295
`
`BANDBIUI-HuY
`
`*To whom correspondence should be addressed
`
`Current Opinion in Investigational Drugs 2002 3(2):295-304
`© PharmaPressLtd ISSN 1472-4472
`
`Inhibitors of mammalian target of rapamycin as novel antitumor agents:
`From bench toclinic
`Shile Huang & Peter J Houghton”
`Address
`increased growth and malignant characteristics of cells. It is a
`Department of Molecular Pharmacology
`
`lipophilic—macrolide, that selectively inhibits =a
`
`
`
`St Jude Children's Research Hospital
`serine/theronine kinase, specifically, the mammalian targetof
`332 N Lauderdale
`rapamycin)
`(mTOR).
`mTOR
`lies
`downstream of
`Memphis
`TN 38105-2794
`phosphatidylinositol 3-kinase (PI3K) in the PI3K signaling
`USA
`pathway. Rapamycin was originally isolated as a fungicide
`Email: peter.houghton @stjude.org
`from the soil bacteria Streptomyces hygroscopicus, collected
`from Easter Island (known as Rapa Nui tothe natives) in the
`South Pacific in 1975 [1,2]. Structurally similar
`to the
`immunosuppresive reagent FK-506 (tacrolimus; Fujisawa
`Pharmaceutical Co Ltd), rapamycin was initially developed
`for transplant rejection [3,4] and was approved by the US
`Food and Drug Administration in September 1999 and the
`European Commission in March 2000. While rapamycin was
`being developed as an immunosuppressant, it was also found
`to exert potent antitumoractivity in vitro and in vivo [5-7].
`However, perhaps because its mechanism of action was
`unknown at that time, rapamycin was not developed as a
`cancer therapeutic.
`
`Rapamycin and its derivatives, CCI-779 and RAD-001, inhibit the
`mammalian target
`of
`rapamycin
`(mTOR), downregulating
`translation of specific mRNAsrequired forcell cycle progression
`from G1 to S phase. Preclinically, mTOR inhibitors potently
`suppress growthandproliferation of numerous tumorcell lines in
`culture or when grownin mice as xenografts. CCI-779 and RAD-
`001 are being developed as antitumor drugs and are undergoing
`clinical
`trials. Clinically, CCI-779 has
`shown evidence of
`antitumor activity but
`induced relatively mild side effects
`in
`The potential for rapamycin as a cancer therapeutic was
`patients. Here we discuss potential antitumor mechanisms and
`refocused, in part, by studies of Dilling et al [8]. These were
`resistance mechanisms of mTOR inhibitors, and summarize the
`
`the to demonstrate potent and_selectivefirst studies
`
`current status of these compounds as novel antitumor agents.
`inhibition of growth by rapamycin. Rapamycin potently
`inhibited
`the growth of
`rhabdomyosarcoma
`cells
`at
`concentrations of approximately 1 ng/ml, whereas human
`colon cancer cells were inhibited only at micromolar
`concentrations in culture (Table 1). Proliferation of many
`rhabdomyosarcomacells is regulated by an autocrine loop
`involving secretion oftype II insulin-like growth factor (IGF-
`Il) and signaling through the type I IGF receptor [9,10].
`Indeed,
`the cell
`lines most sensitive to rapamycin were
`dependent on this autocrine pathway.
`
`i E
`
`FASAPIA
`
`Keywords Antitumor, cell cycle, mammalian target of
`rapamycin (mTOR), rapamycin
`
`Introduction
`Malignant disease is characterized by genetic mutations or
`compensatory changes in cells that result
`in unregulated
`population growth duetoincreased proliferation or decreased
`cell death. Since the early 1950s, extensive chemical synthesis
`and screening programs have resulted in clinical
`trials of
`many potential anticancer agents.
`In some cases, cytotoxic
`agents have significantly increased survival rates in adult
`diseases and notably for children with hematologic as well as
`solid tumors. However, relatively few cytotoxic agents have
`proven to be useful against a wide spectrum of cancers and
`‘ most cause significant toxicity. The reasonsfor the relatively
`pooractivity of cytotoxic agents are numerous. Most do not
`target the transforming event, rather they induce forms of
`damage that lead to necrosis or activation of cell-suicide,
`apoptosis. As a consequence, agents are cytotoxic to both
`malignant tumorcells and normalhealthy cells, often causing,
`severe side effects. Understanding pathways known to be
`critical to the growth and survival of tumors is essential to
`developing potentially selective treatments. Validation of this
`concept has been met with compounds such as imatinib
`(Gleevec, STI-571; Novartis AG). This compound inhibits
`tyrosine kinases such as BCR/ABL in chronic myeloid
`leukemia and c-KIT in gastrointestinal stromal tumors. Less
`success has been met by targeting activated Ras with
`inhibitors of farnesyltransferase.
`
`Wyeth-Ayerst
`Rapamune;
`(sirolimus,
`Rapamycin
`Laboratories; Figure 1), an immunosuppressant, has emerged
`as a potent inhibitor of a signaling pathway that may be
`deregulated in some forms of cancer,
`leading to both
`
`Additional findings from various research groups around
`the world support rapamycin as a good candidate for a
`cancer therapeutic agent. In many malignant cells in culture,
`rapamycin can act as a cytostatic agent by arresting cells in
`G1 phase. Another potential modeof its antitumor action is
`via the induction of apoptosis. Rapamycin potently inhibits
`proliferation
`or
`growth
`of
`cells
`derived
`from
`rhabdomyosarcoma,
`neuroblastoma,
`glioblastoma,
`small
`medulloblastoma,
`cell
`lung
`cancer
` [8,11-16],
`osteoscarcoma [17], pancreatic carcinoma[18,19], breast and
`prostate carcinoma[20-22], murine melanoma and leukemia,
`and B-cell lymphoma[6,23-25] (Table 1).
`
`Despite the antiproliferative effects of rapamycin, it has poor
`water-solubility and stability in solution, precluding.
`its
`formulation for parenteral use as an anticancer agent. Two
`rapamycin ester analogs, CCI-779 (Wyeth-Ayerst Research;
`Figure 1) and RAD-001 (everolimus; Novartis AG; Figure 1),
`with improved pharmaceutical properties, but
`similar
`cellular effects to rapamycin [16,20-22,26,27,28e,29e], are
`currently undergoing antitumor phase III and phase I
`clinicaltrials, respectively. This review will discuss potential
`antitumor and resistance mechanisms of rapamycin andits
`derivatives, and summarize the preliminary data about
`these compounds, from the bench totheclinic.
`
`West-Ward Pharm.
`Exhibit 1017
`Page 001
`
`West-Ward Pharm.
`Exhibit 1017
`Page 001
`
`

`

`296 Current Opinion in Investigational Drugs 2002 Vol 3 No 2
`
`Table 1. Sensitivity of different tumorcell lines to rapamycin.
`Cell Line
`Rhabdomyosarcomacells
`Rh1 (10% serum)
`Rhi (serum free)
`Rh18
`Rh28
`Rh3o
`Colon carcinomacells
`GC/c1
`VRC,/c1
`CaCo
`HCT8
`HCT29
`HCT116
`Smail cell lung cancer cells
`H69
`H345
`H510
`Neuroblastoma cells
`NB-SD
`NS-1643
`NB-EB
`NB-1691
`NB-1382.2
`Glioblastomacells
`si-62
`SJ-G3
`Medullablastomacells
`DAOY
`Osteoblasts-like osteosarcoma cells
`ROS 17/2.8
`Pancreatic cancercells
`Panc-1
`MiaPaCa-2
`Leukemia cells
`RBL-2H3
`B-cell lymphoma cells
`BKS-2
`ge
`‘NFS1.1
`WEHI-279
`Thymomacells
`eid
`
`Rapamycin (IC
`(ng/ml)
`4680
`3.6
`0.1
`8.0
`0.37
`(ng/ml)
`9800
`1280
`1570
`8400
`> 10,000
`> 10,000
`(nM)
`~1
`~1
`~1
`(ng/ml)
`4
`~1
`2.9
`18
`639
`ng/ml
`UB
`> 10,000
`(ng/ml)
`~1
`(nM)
`< 100
`(ng/ml)
`1
`3
`(nM)
`~10
`(ng/ml)
`0.31
`pet
`0.60
`inom)
`
`Reference
`[8]
`
`[8]
`
`(11,12]
`
`[13]
`
`13
`im
`
`[16]
`
`[17]
`
`[18,19]
`
`[23]
`
`[25]
`
`[25]
`
`
`
`
`Antitumor mechanism ofinhibitors of mTOR
`Rapamycin and its analogs, CCI-779 and RAD-001, are the
`most potent and selective inhibitors of mTOR reported so
`far. The three agents share a common mechanism of
`antitumoraction,ie, inhibiting mTOR, which, links mitogen
`stimulation to protein synthesis andcell cycle progression.
`mTOR
`antitumor mechanism of
`To better understand the
`rapamycin and its derivatives, we will briefly review the
`emerging cellular role of mTOR. mTORisreferred to by
`various other names, some of which are derived from its
`binding partner FK-506-binding protein, FKBP12 (discussed
`below). These names are FRAP (FKBP12 and rapamycin-
`associated protein), RAFT1 (rapamycin and FKBP12 target
`1), RAPT1 (rapamycin target 1) and SEP (sirolimus effector
`protein).
`In the mid-1990s, mTOR was identified as a
`mammalian serine/threonine kinase of approximately 289
`kDa in humans, mice and rats
`[30-33]. TOR proteins
`represent a class of evolutionarily conserved kinases in
`eukaryotes.
`In the yeasts, Saccharomyces
`cerevisiae and
`Schizosaccharomyces pombe, two TOR genes, TOR] and TOR2,
`
`have been cloned, which share 67% identity and encode
`proteins of approximately 280 kDa [34-36]. In the fruit fly,
`Drosophila melanogaster,
`a single TOR ortholog,
`termed
`dTOR, has been characterized, sharing 38% identity with
`TOR2 from Saccharomyces cerevisiae [37,38]. mTOR shares
`approximately 45% identity with TORI and TOR2 from the
`yeast Saccharomyces cerevisiae, and 56% identity with dTOR in
`overall sequence [39,40]. Human, mouse and rat mTOR
`proteins share 95% identity at the aminoacid level [40,41].
`mTORcontains a catalytic kinase domain and a FKBP12-
`rapamycin binding (FRB) domain near the C-terminus, and
`up to 20 tandemly repeated HEAT (Huntingtin, EF3, A
`subunit of PP2A and TOR) motifs at the N-terminus, as well
`as FAT (FRAP-ATM-TRRAP) and FATC (FAT C-terminus)
`domains (Figure 2). Since the C-terminus of mTOR shares
`strong homology to the catalytic domain of PI3K, mTOR is
`considered a member of
`the PlIK-related kinase family,
`which also includes MEC1, TEL1, RAD3, MEI-41, DNA-PK,
`ATM, ATR and TRRAP [42¢¢]. Both PI3K and potentially
`protein kinase B (PKB; Akt) lie upstream of mTOR, whereas
`ribosomal p70S6 kinase (p70S6K) and eukaryotic initiation
`factor-4E (eIF-4E) binding protein isoforms (4E-BP1-3) are the
`
`West-Ward Pharm.
`Exhibit 1017
`Page 002
`
`West-Ward Pharm.
`Exhibit 1017
`Page 002
`
`

`

`Inhibitors of mammalian target of rapamycin as novel antitumor agents: From bench to clinic Huang & Houghton 297
`
`
`
`rapamycin
`(Wyeth-Ayerst)
`
`ccl-779
`
`(Wyeth-Ayerst)
`
`
`RAD-001
`(Novartis)
`
`‘cH,
`
`H,C
`
`CH, H.C...
`
`y
`
`cH,
`
`
`
`Figure 1. Molecular structures of rapamycin, CCI-779 and RAD-001. °o
`FRB
`
`z:: J
`
`Po
`|
`
`ey
`=
`i]
`°P|
`
`BINDSBAMIPIED
`
`best characterized downstream mTOR effector molecules.
`Increasing evidence has implicated mTOR as a central
`controller of cell growth and proliferation. mTOR may
`directly or indirectly regulate translation initiation, actin
`organization, membranetraffic and protein degradation,
`protein kinase C signaling, ribosome biogenesis and tRNA
`synthesis, as well as transcription [42ee]. Recent results also
`suggest
`that mTOR may sense
`cellular ATP levels,
`suppressing protein synthesis when ATP levels decrease
`[43].
`
`Specificity of rapamycin action
`Rapamycin inhibits proliferation and growth of many
`tumorcells, which is clearly a consequence of binding
`mTOR. Whetherthis action is a consequence ofinhibiting
`mTORkinase activity per seis less clear. Rapamycin cannot
`directly bind to mTOR.It first has to bind to the 12 kDa
`cytosolic immunophilin, FKBP12,
`found in mammalian
`cells,
`to form the FKBP12-rapamycin complex. The
`complex then interacts with the FRB domain in mTOR
`(Figure
`2),
`and inhibits
`function of mTOR. High
`concentrations of rapamycin together with FKBP12 are
`required to inhibit mTOR kinase activity in vitro and
`mTOR autophosphorylation. However,
`the specificity of
`rapamycin action can be demonstrated in vivo as certain
`mutations in the FRB domain of mTOR affect FKBP12-
`rapamycin binding, and significantly reduce the cellular
`sensitivity of
`rapamycin. The first
`rapamycin-resistant
`alleles, TORI-1 and TOR2-1, identified in a Saccharomyces
`cerevisiae genetic screen were shown to confer dominant
`resistance. These mutant TORproteins lost the ability for
`FKBP-rapamycin
`complex
`binding
`[44].
`Similarly,
`mammalian
`cells
`also
`became
`highly
`resistant
`to
`rapamycin when a mutation (Ser“”lle"”) occurred in the
`FRB domain of mTOR, whichresulted in decreased affinity
`for binding of FKBP12-rapamycin complex [14,45,46]. In
`the yeast, Saccharomyces cerevisiae, decreased RBP1,
`a
`homolog of mammalian FKBP12, or mutation at Tyr”, led
`to decreased binding of
`rapamycin and conferred a
`recessive resistance phenotype[47].
`
`
`Figure 2. Schematic representation of mTOR domains.
`
`[eat|]vem||a
`
`HEAT repeats
`
`ON
`
`HEAT Huntingtin, EF3, A subunit of PP2A and TOR, FAT FRAP-
`ATM-TRRAP, FRB FKBP12-rapamycin binding, CD catalytic
`domain, FATC FAT C-terminus.
`
`Potential models for rapamycin inhibition of mTOR
`Small molecule kinase inhibitors act directly,
`regulating
`kinase activity generally by competition for ATP binding.
`However, whether
`the FKBP12-rapamycin complex or
`rapamycin alonedirectly inhibits the kinase activity of mTOR
`is still controversial. In vitro, rapamycin inhibited the modest
`increase in kinase activity of
`immunoprecipitated mTOR
`inducedbyinsulin [48]. The FKBP12-rapamycin complex also
`inhibited the autokinase activity of mTOR, although a much
`higher concentration of rapamycin was needed invitro than in
`vivo to inhibit
`the activity of mTOR [49]. Conversely,
`treatment of cells with rapamycin did not alter
`the
`autophosphorylationlevel of Ser, and hadlittle or noeffect
`on the kinase activity of immunoprecipitated mTOR [37,49].
`More recently, an alternative model for mTOR function has
`been proposed. Specifically, mTOR may repress phosphatase
`activity associated with downstream targets. The inhibition of
`mTOR induced by bound FKBP12-rapamycin complex, may
`result
`in
`activation of
`this phosphatase, which
`then
`dephosphorylates downstream effector molecules such as
`p70S6K [50ee,51e]. Consistent with this model, we [52,
`Houghton & Huang, unpublished data] have suggested that
`mTORregulates the catalytic subunit of PP2A associated with
`p44/42 mitogen-activated protein (MAP) kinases in some
`
`West-Ward Pharm.
`Exhibit 1017
`Page 003
`
`West-Ward Pharm.
`Exhibit 1017
`Page 003
`
`

`

`298 Current Opinion in Investigational Drugs 2002 Vol 3 No 2
`
`p44/42
`inhibits
`rapamycin
`cells,
`these
`In
`cells.
`phosphorylation on Thr™ following IGF-I
`stimulation.
`However, more studies
`are necessary to confirm the
`generality of this phosphatase model. An alternative modelis
`for mTORto act as a scaffold and for the FKBP12-rapamycin
`complex to disrupt higher order mTOR-protein complexes.
`
`Rapamycin inhibition of mTOR-controlled signaling
`pathways
`Althoughspecific details of how rapamycin inhibits function of
`mTORremain tobe resolved,it has been widely accepted that
`inhibition of mTOR by rapamycin blocks growth factor
`stimulationof 40S ribosomal p70S6 kinase and phosphorylation
`of 4E-BP1 (also designated PHAS-I). This results in a 15 to 20%
`inhibition of overall protein translation and arrests cell cycle
`progression in G1. Consistent with this observation, mTOR
`controls the synthesis of essential proteins involved in cell cycle
`progression (cyclin D1 and ornithinine decarboxylase) [53,54]
`
`[55]. A scheme of mTOR-controlled
`(c-Myc)
`and survival
`signaling pathways based on rapamycin effects is shown in
`Figure 3. 4E-BP1, the suppressorof eIF-4E, has been reported to
`be a direct substrate for mTORin cells [56,57]. In vitro, mTOR
`selectively phosphorylates 4E-BP1 at least at two and possibly
`four Ser/Thr residues (Thr”, Thr“, Thr” and Ser") in the N-
`terminal region [58ee,59]. Phosphorylation of 4E-BP1 appears to
`be an ordered process [58¢e,59,60]. Phosphorylation of Ser
`depends
`on
`phosphorylation
`of
`all
`three Ser/Thr
`phosphorylation sites
`[59,60], whereas mutations of Thr”
`and/or Thr” to Ala(s) prevents phosphorylation of Ser” and
`Thr”, suggesting that phosphorylation of Thr” and Thr”serves
`as a requisite ‘priming’ event[51e]. It appears that mTORalso
`plays a critical role in regulating the phosphorylation of Ser”
`and Thr”.
`In the presence of rapamycin, 4E-BP] becomes
`hypophosphorylated and associates with elF-4E. This prevents
`formation of the elF-4F initiation complex and cap-dependent
`translation of mRNA.
`
`
`Figure 3. Rapamycin-inhibited signaling pathways controlled by mTOR.
`
`
`
`
`IGF-IR
`
`FKB12-rapamycin
`
`
` [ere|[exert_|NR |
`Phosphatase '—Cimtor
`
`A
`(PHAS-1)
`
`4E-BP'1
`
`elF-4E
`
`
`
`insulin-like growth factor, IRS1 insulin receptor substrate 1,
`Arrows representactivation, whereas bars representinhibition. IGF-IR type |
`PI3K phosphatidylinositol 3-kinase, PTEN phosphatase and tensin homolog deleted on chromosometen, PKB/Akt protein kinase B, FKB12-
`rapamycin FK-506-binding protein 12-rapamycin complex, mTOR mammalian target of rapamycin, elF-4E eukaryotic initiation factor-4E,
`4E-BP1 (PHAS-1) elF-4E-binding protein 1, S6 40S ribosomal protein, p70S6K p70S6kinase.
`
`West-Ward Pharm.
`Exhibit 1017
`Page 004
`
`West-Ward Pharm.
`Exhibit 1017
`Page 004
`
`

`

`eagogjul-juy
`
`
`
`__geinoseAcipreg
`
`<Tt
`
`=o
`
`Inhibitors of mammalian target of rapamycin as novel antitumor agents: From bench to clinic Huang & Houghton 299
`
`Ribosomal p70S6K represents the other well characterized
`downstream target of mTOR. Twop70S6 kinases have been
`characterized, namely, p70S6K1 and p70S6K2. The activation
`of both these kinases can be inhibited by rapamycin [61,62].
`mTOR maydirectly or indirectly phosphorylate p70S6K1 at
`Thr” or Thr” [50¢*,63,64,65¢e,66¢,67]. Phosphorylation of
`these two residues is blocked by rapamycin. Furthermore,
`mutation ofeither of these residues can abrogatethe ability of
`rapamycin to inhibit p70S6K activation. p70S6K functions to
`increase translation of 5'
`terminal oligopyrimidine (5'TOP)
`tract mRNAs, primarily coding for
`elements of
`the
`translational machinery,
`such
`as_
`ribosomal
`proteins,
`elongation factors, the poly(A) binding protein [61] and IGF-II
`[68]. Inhibition of mTOR by rapamycin thusselectively causes
`decreased translation of 5'TOP-containing mRNAs.
`
`In addition to pathways controlling translation initiation,
`mTORhas been implicated in regulating the retinoblastoma
`protein (pRb), RNA polymerase (Pol) 1/II/III-transcription
`andtranslation of rRNA and tRNA, and phosphatases (PP2A,
`PP4, PP6) [69]. It seems that these pathwaysare cell type-
`dependent. For example,
`in vascular smooth musclecells,
`rapamycin may act upstream of pRb to slow or arrest cell
`cycle transit [70]. In this model, rapamycininhibits activation
`of cyclin-dependent kinases
`(CDKs), which results
`in
`hypophosphorylation of pRb protein, and inhibits cells
`progressing from G1 to S-phase [70].
`In T-lymphocytes,
`rapamycin induces G1 arrest, in part through inhibition of
`activation of CDK1 (p34) and the formation of the cyclin E-
`p33“ complex [71,72]. G1 arrest by rapamycin may also be
`due to prevention of the degradation of CDK inhibitory
`protein p27""' that occurs whencells are stimulated by growth
`factors [73,74]. This is further supported by the observation
`that p27" deficient fibroblasts are somewhat resistant
`to
`rapamycin as determined by assaying for DNA synthesis [75].
`In NIH3T3cells, rapamycin inhibits the G1 to S transition in
`part through decrease of cyclin D1 mRNAlevel and protein
`stability [76], or delay of the expression of cyclin A [77].
`
`Antitumoractivity of rapamycin
`As previously mentioned, rapamycin has been approved as an
`immunosuppressive drug for organ transplantation by the
`FDA. So far, rapamycin has been used clinically in organ
`transplantation with great success, particularly in kidney
`transplantation [78,79]. This is because rapamycin caninhibit T-
`cell activation and proliferation. Increasing evidence indicates
`that rapamycin is not only a potent immunosuppressant, but
`also a promising antitumor agent. As reviewed above(Table 1),
`rapamycin potently inhibits the growth of many tumorcell
`lines in vitro, and has demonstrated antitumoractivity in both
`xenograft and syngeneic murine tumor models.
`
`However,rather than acting as a cytostatic, rapamycin induces
`cell death under some conditions. Early data show that
`rapamycin induces programmed cell death or apoptosis of B-
`cells [24,25]. Consistent with these findings,
`recent studies
`indicate that rapamycin alone can also induce apoptosis of
`certain rhabdomyosarcoma cells [14,15], and monocyte- and
`CD34-derived dendritic cells [80]. When combined with other
`chemotherapeutic
`agents
`in
`vitro,
`rapamycin enhanced
`cisplatin-induced apoptosis in human small cell lung cancercell
`lines
`[11],
`potentiated
`apoptosis
`of
`the murine T-
`lymphoblastoid cell line S49, induced by dexamethasone [81]
`
`and augmented cisplatin- or camptothecin-induced cytotoxicity
`in DAOY human medulloblastoma cell lines [16]. Rapamycin
`produced
`additive
`cytotoxicity with 5-fluorouracil
`and
`cyclophosphamide in a Colon 38 tumor model [7]. At present,
`little is known about
`the molecular mechanism by which
`rapamycin induces apoptosis of tumorcells. However, Huang
`et al [15] have observed that the responses of malignant and
`normal cells to rapamycin are qualitatively different. When
`treated with rapamycin, cells with wild-type p53 arrest in Gl
`phase and maintain viability. In contrast, when grown under
`autocrine conditions
`(ie,
`serum-free)
`in the presence of
`rapamycin, p53 mutant cells accumulate in Gl phase, but
`progress to S-phase and undergo apoptosis. More than 90% of
`apoptotic Rh30 cells (mutant p53 alleles, Arg”Cys”) were
`BrdU-labeled, suggesting that
`the cells died after initiating
`replication. Thus, rapamycin-induced death appears to be a
`consequence of continued cell cycle progression, suggesting
`that p53 senses inhibition of mTOR and co-operates toreinforce
`a Gl arrest. This model has been further tested using Rh30
`infected with adenovirus expressing wild-type p53 (Ad-p53)
`and p53 +/+ or p53 -/- murine embryofibroblasts (MEFs) [15].
`Restoring the p53-mediated G1 checkpoint by Ad-p53 infection
`causes Rh30 rhabdomyosarcoma cells to arrest
`in Gl and
`prevents rapamycin-induced apoptosis (Figure 4). Similarly,
`when exposed to rapamycin, p53 -/- MEF cells continuecell
`cycle progression, and become apoptotic, whereas p53 +/+
`MEFcells arrest in G1 and remainviable. Exactly whycells that
`fail to arrest in G1 in the presence of rapamycin dieis currently
`unknown.
`
`Mechanismsof resistance to rapamycin
`As shown by Dilling ef al [8], under similar conditions of
`growth, variouscell lines demonstrated several thousand-
`fold differences in sensitivity to rapamycin. The mechanism
`for this intrinsic resistance is under investigation. Cells may
`alsoacquire resistance either with or without mutagenesis.
`
`Mutations in FKBP12, mTOR and p70S6K
`Budding
`yeast
`Saccharomyces
`cerevisiae
`treated with
`rapamycin irreversibly arrested in the G1 phase. However,
`when yeast TORI and TOR2 were genetically mutated to
`TOR1-1 and TOR2-1, these strains were completely resistant
`to the growth-inhibitory effect of rapamycin. These resistant
`alleles encode proteins
`that have reduced affinity for
`binding the FKBP12-rapamycin complex [44]. Also in yeast,
`a
`recessive resistance phenotype was associated with
`decreased RBP1, a homolog of mammalian FKBP12, or a
`mutation altering Tyr”,
`leading to decreased binding of
`rapamycin [47]. In mammalian cells, resistance to rapamycin
`selected after mutagenesis
`is
`related to a dominant
`phenotype consistent with mutation in mTOR [45]. Similar
`to results in yeast, mTOR mutants are associated with
`decreased affinity for binding of the FKBP12-rapamycin
`complex. High-level resistance to rapamycin is obtained
`when a mutant mTOR (Ser™’->lle’), having reduced
`affinity for binding the FKBP12-rapamycin complex,
`is
`expressed [14,46]. mTOR is essential
`for activation of
`ribosomal
`p70S6K1
`through
`phosphorylation
`of
`the
`rapamycin-sensitive
`sites
`at Thr” or Thr”
`[63,64].
`Substitution of either of these residues can also abrogate the
`ability of rapamycin to inhibit p70S6K activation. Whether
`this results in resistance to the growth inhibitory effect of
`rapamycinis less clear, and maybe cell context-specific.
`
`West-Ward Pharm.
`Exhibit 1017
`Page 005
`
`West-Ward Pharm.
`Exhibit 1017
`Page 005
`
`

`

`102 ananse
`
`300 Current Opinion in Investigational Drugs 2002 Vol 3 No 2
`
`Figure 4. Protective effect of the tumor suppressor p53 on rapamycin-induced apoptosis.
`
`@Oo
`
`c 8a2o=—o2G2E>n
`
`Control
`
`Rap 100
`
`Ad-Bgal
`
`
`
`Ad-Bgal + Rap 100
`
`Ad-p63
`
`Ad-p63 + Rap 100
`
`0°
`10° 101 102 103 104100
`
`101
`
`103
`
`104
`
`°a
`2oO
`
`Annexin V-FITC fluorescence
`
`Rh30 rhabdomyosarcoma cells were infected with either adenovirus expressing wild-type p53 (Ad-p53) or Ad-f-galactosidase ([}-gal) at a
`multiplicity of infection of 1. After 24 h, medium wasreplaced with serum-free N2E, andcells were grownfor a further 6 daysin the absence
`or presence of 100 ng/ml rapamycin (Rap 100). Cells were harvested and apoptosis determined by quantitative FACs analysis (ApoAlert).
`Left panels show dualstaining for propidium iodide uptake and annexin V-FITC. Right panels show corresponding distribution of annexin V-
`FITC staining in populationsofcells. (Adapted from Huangetal [15].)
`
`
`Decrease of 4E-BP1 protein expression
`Mechanisms of acquired resistance (withoutuse of mutagens)
`or intrinsic resistance have received less attention. Murine
`BC3H1 cells selected for acquired resistance demonstrated
`reduced levels of p27", and consistent with this, embryo
`fibroblasts with disrupted p27"are relatively resistant
`to
`rapamycin, as determined by inhibition of DNA precursors
`[75]. These data are consistent with G1 arrest, being in part
`due to rapamycin stabilizing this cyclin-dependent kinase
`inhibitor protein. Recently,
`rapamycin-resistant cell
`lines,
`Rh30/Rapal0K and C2, have also been obtained by growing
`
`rhabdomyosarcoma cells in the continuous
`Rh30 parental
`presence of increasing concentrations of rapamycin, without
`prior mutagenesis [82, Huang & Houghton, unpublished
`data]. Whenresistant clones were grown without rapamycin
`for 6 or 10 weeks, they reverted to rapamycin sensitivity (in
`terms of IC,, by growth inhibition assay). Thus, acquired
`rapamycin resistance was unstable.
`
`Analysis of several clones revealed increased levels of the c-
`Myc protein. Of interest, these rapamycin-resistant clones
`exhibited increased anchorage-independent growth in soft
`
`West-Ward Pharm.
`Exhibit 1017
`Page 006
`
`West-Ward Pharm.
`Exhibit 1017
`Page 006
`
`

`

`Inhibitors of mammalian target of rapamycin as novel antitumor agents: From bench to clinic Huang & Houghton 301
`
`agar. Consistent with increased c-Myc,the levels of the 4E-
`BP suppressor proteins bound to eIF-4E were significantly
`lower (approximately 10-fold), as were total cellular levels of
`4E-BP proteins. Steady state levels of 4E-BP transcripts
`remained unaltered, however, the rate of synthesis appeared
`to be decreased in rapamycin-resistant clones [Huang &
`Houghton, unpublished data].
`In clones that reverted to
`rapamycin sensitivity, total levels of 4E-BP1 became similar
`to those in parental cells. In some cases, intrinsic resistance
`also appears to relate to low 4E-BP:eIF4E levels. In colon
`carcinoma cells, very low levels of 4E-BP were detected,
`whereas eIF4E levels were similar to those in sensitive
`
`lines. In contrast, no significant changes were
`tumor cell
`determined for p7056 kinase levels or activity between
`parental andresistant clones.
`
`demonstrate elevated levels of phosphorylated Akt and
`activated p70S6K. While CCI-779 had noeffect on Akt
`activation, as anticipated, it normalized p7OS6K activity.
`Whether,
`loss of PTEN function consistently sensitizes
`tumor
`cells
`to
`rapamycin
`analogs
`remains
`to be
`demonstrated.
`
`Thesepreclinical results have revealed that CCI-779 exhibits
`impressive cytostatic, and in some instances, cytotoxic
`properties, and may be valuable to delay tumor progression
`and to improve survival when used alone, or in combination
`with other chemotherapeutic agents.
`
`
`
`BANOOIUI-QUY
`
`Early data from phase I trials have shown promise of CCI-
`779 in treatment of some cancers [88]. In European phase|
`clinical trials [89], CCI-779 was administered as a weekly 30-
`min intravenousinfusion in 18 patients with different types
`of advanced solid tumors. Doses ranged from 7.5 to 220
`mg/m’/week. After > 8 weekly doses, significant
`tumor
`regressions were observed in twopatients (receiving 15
`mg/m’ /week) with lung metastasis of renal cell carcinomas
`and in one patient (receiving 22.5 mg/m’/week) with a
`neuroendocrine tumor of
`the lung. Additionally,
`two
`patients experienced tumor
`stabilization.
`It
`is unclear
`whether the very high dose levels used in this study are
`necessarytoelicit antitumoractivity. Considering the high
`plasma levels of CCI-779 measured in patients (1000-fold
`greater than required for rapamycin activity in vitro), it is
`conceivable that CCI-779 is acting through a secondary
`mechanism independent of mTOR.
`
`Development of CCI-779 and RAD-001 as
`antitumor agents
`CCI-779
`2,2-
`rapamycin-42,
`inhibitor-779;
`cycle
`(cell
`CCI-779
`of
`is
`an
`ester
`bis(hydroxymethyl)-propanoic
`acid)
`rapamycin (Figure 1), which was developed by Wyeth-
`Ayerst as an antitumor agent. Like rapamycin, CCI-779
`acts
`by
`inhibition
`of mTOR,
`preventing
`the
`phosphorylation
`of
`4E-BPs
`and
`p70S6K [20-22,26].
`However, in contrast to rapamycin, CCI-779 is stable in
`aqueous
`solution, hence
`it
`can be
`formulated
`for
`intravenous administration.
`In preclinical
`tests, CCI-779
`possesses similar antitumor profiles to rapamycin [16,20-
`22,26,28¢,29e]. CCI-779
`potently
`inhibits growth
`of
`numerous cultured human tumor cell
`lines including
`trials were also conducted to
`In the US, phase I clinical
`human breast, prostate, pancreatic and small cell
`lung
`determine the safety and tolerability of CCI-779 [90]. CCI-
`carcinomas, glioblastoma, medulloblastoma, melanoma,
`779 was administeredasa daily intravenous 30-min infusion
`rhabdomyosarcoma and T-cell leukemia, with IC,, values
`for 5 days every 2 weeks. Of45 patients with various types
`in the nanomolar range [16,20-22]. These observations have
`of cancer, nine achieved some evidence of tumor response.
`been further supported by the significant inhibitory effect
`Importantly, results from the above twotrials indicate that
`of CCI-779 on growth of human tumor xenografts in
`CCI-779 is well
`tolerated in patients with only mild side
`athymic nude mice [16,20,21,24]. When combined with
`effects,
`such as acneform rash, mild mucositis,
`some
`cisplatin or camptothecin, CCI-779 showed additive
`thrombocytopenia, and elevated triglyceride and cholesterol
`cytotoxicity in subcutaneous implants of human brain
`levels. Rapamycin and its analogs potently inhibit T-cell
`tumors [16]. Interestingly,
`in vivo CCI-779 also inhibited
`the growth of human U251 malignant glioma cells that
`activation,
`thus the potential
`for
`immunosuppression in
`patients treated with CCI-779 wasanticipated. Interestingly
`were
`resistant
`to rapamycin in
`vitro, although the
`
`CCI-779~—induced evidence_ofonly modest
`
`
`mechanism is unknown [16].
`In addition, more recent
`results
`have
`revealed
`that
`the
`tumor
`suppressor
`immunosuppression. Rapamycins
`also
`inhibit
`insulin
`phosphatase and tensin homolog deleted on chromosome
`signaling, but in the clinical
`trials reported there was no
`ten (PTEN)-mutated or -deficient cancer cells are more
`toxicity consistent with inhibition of insulin signaling. CCI-
`779 is currently in phaseIII clinical trials. In athymic nude
`sensitive to CCI-779 (21 280,29]. PTEN acts as a major
`negative regulator of the PI3K/Akt signaling pathway [83-
`mouse xenografts, apart from breast and prostate cancers,
`85]. Loss of PTEN by deletion or mutation occurs in as
`human glioblastoma
`(U87MG)
`tumors
`and_ pancreatic
`many as 50%of all solid human tumors [86], resulting in
`carcinoma were also very sensitive to CCI-779. Thus, with
`activation of Akt. Conceptually, this could activate mTOR-
`an expanded focus,
`it is probable that clinical
`trials will
`dependent pathways, hence forming the basis for CCI-779
`identify other tumortypesthatare sensitive to CCI-779.
`hypersensitivity of PTEN deficient cells. However,
`it
`is
`unclear if Akt activates mTOR in situ, as mutation of
`putative Akt phosphorylation sites in the C-terminus of
`mTOR does not abrogate insulin stimulation of p70S6K
`activation [87]. Of considerable interest, however,
`is the
`report by Podsypaninaet al [28e] showing inhibition of
`neoplastic transformation in PTEN +/- mice. These
`animals develop spontaneous multifocal complex atypical
`hyperplasia in
`the uterine secretory epithelium that
`progresses
`to neoplastic transformation. Tumor
`cells
`
`RAD-001
`another
`is
`(40-O-(2-hydroxyethyl)-rapamycin)
`RAD-001
`rapamycin analog that is being developed as an antitumor
`agent. Like rapamycin and CCI-779, RAD-001 works by
`inhibition of mTOR, downregulating p70S6 kinase activity
`and decreasing the phosphorylation level of 4E-BP1 [27].
`Preliminary data indicate that in vitro, RAD-001 potently
`inhibits growth of numerous human tumorcell lines, with
`50% inhibition of growth in the femtomolar range [27].
`
`
`
`West-Ward Pharm.
`Exhibit 1017
`Page 007
`
`West-Ward Pharm.
`Exhibit 1017
`Page 007
`
`

`

`302 Current Opinion in Investigational Drugs 2002 Vol 3 No 2
`
`Unlike CCI-779, RAD-001 is a hydroxyethyl ether derivative
`of
`rapamycin, designed for oral administration.
`In vivo
`experimental
`results reveal
`that RAD-001 inhibited the
`growth of human tumor xenografts in nude mice at doses
`ranging from 0.5 to 5.0 mg/kg/day. At these doses, R