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`cancer drug targets.
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`

`
`Aims and Scope
`Current Cancer Drug Targets aims to cover all the latest and outstanding developments in
`the medicinal chemistry, pharmacology, molecular biology, genomics and bioclicmistry of
`contemporary molecular drug targets involved in cancer e.g. disease specific proteins,
`receptors, enzymes, genes.
`Each issue of the journal contains a series oftimely in—deptl1 reviews written by leaders in the
`field covering a range of current topics in drug targets involved in cancer.
`As the discovery, identification, characterization and validation of novel human drug targets
`for anti—cancer drug discovery continties to grow, this Journal will be essential reading for all
`pharmaceutical scientists involved in drug discovery and development.
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`

`
`Mammalian Target of Rapamycin (mTOR) Inhibitors as Anti-Cancer Agents
`
`Current Cancer Drug Targets, 2004, 4, 621-635
`
`621
`
`Ravi D. Rao, Jan C. Buckner and Jann N. Sarkaria*
`
`Department of Oncology, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905, USA
`Abstract: Highly specific signal transduction inhibitors are being developed as anti-cancer agents against an
`array of molecular targets, with the promise of increased selectivity and lower toxicity than classic cytotoxic
`chemotherapy agents. Rapamycin and its analogues are a promising class of novel therapeutics that specifically
`inhibit signaling from the serine-threonine kinase, mammalian target of rapamycin (mTOR). mTOR is a key
`intermediary in multiple mitogenic signaling pathways and plays a central role in modulating proliferation and
`angiogenesis in normal tissues and neoplastic processes. Rapamycin potently inhibits T-cell proliferation, and
`is approved for clinical use as an immuno-suppressant following kidney transplantation. Hyperactivation of
`mTOR signaling has been implicated in tumorigenesis, and promising pre-clinical studies in several tumor
`types suggest that the anti-proliferative and anti-angiogenic properties of rapamycin may be useful in cancer
`therapy. These studies have led to several clinical trials evaluating the safety and efficacy of rapamycin analogs
`in cancer therapy. The goal of this article is to review the mechanism of action of rapamycin as an anti-cancer
`agent, and to review the clinical experience with rapamycin and rapamycin analogs as immunosuppressive and
`anti-neoplastic therapeutic agents.
`Key words: Rapamycin, CCI-779, temsirolimus, RAD-001, everolimus, AP23573, sirolimus, cancer, cytostatic anti-cancer
`drugs, mTOR, renal transplant, immunosuppressive agents.
`
`INTRODUCTION
`
`Rapamycin and its analogues are novel, molecularly
`targeted drugs that are being developed as anti-cancer agents.
`The parent compound, rapamycin (Sirolimus, Rapamune;
`Wyeth-Ayerst) is approved by the Food and Drug
`Administration (FDA) for the prevention of allograft
`rejection following renal
`transplantation, and for
`incorporation into drug-eluting stents to prevent re-stenosis
`following coronary angioplasty. Experience in the transplant
`setting suggests that long-term use of this agent is safe and
`well tolerated. Rapamycin analogues with more favorable
`pharmacokinetic properties are currently being developed as
`anti-cancer drugs. Rapamycin and its analogs inhibit the
`signaling activity of the serine-threonine protein kinase,
`mammalian target of rapamycin (mTOR). mTOR functions
`downstream from multiple growth factor receptor tyrosine
`kinases to promote cell growth and proliferation. Key
`downstream targets of mTOR include p70S6 kinase and
`eukaryotic initiation factor 4E-binding protein (4EBP1),
`which modulate the translation of select mRNA transcripts
`that ultimately impact on cell growth and cell cycle
`progression. More recent data have linked mTOR signaling
`with the cellular response to hypoxia and the expression of
`vascular endothelial growth factor (VEGF), which suggests
`that mTOR may be an important mediator of tumor
`angiogenesis. In tumors that are reliant on mTOR signaling,
`disruption of these key signaling pathways by rapamycin
`results in cell cycle arrest and inhibition of angiogenesis,
`and these effects may account for the anti-neoplastic
`activities of mTOR inhibitors seen in multiple tumor types.
`Based on promising pre-clinical studies, rapamycin and its
`analogs currently are being tested as anti-neoplastic agents,
`
`*Address correspondence to this author at the Department of Oncology,
`Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905,
`USA; E-mail: sarkaria.jann@mayo.edu
`
`both given alone or in combination with conventional cancer
`therapies. In this review, the biology of mTOR signaling
`and the cellular pharmacology of mTOR inhibition will be
`discussed as will the clinical development of mTOR
`inhibitors.
`
`History
`
`Rapamycin is a macrocyclic lactone antibiotic that was
`first isolated from the bacterium Streptomyces hygroscopicus
`found in soil samples taken from Easter Island (called ‘Rapa
`Nui’ by its native inhabitants; hence the name ‘Rapamycin’).
`After its isolation and purification, studies revealed that
`rapamycin was a potent anti-fungal agent and an effective
`immunosuppressant[1]. Subsequent studies demonstrated
`that rapamycin inhibited proliferation in several tissues
`including IL-2 stimulated T-cells, vascular endothelium,
`smooth muscle, and tumor cells. These observations
`prompted the development of rapamycin and its analogs as
`immunosuppressive agents, inhibitors of vascular re-
`occlusion and as anti-cancer agents.
`Rapamycin and three analogs, CCI-779, RAD-001 and
`AP23573, have been developed for human use (Fig. (1)).
`Among
`these, only rapamycin (Sirolimus, Wyeth
`Pharmaceuticals) is currently approved, for preventing
`kidney allograft rejection following renal transplantation and
`in drug-eluting stents to reduce the incidence of re-stenosis
`following coronary artery angioplasty. CCI-779
`(Temsirolimus, Wyeth Pharmaceuticals) is an ester of
`rapamycin, with superior oral bioavailability compared to
`the parent compound rapamycin. This drug is available in
`oral and intravenous formulations, and clinical development
`of this drug is well underway with several phase II and III
`trials being conducted. RAD-001 (Everolimus, Novartis
`Pharmaceuticals) is an orally available hydroxyethyl
`derivative of rapamycin developed by Novartis for
`applications in the transplant, cardiovascular and oncological
`
`1568-0096/04 $45.00+.00
`
`© 2004 Bentham Science Publishers Ltd.
`
`

`
`622 Current Cancer Drug Targets, 2004, Vol. 4, No. 8
`
`Sarkaria et al.
`
`R
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`RAD001, R =
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`Fig. (1). Structure of rapamycin and rapamycin derivatives.
`
`O
`
`settings, and clinical testing for all these indications are
`ongoing. The newest mTOR inhibitory agent to be
`developed
`for clinical use
`is AP23573
`(Ariad
`Pharmaceuticals). Early phase I clinical trials with this
`agent, which is also an analog of rapamycin, are now
`underway.
`
`BIOLOGY OF mTOR
`
`mTOR is a serine-threonine-directed kinase that belongs
`to the family of phosphatidyl-inositol 3-kinase-related
`kinases (PIKK). All members of this PIKK family contain a
`C-terminal kinase domain that shares significant homology
`with that of the phosphatidyl-inositol 3-kinase (PI3K); other
`members of this family include ataxia telangiectasia mutated
`(ATM), ATM and Rad3 related (ATR) and DNA-dependent
`protein kinase (DNA-PK) [2,3]. These latter 3 kinases play
`key roles in orchestrating DNA damage checkpoint responses
`and DNA repair [4]. In contrast, mTOR monitors
`intracellular nutrient and energy availability and promotes
`cell growth and proliferation following mitogenic stimuli,
`dependent upon the availability of requisite nutrients [5].
`Rapamycin is a highly specific inhibitor of mTOR
`function. Rapamycin is unable to bind directly to mTOR,
`but forms a complex with the immunophilin, 12 kDa
`FK506-binding protein (FKBP12; FK-506 is an unrelated
`immunosuppressant); it is this drug-protein complex that
`binds to mTOR through an FKBP12-rapamycin binding
`(FRB) domain[6]. The FRB domain is adjacent to the
`kinase domain in mTOR and formation of this tri-molecular
`complex markedly attenuates downstream signaling from
`mTOR. Interestingly, rapamycin treatment does not inhibit
`mTOR catalytic kinase activity directly, since
`autophosphorylation of mTOR is unaffected by rapamycin
`treatment. Instead, binding of the FKBP12/rapamycin
`complex is thought to prevent interaction of mTOR with its
`kinase substrates and thus prevent downstream signaling [7].
`The interaction of the rapamycin/FKBP12 complex with
`mTOR is highly specific and is so stable that inhibition of
`mTOR by rapamycin is essentially irreversible. The cellular
`and biochemical effects of rapamycin are generally believed
`to result exclusively from inhibition of mTOR signaling
`[8,9].
`
`The mTOR signaling network (Fig. (2)) is important for
`driving cell growth and proliferation in multiple tumor
`types. Several receptor tyrosine kinases (RTKs), including
`the epidermal growth factor receptor (EGFR), platelet-
`derived growth factor receptor (PDGFR) and insulin-like
`growth factor (IGFR) can activate PI-3 kinase activity,
`which, in turn, phosphorylates phosphatidylinositol (PI) on
`the D-3 position [10]. The resulting accumulation of
`phosphatidylinositol-3, 4, 5-triphosphate on the cytoplasmic
`surface of the plasma membrane leads to activation of a
`number of kinase signaling pathways including that
`regulated by protein kinase B (PKB, Akt). Akt stimulates
`mTOR function both through direct phosphorylation of a
`negative regulatory domain within mTOR [11] as well as
`through its effects on the tuberous sclerosis complex-2
`(TSC2) protein [12-15]. TSC2, in a complex with tuberous
`sclerosis complex-1 (TSC1) protein, functions as a GTPase-
`activating protein towards the Rheb1 GTPase. Akt-mediated
`phosphorylation of TSC2 disrupts the TSC1/TSC2 complex
`and relieves inhibition of Rheb1 activity; activated Rheb1
`then can stimulate mTOR phosphorylation and signaling
`(see Fig. (2)) [16-19]. The inhibitory effects of TSC2 on
`mTOR activity are stimulated in nutrient deprived
`conditions by activation of LKB-1, which signals through
`AMP-activated protein kinase (AMPK) to enhance TSC2
`activity [20,21]. Signaling from mTOR also is regulated by
`association with Raptor, which probably functions as a
`scaffolding protein to promote transient association and
`phosphorylation of downstream
`targets
`[22,23].
`Collectively, these data highlight the idea that mTOR
`functions within a molecular complex of multiple proteins
`that regulate its activity [24].
`PI3K-mediated activation of Akt is normally opposed by
`the lipid phosphatase PTEN (phosphatase and tensin
`analogue), which dephosphorylates phosphatidylinositol at
`the D-3 position. Deletion or mutation of the gene encoding
`this tumor suppressor protein commonly occurs in multiple
`tumor types and results in constitutive activation of PI3K-
`dependent signaling pathways that include Akt and activated
`Ras as signaling mediators. Consistent with the potential
`role of the Akt/mTOR signaling pathway in tumorigenesis,
`overexpression of activated Akt and activated Ras in glial
`progenitor cells leads to formation of GBM-like tumors in a
`
`

`
`Mammalian Target of Rapamycin (mTOR) Inhibitors as Anti-Cancer Agents
`
`Current Cancer Drug Targets, 2004, Vol. 4, No. 8 623
`
`Fig. (2). The mTOR signaling network.
`
`transgenic mouse model [25], and these tumors are
`exquisitely sensitive to treatment with rapamycin. Germ-line
`inherited deficiencies in PTEN, TSC or LKB-1 result in
`Cowden’s disease, Tuberous Sclerosis and Peutz-Jeghers
`syndrome, respectively, which are all characterized by the
`development of multiple benign hamartomas. Loss-of-
`function in any one of these three proteins results in
`hyperactivation of mTOR signaling, and this presumably
`accounts for the development of the characteristic
`hamartomatous lesions. Thus, constitutive activation of
`mTOR signaling can be an important contributor to
`tumorigenesis in both benign and malignant tumors.
`
`mTOR-Dependent Signaling Pathways
`
`mTOR signals downstream to multiple protein targets:
`p70 S6 kinase (p70S6K), eukaryotic translation initiation
`factor 4E binding protein (4E-BP1), and the hypoxia-
`inducible transcription factor, HIF-1α. These 3 well-
`characterized downstream signaling targets have been
`implicated in control of hypoxia- and mitogen-induced
`tumor proliferation and disruption of these pathways may
`play an important role in the anti-tumor effects of
`rapamycin. In the sections below, we will describe the
`potential links between the anti-tumor effects of mTOR
`inhibitors and disruption of downstream signaling to
`p70S6K, 4EBP1 and HIF-1α in more detail.
`
`p70S6K
`
`mTOR regulates translation of select mRNA transcripts
`containing 5’-terminal oligopyrimidine (5’TOP) tracts
`
`through phosphorylation of p70S6K. Following PI3K-
`dependent phosphorylation of residues within an auto-
`inhibitory domain, mTOR regulates the phosphorylation of
`Thr-389 [26]. Modification of this residue is essential for
`subsequent phosphorylation of other residues within the
`activation loop of the kinase domain, which allows for full
`catalytic activity. After mitogen stimulation, activated
`p70S6K phosphorylates the S6 component of the 40S
`ribosomal subunit, and this promotes translation of mRNA
`containing 5’TOP [27]. Because transcripts for many
`ribosomal proteins and translation elongation factors contain
`this 5’TOP motif, rapamycin-mediated suppression of
`p70S6K activity may inhibit cell growth and proliferation
`by limiting ribosomal biogenesis and restricting protein
`synthesis capacity.
`
`4EBP1
`
`mTOR modulates protein translation initiation by
`regulating the assembly of the eukaryotic initiation factor 4F
`(eIF4F) complex on the 5’-methyl-GTP cap of mRNA
`transcripts. The eIF4F complex is a heterotrimer composed
`of the mRNA cap-binding protein eIF4E, a scaffolding
`protein eIF4G, and a helicase eIF4A (reviewed in [5]). This
`tripartite complex regulates the rate of cap-dependent protein
`translation by mediating the rate-limiting step of mRNA
`loading onto the small 40S ribosomal subunit. Formation of
`a functional eIF4F complex is controlled by the
`phosphorylation status of an eIF4E binding protein (4E-
`BP1). In nutrient- or growth factor-deprived cells, the
`association of hypophosphorylated 4E-BP1 with eIF4E
`blocks binding of eIF4G to the cap structure and inhibits
`
`

`
`624 Current Cancer Drug Targets, 2004, Vol. 4, No. 8
`
`Sarkaria et al.
`
`cap-dependent translation. In contrast, nutrient- or mitogen-
`induced phosphorylation of 4E-BP1 disrupts association
`with eIF4E and allows formation of a functional eIF4F
`complex [28,29]. 4E-BP1 is phosphorylated on at least 5
`serine or threonine sites, and these mitogen-induced
`modifications are regulated, in part, by mTOR. Several
`studies have suggested that mTOR directly phosphorylates
`all 5 sites on 4E-BP1 [30,31], while others have argued that
`direct mTOR phosphorylation of 2 of the sites (Thr-37 and
`Thr-46) serves as a priming event that allows subsequent
`phosphorylation of the other sites (Ser-65, Thr-70, and Ser-
`83) by other signaling pathways [32]. Rapamycin
`diminishes 4E-BP1 phosphorylation, prevents dissociation
`of 4E-BP1 from eIF4E, and results in inhibition of cap-
`dependent translation. Because translation is less efficient for
`transcripts with complex secondary structures, rapamycin
`preferentially inhibits translation of mRNA transcripts
`containing complex 5’ untranslated regions (UTR) [33].
`Transcripts with complex 5’UTRs whose translation is
`inhibited by rapamycin include key proteins involved in cell
`proliferation and angiogenesis, such as cyclin D1, ornithine
`decarboxylase and vascular endothelial growth factor
`(VEGF).
`
`HIF-1αααα
`
`Hypoxia induces the expression of multiple genes
`containing hypoxia response elements (HREs), and
`transcription from this response element is regulated
`primarily by the HIF-1 transcription factor. HIF-1 is a
`heterodimer composed of HIF-1α and HIF-1β. Under
`normoxic conditions, HIF-1 heterodimer levels are
`undetectable due to rapid degradation of HIF-1α subunit.
`The stability of HIF-1α is regulated through post-
`translational modification of an oxygen-dependent
`degradation (ODD) domain. In the presence of oxygen,
`prolyl hydroxylases modify two conserved proline residues
`(Pro-402 and Pro-564) within
`the ODD [34,35].
`Hydroxylation of these residues promotes HIF-1α
`association with the von Hippel Lindau-containing ubiquitin
`ligase complex and subsequent ubiquitin-mediated
`proteosomal degradation. Because molecular oxygen is
`required for catalysis of this reaction, hypoxic conditions
`prevent hydroxylation of the ODD, which results in
`stabilization of HIF-1α . Other transactivating post-
`translational modifications and dimerization with the HIF-1β
`subunit result in formation of an active HIF-1 transcriptional
`complex. HIF-1 drives expression of genes, such as VEGF,
`which contain HREs within their promoter region. The
`repertoire of hypoxia-inducible genes enables tumor or
`normal tissues to adapt to low oxygen environments and
`include genes involved in oxygen and glucose transport,
`glycolysis, growth-factor signaling, immortalization, genetic
`instability, invasion and metastasis, apoptosis and pH
`regulation [36].
`Signaling through the PI3K/mTOR pathway regulates
`HIF-1α expression and activity [37]. The link between PI3K
`signaling and HIF-1α activity was first established in Ras-
`transformed cells, where hypoxia-induced signaling to HIF-1
`was blocked by genetic or pharmacological inhibition of
`PI3K activity [38]. Subsequent studies have demonstrated
`that restoration of wild-type PTEN function, expression of a
`
`dominant-negative Akt construct, or treatment with
`rapamycin blocks hypoxia and mitogen-induced HIF-1
`signaling [39-41]. Moreover, a recent study demonstrated
`that rapamycin blocks both hypoxia-induced HIF-1α
`accumulation and transactivation, and that this effect of
`rapamycin is specifically due to pharmacological inhibition
`of mTOR [42]. Collectively, these data suggest the existence
`of a PI3K/Akt/mTOR signaling pathway that regulates HIF-
`1α expression, stability or activation.
`
`ANTI-TUMOR EFFECTS OF RAPAMYCIN
`
`The central role for mTOR in modulating cell
`proliferation in both tumor and normal cells and the
`importance of mTOR signaling for the hypoxic response
`suggests that rapamycin-based therapies may exert anti-
`tumor effects primarily through either inhibition of tumor
`cell proliferation or suppression of angiogenesis. Pre-clinical
`and early clinical results demonstrate that only a subset of
`tumors will respond to rapamycin-based therapies. In the
`following sections, we will review the pre-clinical efficacy
`data, the evidence supporting the anti-angiogenic and
`cytostatic properties of rapamycin, and discuss potential
`mechanisms of resistance to mTOR inhibition.
`Pre-clinical studies have demonstrated efficacy of
`rapamycin analogs and the parent compound in multiple
`tumor types. In the National Cancer Institute 60 tumor cell
`line panel, both rapamycin (NSC 226080) and CCI-779
`(NSC 683864) demonstrated growth inhibitory activity
`against a broad spectrum of tumors with a subset of
`leukemia, lung, brain, prostate, breast, renal and melanoma
`tumor cell lines being inhibited at low nanomolar
`concentrations (http://dtp.nci.nih.gov/). Early animal studies
`at the NCI and at Ayerst Research Laboratories demonstrated
`modest growth inhibitory properties of rapamycin in murine
`tumor models of B16 melanoma, P388 lymphocytic
`leukemia, EM ependymoblastoma, CD8F1 breast carcinoma,
`Colon 38, CX-1 and 11/A colon cancer models [43].
`Subsequent published studies have documented significant
`tumor growth inhibition with rapamycin or CCI-779
`treatment of DAOY medulloblastoma, U251 or SF295
`glioma, PC-3, DU-145, LAPC4, or LAPC9 prostate
`carcinoma, and Rh-18 rhabdomyosarcoma human tumor
`xenografts [44-47]. These data have provided the impetus for
`development of mTOR inhibitor therapy in a variety of
`tumor types.
`Treatment of tumor cells in vitro with rapamycin results
`in an accumulation of cells in the G1 phase of the cell cycle.
`Similarly, rapamycin treatment in tumor-bearing animals
`results in decreased tumor cell proliferation as indicated by
`BrdU labeling index [48]. At the molecular level, this drug-
`induced cell cycle arrest is associated with an accumulation
`of the cyclin-dependent kinase inhibitor, p27kip1, decreased
`expression of cyclinD1, and a corresponding decrease in
`phosphorylation of the retinoblastoma protein. CyclinD1
`mRNA contains a complex 5’UTR, and reduction in
`cyclinD1 levels presumably results from rapamycin-mediated
`inhibition of 4EBP1 phosphorylation and the resulting
`inhibition of eIF4F function. Likewise, the mRNA encoding
`for multiple oncogenes or proteins involved in DNA
`metabolism and S-phase progression contain complex
`
`

`
`Mammalian Target of Rapamycin (mTOR) Inhibitors as Anti-Cancer Agents
`
`Current Cancer Drug Targets, 2004, Vol. 4, No. 8 625
`
`5’UTRs and are regulated by mTOR [49]. Although
`rapamycin can induce apoptosis in select tumor models,
`rapamycin treatment typically slows tumor growth but does
`not induce tumor regression, suggesting that increased tumor
`cell loss through apoptosis or other mechanisms of cell
`death likely are not major contributors to drug effect in most
`tumors. Collectively, these data support the idea that
`rapamycin exerts a cytostatic effect on tumor cell growth.
`Rapamycin also has cytostatic effects on normal and
`tumor vasculature. As discussed above, mTOR plays an
`important role in modulating the cellular response to
`hypoxia through stabilization and activation of HIF-1α.
`HIF-1 drives expression of hypoxia-responsive genes, and
`these genes include those such as vascular endothelial
`growth factor (VEGF) that are important for tumor
`angiogenesis. Translation of VEGF mRNA also is regulated
`in an mTOR-dependent manner via its complex 5’UTR.
`Consistent with these mechanisms of regulation, treatment
`of tumor-bearing animals with rapamycin results in
`decreased expression of VEGF mRNA in tumors [50] and
`decreased circulating levels of VEGF protein [51]. VEGF
`drives endothelial proliferation through interaction with its
`cognate receptor tyrosine kinases, VEGF receptor 1 and 2,
`and these receptors can signal downstream through the
`PI3K/Akt pathway to mTOR. Thus, proliferation of smooth
`muscle and endothelial cells is inhibited by mTOR
`inhibition [52,53], and this effect is likely related to the
`efficacy of rapamycin-eluting stents in preventing vascular
`
`re-occlusion. In animal models, rapamycin inhibits tumor
`growth and neo-vascularization of CT-26 colon tumors
`grown in a dorsal skin-fold model and suppresses serum
`levels of VEGF in tumor bearing animals[51]. Thus the anti-
`angiogenic effects may contribute to the efficacy of mTOR
`inhibitors in cancer therapy.
`The anti-tumor effects of rapamycin therapies are likely
`secondary to both inhibition of tumor cell proliferation and
`inhibition of tumor angiogenesis. The relative contribution
`of these two effects in any given tumor may be difficult to
`delineate. However, pre-clinical and clinical data suggest that
`that only a subset of tumors will respond to rapamycin; one
`of the challenges will be to identify patients most likely to
`respond to this class of agents. Future studies delineating
`the relative contributions of the anti-proliferative and anti-
`angiogenic effects to the overall anti-tumor efficacy of
`rapamycin will be invaluable for identifying the relevant
`cellular targets that are responsible for the anti-tumor effects
`of rapamycin.
`
`COMBINATIONS OF MTOR ANTAGONISTS WITH
`OTHER ANTI-CANCER AGENTS
`
`Angiogenesis and tumor cell proliferation have been
`implicated as important mediators that can influence the
`efficacy of traditional cytotoxic cancer therapies; therefore,
`much research effort has focused on the evaluation of
`
`Fig. (3). Rapamycin enhances the efficacy of radiation in U87 xenografts. Nude mice with established U87 flank xenografts were
`randomized into four treatment groups: 1) placebo, 2) radiation only (4 Gy x 4), 3) rapamycin only (1 mg/kg), or 4) radiation and
`rapamycin. The tumor re-growth for each treatment group is shown. Data points represent the mean relative tumor volume ± SE.
`Treatment was initiated on Day 0 with the first injection of rapamycin (Rap). The schedule for Rap and radiation (RT) treatments is
`depicted below the x-axis. Highly similar results were obtained in two independent experiments. (Reproduced with permission,
`Cancer Res., 2002, 62, 7291-7297).
`
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`Sarkaria et al.
`
`combinations of mTOR inhibitors with standard cancer
`therapies. Rapamycin potentiates cisplatin induced apoptosis
`in multiple cell lines including HL-60 leukemia cells and
`SKOV3 ovarian cancer cells [54]. Likewise, insulin-like
`growth factor-induced resistance to cisplatin in Rh30
`rhabdomyosarcoma cells can be reversed by rapamycin
`treatment [55]. Geoerger et al. demonstrated
`that
`combinations of rapamycin with either cisplatin or
`camptothecin provides additive growth inhibition in the
`rapamycin-sensitive DAOY medulloblastoma cell line but
`not in the rapamycin-resistant D283 cell line [46].
`Consistent with this in vitro data, CCI-779 therapy provides
`for additive tumor growth inhibition in animals when
`combined with doxorubicin or cisplatin in PC3 or DAOY
`xenografts, respectively [46]. Several other investigators
`have confirmed the in vitro effect of rapamycin on
`sensitizing cancer cells to chemotherapeutic agents such as
`adriamycin, VP-16, and cisplatin1 and hormonal therapies
`such as tamoxifen2 dexamethasone3, and the anti-estrogen
`ERA-9234. Rapamycin also has been combined with other
`molecularly targeted therapeutics. In our laboratory, we
`found that the combination of rapamycin with the EGFR
`inhibitor EKI-785 resulted in synergistic growth inhibition
`(unpublished data), and similar results were reported with
`the combination of RAD-001 and the VEGFR/EGFR
`inhibitor AEE7885. Likewise, rapamycin combined with the
`bcr/abl inhibitor Imatinib mesylate (Gleevec) provided
`synergistic growth inhibition of chronic myeloid leukemia
`(CML) cell lines6. Taken together, these data suggest that
`mTOR-dependent signaling may be important for resistance
`to chemotherapy-induced apoptosis and provides a rationale
`for
`the combination of rapamycin with specific
`chemotherapy agents.
`Rapamycin also can enhance the efficacy of radiation
`therapy. Based on significant pre-clinical and clinical data
`demonstrating that tumor proliferation during fractionated
`radiotherapy contributes to clinical radiation resistance
`
`1.Savaraj, N.; Wu, C.; Wangpaichitr, M.; Lampidis, T.; Robles, C.; Furst,
`A.; Feun, L. Circumvention of drug resistance in small cell lung cancer by
`mTOR inhibitor. Proc. Am. Assoc. Cancer Res. 2003, 44, 2nd ed., Abstract
`# 3704.
`2.deGraffenried, L.; Friedrichs, W.; Fulcher, L.; Silva, J.; Roth, R.;
`Hidalgo, M. The mTOR inhibitor, CCI-779, restores tamoxifen response in
`breast cancer cells with high Akt activity. Eur. J. Cancer 2002, 38(Suppl.
`7), Abstract # 528.
`3.Yan, H.; Shi, Y.; Frost, P.; Hoang, B.; Gera, J.; Lichtenstein, A. The
`mTOR inhibitor rapamycin sensitizes multiple myeloma cells to apoptosis
`induced by dexamethasone. Blood 2003, 102, Abstract # 3453.
`4.Zhang, T.; Sadler, T.; Annable, M.; Achilleos, P.; Frost, P.; Greenberger,
`L. Combination therapy for treating breast cancer using the antiestrogen,
`ERA-923 and the mTOR inhibitor, CCI-779. Proc. Am. Assoc. Cancer Res.
`2003, 44, 2nd ed., Abstract # 3715.
`5.Goudar, R.; Keir, S.; Hjelmeland, M.; Conrad, C.; Traxler, P.; Lane, H.;
`Wang, X.; Bigner, D. D.; Friedman, H. S.; Rich, J. N. Combination therapy
`of inhibitors of the epidermal growth factor/ vascular endothelial growth
`factor receptor 2 (AEE788) and the mammalian target of rapamycin
`(RAD001) offers improved glioblastoma tumor growth inhibition. EORTC-
`NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics.
`2003, Abstract # A88.
`6.Halbur, L.; Ly, S. C.; Ong, T. Inhibition of mTOR by Rapamycin
`Enhances Killing of CML Cells by Imatinib Mesylate and Overcomes
`Certain Forms of Imatinib R