Tumor Biology

, Volume 34, Issue 4, pp 1991–2002

Targeting the PI3K/AKT/mTOR signaling pathway in glioblastoma: novel therapeutic agents and advances in understanding

Authors

  • Arshawn Sami
    • Department of Radiology and Imaging SciencesEmory University School of Medicine
    • Department of PathologyNew York Medical College
    • Department of NeurosurgeryNew York Medical College
Review

DOI: 10.1007/s13277-013-0800-5

Cite this article as:
Sami, A. & Karsy, M. Tumor Biol. (2013) 34: 1991. doi:10.1007/s13277-013-0800-5

Abstract

Glioblastoma multiforme (GBM) is a grade IV astrocytoma with a median survival of 12 months despite current multi-modal treatment options. GBM is distinguished clinicopathologically into primary and secondary subtypes. Mutations of phosphatase and tensin homolog, and subsequent upregulation of the downstream protein kinase B/mammalian target of rapamycin (mTOR) signaling pathway, are commonly seen in primary GBM and less predominantly in secondary GBM. While investigations into targeted treatments of mTOR have been attempted, feedback regulation within the mTOR signaling pathway may account for therapeutic resistance. Currently, rapamycin analogs, dual-targeted mTOR complex 1 and 2 agents as well as dual mTOR and phosphatidylinositol-3 kinase-targeted agents are being investigated experimentally and in clinical trials. This review will discuss the experimental potential of these agents in the treatment of GBM and their current stage in the GBM drug pipeline. Knowledge obtained from the application of these agents can help in understanding the pathogenesis of GBM as well as delineating subsequent treatment strategies.

Keywords

GlioblastomamTORAKTPI3KTargeted therapy

Introduction

Glioblastoma multiforme (GBM) is a disease with uniformly poor prognosis, which has been traditionally categorized into primary and secondary subtypes based on clinicopathological features. Common alterations in primary GBM include phosphatase and tensin homolog (PTEN), phosphatidylinositol-3 kinases (PI3K), and epidermal growth factor receptor (EGFR) seen in approximately 30 % of GBMs each [1]. Furthermore, resultant deregulation of the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway from these alterations has shown to be an important determinant of gliomagenesis [24]. However, clinical trials with mTOR inhibitor rapamycin and its analogs have failed to achieve a significant therapeutic effect. Mechanisms that have been suggested to account for this therapeutic resistance include feedback loops, parallel signaling pathways, and limited drug targeting of mTOR [510]. Recent development of second-generation mTOR-targeted agents such as dual mTOR complex 1 (mTORC1)/mTOR complex 2 (mTORC2) or dual mTOR/PI3K inhibitors have been proposed, with several undergoing clinical trial currently in progress [11, 12]. Studies have also highlighted the close interaction between the tumor suppressor p53 and PTEN/AKT/mTOR signal transduction pathways implicating a complex regulatory network, which may affect tumor development and therapeutic targeting [13, 14]. This review is intended to discuss the recent findings regarding targeting of the mTOR signaling pathway in GBM. (Fig. 1)
https://static-content.springer.com/image/art%3A10.1007%2Fs13277-013-0800-5/MediaObjects/13277_2013_800_Fig1_HTML.gif
Fig. 1

Targeting the mTOR signaling pathway. The mTOR protein exists in two protein complexes, namely mTORC1 (mTOR, PRAS40, LST8, Deptor, and Raptor) and mTORC2 (mTOR, mSIN1, Protor, LST8, Deptor, and Rictor). Feedback inhibition stemming from sustained inhibition of mTORC1 signaling promotes activation of the mTORC2/AKT and ERK1/2 pathways via IRS/PI3K. ERK1/2 feedback on mTOR pathway signaling can occur by inhibiting TSC1. TP53 can influence the mTOR pathway by regulating PTEN, TSC2, and PI3K expression as well as other mechanisms. The mTOR pathway can influence p53 levels by regulating MDM2 expression downstream of AKT. Mutant p53 (mtp53) alterations include: dominant-negative inhibition (inhibition of wild-type p53 by tetramerization), gain-of-function (genetic transactivation at non-wild-type p53 response elements), and loss-of-function (loss of endogenous wild-type p53 function). Drugs and their targeting of mTORC1, mTOR/PI3K, and mTORC1/mTORC2 are shown. AKT protein kinase B, ATRA all-trans retinoic acid, ATO arsenic trioxide, Deptor DEP domain containing MTOR-interacting protein, ERK1/2 extracellular regulated kinase/mitogen activated protein kinase, FKBP FK506-binding protein 12, Grb growth factor receptor-bound protein, IRS insulin response signal, PDK1 3-phosphoinositide-dependent kinase 1, PI3K phosphoinositide 3-kinase, PIP2 phosphoinositol-2-phosphate, PIP3 phosphoinositol-3-phosphate, PTEN phosphatase and tensin homolog, MEK1/2 MAPK kinase, mTOR mammalian target of rapamycin, Raptor regulatory-associated protein of mTOR, Rictor Rapamycin-insensitive companion of mTOR, RTK tyrosine receptor kinase, SOS son of sevenless homolog, sox2 SRY (sex-determining region Y)-box 2, TSC1/TSC2 tuberous sclerosis protein 1/tuberous sclerosis protein 2

mTOR pathway signaling and regulation

Regulation of cellular growth and proliferation in GBM occurs via integration of growth factor signaling, intracellular metabolic requirements, and extracellular signaling molecules through the PTEN/AKT/mTOR signaling pathway. Membrane-bound RTK, including platelet-derived growth factor receptor (PDGFR), EGFR, and insulin-like growth factor 1 receptor (IGFR), receive growth factor and nutrient inputs resulting in receptor phosphorylation and membrane recruitment of PI3K [15, 16]. PI3K phosphorylates phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) in order to further recruit AKT to the membrane where AKT can be phosphorylated at Thr308 by phosphoinositide-dependent protein kinase 1 and at Ser473 by mTORC2/rapamycin-insensitive companion of mTOR (Rictor) for full AKT activation. Mutations of various class 1A isoforms of PI3K, namely PIKCA and PIK3R1, can be mutated and result in constitutive pathway activation [1719]. PTEN dephosphorylation of PIP3 to PIP2 acts as a major inhibitor of the pathway and when mutated or lost, cannot be compensated for by any other enzyme. Mutation of PTEN results in constitutive activation of the AKT pathway, which can influence multiple downstream targets involved in angiogenesis, cell survival, invasion, proliferation, cell cycle, and gliomagenesis. Some targets for AKT include MDM2, glycogen synthase kinase 3 beta, p21, p27, Bad, Raf1, tuberous sclerosis protein (TSC)1, eNOS, and I-kappaB kinase [16, 20, 21]. Activation of AKT can also occur by constitutive activation of upstream growth factor receptors, mutations in the catalytic PI3K subunit, or overexpression of AKT isoforms but these are uncommon mechanisms in GBM [22].

One of the major downstream signaling targets of AKT is mTOR, a critical effector of cell signaling pathways generally deregulated in a variety of cancers including GBM [2325]. The mTOR protein exists in two multiprotein complexes, mTORC1 and mTORC2. The mTORC1 is comprised of regulatory-associated protein of mTOR (Raptor), mTOR, mammalian LST8/G-protein β-subunit like protein (GβL), PRAS40, and DEP domain containing mTOR-interacting protein (DEPTOR). Furthermore, mTORC2 is comprised of Rictor, mTOR, GβL, and mammalian stress-activated protein kinase interacting protein 1 (mSIN1). Activation of mTORC1 results in phosphorylation of S6 kinases (S6K1 and S6K2) and eukaryotic initiation factor 4B that control translation as well as programmed cell death 4 protein (PDCD4) [2628]. MTORC1 activation also phosphorylates 4E binding proteins, which are a family of proteins involved in repressing cap-dependent mRNA translation of cyclins, MYCN, Bcl-xl, S6, PDCD4, and others [29, 30]. MTORC1 integrates various inputs including growth factors, energy status, oxygen, and amino acid levels to regulate cell growth by promoting biosynthesis of proteins, lipids, and organelles as well as limiting catabolic autophagic activity [31]. Activation of mTORC2 results in phosphorylation of AKT, SGK1, and PKC involved in cell survival, metabolism, proliferation, and cytoskeletal organization.

Pathway crosstalk and feedback

Complex upstream and downstream regulation of mTOR suggests this molecule serves as a focal point in metabolic regulation as well as a regulator of various pathological processes. Regulation of the mTORC1 complex occurs via the Ras-related GTPase (Rag) and Ras homolog enriched in brain (Rheb), as well as factors that signal through mTORC1 directly [32, 33]. Rag is involved in sequestering mTORC1 to the endomembrane, which is required for mTORC1 activation [34], and regulation of Rheb occurs via the TSC composed of TSC1 (hamartin) and TSC2 (tuberin) [33]. TSC1 stabilizes the complex, while TSC2 acts as a GTPase-activating protein for Rheb. TSC2 normally inhibits Rheb by converting it into its inactive GDP-bound state, thus preventing Rheb from interacting with mTORC1 to stimulate its activity [35]. Various upstream signaling molecules impinge on TSC1/TSC2 including those involved in signaling of metabolism and hypoxia, such as AMP-activated protein kinase (AMPK), PI3K/AKT, mitogen-activated protein kinase (MAPK) (ERK1/2) [11, 24, 36].

Recent studies have highlighted the interconnections between the p53 and mTOR pathways in a tissue-specific manner [13, 14]. Glucose starvation has been shown to induce AMPK-mediated phosphorylation of specific sites in the N-terminus, such as Ser15, where further phosphorylation at adjacent sites (Ser20, Ser33, Ser46, and Thr18) can promote p53-mediated transcription [37]. In addition, induction of protein phosphatase 2A in an mTOR-dependent manner can generate a Ser15-specific p53 phosphatase that downregulates p53 [38]. Thus, AMPK forms a rapid positive feedback loop while mTOR forms a rapid negative feedback loop in order to regulate p53 expression. Furthermore, AMPK can suppress mTOR by phosphorylating upstream TSC2 [39] and downstream AKT, which has been shown to activate MDM2 [31], suggesting another pathway of p53 regulation. Similarly to how p53 regulates cell cycle, activated AKT can also inhibit FOXO transcription factors involved in cell cycle regulation [40].

While these loops act in a rapid manner (few hours), p53 interaction with the mTOR signaling pathway also occurs on a slower time scale (12–24 h). Various studies have indicated that p53 response elements exist for IGF-BP3 [41], PTEN [42], AMPKβ1 [37], TSC2 [37], and Sestrin1/2 [43]. Expression of IGF-BP3 results in binding of free insulin-like growth factor 1 (IGF-1), which downregulates IGF-1/AKT signaling [41]. PTEN degrades PIP3 to PIP2 in order to downregulate AKT, resulting in increased TSC1/2 activity and decreased mTORC1 activity [42]. AMPKβ1 is the scaffold protein for AMPK, resulting in increased AMPK formation and activity thus resulting in reduced TSC1/2 activity [37]. Sestrin1/2 upregulation can act through an AMPK-TSC2 pathway to inhibit mTORC1 [43]. Both rapid and slow interactions between p53 and mTOR serve to regulate multiple cell processes coordinating cell cycle, transcription, and translation. Upregulation of the p53 pathway generally suppresses the mTOR pathway and vice versa, indicating precise control of DNA synthesis and protein translation activities in cells. Disruption of either pathway may show complex feedback-mediated dysregulation during neoplastic development.

mTOR signaling aberrations in GBM

PTEN mutation or loss of heterozygosity (LOH) is a common finding in GBM, which may promote tumorigenesis via upregulation of the AKT signaling pathway. PTEN is the second most common sporadically mutated tumor suppressor (p53 being the first), with mutations of PTEN occurring in 15–40 % of GBM [1, 44]. PTEN mutations are found in approximately 25 % of primary GBM and 5 % of secondary GBM while 10q LOH is similar between subtypes [45]. Furthermore, increased AKT phosphorylation is seen in most GBM tumors despite the lack of detected PTEN mutation in many cases, suggesting that inactivation of PTEN may be more predominant than previously believed [46]. This may be due to prior incomplete evaluation of PTEN mutations as well as epigenetic silencing of PTEN promoters, which have been demonstrated in GBM and low-grade glioma [47, 48]. Recent studies have also suggested that microRNAs (miR), small mRNA silencing molecules, may play a role in silencing PTEN or other components of the mTOR signaling pathway [49]. These mIRs include miR-21 and miR-26a/b, which target PTEN, as well as miR-7, miR-128, and miR-146-5p, which can target EGFR in GBM. Haploinsufficiency of PTEN or other mechanisms of PI3K activation may also be involved in promoting LKB1/AKT signaling.

PTEN and its downstream mTOR signaling pathway may regulate cancer stem cell (CSC) self-renewal and differentiation. PTEN deletion has been shown to increase cell size and proliferation, as well as decrease apoptosis of neural progenitor cells [50, 51]. Furthermore, abnormal migration of progenitor cells with PTEN mutation has been observed and thereby resulting in dysplasia of the cerebellum and hippocampus [52]. This may contribute to gliomagenesis; however, PTEN mutation alone is unable to produce neoplasia and often requires additional mutations, such as p53, in order to influence gliomagenesis [3, 53, 54]. PTEN loss diminished growth factor-dependent proliferation and increased G0–G1 cycling of neural stem cells (NSCs) [2]. Furthermore, PTEN loss upregulated genes involved in cell cycle (cyclins) and DNA replication (Ki-67, DNA primases) as well as genes involved in stemness, cell differentiation, and metabolism of NSCs. Evaluation of NSCs showed greater neurosphere formation and self-renewal with PTEN loss, but no alteration in differentiation [51, 52]. In PTEN-null GBM cell lines, reexpression of PTEN showed suppression of proliferation due to G1 cellular arrest dependent on p27kip1 expression as opposed to induction of apoptosis [55, 56]. Reduced in vivo tumor growth was also seen upon reconstitution of PTEN in GBM cells due to reduced AKT-dependent angiogenesis [57]. Other attributes of CSCs such as dissemination are also regulated by PTEN. Inhibitors of PI3K or overexpression of PTEN show reduced in vitro cell migration, invasiveness, and expression of MMP2 and MMP9 [58, 59].

First-generation mTOR-targeted agents in GBM

Targeting of mTOR in GBM has shown promise as a mode of therapy but has largely failed clinical trials. Identification of mechanisms of resistance to mTOR inhibitors has not only elucidated signaling mechanisms in cancers but has led to the development of newer, more potent inhibitors. Furthermore, the role of this significant pathway in GBM formation and dissemination suggests that targeted therapies may continue to be a promising area of investigation.

Rapamycin was identified as an antifungal in 1975 from the soil fungus Streptomyces hygroscopicus on the coast of Easter Island, also known as Rapa Nui, and underwent development as an immunosuppressant by Wyeth in 1997 prior to being investigated for antitumor properties [11, 60]. Various rapamycin analogs (Rapalogs) have been generated with better hydrophilic solubility and bioavailability, including temsirolimus (CCI-779, Wyeth), everolimus (RAD001, Novartis), ridaforolimus (AP235373, Ariad Pharmaceuticals/Merck), and nab-rapamycin (Abraxis Bioscience) (Table 1) [5, 6, 6164]. Rapamycin binds to FK-binding protein 12 (FKBP12) and inhibits the FKBP12-rapamycin domain of mTOR through allosteric inhibition [11]. Rapamycin predominantly inhibits mTORC1; however, prolonged exposure in some cells can inhibit mTORC2 [65]. Rapamycin has shown clinical success in mantle cell lymphoma, renal cell carcinoma, and endometrial cancers, but has largely failed in trials on breast carcinoma, and GBM [66]. Phase II studies of rapamycin in GBM demonstrated significant radiographic improvement of disease; however, overall survival and progression-free survival was not significantly improved despite accounting for responding patients, previous treatments, and biomarkers (pp70S6K, pAKT) of rapamycin efficacy [5, 6].
Table 1

First-generation mTOR-targeted agents

Agent name

Company

Clinical trial stage

Tumor type

References

Temsirolimus (Torisel), CCI-779

Wyeth

Phase 2

Glioblastoma

[5, 6, 12, 61, 79]

Sirolimus (Rapamune), Rapamycin

Wyeth

Phase 1

Glioblastoma

 

Phase 1

Astrocytoma

[63]

Everolimus (Afinitor), RAD-001

Novartis

Phase 1

Glioblastoma

[62, 80]

Phase 1

Astrocytoma

[63]

Ridaforolimus, AP23573, MK-8669, Deforolimus

Ariad Pharmaceuticals/ Merck

Phase 1

Glioblastoma

[64]

Nab-rapamycin

Abraxis Bioscience

   

Recent studies have elucidated a variety of mechanisms regarding therapeutic resistance to rapamycin and Rapalogs, including negative feedback loops, multiple parallel signaling pathways in GBM, and lack of specificity in targeting mTOR. Tumor treatment with rapamycin suppressed mTORC1 activation but paradoxically induced activation of mTORC2 via a negative S6K1/insulin receptor substrate 1 (IRS-1)/PI3K feedback loop [79, 67, 68]. S6K1, downstream of mTORC1, inhibits IRS-1 which mediates suppression of PI3K where PI3K is a focal point regulating multiple downstream molecules, including the Ras/ERK as well as PTEN/AKT signaling pathways [9, 69]. Rapamycin treatment in GBM can induce AKT activation via a loss of negative feedback thereby suggesting a mechanism for therapeutic resistance [70]. Hyperactivation of PI3K and its downstream effectors can upregulate signaling pathways affecting a variety of pro-tumorigenic functions, including cell cycle, growth, proliferation, self-renewal, and migration. Some studies have shown that activation of AKT can occur by other convergent pathways. One mechanism includes autocrine secretion of IGF-1 which suggests that inhibition of multiple targets in the PI3K/AKT/mTOR signaling pathway may be necessary for therapy [71]. Oncogenic dependence on AKT signaling is not seen in GBM, and parallel signaling pathways may be utilized to resist targeted therapy [69, 72]. In addition, rapamycin has been shown to reduce approximately 50 % of overall protein translation mediated by mTOR [73]. However, reduction in translation has been shown to depend on 5′UTR secondary structures and internal ribosome entry sites in translated mRNA, implicating a more complex regulation in oncogenic signaling.

Despite limitations in treatment, Rapalogs have been evaluated in combination with other targeted and chemotherapeutic agents (Table 1). Recent studies highlighted the potential synergism between EGFR and mTOR inhibition, and various clinical trials have investigated combinations such as gefitinib and everolimus [61, 62]. Furthermore, a combination of EGFR inhibitors erlotinib or gefitinib and traditional radiochemotherapy has been evaluated [7476]. However, these combination therapies have failed to result in long-standing improvements in prognosis. One possible mechanism may involve mutations in PTEN, which may promote resistance to EGFR inhibitors [77]. Along with feedback loops and parallel mitotic signaling pathways, multiple methods of overcoming targeted treatment are possible [8, 9]. High levels of EGFR and low levels of activated AKT are associated with improved response to EGFR inhibitors, suggesting improved patient stratification may be necessary for further trials [78]. And despite the improvement in Rapalog pharmacokinetics, limited drug targeting of tumors remains [10]. Recent studies have evaluated the combination of radiation and temozolomide treatment Rapalogs (e.g., temsirolimus, everolimus) showing increased immunotoxicity which may limit therapeutic dosages as well as necessitate novel dosing strategies [79, 80].

Second-generation mTOR-targeted agents in GBM

Due to the limitations in therapy with Rapalogs, more specific inhibitors as well as dual inhibitors have been evaluated (Tables 2 and 3). Newer ATP-competitive mTOR inhibitors have demonstrated greater potency in inhibiting mTORC2 and upstream kinase PI3K along with mTORC1 [11]. However, these agents, especially targeting PI3K, have been met with some limitation in predominantly inducing growth arrest as opposed to inducing cell death [81].
Table 2

Dual PI3K/mTOR inhibitors currently being evaluated in GBM

Agent name

Company

Clinical trial stage

Tumor type

References

PI-103

Merck

Phase 0

Glioblastoma

[88, 90, 91, 124]

XL-765

Exelixis

Phase 1

Glioblastoma

[12, 97]

Phase 0

Glioblastoma

[97]

NVP-BEZ235

Novartis

Phase 0

Glioblastoma

[11, 9296]

PKI-587

Wyeth-Ayerst

Phase 0

Glioblastoma

[98]

PKI-179

Wyeth

Phase 0

  

GSK-2126458

GlaxoSmithKline

Phase 0

  

SF-1126

Semafore

Phase 0

  

GDC-0980

Genentech

Phase 0

  

PF04691502

Pfizer

Phase 0

  

PWT-33597

Pathway Therapeutics

Phase 0

  

GNE-477

Genentech

Phase 0

  

WJD-008

    
Table 3

Dual mTORC1/mTORC2 inhibitors currently being evaluated in GBM

Agent name

Company

Clinical trial stage

Tumor type

References

KU0063794

AstraZeneca

Phase 0

Glioblastoma

[88]

AZD-8055

AstraZeneca/KudOS

Phase 1

Glioblastoma

[12, 99]

WAY-600

Wyeth

Phase 0

Glioblastoma

[100]

WYE-687

WYE-354

INK-128

Intellikine

Phase 0

  

OSI-027

OSI Pharmaceuticals

Phase 0

  

XL-388

Exelixis

Phase 0

  

P2281

Piramal Life Science

Phase 0

  

P529 (Palomid 529)

Paloma Pharmaceuticals

Phase 0

  

Torin1

Gray/Sabatini Laboratory

Phase 0

  

PP242

University of California

Phase 0

  

PP30

University of California

Phase 0

  

WYE-125132 (WYE-132)

Wyeth

Phase 0

  

PI103

The dual mTOR/PI3K inhibitor PI103 (Merck) was the first mTOR inhibitor that also possessed simultaneous target inhibition and also inhibited mTOR in an ATP-competitive manner [82]. PI103 is a potent, cell-permeable, ATP-competitive inhibitor of the PI3K family that has shown potent synergy with the EGFR inhibitor erlotinib in glioma tumor suppression without observable toxicity [83]. However, the poor pharmacokinetic properties of PI103 have led this drug to serve as a lead compound for the development of other agents [84]. Compounds developed from PI103 included a variety of mTOR and PI3K inhibitors showing potent effects in GBM including: KU0063794, a dual mTORC1/mTORC2 inhibitor; GDC-0941, a PI3K inhibitor, and NVP-BEZ235 [8588] (Table 4). PI103 has been shown the ability to induce autophagy in glioma and thereby support therapeutic resistance through this self-digestion mechanism [89]. However, this study showed that PI103 synergized with the inhibition of autophagosome maturation to promote glioma apoptosis via a Bax-dependent intrinsic mitochondrial pathway. PI103 has also been shown to combine synergistically with doxorubicin to increase GBM apoptosis and reduce colony formation of GBM stem cells [90]. Other studies have similarly shown reduced GBM cell proliferation after PI103 treatment [91].
Table 4

PI3K inhibitors currently being evaluated in GBM

Agent name

Company

Clinical trial stage

Tumor type

References

Perifosine (KRX0401)a

Aeterna Zentaris Inc./ Keryx Biopharmaceuticals

Phase 1

Glioblastoma

[12, 112115]

XL-147

Exelixis

Phase 1

Glioblastoma

[12]

XL-765

Wortmannin

 

Phase 0

Glioblastoma

[106]

LY294002

 

Phase 0

Glioblastoma

[104, 105]

PX-866

Oncothyreon

Phase 0

Glioblastoma

[108110]

AMG-511

Amgen

Phase 0

Glioblastoma

[119]

NVP-BKM120

Novartis

Phase 0

Glioblastoma

[111]

ZSTK-474

Zenyaku Kogyo Co.

Phase 0

Glioblastoma

[120]

CH5132799

Chugai Pharmaceutical Co.

Phase 0

Glioblastoma

[118]

GDC-0941

Genentech

Phase 0

Glioblastoma

[117]

IC-87114

ICOS Co.

Phase 0

Glioblastoma

[121]

AS-605240

 

Phase 0

Glioblastoma

[122]

GNE-317

Genentech

Phase 0

Glioblastoma

[116]

P-529

Paloma Pharmaceuticals

Phase 0

Glioblastoma

[123]

TGX-221

Kinacia Pty Ltd

Phase 0

  

INK-1117

Takeda

Phase 0

  

IPI-145

Infinity Pharmaceuticals

Phase 0

  

TG100-115

    

CAL-263

Calistoga Pharmaceuticals

   

CUDC-907

Curis Inc.

   

AEZS-136

Aeterna Zentaris

   

aPerifosine has shown dual PI3K and AKT inhibitor effects

NVP-BEZ235

NVP-BEZ235 (Novartis), a simultaneous PI3K family and mTOR inhibitor, has shown efficacy in GBM and is currently in phase 1 trials for a variety of solid tumors [12, 92]. In one study, NVP-BEZ235 demonstrated suppression of mTORC1 (e.g., S6K1, S6, and 4EBP1) and mTORC2 (e.g., AKT) downstream components resulting in cell cycle arrest and induced autophagy [11]. Furthermore, this study showed a decreased expression of vascular endothelial growth factor as well as an antiangiogenic effect in vivo. In another study, NVP-BEZ235 showed inhibited in vivo glioma tumor proliferation and improved antitumor effects compared to rapamycin analogs [87]. NVP-BEZ235 also reduced sphere formation and expression of stem cell markers in cancer stem-like cells of GBM [93, 94]. Furthermore, this group showed increased expression of differentiation markers and decreased tumorigenicity in orthotopic and heterotopic zenograft models. Combination therapies with NVP-BEZ235 have also been explored. One strategy to overcome resistance to dual PI3K-mTOR inhibition utilized NVP-BEZ235 with autophagy inhibitor chloroquine to show a synergistic increase in in vivo tumor apoptosis [89]. In another study, NVP-BEZ235 was shown to inhibit the DNA repair proteins ATM and DNA-PKC in GBM thought to mediate resistance to ionizing radiation [95]. Furthermore, this study showed that NVP-BEZ235 inhibited double strand DNA break repair in vivo and may thereby have a synergistic relationship with radiotherapy in humans. NVP-BEZ235 has also been shown to sensitize xenograft GBM tumor models to radiation, which depended on its ability to induce autophagy [96].

XL-765

XL-765 (Exelixis), a dual PI3K/mTOR inhibitor, was shown in combination with temozolomide to possess potency against GBM [97]. This study showed inhibition of in vitro and in vivo tumor cell growth along with downregulation of PI3K and mTOR signaling. Furthermore, the in vivo animal tumor models showed significantly tumor reduction and improved survival with monotherapy as well as a synergistic effect with combinatorial therapy. XL-765 is also currently undergoing trials in combination with radiotherapy and temozolomide for GBM as well as in subjects with recurrent GBM [12].

Other PI3K/mTOR-targeted agents

The dual PI3K/mTOR inhibitor PKI-587 (Merck) has been investigated in a variety of cancers and has been shown to suppress tumor growth and AKT phosphorylation in glioma in vivo xenograft models [98]. But limited evaluation of this compound has been seen in clinical trial for GBM. Various dual PI3K/mTOR-targeted agents that have been explored in other cancers but not yet in laboratory or clinical studies with GBM include: GSK2126458 (GlaxoSmithKline), SF1126 (Semafore), GDC0980 (GlaxoSmithKline), and PF04691502 (Pfizer).

Other mTORC1/mTORC2-targeted agents

The dual mTORC1/mTORC2 inhibitor KU0063794 (AstraZeneca) demonstrated inhibition of S6 and induced autophagy more potently than rapamycin but did not induce apoptosis [89]. However, this study showed that the addition of PI3K inhibitor PIK90 and KU0064794 induced apoptosis. AZD-8055 (AstraZeneca/KudOS) showed efficacy in reducing S6 phosphorylation at Ser235/236 and AKT at Ser473 as well as reduction of in vivo tumor growth [99]. Furthermore, current clinical trials of this drug in adults with recurrent gliomas are under investigation (clinicaltrials.gov). Similarly, the drugs WAY-600, WYE-687, and WYE-354 (Wyeth) were shown to inhibit in vivo GBM tumor growth as well as suppress S6KT389 and AKTSer473 activation [100]. Other dual mTORC1/mTORC2 inhibitors that have not yet undergone laboratory or clinical studies in GBM include INK128 (Intellikine), OSI027 (OSI Pharmaceuticals), XL388 (Exelixis), P2281 (Piramal Life Science), P529 (Paloma Pharmaceuticals), Torin 1 (Gray/Sabatini Laboratory), PP242 (University of California), PP30 (University of California), and WYE-125132/WYE-132 (Wyeth).

PI3K-targeted agents in GBM

Initially discovered as a potent oncovirus mediator of tumorigenesis, PI3K has emerged as a potential target in GBM due to tumor resistance to Rapalogs in clinical trials as well as its critical role in gliomagenesis [17, 18]. Similarly, AKT inhibitors have also emerged as potential therapeutic targets due to their regulation of multiple downstream pathways [101]. Combined treatments of mTOR and these targets have been suggested to overcome the therapeutic resistance of single inhibitors but may be limited by overall toxicity due to the critical upstream position of PI3K and AKT.

PX-866

Previous studies evaluating the PI3K inhibitors LY294002 and Wortmannin demonstrated potent reduction of cancer cell proliferation [102, 103]. Despite LY294002 showing efficacy in priming apoptosis [104] or synergizing with temozolomide in GBM [105], it has significant toxicity thereby limiting their clinical efficacy. Similarly, Wortmannin has shown synergism with radiation in suppressing spheroid growth of GBM, but possesses significant clinical toxicity [106]. A second-generation PI3K inhibitor that has been developed with improved bioavailability and minimal toxicity is PX-866 (Oncothyreon) [107]. PX-866 was shown to inhibit GBM proliferation, increase animal survival after orthotopic xenografted tumors, and decrease both signaling downstream of mTORC1 and mTORC2 [108]. Furthermore, this study showed that PX-866 reduced GBM angiogenesis and invasion, induced autophagy, as well as normalized the tumor metabolic profile (e.g., choline/NAA ratio) as assessed by magnetic resonance spectroscopy. In another study, the combination of polynuclear platinum-based therapy (e.g., BBR3610) with the PI3K inhibitor PX-866 was shown to synergistically inhibit glioma cell proliferation and migration well as improved survival in orthotopic xenografted tumor models [109]. Furthermore, these combined agents induced autophagy but not apoptosis, as well as decreased pAKT and pBad expression. Interestingly, the results of these studies were reported in a variety of GBM cell lines with different PTEN and p53 mutations, thereby suggesting that PX-866 may show clinical potency in a variety of tumor types. PX-866 was also shown to inhibit cell proliferation of GBM in both monolayer as well as three-dimensional spheroids [110]. Currently, PX-866 is in clinical trials for recurrent or progressed GBM [12].

NVP-BKM120

A recent pan-PI3K inhibitor, NVP-BKM120 (Novartis), has been investigated in GBM. A recent study showed the ability of BKM120 to inhibit GBM cell proliferation and improve median survival of an in vivo tumor model [111]. Furthermore, this study showed that BKM120 induced apoptosis regardless of PTEN or EGFR status and instead generated apoptosis in p53 wild-type cells vs. mitotic failure in cells with p53 mutation.

Perifosine

Perifosine (KRX0401, Aeterna Zentaris Inc./Keryx Biopharmaceuticals) is a dual PI3K/AKT inhibitor, which has shown limited success in clinical trials for a variety of tumors including colorectal cancer and multiple myeloma. In one study, perifosine can inhibit both AKT and MAPK signaling in mouse glial progenitors in vitro causing cell cycle arrest in G1 and G2 [112]. However, despite impacts on normal cells, this study showed that perifosine synergized with temozolomide to suppress tumor growth of a xenograft model, suggesting that a targeted tumor therapy could be feasible. In a study using the in vivo ANLucBCLuc apoptosis imaging reporter in GBM, perifosine was seen to increase bioluminescence intensity in a dose-dependent manner thereby suggesting both a potential mechanism as well as potency [113].

Studies of perifosine in combination with adjuvant radiochemotherapeutic approaches have also been evaluated in order to assess synergistic combinations. In an in vivo model of pediatric platelet-derived growth factor (PDGF)-driven brainstem glioma using the RCAS/tv-a retroviral system, perifosine was seen to induce terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive nuclei [114]. However, in this study using a xenograft model, perifosine failed to prolong survival similarly to 10 Gy of radiotherapy and also failed to synergize with radiation. In a further study of PDGF-driven primary glioma cells generated with the RCAS/tv-a retroviral system, perifosine in combination with temsirolimus showed efficacy in suppressing cell proliferation of PTEN-intact and PTEN-null cells [115]. Furthermore, in vivo-generated tumors underwent combined perifosine and temsiroliums treatments, which showed decreased tumor proliferation, increased cell death as well as decreased AKT and mTOR signaling. Similarly to previous findings, single treatment with perifosine failed to demonstrate a significant in vivo reduction in tumor proliferation. Currently, perifosine in combination with temsirolimus is undergoing clinical trials in humans for recurrent or progressive GBM [12].

Other PI3K-targeted agents

The novel PI3K inhibitor GNE-317 (Genentech) showed efficacy in reducing cell proliferation in a GBM in vitro and in vivo model [116]. Furthermore, GNE-317 was effective in suppressing pAKT and pS6 activity in orthotopically transplanted brain tumors supporting an ability to cross the blood–brain barrier. Recently, the PI3K inhibitor GDC-0941 in combination with vitamin B10 was shown to inhibit GBM by stimulating expression of Transcription factor EB, involved in lysosomal biogenesis, as well as LAMP-1 and cathepsin B, involved in lysosomal membranes [117]. Moreover, lysosomal membrane permeabilization correlated with expression of Bax, loss of mitochondrial membrane potential, activated capsase-3, and increased cell apoptosis. A recently characterized PI3K inhibitor, CH5132799 (Chugai Pharmaceutical Co.), was shown in in vitro as well as in vivo tumor models to inhibit GBM proliferation [118]. Two new PI3K agents, AMG-511 (Amgen) and ZSTK474 (Zenyaki Kogyo Co.), were recently described to be a potent pan class I PI3K inhibitor that inhibited GBM tumor growth in a xenograft model [119, 120]. PI3K inhibitors have also been suggested to synergize with radiation in the treatment of GBM with inhibitors of p110α being more significant than p110β in suppressing cell proliferation and in vivo tumor growth [81]. The PI3K p110δ inhibitor IC-87114 (ICOS Co.) was shown in one study to suppress glioma cell migration but not invasion [121]. A PI3Kγ inhibitor AS-605240 was also effective in inhibiting proliferation of human and rodent glioma cell lines [122]. PI3K inhibitors XL-147 and XL-765 (Exelixis) have recently completed phase I trials in GBM [12]. The PI3K inhibitor P529 (Paloma 529, Paloma Pharmaceuticals) demonstrated inhibited mTORC1 and mTORC2 complex formation along with reducing in vivo tumor growth, vascularity, and angiogenesis [123].

Conclusion

Since the discovery of PTEN mutations and upregulation of the mTOR signaling pathway in GBM, various approaches towards targeted therapy have been attempted but have thus far been unsuccessful in generating a lasting outcome. Further therapies have been designed to target multiple molecules in the mTOR signaling pathway including mTOR, PI3K, and AKT. Future in vitro and in vivo experiments as well as clinical trials are warranted to assess the success of these approaches. Ultimately, clinical trials will serve to resolve the efficacy of such therapeutic approaches as well as aid in the design of more effective, personalized treatments.

Acknowledgments

The authors received no financial support for the research, authorship, and/or publication of this article.

Conflicts of interest

None

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© International Society of Oncology and BioMarkers (ISOBM) 2013