Distinct signaling mechanisms of mTORC1 and mTORC2 in glioblastoma multiforme: A tale of two complexes

Advances in Biological Regulation 57 (2015) 64e74

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Distinct signaling mechanisms of mTORC1 and mTORC2 in glioblastoma multiforme: A tale of two complexes Meena Jhanwar-Uniyal*, John L. Gillick, Jayson Neil, Michael Tobias, Zachary E. Thwing, Raj Murali Department of Neurosurgery, New York Medical College, Valhalla, NY 10595, USA

a b s t r a c t Keywords: mTOR mTORC1 mTORC2 S6K 4E-BP1 GBM

Mechanistic target of rapamycin (mTOR) is a serineethreonine kinase that functions via two multiprotein complexes, namely mTORC1 and mTORC2, each characterized by different binding partners that confer separate functions. mTORC1 function is tightly regulated by PI3-K/Akt and is sensitive to rapamycin. mTORC2 is sensitive to growth factors, not nutrients, and is associated with rapamycin-insensitivity. mTORC1 regulates protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. Also, mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt and SGK. Recent evidence has suggested that mTORC2 may play an important role in maintenance of normal as well as cancer cells by virtue of its association with ribosomes, which may be involved in metabolic regulation of the cell. Rapamycin (sirolimus) and its analogs known as rapalogues, such as RAD001 (everolimus) and CCI-779 (temsirolimus), suppress mTOR activity through an allosteric mechanism that acts at a distance from the ATP-catalytic binding site, and are considered incomplete inhibitors. Moreover, these compounds suppress mTORC1-mediated S6K activation, thereby blocking a negative feedback loop, leading to activation of mitogenic pathways promoting cell survival and growth. Consequently, mTOR is a suitable target of therapy in cancer treatments. However, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. In recent years, new pharmacologic agents have been

* Corresponding author. Tel.: þ1 914 594 2513; fax: þ1 914 594 4002. E-mail address: [email protected] (M. Jhanwar-Uniyal).

http://dx.doi.org/10.1016/j.jbior.2014.09.004 2212-4926/© 2014 Published by Elsevier Ltd.

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developed which can inhibit these complexes via ATP-binding mechanism, or dual inhibition of the canonical PI3-K/Akt/mTOR signaling pathway. These compounds include WYE-354, KU003679, PI-103, Torin1, and Torin2, which can target both complexes or serve as a dual inhibitor for PI3-K/mTOR. This investigation describes the mechanism of action of pharmacological agents that effectively target mTORC1 and mTORC2 resulting in suppression of growth, proliferation, and migration of tumor and cancer stem cells. © 2014 Published by Elsevier Ltd.

Introduction The mechanistic target of rapamycin TOR (mTOR; AKA Mammalian target of Rapamycin) is a 289kDa atypical serine/threonine (S/T) protein kinase and is a member of the phosphoinositide 3-kinaserelated kinase (PI3-K), which is conserved in all eukaryotes (Laplante and Sabatini, 2012; Russell et al., 2011). Primarily, mTOR activity has been linked to cellular growth, proliferation, survival, migration, protein translation, and metabolic processes (Sabatini, 2006). mTOR and associated proteins in its signaling pathway are often deregulated in many human cancers, and contribute to both cancer pathogenesis and therapy resistance (Darb-Esfahani et al., 2009; Rosenwald, 2004; Chan et al., 2004). Structurally, the C-terminus contains a kinase domain that places mTOR in the PI3-K family, while its N-terminus contains numerous huntingtin elongation factor-3-protein phosphatase-2A TOR1 (HEAT) repeats, which mediate the majority of interactions with its associated proteins. Two major multiprotein complexes comprise mTOR, namely mTORC1 and mTORC2. mTORC1 complex is formed by regulatory associated protein of mTOR (Raptor), proline-rich Akt substrate 40 kDa (PRAS40), mammalian lethal with Sec-13 protein 8 (mLST8, also known as GbL) and DEP domain TOR-binding protein (Deptor), and is sensitive to rapamycin (Loewith et al., 2002; Laplante and Sabatini, 2012). Raptor is indispensable for the kinase activity of mTORC1 in vitro and in vivo, although it does not possess any enzymatic activity by itself (Kim et al., 2002; Guertin and Sabatini, 2007), it promotes formation of this complex. mTORC1 is a sensor to cellular responses such as growth factors and insulin, and in turn, controls cell growth in response to these stimuli. The targets of mTORC1 have been characterized in detail: the ribosomal S6 kinases (S6K1 and S6K2) and the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BP1). Furthermore, mTORC1 associates with the eukaryotic initiation factor 3 (eIF3) complexes and regulates protein translation initiation via phosphorylation of these substrates. Among its many functions, mTORC1 is known to promote protein translation through activation of p70 S6 Kinase (S6K), and inhibition of eukaryotic initiation factor 4E binding protein (4EBP1) (Jacinto and Hall, 2003; Sabatini, 2006), as well as enhancing RNA translation via S6 ribosomal protein (Volarevic and Thomas, 2001). Activated S6K regulates protein synthesis through phosphorylation of the 40S ribosomal subunit, and thereby increasing the translational efficiency of a class of mRNA transcripts with a 50 -terminal oligopolypyrimidine. Moreover, S6K can also regulate translation via phosphorylation of eIF4B at Ser422, causing eIF4B to associate with eIF3, thus promoting eIF4F complex formation. mTOR also phosphorylates 4E-BP1, which releases eIF4E from 4E-BP1, allowing eIF4E to associate with eIF4G and other relevant factors to promote cap-dependent translation, regulating protein synthesis. S6K exists in two distinct S6 kinases, p90 S6K and p70/85 S6K. The latter kinase consists of two isoforms, one 70-kD cytoplasmic isoform (p70 S6K) and the other nuclear (p85 S6K). Both isoforms appear to phosphorylate the S6 protein and mediate translation of polypyrimidine tract mRNA. Furthermore, studies have shown that the translational control of nuclear S6K regulates DNA synthesis, in particular the transition from G1 to S phase. Recent studies have shown that the growth factor receptor-bound protein 10 (GRB10) was identified as an mTORC1 substrate (Hsu et al., 2011; Yu et al., 2011). mTORC1 directly phosphorylates and simultaneously stabilizes GRB10, leading to feedback inhibition of the PI3-K pathway. Identification of

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the mTORC1-GRB10 interaction complements a known negative feedback loop in which mTORC1 activation can inhibit the PI3-K pathway by S6K-mediated phosphorylation and degradation of insulin receptor substrate-1 (IRS-1), and it augments our understanding of the underlying mechanisms by which mTORC1 inhibits PI3-K/Akt signaling (O'Reilly et al., 2006). Another associated protein, PRAS40, has an inhibitory effect when it binds to mTORC1 suppressing its activation in response to growth factor depletion. The significance of activated PRAS40 expression is evident from the analysis of the patients, which have gone through treatment with rapamycin and displayed activated levels of Akt and therapeutic resistance (Cloughesy et al., 2008), suggesting that phospho-PRAS40 can function as a surrogate marker for activated Akt. Furthermore, PRAS40 contains an mTOR signaling motif, and overexpression of PRAS40 is capable of competing with other mTORC1 targets for phosphorylation (Laplante and Sabatini, 2012). Another partner of mTOR, mLST8, as part of the TORC1 complex; is involved in its activation by amino acids. However, it is considered dispensable for other mechanisms of TORC1 activation. Deptor, which binds mTOR, is a recently described inhibitor of mTOR that is capable of inhibiting both TORC1 and TORC2 activity, although the upstream regulators of Deptor remain unknown (Russell et al., 2011). Upstream of mTORC1 and mTORC2, the GTPase termed Ras homolog enriched in brain (Rheb) is found to activate mTORC1 via direct binding to mTOR (Reiling and Sabatini, 2006). Activation of upstream Akt inhibits the Tuberculin Sclerosis 1/2 (TSC 1/2) heterodimer complex that allows Rheb to activate mTORC1 (Sabatini, 2006). Downstream of mTORC1, the kinase S6K directly phosphorylates multiple targets including programmed cell death protein 4 (PDCD-4), a tumor suppressor (Dorrello et al., 2006). mTORC1 substrate S6K inhibits phosphorylation of insulin receptor substrate-1 (IRS-1), thus via this signaling pathway it has inhibitory control on PI3-K and Akt (Harrington et al., 2004). Alternatively, the inhibition of Akt by activated mTOR can occur via different mechanisms, since inhibition of Akt is in the presence of several growth factors and not exclusively in the presence of IGF-1 (Tamburini et al., 2008). The mechanism by which rapamycin binds directly to mTOR is via forming a complex with a small protein FK506-binding (FKBP12), of only 12 kDa, with the resulting dimer being a potent inhibitor of mTOR activity (Choi et al., 1996). mTORC2 has been linked to cytoskeletal reorganization and cell survival through Akt (Jacinto et al., 2004). This pathway is crucial to many diverse physiological functions including cell cycle progression, transcription, translation, differentiation, apoptosis, motility, and metabolism (Guertin and Sabatini, 2007). mTORC2 is comprised of rapamycin-insensitive companion of mTOR (Rictor), stress-activated protein kinase-interacting protein 1 (Sin1) and protein-binding Rictor (Protor) (Jacinto et al., 2004; Sarbassov et al., 2004; Gulati et al., 2009; Jhanwar-Uniyal et al., 2013). The complex formed between Rictor and mTOR regulates the further substrate recruitment by mTORC2. Notably, the Rictorcontaining mTOR complex is not bound by FKBP12-rapamycin, thus does not affect S6K, a downstream substrate of mTORC1. A well-described substrate of mTORC2, Akt, phosphorylates Protein Kinase C a (PKCa), regulates the actin cytoskeleton and various other cellular functions (Jhanwar-Uniyal et al., 2013). Protor, another mTORC2 binding protein, binds mTORC2 through Rictor and its stability is dependent on the expression of other mTORC2 components; while Protor alone is dispensable for mTORC2 catalytic activity, but plays an important role in regulation of serum glucocorticoid protein kinase (SGK1) activity. Another mTORC2 binding partner, Sin1, is required for mTORC2 function by promoting Rictor-mTOR binding (Pearce et al., 2011). Recent evidence has suggested that Sin1 plays an important role in cancer development, since the mutation in its key domain disrupts mTORC2 leading to the sustained activation of Akt (Liu et al., 2014). The binding of Rictor and Raptor to mTOR is mutually exclusive. Furthermore, increases in mTOR activity by phosphorylation of Sin1 prevent its lysosomal degradation. The kinase domain of mTOR is required for the functional activity of mTORC2, and it controls the integrity of mTORC2 by maintaining the protein stability of Sin1 (Chen and Sarbassov dos, 2011). mTORC2 shares some common subunits with mTORC1, including mTOR, Deptor and mLST8. Further studies have shown that mLST8 is necessary for maintaining the Rictor-mTOR interaction also in mTORC2 complex, implying that mLST8 might be an important molecule for both mTOR complexes and may regulate the dynamic equilibrium of these complexes in mammalian cells. Recent investigations have demonstrated that the upstream signaling by growth factors, insulin, and other stimuli, increases PI3-K signaling, activating mTORC2; by promoting its association with ribosomes. Furthermore, it appears that mTORC2 phosphorylates AktThr450 besides its well-known site AktSer473 (Zinzalla et al., 2011). In addition, Akt phosphorylation via stimulus of mTORC2/ribosome

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binding, which is induced by PI3-K signaling in cancer cells, can be disrupted by inducing apoptosis in PTEN-deficient cells (Zinzalla et al., 2011). In this regard, there may be an interaction between mTORC1 and mTORC2. Recent investigations have suggested that mTORC2 activation can be achieved via a PI3K-independent mechanism, which involves the pathway concerning the YAP/HIPPO, Notch, and small G-protein Rac1. The presence of cancer stem cells (CSCs) was demonstrated in many tumors including GBM through the identification of specific antigenic markers and shown to possess three distinct properties: selfreplication, ability to differentiate into multiple lineages and extensive proliferative potential (Zeppernick et al., 2008; Piccirillo et al., 2009; Mangiola et al., 2007). The aberrance of several development pathways has been implicated in the creation and maintenance of CSCs. One such pathway is PI3-K/PTEN, a family of the lipid/Akt/mTOR signaling cascade. PTEN phosphatase, which works in an antagonistic manner from PI3-K, is often deregulated in GBM and other tumors. Several investigations have indicated that PTEN plays an important role in stem cell regulation. In particular, recent studies have suggested that PTEN plays a pivotal role in maintenance of side population (SP) of leukemic stem cells, perhaps through its role in regulation of PI3-K/Akt/mTOR pathway (Fragoso and Barata, 2014). Recently, the role of mTOR signaling in the maintenance of GBM CSCs has been addressed (Jhanwar-Uniyal et al., 2011, 2013). mTOR hyperactivation in both embryonic and adult stem cells led to the differentiation and depletion of the stem cell population. Furthermore, persistent activation of mTOR in normal epithelial stem cells results in exhaustion of these stem cells (Castilho et al., 2009). Several studies have revealed that cellular stress, cytokines, or activation of the mTOR pathway is able to increase the expression of CSC surface markers and phenotypes in certain bulk tumor cells (Matsumoto et al., 2009; You et al., 2010; Platet et al., 2007; McCord et al., 2009) indicating that cell-extrinsic environmental factors may reprogram conventional tumor cells to cells with stem cell-like properties. In human glioma cells, activation of HIF-1a, a positive downstream target of mTOR (Hudson et al., 2002), enhances CD133þ glioma-derived cancer stem cell expansion by increasing selfrenewal activity and inhibiting cell differentiation (Soeda et al., 2009). Recurrence of GBM is shown to be largely associated with regeneration of tumor from remaining CSCs after initial treatment. Thus, targeting CSCs is an extremely important aspect of the clinical treatment of GBM. One proposed mechanism for targeting CSCs is to first induce differentiation, thus making the cells more amenable to other therapeutic agents. A recent study by Friedman et al. (2013) examined this approach by illustrating that mTOR inhibition alone in combination with differentiating agent, all-trans retinoic acid (ATRA), can target CSCs. The results demonstrated that ATRA caused differentiation of CSCs, as evident by the loss of stem-cell marker nestin expression. Treatment of GBM cells with rapamycin leads to nuclear localization of nestin. This synergism of differentiation and mTOR inhibition may be mediated by the MEK1/2-ERK1/2 pathway. This is of particular interest because resistance to the gold standard chemotherapeutic agent for GBM, temozolomide, was found to be mediated by MEK-ERK induced activation of O(6)-methylguanine DNA methyltransferase (MGMT) (Shimizu et al., 2012). The effectiveness of inhibiting both ERK1/2 and mTOR was examined in other cancers. A phase I trial of 236 patients with advanced colorectal cancer treated with a PI3-K inhibitor, a MAPK inhibitor, or a combination of the two (Brescia et al., 2013) showed that dual inhibition was superior in efficacy compared to inhibition of a single pathway alone. This may also provide an explanation for only marginal benefits seen in an early phase trial of mTOR inhibitor (temsirolimus) as a sole treatment modality for recurrent GBM (Galanis et al., 2005). Rapamycin and its chemically related compounds (also known as rapalogues) have recently been considered for treatment of cancer (Sokolosky et al., 2011). Clinical trials using mTOR inhibitors, rapamycin and its analogs (CCI-779/temsirolimus, RAD001/everolimus, and AP23573) have shown promising, yet challenging results in the management of various tumors including GBM. PTEN, a negative regulator of PI3-K (Cloughesy et al., 2008), is deregulated in GBM. In a clinical trial for PTENdeleted GBM patients, in which recurrent GBM tumors were detected for deletions of PTEN, patients were treated daily with rapamycin to evaluate its maximal tolerated dose and efficacy. Rapamycin successfully reduced tumor cell proliferation in half of these treated patients (Cloughesy et al., 2008). However, in this phase I trial, while rapamycin displayed some promising results, it faced some inherent challenges in targeting the mTOR pathway as a significant number of patients showed increased levels of activated Akt and PRAS40. Rapamycin treatment led to an activation of Akt due to

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loss of negative feedback, and decreased progression-free survival. The majority of rapamycin clinical trials involved the implementation of rapamycin as a chemotherapeutic agent in combination with other anti-tumor agents. In consecutive phase I and phase II trials, the combined effect of rapamycin with gefitinib or erlotinib, showed a maximum tolerated dose of 500 mg gefitinib and 5 mg sirolimus for patients with an acceptable toxicity profile (Reardon et al., 2006). However, a phase II trial with this particular drug combination showed only minor effects as demonstrated by a progression-free survival rate of 3.1% (Reardon et al., 2010). Temsirolimus is an analog of rapamycin, which also inhibits mTOR, and has demonstrated clinical efficacy in the treatment of non-CNS cancers including renal cell carcinoma and mantle cell lymphoma (Dancey et al., 2009). Several other phase II clinical trials have investigated the efficacy of temsirolimus as a novel chemotherapeutic agent for GBM. A weekly administration of temsirolimus to forty-three patients showed no improvement in clinical outcomes, implying the drug had no clinical efficacy (Chang et al., 2005). In contrast, another phase II trial showed 36% radiologic evidence of improvement following temsirolimus treatment in a trial of 65 patients, with a significantly longer progression-free survival time (Galanis et al., 2005). These findings indicate the need for further investigation to establish the accurate efficacy of these agents. Despite significant therapeutic advances, GBM remains incurable with these treatments, perhaps due to the activation of mitogenic pathways, and RAS/ERK1/2 activation via feedback loops (Albert et al., 2009). In recent years, several small molecules have been identified that directly inhibit mTOR by targeting the ATP-binding site; these include PP242, P30, and NVP-BEZ235. Two of these molecules, PP242 and P30, are the first potent, selective, ATP-competitive inhibitors of mTOR. Unlike rapamycin, these molecules inhibit both mTORC1 and mTORC2, and unlike the PI3-K family inhibitors such as LY294002, these molecules inhibit mTOR with a high degree of selectivity relative to the PI3-Ks and protein kinases. To distinguish these molecules from the allosteric mTORC1 inhibitor rapamycin, the term “TORKinibs” was coined. The dual role of mTOR within the PI3-K/Akt pathway as both an upstream activator of Akt and the downstream effector of cell growth and proliferation has excited interest in active-site inhibitors of mTOR. Here, we describe the functional interaction of mTORC1 and mTORC2, and its role in therapeutic targeting in cancers and their resistance. Results and discussion Rapamycin (sirolimus) and rapalogues, including RAD001 (everolimus) and CCI-779 (temsirolimus), suppress mTOR activity via an allosteric mechanism, acting distinctly from ATP-catalytic binding site inhibitors. However, the disadvantages of rapamycin and rapalogues are that they primarily inhibit mTORC1-mediated S6K activation, and block a negative feedback loop, which leads to activation of PI3K/Akt and Ras/MEK/ERK signaling pathways, in turn promoting cell survival and growth. Recently defined novel small ATP-binding site molecules have been identified that directly inhibit mTOR, unlike rapamycin, which is an allosteric inhibitor. In addition, novel ATP-binding compounds with pyrazolopyrimidines are shown to inhibit members of the PI3-K family, including mTOR. One such compound, PP242, is an ATP-competitive inhibitor of mTOR, which shows potent and selective inhibition of mTORC1 and mTORC2 (Feldman et al., 2009). In this investigation we observe that PP242 treatment abolished the activation of mTORC1 substrate S6K and 4E-BP1 (Fig. 1A). ATP-binding inhibitor, PP242, effectively suppressed mTORC1 activity, and decreased GBM cell growth (Fig. 1A and B). The analysis of cell proliferation and growth showed that PP242 was more effective than rapamycin in suppressing GBM cell proliferation (Fig. 1B and C). Significant suppression in S-phase entry was achieved by using the other mTORC1/2 inhibitor, KU-0063794, which was more pronounced than using the sole PI3-K inhibitor, LY-294002, and PI3-K/mTORC1 inhibitor, PI-103 (Fig. 2A). The migration of the GBM cell was suppressed more by PP242 than rapamycin. In order to establish that these compounds effectively block mTORC2 activity, we studied their effect on migration; we observed that in comparison to rapamycin, PP242 was effective in suppressing GBM cell migration. The combined inhibitor of PI3-K/mTORC1, PI-103, was nearly ineffective in suppressing the migration (Fig. 2B). We have provided evidence that both mTORC1 and mTORC2 complexes will need to be targeted in order to effectively block mTOR activity in GBM; however it should be noted that prolonged treatment with rapamycin may lead to activation of ERK pathway and mTORC2 complex, which promotes cellular growth and motility in GBM cells (Chang et al., 2005; Jhanwar-Uniyal et al., 2013). Such activation was

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Fig. 1. A. A comparison of treatment with rapamycin (RAPA), ATP-binding inhibitor PP242, and dual inhibitor of PI3-K/mTOR pathway PI-103 to investigate downstream targets of mTOR pathway 4E-BP1 and S6K. The immunoblotting analysis shows that PP242 completely abolished the activation of S6K. RAPA was also effective, albeit to a lesser extent. Treatment with PI-103 was ineffective in suppressing S6K activation. B. A comparison of RAPA with two inhibitors of ATP binding (PP242 and KU-0063794) showed that PP242 was most effective in reducing the cell viability. C. A comparison of cell viability over the course of 4 days suggested that PP242 and WYE354 significantly reduced cell viability effectively. KU was effective, but to a lesser degree.

not seen when cells were treated with combined inhibitors of mTORC1 and mTORC2 (unpublished observation). Administration of rapamycin caused an increased sensitivity to radiation of a U87 xenograft and significantly increased the re-growth of tumor, which was attributed to rapamycin's effect on decreased cell proliferation and cell cycle arrest (Eshleman et al., 2002). The best-known substrates of mTORC1 are S6K and 4E-BP1; the main substrates of mTORC2 are Akt and related kinases (Sabatini, 2006). Mechanistically, rapamycin and rapalogues carry two major disadvantages: First, these compounds suppress mTORC1-mediated S6K activation, which in turn blocks a negative feedback loop, leading to the activation of PI3-K/Akt pathway promoting cell survival in many cancer cells. Second, rapamycin is an incomplete inhibitor of mTORC1, reducing phosphorylation of 4E-BP1 only partially in most cell contexts. Activating mutations of PI3-K/mTOR are commonly found in GBM patients' phosphorylation of key signaling proteins in the PI3-K pathway (The Cancer Genome Atlas, 2008; Parsons et al., 2008). PI3-K and mTOR pathway in GBM can also be

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Fig. 2. A. These inhibitors via suppressing entry of cells into S-phase. Here, we analyzed S-phase entry via analyzing EdU incorporation (Alexa staining). As expected, the number of cells entering S-phase was significantly higher in PDGF-treated cells. Treatment with KU-0063794 dramatically reduced the number of cells entering into S-phase as shown by lower levels of green staining. PI3-K inhibitor, LY-294002, also reduced S-phase entry but much less than ATP-binding inhibitor KU-0063794. Also shown here, dual inhibitor of PI3-K/mTORC1, PI-103, reduces entry of cells into S-phase. B. The results of the cellular migration assay revealed that PP242 completely blocked the migration of GBM cells. RAPA also decreased the cellular migration, but to a lesser extent. However, PI-103 was ineffective in reducing cellular migration as its levels are close to the control during the 5-day period. The top panel shows the integrity of the cells using F/actin staining showing no significant loss in the cytoskeleton. The bottom panel shows the spared area of scratch wound migration following two treatments with WYE demonstrated complete inhibition of migration as the spared area remained intact following 24 h after treatment while the control treatment had reduced the gap. C. A schematic representation of mTOR pathway showing the components of mTORC1 and mTORC2 pathway. Also shown are the chemical structures of inhibitors of this pathway used in this study. (For example, rapamycin (inhibitor of mTORC1), WYE-354 and KU-006379 (ATP-binding inhibitors), and PI-103 (dual inhibitor of PI3-K/mTORC1)).

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activated by amplification of EGFR due to the presence of an activating mutation, among which the most commonly seen occurs at EGFRvIII. More importantly, c-MET and PDGFRa, who possess a paramount role in gliomagenesis, also activate the PI3-K pathway. Upon activation, mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2. mTORC1 displays sensitivity to rapamycin and rapalogues, and mTORC2 is considered resistant to rapamycin and insensitive to nutrient signals. However, recent evidence points to mTORC2's role in metabolic control of cells (Zinzan et al., 2011). Raptor is the major subunit of mTORC1, the second subunit of the mTORC1 complex, mLST8, is considered to bind the kinase domain of mTOR and positively regulate its kinase activity. mLST8 plays a crucial role in maintaining the interaction between mTOR and either Raptor or Rictor, which is part of the mTORC2 complex. mLST8 shuttles mTOR between the two complexes and sustains the intracellular equilibrium of mTORC1 and mTORC2 (Guertin et al., 2006; Kim et al., 2003; Zeng et al., 2007). Whereas PRAS40 inhibits the mTORC1 activity via Raptor, another protein, Deptor interacts directly with mTOR in both mTORC1 and mTORC2, and is considered as a naturally occurring inhibitor of both complexes. Overexpression of Deptor leads to decreased S6K phosphorylation and mTORC2-mediated signaling back to Akt, monitored by S473 phosphorylation and siRNA inhibition of Deptor (Peterson et al., 2009). In another observation, KRAS mutation is shown to define the sensitivity of PP242, as cells with KRAS mutation display resistance to PP242 treatments, where the degree of resistance was defined by estimating the phosphorylation levels of translational repressor 4E-BP1 (Ducker et al., 2014). We show here that selective, active site mTOR kinase inhibitors have potent effects in suppressing cancer cell proliferation and migration. Active-site inhibition of mTOR addresses rapamycin-resistant mTORC1 outputs and prevents activation of Akt resulting from feedback regulation. mTORC1/2 inhibition caused selective growth suppression of cancer cells, due to the inhibition of the PI3-K/Akt/mTOR pathway. Rapamycin incompletely inhibits mTOR in that it binds a non-catalytic binding site, leaving the active site unchanged. On the other hand, PP242 represents a member of a new generation of compounds that inhibit mTOR via an ATP-competitive mechanism. Compared with rapamycin, PP242 more efficiently inhibits mTORC1 as evidenced by diminished S6K phosphorylation. Unlike rapamycin, PP242 nearly abolishes activation of Akt, thus inhibiting the mTORC2 complex (unpublished observation). Notably, prolonged exposure to allosteric mTOR inhibitor rapamycin caused activation of a mitogenic pathway due to alteration of signaling feedback loops (Albert et al., 2009). At the range of IC50 dose (2.2 mM) of PP242, GBM cell proliferation was strongly suppressed in vitro. PP242 is more effective in preventing the occurrence of negative feedback loops by enhancing its inhibitory effect on mTORC2. Therefore, PP242 is a more potent inhibitor of proliferation and migration of GBM cells. Our findings indicate that ATP kinase mTOR inhibitors may have far superior effects by virtue of their ability to fully inhibit rapamycin sensitive and insensitive complexes. Dual inhibitor of PI3-K/mTORC1, PI-103, partially suppressed S6K phosphorylation, but did not suppress 4E-BP activation (Fig. 1A). This compound demonstrated results similar to PP242 as it reduced cell migration when compared to the control. We also studied WYE-354 (mTORC1/2 inhibitor), which suppressed cell proliferation with the same degree as PP242 over the period of 4 days, while KU-0063794 was less effective (Fig. 1C). In our study, KU-0063794 suppressed cell viability, more than rapamycin, but noticeably less than PP242 (Fig. 1B). While structurally KU-0063794 and WYE-354 are distinct, mechanistically they suppress the growth of cancer cells by G1 arrest. The existence of an advantage of mTORC1/2 specific inhibitors over panePI3-K/mTORC1/2 pathway inhibitors remains to be fully defined. Several studies have provided evidence that PI3-K/mTORC1/2 inhibitors (PI-103) have effectively suppressed downstream pathways in many cancers (Knight et al., 2006). PI3-K has numerous roles in cell survival, differentiation, metabolism and migration, some of which are independent of Akt and mTOR. Findings of this study suggest that whereas mTORC1/2 and mTORC1 inhibitors affected proliferation and migration, the combined mTORC1 and mTORC2 inhibitors were most effective. The induction of mRNA translation by mTORC1 is mediated by the interaction of downstream S6K and the eukaryotic translation initiator factor 4E-BP1. Feedback loop regulation of PI3-K/mTOR signaling has a significant impact on therapeutic responses, in particular on the monotherapy groups. For example, activation of S6K by mTORC1 causes feedback inhibition of IGF1/insulin signaling by phosphorylating IRS-1 (insulin receptor substrate 1), causing IRS-1 degradation, and leading to decreased PI3-K signaling and reduced AktT308 phosphorylation. However, rapalogue-

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induced inhibition of mTORC1 consequently inhibits S6K phosphorylation, releasing negative feedback, inducing AktT308 re-phosphorylation and thus increasing mTORC2 activation (Jhanwar-Uniyal et al., 2011, 2013). Under physiologic conditions, S6K can inhibit Akt through feedback via IRS-1 (Sabatini, 2006); however, below threshold levels of S6K achieved following inhibition of the mTOR pathway can be tumorigenic (Riemenschneider et al., 2006). However, it is difficult to assess the degree of ultimate threshold level of S6K that is responsible for tumorigenic action (Fig. 2C). There may be alternative ways by which Akt is regulated by mTORC1 (Kubica et al., 2008). We previously demonstrated that ATP-binding inhibitors, unlike rapamycin, remain less effective in releasing this feedback loop (unpublished observation). Summary mTOR integrates extracellular growth signals and nutrients to regulate cell proliferation, migration, growth, autophagy, metabolism, and survival. mTOR protein kinase interacts with multiple proteins via its two distinct multiprotein complexes, mTORC1 and 2, to regulate the aforementioned functions. This pathway is deregulated in multiple tumor types. Inhibition of this pathway carries a significant therapeutic value. Rapamycin and its derivatives, which inhibit via an allosteric mechanism, are found to be incomplete inhibitors of mTORC1, and a major activator of mitogenic pathways. Therefore, development of novel ATP-binding compounds that inhibit mTORC1/2 in physiological as well as molecular levels would provide a selective inhibition of this activated pathway. Importantly, the development of ATP-binding inhibitors would prove to be more effective by their ability to dephosphorylate activated PRAS40, thereby reinstating its intrinsic inhibition of mTORC1. Acknowledgments We gratefully acknowledge the funds from Advanced Research Foundation. We thank Archana Marathi for technical help. References Albert L, Karsy M, Murali R, Jhanwar-Uniyal M. Inhibition of mTOR activates the MAPK pathway in glioblastoma multiforme. Cancer Genomics Proteomics 2009;6:255e61. Brescia P, Ortensi B, Fornasari L, et al. CD133 is essential for glioblastoma stem cell maintenance. Stem Cells 2013;31(5):857e69. Castilho RM, Squarize CH, Chodosh LA, et al. mTOR mediates Wnt-induced epidermal stem cell exhaustion and aging. Cell Stem Cell 2009;5(3):279e89. Chan JA, Zhang H, Roberts PS, et al. Pathogenesis of tuberous sclerosis subependymal giant cell astrocytomas: biallelic inactivation of TSC1 or TSC2 leads to mTOR activation. J Neuropathol Exp Neurol 2004;63(12):1236e42. Chang SM, Wen P, Cloughesy T, et al. Phase II study of CCI-779 in patients with recurrent glioblastoma multiforme. Investig New Drugs 2005;23:357e61. Chen CH, Sarbassov dos D. The mTOR (mammalian target of rapamycin) kinase maintains integrity of mTOR complex 2. J Biol Chem 2011;286(46):40386e94. Choi J, Chen J, Schreiber SL, et al. Structure of the FKBP12erapamycin complex interacting with the biding domain of human FRAP. Science 1996;273(5272):239e42. Cloughesy TF, Yoshimoto K, Nghiemphu P, et al. Antitumor activity of rapamycin in a phase I trial for patients with recurrent PTEN-deficient glioblastoma. PLoS Med 2008;5:e8. Dancey JE, Curiel R, Purvis J. Evaluating temsirolimus activity in multiple tumors: a review of clinical trials. Semin Oncol 2009; 36(Suppl. 3):S46e58. Darb-Esfahani S, Faggad A, Noske A, et al. Phospho-mTOR and phospho-4EBP1 in endometrial adenocarcinoma: association with stage and grade in vivo and link with response to rapamycin treatment in vitro. J Cancer Res Clin Oncol 2009;135(7): 933e41. Dorrello NV, Peschiaroli A, Guardavaccaro D, et al. S6K1- and beta TRCP-mediated degradation of PDCD4 promotes protein translation and cell growth. Science 2006;314(5798):467e71. Ducker GS, Atreya CE, Simko JP, et al. Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors. Oncogene 2014;33:1590e600. Eshleman JS, Carlson BL, Mladek AC, Kastner BD, Shide KL, Sarkaria JN. Inhibition of the mammalian target of rapamycin sensitizes U87 xenografts to fractionated radiation therapy. Cancer Res 2002;62:7291e7. Feldman ME, Apsel B, Uotila A, et al. Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol 2009;7:e38. Fragoso R, Barata JT. PTEN and leukemia stem cells. Adv Biol Regul 2014;56:22e9. Friedman MD, Jeevan D, Tobias M, et al. Targeting cancer stem cells of glioblastoma multiforme using mTOR inhibitors and differentiating agent all-trans retinoic acid. Oncol Rep 2013;30(4):1645e50.

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