6). TSC1 and TSC2 encode the proteins hamartin and tuberin, respectively, which together form the TSC (Tsc1-Tsc2; (7,8)). Tsc1-Tsc2 acts as an inhibitor of Ras homolog enriched in brain (Rheb) - mammalian Target of Rapamycin (mTOR) complex 1 (mTORC1) signaling pathway (9). Indeed, strong evidence coming from cellular and animal models, as well as preliminary observations in patients, point to hyperactivation of mTORC1 as the major cause of TSC pathology (10). Consequently, mTOR inhibitors, especially rapamycin and its derivatives (rapalogs), are used in

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clinical practice to target several TSC symptoms (e.g., tumorigenesis, epilepsy). However, rapalogs, although quite effective, need to be administered chronically and likely for a lifetime. Furthermore, mTORC1 is not the sole target of Tsc1-Tsc2Rheb signaling cascade (11). Therefore, based on recently gained knowledge, several researchers started to search for new, supportive or alternative strategies to fight neurological manifestations and tumorigenesis in TSC. These novel therapeutic approaches are the main subject of this mini-review.

Clinical Manifestations of TSC sire -Magloire TSC was first described by French physician De Bourneville in 1880. The first diagnostic criteria, proposed by Heinrich Vogt (Vogt triad) in 1908, were epilepsy, mental retardation, and facial angiofibroma. Nowadays, TSC is a well described multisystem disorder, primary manifestations of which are benign tumors and lesions in various organs of the body, for example, brain (cortical tubers, subependymal nodules, subependymal giant cell astrocytomas (SEGAs), retinal hamartomas), skin (facial angiofibromas), heart (cardiac rhabdomyoma), and kidneys (renal angiomyolipoma). Additionally, several neurological symptoms have been described in TSC patients, including epilepsy (in 80–90% of affected individuals), intellectual disability (60%), and ASD (25–50%; (1,2,12)). In some patients there are also additional neuropsychiatric problems, such as anxiety, depression, attention-deficit/hyperactivity disorder (ADHD) and aggressive/disruptive behavior (13). Interestingly, it is still unknown how, and to what extent neuroanatomical and neurological symptoms are linked and affect disease progression. Correlations between particular neurological problems are also still debated. For example, cortical tubers are seen in more than 90% of TSC individuals, and epilepsy is present in 80–90% of patients. Multifactorial analyses of epilepsy, cognition, and tuber load in 61 TSC patients showed that patients with a higher tuber/brain proportion were characterized by a younger epilepsy onset and lower cognition index (14). Larger cortical lesions were also shown to be associated with more severe epilepsy and worse cognitive outcome in TSC patients, compared with small- or medium-sized tubers (15). Finally, surgical resection of epileptogenic foci is considered a therapeutic option in TSC patients (16). However, epilepsy and cortical tubers do not always coexist, and there are seizure-free patients with numerous cortical tubers, and patients with severe epilepsy who have no cortical lesions that are visible on brain magnetic resonance imaging. Over the last decade several animal models of TSC (Ecker rats, conditional knockout mice, mice with virally induced gene knockdown, zebrafish and fruit fly) have been established. While there is no single animal model which reproduces all TSC features, major symptoms like tumorigenesis, brain lesions, disorganization of brain laminar architecture, hypomyelination, epilepsy and social behavior deficits have been reproduced (17–22). These models have been successfully used

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to (i) study molecular and cellular mechanisms of TSC (ii) develop therapeutic strategies (iii) understand the relationship between brain tumorigenesis and epilepsy, as well as (iv) to search for brain lesion-independent epilepsy triggers.

Molecular Biology of TSC TSC is caused by the lack of functional Tsc1-Tsc2 complex due to mutations in TSC1 or TSC2 genes, which encode hamartin (Tsc1, 140 kDa) and tuberin (Tsc2, 200 kDa) respectively ((7,8); Fig. 1). Recently, a third component of Tsc1-Tsc2, TBC1D7, was described, and its likely role is to additionally stabilize the complex (23,24). Initially, Tsc1-Tsc2 was thought to be a GTPase activating protein (GAP) for Ras-related protein 1 (Rap1) and Rab5 (25, 26), but soon after Rheb was described as its canonical and best studied target (27). GAP activity of Tsc1-Tsc2 is provided by tuberin, which contains a C-terminal GAP domain. Since no other functional domains have been identified in tuberin or hamartin, and the lack of hamartin results in degradation of tuberin, hamartin is considered to stabilize the complex. However, poor conservation of the Nterminal tuberin-binding domain of hamartin led some researchers to postulate that hamartin has additional functions outside Tsc1-Tsc2 (11). Tsc1-Tsc2 is situated at the crossroads of several important signaling pathways and is heavily regulated by phosphorylation (Fig. 1). Tuberin undergoes inhibitory phosphorylation of tuberin by Akt, extracellular signal–regulated kinases (ERKs) and 90 kDa ribosomal S6 kinase in response to trophic factors, and hamartin undergoes inhibitory phosphorylation by IjB kinase b (IKKb) in response to cytokines (28–31). However, insufficient cellular energy levels, hypoxia or increased stress result in an activating phosphorylation of tuberin by AMP-activated protein kinase (32). Interestingly, how exactly inhibitory phosphorylation blocks Tsc1-Tsc2 activity has remained obscure for many years. Neither tuberin phosphorylation by Akt nor by ERKs affected its GAP activity in vitro or reproducibly changed its stability (8). However, it was recently demonstrated that Akt-driven phosphorylation prevents Tsc1-Tsc2 localization to intracellular membranes (e.g., surface of lysosomes) where Rheb resides, which allows the subsequent activation of Rheb and mTORC1 (33). ERKs, on the other hand, are suggested to stimulate Tsc1-Tsc2 complex dissociation (30). Understanding how Akt and ERKs inhibit Tsc1-Tsc2 is of practical importance, since it has been postulated that in TSC these events contribute to inhibiting the remaining activity of the complex, which leads to further hyperactivation of mTORC1 (either in heterozygous cells or in cells where mutated proteins form Tsc1-Tsc2 with some activity retained). Along with the notion that loss of Tsc1-Tsc2 GAP activity causes TSC, hyperactivation of mTORC1 pathway (Fig. 1) is observed in cellular and animal models of TSC (e.g., 19) and, more importantly, in patient tissues. mTORC1 is one of two protein complexes formed by mTOR, a serine/threonine kinase

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FIG 1

Druggable canonical and non-canonical Tsc1-Tsc2 signaling and functions. mTORC1 is the main downstream target of Tsc1Tsc2 that can be targeted with rapalogs. On the other hand, evidence exists that mTORC2 is downregulated in TSC, thus its specific activator (e.g., A-443654) can be used to return its activity. Some cellular activities of Tsc1-Tsc2 does not require mTORC1, for example, secretion of VEGF, secretion and activation of MMPs, primary cilia and aggresome formation, activation of Notch and PAK2 signaling. Targeting MMPs, Notch signaling (with DAPT) and PAK2 can be used as potential therapeutic options. Finally, upstream inhibitors of Tsc1-Tsc2, for example, ERKs, can be targeted to reverse TSC pathology (42,70).

which controls almost every aspect of cellular metabolism (34,35). Another mTOR complex is mTORC2 (36). These complexes have different protein composition, non-overlapping sets of substrates and are differentially regulated by Tsc1-Tsc2 (35, 37). Among a myriad of cellular processes controlled by mTORC1 (35), the ones most extensively studied in context of TSC are positive regulation of translation initiation, regulation of transcription and inhibition of autophagy (10,38). mTORC2 is mostly known for its regulation of AGC (PKA, PKG, PKC) kinases, among which Akt and PKCa are the most extensively studied. In addition, mTORC2 controls actin cytoskeleton dynamics via PKCa or small GTPases of the Rho family (36). Vast majority of studies performed to date indeed revealed mTORC1 upregulation and downregulation of mTORC2 in cellular and animal models of TSC (e.g., 20, 21, 37). While contribution of mTORC2 deficiency to TSC is not understood, the degree of mTORC1 hyperactivation correlates well with the severity of TSC symptoms, at least in the central nervous system. For example, mice lacking Tsc2 in cells of glial origin (Tsc2GFAP:Cre) have higher phosphorylation of ribosomal protein S6 than Tsc1GFAP:Cre KO mice, which indirectly proves higher mTORC1 activation (39). At the same time more severe epilepsy is observed in Tsc2GFAP:Cre KO mice (39). Altogether, this suggests that more severe symptoms are associated with stronger mTORC1 hyperactivation. Furthermore, Kwiatkowski group created genetically engineered mice with 35 or 20% of physiological tuberin level (40) and showed that this difference was sufficient to increase mTORC1 activation and aggravate

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the neuroanatomical and behavioral abnormalities. However, more direct evidence of a causal link between mTORC1 hyperactivity and TSC pathology comes from experiments with the use of rapalogs. In TSC models, these drugs correct mTORC1 signaling and reverse cellular hypertrophy and high levels of cellular stress (e.g., ER stress, oxidative stress) typical for TSC (18,20,41,42). In animal TSC models rapalogs prevent disorganization of the brain cortex and hippocampus, tumor (or tumor-like cell masses) development, they also reduce the severity of seizures and correct social behavior deficits (e.g., 18–21, 43). Consequently, as discussed in the last section of this mini-review, rapalogs are already approved or are in clinical trials to treat a variety of TSC symptoms (see below). Yet, several studies clearly show that upon rapamycin discontinuation tumors regrow (including brain and kidney tumors) and epilepsy returns (44). Thus, there is an urgent need for alternative or supportive therapies to cure both brain lesions (SEGA) and epilepsy, which are among most threatening TSC symptoms. Although mTORC1 seems to be the main downstream target of Tsc1-Tsc2, evidence exists that some cellular activities of Tsc1-Tsc2 might not require Rheb or mTORC1 [e.g., secretion of vascular endothelial growth factor (VEGF; 11, 45)], secretion and activation of matrix metalloproteinases (MMPs; 11, 46), activation of mTORC2 (11, 37). TSC2 was also reported to act as a transcription factor (47). In addition, the function of Rheb is not narrowed to mTORC1 activation. Its postulated mTORC1independent functions include primary cilia and aggresome

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formation, and activation of Notch or PAK2 signaling (11,48). These incidental observations should be kept in mind when considering TSC cellular pathology and potential therapeutic strategies (e.g., Notch, PAK2, and MMPs inhibitors, Fig. 1).

New Approaches Towards Therapy of Epilepsy and Tumors in TSC In this last part of our review, we discuss the potential advantages and caveats of rapalog use and the need for alternative or supportive therapy to cure TSC. The most troublesome TSC symptoms are epilepsy, SEGA, and renal angiomyolipomas. In the majority of TSC patients, epilepsy begins in the first year of life, with a median onset between the third and fifth month of life, and is very difficult to handle. Furthermore, the early presentation of seizures and their poor control are established risk factors for poor neuropsychological outcome in TSC patients (14,49,50). SEGA is a major cause of mortality and morbidity in children and adolescents suffering from TSC. Despite being histologically benign, these tumors grow inside brain ventricles and may obstruct the flow of cerebrospinal fluid, causing hydrocephalus and associated neurological symptoms, including worsening of the seizure burden, visual loss, and disturbed consciousness, among others. Renal angiomyolipomas are also benign tumors but they have the risk of rupturing with bleeding or damaging the surrounding tissue during their growth. Taking into consideration these three most life threatening symptoms of TSC, the major effort is now to use our understanding of TSC molecular biology to target tumorigenesis and epileptogenesis. Although mTOR inhibitors are most thoroughly studied in this context, also new approaches, which take into consideration the changed metabolom of TSC cells, are investigated.

mTOR Inhibitors Given the established role of mTOR pathway abnormalities in TSC, mTOR inhibitors (e.g., rapamycin [sirolimus] and its derivative, everolimus [RAD001]) have been studied in TSC patients since 2006. In the first case series of five SEGAs associated with TSC, rapamycin significantly reduced tumor volume. A similar effect was observed with kidney angiomyolipomas (51), facial angiofibromas (52), and lymphangioleiomyomatosis (53). The randomized, Phase 3, placebo-controlled, double-blind clinical trials EXIST-1 (study of the safety and efficacy of everolimus in TSC-associated SEGAs) and EXIST-2 (study of the safety and efficacy of everolimus in TSC-associated kidney angiomyolipomas) demonstrated beneficial effects of everolimus on both TSC manifestations. Based on these data, everolimus was approved by the EMA and FDA for two indications in TSC. Everolimus is indicated for the treatment of adult patients with renal angiomyolipoma (AML) associated with TSC who are at risk of complications (based on such factors as tumor size, the presence of aneurysm, and the presence of multiple or bilateral

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tumors) but who do not require immediate surgery. Everolimus is also indicated for the treatment of patients, regardless of age, with SEGAs that require therapeutic intervention but are not amenable to surgery. Both rapamycin and everolimus have also been shown to reduce the severity of epilepsy in TSC patients (54,55). Both agents are also being studied in TSC-associated neuropsychiatric problems, particularly autism and neurocognitive deficits (clinicaltrials.gov). Treatment with mTOR inhibitors, however, is associated with a significant risk of adverse events, including increased risk of infections (stomatitis is the most frequent), acne, amenorhoea, disturbed wound healing, and laboratory abnormalities (56). Considerations of these effects are important because it is widely accepted that treatment with mTOR inhibitors should be longlasting. Withdrawal from mTOR inhibitor treatment was associated with tumor regrowth (e.g., SEGA, kidney AML, cardiac rhabdomyoma, and skin lesions) in a vast majority of patients.

Alternative Therapeutic Strategies Treatment with mTOR inhibitors for TSC-associated epilepsy is difficult because epilepsy begins in infancy, and the risk of severe adverse reactions in this age group is high. Moreover, the safety and efficacy of mTOR inhibitors in infants have not been proven. Thus, epilepsy in TSC has been targeted for a long time by several different approaches. Epilepsy in TSC patients is frequently drug-resistant. The current therapeutic options include antiepileptic drugs, steroids, surgical treatment (e.g., removal of cortical tubers), vagus nerve stimulation, and a ketogenic diet (57). Among the antiepileptic drugs, vigabatrin appears to be particularly effective in TSC compared with both the effects of other drugs and its effects in non-TSC epilepsy. In fact, early treatment with vigabatrin that begins before the onset of clinical seizures but after the onset of epileptiform discharges on EEG prevents both seizures and intellectual disability (58). The reasons behind the outstanding efficacy of this drug in TSC are not exactly clear. Vigabatrin is an irreversible inhibitor of GABA transaminase. Hence, it increases the levels of available GABA at the synapse. Indeed, an imbalance between excitatory and inhibitory transmission has been reported in TSC models (59) and brain tissue that was obtained from TSC patients during surgery. In fact, neuronal excitation/inhibition ratio stabilization appears to be sufficient to prevent spontaneous seizures in Tsc11/2 mice. Lozovaya et al. (59) showed that unprovoked and transient epilepsy in Tsc11/2 neonates is likely an effect of the upregulation of GluN2C expression and subsequent aberrant GluN2C-mediated currents in the cortex. Similar disturbances in GluN2C transmission were observed in tubers and the perituber area that were resected from patients’ brains (59). Consequently, the GluN2C/D antagonist UBP141 rescued Tsc11/2 animals from early seizures and reversed aberrant transmission in patients’ specimens. The authors concluded that UBP141, similar to vigabatrin, can be used as an antiepileptic drug for the treatment of early epilepsy in TSC (59). However, Zhang et al. (60) used

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FIG 2

Cellular anti-ROS defence as an alternative therapeutic target in TSC. A. Diagram of GSH synthesis pathway with indicated potential therapeutic targets and drugs used to kill “TSC cells.” B. Model of cell death induction in “TSC cell” by drugs targeting cellular anti-ROS defence. See text for more details.

Tsc1GFAP:Cre mouse-derived cells and found that vigabatrin may have additional actions, in which it also decreases rpS6 phosphorylation. This observation prompted the authors to hypothesize that the inhibition of mTOR by vigabatrin accounts for its efficacy in the treatment of TSC epilepsy. The same research group suggested that the positive effects of a ketogenic diet might also be explained by mTOR inhibition, in which they found that ketons downregulate mTOR activity in the brain and prevent its activation in response to kainic acid (61). Epilepsy treatment in TSC patients relies mostly on approaches that were previously proven to be effective in antiepileptic therapy, and real progress has been made in the search for alternative approaches to the treatment of TSCrelated tumors. In our opinion, the major reason behind the search for alternative approaches stems from a serious disadvantage of rapalog-based therapy, which relies on the inhibition of tumor cell growth/proliferation rather than its elimination. Several groups have begun to search for compounds that either alone or combined selectively increase the cell death of TSCdeficient cells. Notably, however, a few of these compounds have been tested in TSC-related brain tumor cells (e.g., SEGAs). Nevertheless, we discuss these below because these approaches quite likely also work in SEGA cells. Two major strategies are used to target TSC-related tumors. The first strategy includes compounds that act cooperatively with rapamycin and decrease tumor cell survival. The postulated reasons for the lack of efficacy of rapalogs in killing TSC tumor cells are (i) increased activation of Akt signaling due to the inhibition of negative feedback from mTOR to trophic factor receptors and (ii) increased autophagy. Both of these may indeed result from prolonged mTORC1 inhibition (62) and have positive effects on cell survival, although autophagy was shown to play a dual role in cancer cells. The beneficial effects of autophagy include the recycling of nutrients and clearance of damaged proteins and organelles. Thus, although rapamycin slowed TSC tumor cell growth/proliferation, it also increased their capacity to cope with low-nutrient conditions

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and increased oxidative stress. Indeed, Alayev et al. (63) showed that resveratrol treatment in TSC2-null cells (Tsc22/2 MEFs, ELT3 cells, Tsc2-null Eker rat uterine leiomyomaderived smooth muscle cells, and human LAM-derived TSC2null 621-101 cells) blocked rapamycin-induced autophagy and increased apoptosis. Additionally, a combination of rapamycin and resveratrol efficiently decreased the survival of ELT3 cells in vivo. Resveratrol is a compound that is abundantly present in grapes and berries and likely can be safely used in patients. Other approaches to kill TSC tumor cells take advantage of changes in the metabolism of these cells and therefore should not be compatible with rapamycin treatment. The Henske group performed metabolomic profiling of TSC2-null cells with different treatments (e.g., chloroquine and estradiol) and found important differences between TSC2-null and wildtype cells that can be pharmacologically targeted (64–66). For example, Parkhitko et al. (65) found that in TSC22/2 MEFs, chloroquine (which is used to inhibit residual autophagy) induced higher glucose consumption and the presence of pentose phosphate pathway metabolites (e.g., octulose-1-8-biphosphate and ribose-5-phosphate). Consequently, the combined treatment of TSC2-null cells with chloroquine and 6-aminonicotinamide (a competitive inhibitor of glucose-6-phosphate dehydrogenase) led to a dramatic and selective decrease in their proliferation and inflammasome activation that could lead to cell death. The two studies discussed above suggest that targeting autophagy in combination with different drugs might be beneficial for killing TSC2-deficient cells. However, a recent study by Liang et al. (67) suggested that the inhibition of autophagy contributes to TSC pathology, at least in the kidneys. These authors showed that the lack of either Tsc1 or Tsc2 led to changes in the transcriptional program within the Hippo pathway through the accumulation of Hippo-Yes-associated protein 1 (YAP), that would be otherwise degraded via the autolysosomal pathway. Consequently, the authors used Verteprofin, a YAP inhibitor, and induced the death of Tsc12/2 cells

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in vivo in a mosaic kidney, which prevented kidney overgrowth and the formation of perivascular epithelioid cell neoplasms (67). Another series of experiments used middle to large pharmacological or siRNA screens to find new potential druggable targets in TSC cells, and all three ended up with the same general target: anti-reactive oxygen species defense (42,68,69). Li et al. (68) performed a medium small-molecule library screen to search for compounds that selectively kill Tsc22/2 MEFs that are pretreated with Hsp90 inhibitor to induce cellular stress due to unfolded protein or ER-stress responses. They found that bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2yl)ethyl sulfide (BPTES), an inhibitor of glutaminase (GLS; i.e., an enzyme that is critical for glutamate synthesis from glutamine), results in the cell death of 17-AAG (Hsp90 inhibitor)-sensitized TSC2-null cells (Fig. 2). No such effect was observed in wildtype MEFs. Combined Hsp90 and GLS inhibition induced the cell death of ELT3 cells both in vitro and upon grafting to SCID (severe combined immunodeficiency) mice in vivo. Glutaminederived glutamate can be further converted to a-keto-glutarate and enter the tricarboxylic acid (TCA) cycle or be used for cglutamylcysteine synthesis, which is a precursor of glutathione (GSH). Further work by Li and coworkers showed that targeting the latter process led to the cell death of Tsc2-null cells with inhibited Hsp90 (68). Independent of the Blenis group, the Henske group screened a large library of 6700 compounds using 621-101 cells, including FDA-approved drugs, drugs in clinical trials, and drugs with known mechanisms of action, to identify agents that specifically kill TSC2-null cells. In contrast to the Blenis study, no additional treatment was used. The top hit was chelerythrine, which induced reactive oxygen species production and GSH depletion specifically in different types of TSC2-null cells. Importantly, chelerythrine inhibited tumor formation upon ELT3 cell grafting to SCID mice. These two studies showed that drugs that are capable of inducing oxidative stress can effectively and selectively kill TSC2-null cells outside the nervous system. The third study mentioned above was performed in our laboratory with the aim of identifying proteins that contribute to the hypertrophy of neurons with Tsc2 knockdown (42). In this screen, we identified c-glutamylcysteine ligase (GCLC), a key enzyme in GSH synthesis (Fig. 2). GCLC expression is increased in dysplastic neurons and balloon cells in both cortical tubers and SEGA cells. The inhibition of GCLC by L-BSO application blocked the growth of cells that were derived from patients’ SEGAs in the short term and led to the subsequent death of these cells. Closer biochemical analysis suggested that L-BSO treatment likely induced genotoxic stress and subsequent cell death as revealed by PARP cleavage. Interestingly, we found that L-BSO specifically in TSC2-deficient cells also decreased the activity of mTORC1. In addition to the major hits that were identified in the medium and high-throughput screens described above, several other potential targets were identified. For example, combined therapy with Hsp90 inhibition and targeting nucleotide synthesis and EGFR, p38, and MEK signaling could be considered

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(68). The Henske group showed that selective serotonin reuptake inhibitors (i.e., known antidepressants) were next to chelerythrine in the selective killing of “TSC” cells (69). Although these new findings await further testing in the context of the nervous system and in humans, the described studies of TSC-related tumors show that there are several unexplored approaches beyond rapamycin for the treatment of nervous system symptoms associated with mTOR-related diseases.

Conclusions TSC is a multisystemic disease, with several life threatening symptoms, against which there is currently no ultimate cure. A way of finding novel approaches for the treatment of TSC is to analyze the molecular mechanisms underlying its pathology. The disruption of mTOR signaling, which is the main cause of TSC-related abnormalities, prompted the use of mTOR inhibitors in the treatment of TSC, however the use of rapamycin and its derivatives has been proven successful only in curbing the epilepsy or overgrowth of tumors, but not in eliminating them. However, over the last few years our understanding of TSC cells metabolism, as well as Tsc1-Tsc2 functions outside mTORC1 signaling, have greatly expanded. Consequently, several attempts to exploit the molecular properties of cells with disrupted Tsc1-Tsc2 have been undertaken in order to correct or to eliminate these cells. The potential drugs tested included compounds which act jointly with rapamycin to block autophagy and increase apoptosis (e.g., resveratrol), as well as compounds which take advantage of changed metabolism in Tsc1-Tsc2 depleted cells to induce apoptosis (such as chloroquine or verteprofin). A promising new target for therapy also seems to be the defense against reactive oxygen species, which is disrupted in TSC cells and can therefore be exploited as a therapeutic target. However, in spite of all these exciting new possibilities, further studies of cell biology of TSC cells (e.g., with with the use of new exciting models based on reprogramming technology) and thorough testing of selected compounds are required.

Acknowledgments This work was partially supported by a Polish National Science Centre OPUS grant (2012/05/B/NZ3/00429) and Sonata Bis grant (2012/07/E/NZ3/00503) to JJ and the European Community’s Seventh Framework Programme (FP7/2007-2013; EPISTOP, grant agreement no. 602391) to KK and JJ. The work of JZ was supported by FP7 grant no. 316125 (“Fishmed”) and National Science Centre grant no. 2015/17/D/ NZ3/03735. She was also a recipient of a START fellowship from the Foundation for Polish Science. JJ, KS, and JZ are recipients of the Foundation for Polish Science “Mistrz” Professorial Subsidy and Fellowship, respectively.

Tuberous Sclerosis Complex

Authors’ Contributions KS, KK, AJK, JZ and JJ wrote the manuscript.

References [1] Crino, P. B., Nathanson, K. L., and Henske, E. P. (2006) The Tuberous Sclerosis Complex. N. Engl. J. Med. 355, 1345–1356. [2] Curatolo, P., Bombardieri, R., and Jozwiak, S. (2008) Tuberous sclerosis. Lancet 372, 657–668. [3] Osborne, J. P., Fryer, A., and Webb, D. (1991) Epidemiology of tuberous sclerosis. Ann. N. Y. Acad. Sci. 615, 125–127. [4] European Chromosome 16 Tuberous Sclerosis Consortium. (1993) Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 75, 1305–1315. [5] van Slegtenhorst, M., de Hoogt, R., Hermans, C., Nellist, M., Janssen, B., et al. (1997) Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 277, 805–808. [6] Tyburczy, M. E., Dies, K. A., Glass, J., Camposano, S., Chekaluk, Y., et al. (2015) Mosaic and Intronic Mutations in TSC1/TSC2 Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing. PLoS Genet 11, e1005637. [7] van Slegtenhorst, M., Nellist, M., Nagelkerken, B., Cheadle, J., Snell, R., et al. (1998) Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products. Hum. Mol. Genet. 7, 1053–1057. [8] Huang, J., and Manning, B. D. (2008) The TSC1-TSC2 complex: a molecular switchboard controlling cell growth. Biochem. J. 412, 179–190. [9] Inoki, K., Li, Y., Xu, T., and Guan, K. L. (2003) Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 17, 1829– 1834. [10] Curatolo, P. (2015) Mechanistic Target of Rapamycin (mTOR) in Tuberous Sclerosis Complex-Associated Epilepsy. Pediatr. Neurol. 52, 281–289. [11] Neuman, N. A., and Henske, E. P. (2011) Non-canonical functions of the tuberous sclerosis complex-Rheb signalling axis. EMBO Mol. Med. 3, 189–200. [12] Curatolo, P., Moavero, R., Roberto, D., and Graziola, F. (2015) Genotype/Phenotype Correlations in Tuberous Sclerosis Complex. Semin. Pediatr. Neurol. 22, 259–273. [13] Muzykewicz, D. A., Newberry, P., Danforth, N., Halpern, E. F., and Thiele, E. A. (2007) Psychiatric comorbid conditions in a clinic population of 241 patients with tuberous sclerosis complex. Epilepsy Behav. 11, 506–513. [14] Jansen, F. E., Vincken, K. L., Algra, A., Anbeek, P., Braams, O., et al. (2008) Cognitive impairment in tuberous sclerosis complex is a multifactorial condition. Neurology 70, 916–923. ndez-Moneo, J. L., Pascual-Pascual, S. I., [15] Pascual-Castroviejo, I., Herna ~ o, J., Gutie rrez-Molina, M., et al. (2013) Significance of tuber size for Vian complications of tuberous sclerosis complex. Neurol. Barc. Spain 28, 550–557. [16] Fallah, A., Rodgers, S. D., Weil, A. G., Vadera, S., Mansouri, A., et al. (2015) Resective Epilepsy Surgery for Tuberous Sclerosis in Children: Determining Predictors of Seizure Outcomes in a Multicenter Retrospective Cohort Study. Neurosurgery 77, 517–524; discussion 524. [17] Goorden, S. M., van Woerden, G. M., van der Weerd, L., Cheadle, J. P., and Elgersma, Y. (2007) Cognitive deficits in Tsc11/- mice in the absence of cerebral lesions and seizures. Ann. Neurol. 62, 648–655. [18] Meikle, L., Pollizzi, K., Egnor, A., Kramvis, I., Lane, H., et al. (2008) Response of a neuronal model of tuberous sclerosis to mTOR inhibitors. J. Neurosci. Off. J. Soc. Neurosci. 28, 5422–5432. [19] Ehninger, D., Han, S., Shilyansky, C., Zhou, Y., Li, W., et al. (2008) Reversal of learning deficits in a Tsc21/- mouse model of tuberous sclerosis. Nat. Med. 14, 843. [20] Goto, J., Talos, D. M., Klein, P., Qin, W., Chekaluk, Y. I., et al. (2011) Regulable neural progenitor-specific Tsc1 loss yields giant cells with organellar dysfunction in a model of tuberous sclerosis complex. Proc. Natl. Acad. Sci. U S A 108, E1070–E1079.

Switon et al.

[21] Magri, L., Cambiaghi, M., Cominelli, M., Alfaro-Cervello, C., Cursi, M., et al. (2011) Sustained activation of mTOR pathway in embryonic neural stem cells leads to development of tuberous sclerosis complex-associated lesions. Cell Stem Cell 9, 447–462. [22] Zhou, J., Shrikhande, G., Xu, J., McKay, R. M., Burns, D. K., et al. (2011) Tsc1 mutant neural stem/progenitor cells exhibit migration deficits and give rise to subependymal lesions in the lateral ventricle. Genes Dev. 25, 1595– 1600. [23] Dibble, C. C., Elis, W., Menon, S., Qin, W., Klekota, J., et al. (2012) TBC1D7 is a third subunit of the TSC1-TSC2 complex upstream of mTORC1. Mol. Cell 47, 535–546. [24] Qin, J., Wang, Z., Hoogeveen-Westerveld, M., Shen, G., Gong, W., et al. (2016) Structural Basis of the Interaction between Tuberous Sclerosis Complex 1 (TSC1) and Tre2-Bub2-Cdc16 Domain Family Member 7 (TBC1D7). J. Biol. Chem. 291, 8591–8601. [25] Wienecke, R., K€ onig, A., and DeClue, J. E. (1995) Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity. J. Biol. Chem. 270, 16409–16414. [26] Xiao, G. H., Shoarinejad, F., Jin, F., Golemis, E. A., and Yeung, R. S. (1997) The tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein (GAP) in modulating endocytosis. J. Biol. Chem. 272, 6097–6100. [27] Tee, A. R., Manning, B. D., Roux, P. P., Cantley, L. C., and Blenis, J. (2003) Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Curr. Biol. 13, 1259–1268. [28] Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K. L. (2002) TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4, 648–657. [29] Manning, B. D., Tee, A. R., Logsdon, M. N., Blenis, J., and Cantley, L. C. (2002) Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol. Cell 10, 151–162. [30] Ma, L., Chen, Z., Erdjument-Bromage, H., Tempst, P., and Pandolfi, P. P. (2005) Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell 121, 179–193. [31] Lee, D. F., Kuo, H. P., Chen, C. T., Hsu, J. M., Chou, C. K., et al. (2007) IKK beta suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway. Cell 130, 440–455. [32] Inoki, K., Zhu, T., and Guan, K. L. (2003) TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590. [33] Menon, S., Dibble, C. C., Talbott, G., Hoxhaj, G., Valvezan, A. J., et al. (2014) Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell 156, 771–785. [34] Laplante, M., and Sabatini, D. M. (2012) mTOR signaling in growth control and disease. Cell 149, 274–293. [35] Malik, A. R., Urbanska, M., Macias, M., Skalecka, A., and Jaworski, J. (2013) Beyond control of protein translation: what we have learned about the noncanonical regulation and function of mammalian target of rapamycin (mTOR). Biochim. Biophys. Acta 1834, 1434–1448. [36] Oh, W. J., and Jacinto, E. (2011) mTOR complex 2 signaling and functions. Cell Cycle 10, 2305–2316. [37] Huang, J., Dibble, C. C., Matsuzaki, M., and Manning, B. D. (2008) The TSC1-TSC2 Complex Is Required for Proper Activation of mTOR Complex 2. Mol. Cell. Biol. 28, 4104–4115. [38] Lipton, J. O., and Sahin, M. (2014) The neurology of mTOR. Neuron 84, 275–291. [39] Zeng, L. H., Rensing, N. R., Zhang, B., Gutmann, D. H., Gambello, M. J., et al. (2011) Tsc2 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation in a mouse model of tuberous sclerosis complex. Hum. Mol. Genet. 20, 445–454. [40] Yuan, E., Tsai, P. T., Greene-Colozzi, E., Sahin, M., Kwiatkowski, D. J., et al. (2012) Graded loss of tuberin in an allelic series of brain models of TSC correlates with survival, and biochemical, histological and behavioral features. Hum. Mol. Genet. 21, 4286–4300.

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[41] Di Nardo, A., Kramvis, I., Cho, N., Sadowski, A., Meikle, L., et al. (2009) Tuberous sclerosis complex activity is required to control neuronal stress responses in an mTOR-dependent manner. J. Neurosci. 29, 5926–5937. [42] Malik, A. R., Liszewska, E., Skalecka, A., Urbanska, M., Iyer, A. M., et al. (2015) Tuberous sclerosis complex neuropathology requires glutamatecysteine ligase. Acta Neuropathol. Commun. 3. [43] Zeng, L. H., Xu, L., Gutmann, D. H., and Wong, M. (2008) Rapamycin prevents epilepsy in a mouse model of tuberous sclerosis complex. Ann. Neurol. 63, 444–453. [44] Weidman, D. R., Pole, J. D., Bouffet, E., Taylor, M. D., and Bartels, U. (2015) Dose-level response rates of mTor inhibition in tuberous sclerosis complex (TSC) related subependymal giant cell astrocytoma (SEGA). Pediatr. Blood Cancer 62, 1754–1760. [45] Brugarolas, J. B., Vazquez, F., Reddy, A., Sellers, W. R., and Kaelin, W. G. (2003) TSC2 regulates VEGF through mTOR-dependent and -independent pathways. Cancer Cell 4, 147–158. [46] Lee, P. S., Tsang, S. W., Moses, M. A., Trayes-Gibson, Z., Hsiao, L. L., et al. (2010) Rapamycin-insensitive up-regulation of MMP2 and other genes in tuberous sclerosis complex 2-deficient lymphangioleiomyomatosis-like cells. Am. J. Respir. Cell Mol. Biol. 42, 227–234. [47] Pradhan, S. A., Rather, M. I., Tiwari, A., Bhat, V. K., and Kumar, A. (2014) Evidence that TSC2 acts as a transcription factor and binds to and represses the promoter of Epiregulin. Nucleic Acids Res. 42, 6243–6255. [48] Alves, M. M., Fuhler, G. M., Queiroz, K. C. S., Scholma, J., Goorden, S., et al. (2015) PAK2 is an effector of TSC1/2 signaling independent of mTOR and a potential therapeutic target for Tuberous Sclerosis Complex. Sci. Rep. 5, 14534. [49] Cusmai, R., Moavero, R., Bombardieri, R., Vigevano, F., and Curatolo, P. (2011) Long-term neurological outcome in children with early-onset epilepsy associated with tuberous sclerosis. Epilepsy Behav. 22, 735–739. [50] Humphrey, A., Neville, B. G. R., Clarke, A., and Bolton, P. F. (2006) Autistic regression associated with seizure onset in an infant with tuberous sclerosis. Dev. Med. Child Neurol. 48, 609–611. [51] Bissler, J. J., Kingswood, J. C., Radzikowska, E., Zonnenberg, B. A., Frost, M., et al. (2013) Everolimus for angiomyolipoma associated with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis (EXIST-2): a multicentre, randomised, double-blind, placebo-controlled trial. Lancet Lond. Engl. 381, 817–824. zwiak, S., Sadowski, K., Kotulska, K., and Schwartz, R. A. (2016) Topical [52] Jo Use of Mammalian Target of Rapamycin (mTOR) Inhibitors in Tuberous Sclerosis Complex-A Comprehensive Review of the Literature. Pediatr. Neurol. doi:10.1016/j.pediatrneurol.2016.04.003 [53] Davies, D. M., de Vries, P. J., Johnson, S. R., McCartney, D. L., Cox, J. A., et al. (2011) Sirolimus therapy for angiomyolipoma in tuberous sclerosis and sporadic lymphangioleiomyomatosis: a phase 2 trial. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 17, 4071–4081. [54] Franz, D. N., Agricola, K., Mays, M., Tudor, C., Care, M. M., et al. (2015) Everolimus for subependymal giant cell astrocytoma: 5-year final analysis. Ann. Neurol. 78, 929–938.  ski, D., [55] Kotulska, K., Chmielewski, D., Borkowska, J., Jurkiewicz, E., Kuczyn et al. (2013) Long-term effect of everolimus on epilepsy and growth in children

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[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

under 3 years of age treated for subependymal giant cell astrocytoma associated with tuberous sclerosis complex. Eur. J. Paediatr. Neurol. 17, 479–485. zwiak, S. (2016) Management of side Sadowski, K., Kotulska, K., and Jo effects of mTOR inhibitors in tuberous sclerosis patients. Pharmacol. Rep. 68, 536–542. zwiak, S., and Nabbout, R., & TSC Consensus Meeting for Curatolo, P., Jo SEGA and Epilepsy Management (2012) Management of epilepsy associated with tuberous sclerosis complex (TSC): clinical recommendations. Eur. J. Paediatr. Neurol. 16, 582–586. zwiak, S., Kotulska, K., Doman  ska-Pakieła, D., Lojszczyk, B., Syczewska, Jo M., et al. (2011) Antiepileptic treatment before the onset of seizures reduces epilepsy severity and risk of mental retardation in infants with tuberous sclerosis complex. Eur. J. Paediatr. Neurol. 15, 424–431. Lozovaya, N., Gataullina, S., Tsintsadze, T., Tsintsadze, V., PallesiPocachard, E., et al. (2014) Selective suppression of excessive GluN2C expression rescues early epilepsy in a tuberous sclerosis murine model. Nat. Commun. 5, 4563. Zhang, B., McDaniel, S. S., Rensing, N. R., and Wong, M. (2013) Vigabatrin inhibits seizures and mTOR pathway activation in a mouse model of tuberous sclerosis complex. PloS One 8, e57445. McDaniel, S. S., Rensing, N. R., Thio, L. L., Yamada, K. A., and Wong, M. (2011) The ketogenic diet inhibits the mammalian target of rapamycin (mTOR) pathway. Epilepsia 52, e7–e11. Alayev, A., Berger, S. M., and Holz, M. K. (2015) Resveratrol as a novel treatment for diseases with mTOR pathway hyperactivation. Ann. N. Y. Acad. Sci. 1348, 116–123. Alayev, A., Sun, Y., Snyder, R. B., Berger, S. M., Yu, J. J., et al. (2014) Resveratrol prevents rapamycin-induced upregulation of autophagy and selectively induces apoptosis in TSC2-deficient cells. Cell Cycle (Georget Tex) 13, 371–382. Li, C., Lee, P. S., Sun, Y., Gu, X., Zhang, E., et al. (2014) Estradiol and mTORC2 cooperate to enhance prostaglandin biosynthesis and tumorigenesis in TSC2-deficient LAM cells. J. Exp. Med. 211, 15–28. Parkhitko, A. A., Priolo, C., Coloff, J. L., Yun, J., Wu, J. J., et al. (2014) Autophagy-dependent metabolic reprogramming sensitizes TSC2-deficient cells to the antimetabolite 6-aminonicotinamide. Mol. Cancer Res. 12, 48–57. Priolo, C., Ricoult, S. J. H., Khabibullin, D., Filippakis, H., Yu, J., et al. (2015) Tuberous sclerosis complex 2 loss increases lysophosphatidylcholine synthesis in lymphangioleiomyomatosis. Am. J. Respir. Cell Mol. Biol. 53, 33–41. Liang, N., Zhang, C., Dill, P., Panasyuk, G., Pion, D., et al. (2014) Regulation of YAP by mTOR and autophagy reveals a therapeutic target of tuberous sclerosis complex. J. Exp. Med. 211, 2249–2263. Li, J., Csibi, A., Yang, S., Hoffman, G. R., Li, C., et al. (2015) Synthetic lethality of combined glutaminase and Hsp90 inhibition in mTORC1-driven tumor cells. Proc. Natl. Acad. Sci. U S A 112, E21–E29. Medvetz, D., Sun, Y., Li, C., Khabibullin, D., Balan, M., et al. (2015) Highthroughput drug screen identifies chelerythrine as a selective inducer of death in a TSC2-null setting. Mol. Cancer Res. 13, 50–62. Tyburczy, M. E., Kotulska, K., Pokarowski, P., Mieczkowski, J., Kucharska, J., et al. (2010) Novel proteins regulated by mTOR in subependymal giant cell astrocytomas of patients with tuberous sclerosis complex and new therapeutic implications. Am. J. Pathol. 176, 1878–1890.

Tuberous Sclerosis Complex