•
`
`American Society of Clinical
`Oncology
`Educational Book
`36th Annual Meeting
`Spring 2000
`
`EDITOR
`Michael C. Perry, MD, Columbia, MO
`
`Editorial Projects Manager
`Rich Harrington, Chestnut Hill, MA
`
`Editorial Assistant
`Lindsay McOmber, Chestnut Hill, MA
`
`© 2000 American Society of Clinical Oncology
`Alexandria, VA
`
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`Dancey 2000
`Page 001
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`

`

`Rapa:m.ycin - S ensi ti ve Signal-Transduction
`Pathways: Protein Translation Control of
`Cell Proliferation
`
`By Janet E. Dancey, MD
`
`Abstract: Rapamycin Is a natural product with antimi(cid:173)
`crobial, immunosuppressant, and antitumor activities
`owing to its modulation of signal-transduction pathways
`linking mitogenic stimuli to the synthesis of specific
`proteins needed for cell-cycle progression from the G1 to
`the 8 phase. Rapamycin and its soluble analog CCI-779
`have in vitro and in vivo anti proliferative activity against
`a broad range of human tumor-cell lines, and CCI-779 is
`just entering clinical trials to determine Its value in
`cancer therapy. The pharmacologic action of rapamycin
`is mediated through its binding to the intracellular pro(cid:173)
`tein FK506 binding protein 12 and subsequent inhibition
`of the protein kinase mammalian target of rapamycin
`(mTOR). mTOR signals to two separate pathways, each of
`which controls the translation of specific mRNAs. One
`rapamycin-sensitive pathway affects the activity of the
`
`C ELL PROLIFERATION requires the synthe(cid:173)
`
`sis of proteins necessary for entry into and
`transit through the cell cycle. 1 Thus, regulatory
`mechanisms to increase translation of these pro(cid:173)
`teins are necessary to promoting cell prolifera(cid:173)
`tion. As an anticancer treatment strategy, target(cid:173)
`ing translational regulation has been a relatively
`un exploited area of therapeutic development.
`However, the identification of the antiprolifera(cid:173)
`tive effects of the agent rapamycin (sirolimus)
`and the identification of its novel mechanism of
`action have renewed interest in targeting the
`translational regulatory apparatus as a therapeu(cid:173)
`tic strategy.
`Rapamycin, a natural product, has antimicro(cid:173)
`bial, immunosuppressant, and antitumor activi(cid:173)
`ties that result from the modulation of signal
`transduction pathways that link mitogenic stim(cid:173)
`uli to the synthesis of specific proteins needed for
`cell-cycle progression from the Gl to the S phase.
`
`Address correspondence to Janf't Dancey, MD. Investiga(cid:173)
`tionezZ Drug Branch, Cancel' Treatment Evaluation Program,
`Division ofCelTlcer Treatment and Diagnosis, National Cancer
`Institute, EPN 715, 6130 Executive Blvd, Rockville, MD 20854;
`email danceyj@ctep.nci_ nih.goll.
`© 2000 by American Society of Clinical Oncology.
`1092-9118/00/68-7.5
`
`68
`
`408 ribosomal protein 86 kinase p70s0k, and the other
`affects the function of eukaryotic initiation factor 4E(cid:173)
`binding protein-1 (4E-BP1), also known as phosphory(cid:173)
`lated heat- and acid-stable protein, The observations by
`several groups that the inhibition of mTOR- mediated
`p70 sek and 4E-BP1 phosphorylation by rapamycin were
`coupled to growth arrest in the G1 phase led to the
`hypothesis that the anti proliferative properties of rapa(cid:173)
`mycin result from its effects on the regulation of protein
`translation affected by these targets. However, the pre(cid:173)
`cise mechanisms of cell-cycle arrest are as yet unknown.
`This review will focus on recent advances in the under(cid:173)
`standing of the mechanisms by which rapamycln inhibits
`cell growth and the issues surrounding the development
`of this type of agent as a potential treatment for cancer.
`
`Rapamycin is in late phase III clinical trials as an
`immunosuppressive drug for organ and bone mar(cid:173)
`row transplant recipients, and its ester analog,
`CCI-779, is being evaluated in early clinical trials
`as a therapeutic agent against cancer. The immu(cid:173)
`nosuppressant effects of rapamycin result from
`its inhibition of the biochemical events required
`for the progression ofinterleukin-2-stimulated T
`cells from the Gl to the S phase of the cell cycle.:.!
`However, the growth-inhibitory actions of rapa(cid:173)
`mycin and its analog are not restricted to lym(cid:173)
`phoid cells, as these agents also have cytostatic or
`cytotoxic activities against solid tumor cell lines.
`In addition to its possible clinical utility, rap amy(cid:173)
`cin is a useful pharmacologic probe for studies of
`the signal-transduction pathways that govern
`translation. This review will focus on recent ad(cid:173)
`vances in the understanding of the mechanisms of
`cell-growth inhibition by rapamycin and the is(cid:173)
`sues surrounding the development of this class of
`agent as a potential mode of therapy in the
`treatment of cancer.
`
`THE DISCOVERY OF RAPAMYCIN AND ITS
`ANTIPROLIFERATIVE ACTIVITY
`
`Rapamycin, a macrolide, was first identified as
`a fungicide produced by the bacteria Streptomyces
`hygroscopicU8, which had been isolated from soil
`samples from Easter Island. 3
`,4 Although it was
`
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`CANCER THERAPIES TARGETING TRANSlATION
`
`originally identified as an antifungal agent, sub(cid:173)
`sequent studies demonstrated impressive antitu(cid:173)
`mor and immunosuppressant activities. Rapamy(cid:173)
`cin was originally evaluated by the National
`Cancer Institute (NCD in the late 1970s. At that
`time, it was found to have antiproliferative activ(cid:173)
`ity in a variety of murine tumor systems, includ(cid:173)
`ing B16 melanoma and P388 leukemia models.5.6
`Rapamycin has since been shown to inhibit the
`growth of B-celllymphoma celllines,7 and small(cid:173)
`cell lung cancer cell lines.8 In addition to its
`growth-inhibiting effects, rapamycin inducedp53
`independent apoptosis of childhood rhabdomyo(cid:173)
`sarcoma cell lines. 9 Treatment with rapamycin
`inhibited cyclin Dl expression and proliferation of
`MiaPaCa-2 and Panc-l human pancreatic cancer
`cell lines. 10 Rapamycin also augmented cisplatin(cid:173)
`induced apoptosis in murine T-cell lines, the hu(cid:173)
`man promyelocytic cell line HL-60, and the hu(cid:173)
`man ovarian cancer cell line SKOV3,u These
`data suggest that the anti proliferative action of
`rapamycin may be an important component of the
`pathway that prevents cell death and that rapa(cid:173)
`mycin may enhance the efficacy of selected cyto(cid:173)
`toxic agents.
`
`THE TARGET OF RAPAMYCIN
`Our understanding of molecular mechanisms
`underlying the biologic effects of rapamycin has
`advanced considerably in recent years (Fig 1).
`Similar to other natural immunosuppressants
`such as cycIosporine and FK506, rapamycin binds
`to members of the ubiquitous immunophilin fam(cid:173)
`ily of FK506 binding proteins (FKBPs), inhibiting
`their enzymatic activity as prolyl isomerases. 12,13
`Although this enzymatic function is important for
`altering protein conformation, it is, surprisingly,
`not relevant to the action of rapamycin.14 Al(cid:173)
`though there are many members of the FKBP
`family, biochemical and genetic studies suggest
`that FKBP12 is the most relevant binding protein
`for the pharmacologic activity of rapamycin in
`eukaryotic cells. Yeast mutants lacking FKBP12
`are viable and resistant to rapamycin toxicity,
`indicating that both the protein and the drug are
`required for rapamycin action. 14 Overexpression
`of FKBP12 in mammalian cells increases their
`sensitivity to rapamycin, and cell lines with re(cid:173)
`duced levels of FKBP12 are rapamycin-resistant,
`providing further evidence for a model in which
`the cellular effects of rapamycin result from its
`
`69
`
`BAD
`1
`Apoptosis
`
`mTOR
`
`I- [ Rapamycin~D
`
`PI-3 kinase I- PTEN
`~
`... - - PKBfAkt
`1
`/~
`I
`\
`
`4E-BP-IIPI-IAS-1
`
`1'70 S6 kinase
`
`Translation
`
`GI
`Fig 1. Rapamycin-sensitive signaling pathways. The target
`of rapamycin kinase (TOR) functions to regulate the activities of
`the translational regulators 4E-BP 1 /PHAS-. and p70 S6 kinase.
`Rapamycin binds to FKBP12 and the complex inhibits mTOR.
`
`binding to FKBP12. 15 Thus, rapamycin may ac(cid:173)
`tually be considered a "pro drug" for the active
`agent at the cellular level, the FKBP12-rapamy(cid:173)
`cin complex.
`The target of the rapamycin-immunophilin
`complex was initially identified in yeast and con(cid:173)
`firmed in mammalian cells. In mammalian cells,
`the complex interacts with a large polypeptide
`kinase of 290 kd, termed mammalian target of
`rapamycin (mTOR)16 (also known as FRAP,17
`RAFTl,18 and RAPT1 19
`), blocking its activity.
`The yeast TOR proteins exhibit a high degree of
`overall sequence identity (> 40%) to mTOR, with
`even greater identity (> 65%) observed in their
`carboxy-terminal catalytic domains (reviewed
`in2o). The high level of sequence conservation,
`together with the strikingly similar effects of
`rapamycin on yeast and mammalian cell growth,
`suggests that TOR proteins are important to cell
`function because they have been highly conserved
`during eukaryotic evolution.
`Yeast and mammalian TOR proteins are mem(cid:173)
`bers of a recently described family of protein
`
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`70
`
`kinases called phosphoinositide 3 kinase (PI3K)(cid:173)
`related kinases (PIKKs). Members of this family
`are involved in a range of essential cellular func(cid:173)
`tions, including cell-cycle progression, cell-cycle
`checkpoints, DNA repair, and DNA recombina(cid:173)
`tion.21 ,22 Among these PIICK family members are
`the cell-cycle regulatory protein kinases ataxia
`telangiectasia mediated, ataxia telangiectasia re(cid:173)
`lated, and DNA-dependent protein kinase cata(cid:173)
`lytic subunit. In contrast to the TOR proteins,
`ataxia telangiectasia mediated, ataxia telangiec(cid:173)
`tasia related, and DNA-dependent protein ki(cid:173)
`nases participate in cell-cycle checkpoints that
`govern cellular responses to DNA damage.21 The
`PIKK family members share a carboxy-terminal
`catalytic domain that bears significant sequence
`homology to the lipid kinase domains of PI3Ks,
`although no intrinsic lipid kinase activity has
`been described for mTOR.
`
`REGULATION OF PROTEIN SYNTHESIS
`BY mTOR
`Stimulation of quiescent cells with growth fac(cid:173)
`tors leads to a dramatic increase in the transla(cid:173)
`tion of a subset of mRNAs whose protein products
`are required for progression through the G I
`phase of the cell cycle. 23 mTOR regulates key
`pathways affecting the efficiency of protein trans(cid:173)
`lation, The upstream signaling pathway that cou(cid:173)
`ples growth-factor-receptor occupancy to mTOR
`protein activation is only partially understood.
`mTOR is a phosphoprotein, and its phosphoryla(cid:173)
`tion state, as well as its catalytic activity, have
`been reported to be modulated by the mitogen(cid:173)
`activated phosphatidylinosol (PI) 3 kinase-Pro(cid:173)
`tein Kinase B (PKB)/Akt. 24
`,25 PI3 kinase and Akt
`are considered to be proto-oncogenes, and the
`pathway is inhibited by the tumor suppressor
`genePTEN. 26 Although other signaling pathways
`are activated downstream of PI3K, the Akt path(cid:173)
`way is of particular interest because of its role in
`inhibiting apoptotic pathways and promoting cell
`proliferation (reviewed in26,27). In mammalian
`cells, activated mTOR signals to two separate
`pathways that control translation of specific sub(cid:173)
`sets of mRNAs. These are the 40S ribosomal
`protein S6 kinase, p70s6 k,8 and the eukaryotic
`initiation factor (eIF)-4E-binding protein-I C4E(cid:173)
`BPI), also known as PHAS-I (phosphorylated
`heat- and acid-stable protein).28,29 Among subset
`mRNAs regulated by these pathways are those
`
`JANET E. DANCEY
`
`encoding components of the protein synthesis
`machinery itself.
`Several recent reports indicated that activation
`of either PI3K or Akt is sufficient to induce the
`phosphorylation of both p70s6k and 4E-BPI/
`PHAS-I through mTOR.30,31 Treatment of acti(cid:173)
`vated PI3K or Akt expressing cells with rap amy(cid:173)
`the p70s6k and 4E-BPl/PHAS-I
`cin blocks
`phosphorylation, indicating that mTOR is re(cid:173)
`quired for these responses.29,32 In addition, there
`is evidence that Akt phosphorylates the carboxyl
`terminus of mTOR, contributing to its activa(cid:173)
`tion. 24,25 These results clearly link the PI3K-Akt
`pathway to p70s6k and 4E-BPl/PHAS-l transla(cid:173)
`tional control pathways through mTOR.
`Compared with its upstream effectors, the
`downstream actions of mTOR on translation are
`more fully characterized. For the subset of mR(cid:173)
`NAs that contain regulatory elements located in
`the 5'-untranslated regions, the binding of the
`mRNA to the ribosomal subunit and the efficient
`initiation of translation is mediated by the mul(cid:173)
`tisubunit eukaryotic initiation factor-4 (eIF-4l
`complex.33 4E-BPl/PHAS-I is a low-molecular(cid:173)
`weight protein that inhibits the initiation of
`translation through its association with eIF-4E,
`the mRNA cap-binding subunit of the elF -4F
`complex.23 Binding of 4E-BPs to eIF-4E is depen(cid:173)
`dent on the phosphorylation status of 4E-BP. In
`quiescent cells, 4E-BPI/PHAS-I is relatively un(cid:173)
`derphosphorylated and binds tightly to elF -4E
`(reviewed in34
`). Stimulation of cells by hormones,
`mitogens, growth factors, cytokines, and G-pro(cid:173)
`tein-coupled agonists results in 4E-BPl/PHAS-I
`phosphorylation through the action ofmTOR and,
`possibly, other kinases, which promotes the dis(cid:173)
`sociation of the 4E-BPI/PHA8-I-eIF-4E complex
`(Fig 2). eIF-4E can then bind to the eIF-4F com(cid:173)
`plex, and this interaction will then lead to an
`increase in translation rates. Conversely, growth(cid:173)
`factor deprivation or treatment with rapamycin
`results in 4E-BPlIPHAS-l dephosphorylation, an
`increase in eIF-4E binding, and a concomitant
`decrease in translation.34
`The second downstream target of mTOR is
`p70s6k, the kinase that phosphorylates the 40S
`ribosomal protein S6. In response to mitogenic
`stimuli, p70s6k phosphorylates 86 on multiple
`sites, and these modifications favor the recruit(cid:173)
`ment of the 40S subunit into actively translating
`polysomes35 and enhance the translation of mR-
`
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`CANCER THERAPIES TARGETING TRANSlATION
`
`71
`
`I ~;~t~~ / Rapamycin ---£rJ
`~,:;:\
`[4i~~
`~ /
`13~~!J)@ ~@ I ActiveeIF4EI
`1
`(~'j1E£AUG - AAAAA
`
`[I~11Ctiv~~
`
`-,_±~1-,/
`[eIF4F Compi;] --~ ~~~ation ofTransla~
`Fig 2. Activation of mTOR leads to the phosphorylation of
`4E-BP l/PHAS·l. eIF-4E is released and then binds to the eIF-4G
`scaffolding protein of the eIF-4F complex at the 5' cap site of the
`mRNA template. The eIF·4F complex contains a 7·methyl(cid:173)
`guanosine cab-binding subunit called eIF-4E, an RNA helicase
`called eIF-4A, and a multifunctional scaffolding protein eIF-4G
`that bridges the 405 ribosome and the mRNA, while also
`binding eIF-4E, eIF-4A, e1F-3, and the poly(A)-binding protein.
`
`NAs bearing 5' terminal oligopolypyrimidine
`tracts. Although these transcripts represent only
`100 to 200 genes, they can encode up to 20% of the
`cell's mRNA. 86
`Rapamycin treatment triggers the rapid de(cid:173)
`phosphorylation and inactivation ofp70s6k in mi(cid:173)
`togen-stimulated cells. Although p70s6k activa(cid:173)
`tion involves a complex series of phosphorylation
`events catalyzed by multiple protein kinases, the
`prompt inhibitory effect of rapamycin suggests
`that persistent stimulatory input from mTOR
`leads to the activated state of p70s6k.36 The exact
`nature of the input supplied by mTOR is unclear;
`however, a recent study suggested that the mTOR
`phosphorylates and suppresses the activity of a
`type 2A protein phosphatase bound directly to
`p70s6k.87
`The observations by several groups that the
`inhibition of mTOR-mediated p70s6k and 4E-BPl
`phosphorylation by rapamycin were coupled to
`growth arrest led to the hypothesis that the
`anti proliferative properties of rapamycin are a
`-40
`result of its effects on translational contro1.38
`Inhibition of these key signaling pathways results
`in the inefficient translation of the mRNAs of
`proteins such as cyclin D141 and ornithine decal'(cid:173)
`boxylase,42 which are important for cell-cycle pro(cid:173)
`gression through the G1 phase. However, in ad(cid:173)
`dition to its actions on p70s6k and 4E-BPll
`PHAS-1, rapamycin prevents cyclin-dependent
`
`kinase activation and retinoblastoma protein
`(pRb) phosphorylation.43-46 Rapamycin
`also
`seems to accelerate the turnover of cyclin Dl,
`both at the mRNA and protein levels, resulting in
`a deficiency of active cdk4lcyclin Dl complexes
`required for pRB phosphorylation and the release
`of E2F transcription factor and increased associ(cid:173)
`ation of p27kip1 with cyclin E/cdk2. These two
`events, along with the inhibition of translation of
`other mRNAs, can certainly explain the observed
`inhibition at the GllS phase transition.40,47 How(cid:173)
`ever, cells derived from mice in which the p27
`gene has been disrupted by homologous recombi(cid:173)
`nation are only partially rapamycin-resistant, in(cid:173)
`dicating that rapamycin can inhibit cell-cycle pro(cid:173)
`gression by p27-independent mechanisms.48
`Whether the effects of rapamycin on cyclin cdks
`and cdk inhibitor p27 are mediated through its
`inhibition of translation remains to be defined
`with precision in different cell types. Thus, al(cid:173)
`though the direct target of rapamycin has been
`identified, the downstream pathway from the
`target to the inhibition of cell-cycle progression
`requires further study.
`
`CLINICAL DEVELOPMENT
`Unfortunately, poor aqueous solubility and in(cid:173)
`stability compromised the development of rapa(cid:173)
`mycin as an anticancer agent. However, the NCI,
`in collaboration with Wyeth-Ayerst, examined
`several derivatives of rapamycin and selected one
`agent, CCI-779, for further development, on the
`basis on its mechanism of action and favorable in
`vitro and in vivo efficacy and toxicity data.
`CCI-779 is a soluble ester analog ofrapamycin
`with impressive in vitro and in vivo cytostatic
`'activity. Results from the NCI human tumor cell
`line screen showed that CCI-779 and its parent
`compound, rapamycin, share a mechanism of ac(cid:173)
`tion that is distinct from those of other cancer
`therapeutic agents. The two agents are similar:
`the Pearson correlation coefficient of the in vitro
`anti proliferative activities and potencies of the
`two agents across the 60-cellline screen is .86. In
`vitro, human prostate and breast cancer lines as
`well as CNS, melanoma, small-cell lung carci(cid:173)
`noma, and T-cell leukemia human tumor lines
`were among the most sensitive to CCI-779 with a
`50% inhibitory concentration of less than 10- 8
`M.49 Platelet-derived growth factor stimulation of
`the human glioblastoma line T98G was markedly
`
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`72
`
`inhibited (50% inhibitory concentration of ap(cid:173)
`proximately 1 pM), consistent with its proposed
`mechanism of action as an inhibitor of signal(cid:173)
`transduction pathways. Growth-inhibited cells
`were arrested in the G 1 phase, and growth-inhib(cid:173)
`itory effects were blocked by the FKBP inhibitory
`molecule ascomycin, suggesting that the mecha(cid:173)
`nism of action of CCI-779 is similar to that of
`rapamycin. 49 In vivo human tumor xenograft
`studies showed significant tumor growth inhibi(cid:173)
`tion, suggesting that CCI-779 should be devel(cid:173)
`oped as a cytostatic rather than a cytotoxic agent.
`Several intermittent dosing regimens of CCI-779
`were effective in these animal models. These
`findings are important because preclinical studies
`have shown that when given intermittently, the
`immunosuppressive effects of CCI-779 resolve
`within 24 hours after the last dose.
`Given its proposed properties as a cytostatic
`agent, CCI-779 may be of value in delaying time
`to tumor progression and in increasing survival in
`patients when used alone or in combination with
`other anticancer agents. Two schedules are cur(cid:173)
`rently being evaluated in phase I trials: a weekly
`schedule and a daily for 5 days every 2 weeks
`schedule. Both phase I trials were designed with
`the traditional phase I objectives of determining
`the maximum-tolerated dose and dose-limiting
`toxicities of the agent. Preliminary results from
`the phase I study of the weekly 30-minute infu(cid:173)
`sion have recently been reported. 50 Twelve pa(cid:173)
`tients treated at dose levels of 7.5 mg/m2 to 60
`mg/m2/wk did not experience dose-limiting toxic(cid:173)
`ities. Interestingly, one partial response and one
`minor response were observed in two patients
`with renal cell carcinoma and lung metastases
`treated at 15 mg/m2
`. Only mild grade 1-2 skin
`reactions and mucositis were seen. The range of
`skin reactions has been described as dryness,
`urticaria, eczema-like lesions, and erythematous
`papules that did not seem to worsen with re(cid:173)
`peated dosing. Because CCI-779 is structurally
`similar, some of the adverse effects reported in
`clinical trials of rapamycin may also be seen
`during the clinical evaluation ofCCI-779. Among
`the most common adverse events that occur as a
`result of oral dosing of rapamycin are hyperlipid(cid:173)
`emia; elevated lactate dehydrogenase levels; hy(cid:173)
`pophosphatemia; hypokalemia; reduced WBC,
`RBC, and platelet counts; stomatitis; and arthral-
`
`JANET E. DANCEY
`
`gias. Phase II studies of a broad range of tumor
`types, sponsored by the NCI and Wyeth-Ayerst,
`will be initiated once phase I studies have been
`completed.
`The observation that CCI-779 induced tumor
`regression in patients treated at relatively non(cid:173)
`toxic doses on the phase I study is particularly
`noteworthy. On the basis of the preclinical re(cid:173)
`sults, it was thought that CCI-779 would act as a
`cytostatic agent, and phase I studies were de(cid:173)
`signed with the traditional objectives of determin(cid:173)
`ing maximum-tolerated dose and dose-limiting
`toxicities. IfCCI-779 is biologically active at lower
`doses, it may not be necessary to treat patients
`with higher doses of this agent that place them at
`risk for toxicity. Ideally, once the biologically
`effective dose to affect the rapamycin target(s) is
`achieved, further dose escalation is unnecessary.
`Defining and limiting drug concentrations to the
`biologically effective range would prevent cross(cid:173)
`reactions with other molecules that cause toxicity
`at higher drug concentrations. In addition, mea(cid:173)
`suring the drug's effect on its target(s) could
`potentially be used as a surrogate efficacy end
`point, assuming there is a strong correlation
`between target modulation and cell survival 01'
`proliferation.
`Unfortunately, determining the optimal biolog(cid:173)
`ically active dose in patients is problematic. Pre(cid:173)
`clinical studies using animal models could define
`plasma and tumor drug concentrations that per(cid:173)
`turb the important cellular target(s) of rapam(cid:173)
`cyin. In turn, the drug's effect on its targets(s)
`should be correlated with any antiproliferative or
`apoptotic effects. Although preclinical studies can
`be helpful in developing assays of target effects
`and defining active drug concentrations, the opti(cid:173)
`mal dose, in principle, should be defined in hu(cid:173)
`man tumor tissue. In practice, this requires a
`valid and reliable assay of the drug's effect on the
`intended target and that patients with accessible
`lesions agree to undergo repeated biopsies. Al(cid:173)
`though these requirements increase the complex(cid:173)
`ity and cost of drug development, efforts to ac(cid:173)
`quire these data can, nonetheless, be amply
`rewarded because information resulting from
`such studies may facilitate subsequent clinical
`development. For CCI-779, assays to determine
`the 4E-BPI/PHAS-1 and/or p70s6k phosphoryla(cid:173)
`tion status may be helpful in defining a pharma-
`
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`CANCER THERAPIES TARGETING TRANSLATION
`
`cologie ally active dose. However, it is possible
`that effects observed at these molecular targets
`may not correlate with antiproliferative effects.
`In fact, there is evidence that cell-cycle progres(cid:173)
`sion and translation can proceed despite the de(cid:173)
`phosphorylation of 4E-BPIIPHAS-I and the inac(cid:173)
`tivation ofp70,,6k by rapamycin. 51 52 These results
`suggest that either these pathways are not the
`only mechanisms by which cell-cycle progression
`is regulated, or-under certain conditions(cid:173)
`mTOR-4E-BPIIPHAS-1 and mTOR_p70s6k path(cid:173)
`ways are redundant. 51,52 Thus, assessing drug
`effects by use of these targets may assist re(cid:173)
`searchers in determining a pharmacologically ac(cid:173)
`tive dose but may not predict drug efficacy either
`because the assays are assessing targets that are
`not related to drug effects on proliferation or
`because downstream factors are rendering the
`cells resistant. In the absence of data, pharmaco(cid:173)
`kinetic studies may be helpful because doses that
`provide concentrations in humans which are ac(cid:173)
`tive in preclinical models are likely to be effective.
`In addition to a definition of the appropriate
`dose, choosing the appropriate efficacy end point
`for phase II studies is also problematic. Preclini(cid:173)
`cal data suggested that CCI-779 would prevent or
`delay the growth of tumors rather than induce
`tumor regressions. Solely on the basis of the
`preclinical results, one could argue that efficacy
`end points other than response should be used in
`phase II trials of CCI -779. Although it is possible
`that by reducing the rate of cell proliferation
`relative to the rate of apoptosis could lead to a
`reduction in tumor mass over time, cytostatic
`agents are more likely to cause prolonged disease
`stabilization rather than objective tumor re(cid:173)
`sponse. Possible surrogate phase II end points
`that have been proposed for the evaluation of
`other cytostatic agents include time to progres(cid:173)
`sion, changes in tumor markers, target inhibition,
`and positron emission tomography scan-assayed
`indices of cell proliferation, as well as the propor(cid:173)
`tion of patients with early disease progression
`and an assessment of clinical benefit (reviewed
`in53
`). Unfortunately, none of these proposed end
`points has been shown to correlate with patient
`benefit.
`Given our current understanding of the mech(cid:173)
`anism of action of rapamycin, a number of hy(cid:173)
`potheses regarding the molecular abnormalities
`that may correlate with the efficacy of CCI-779
`
`73
`
`can be generated. On the basis of preclinical
`results in studies of glioma,49 small-cell carcino(cid:173)
`ma, 8 and rhabdomyosarcoma, 9 tumors that rely
`on paracrine or autocrine stimulation of receptors
`that trigger the PI3K1AktimTOR pathway or tu(cid:173)
`mors with mutations that cause constitutive acti(cid:173)
`vation of the PI3/ Akt pathway may depend on
`rapamycin-sensitive pathways for growth. In fact,
`abnormal activation of this pathway is relatively
`common because mutations of the tumor-suppres(cid:173)
`sor gene PTEN, which encodes for a lipid phos(cid:173)
`phatase that inhibits PI3K-dependent activation
`of PKB/Akt, occur in multiple tumor types with a
`frequency approaching that of p53. 26 Deletion or
`inactivation of PTEN results in unregulated Akt
`activity. Thus, the presence of PTEN mutations
`may also predict for the activity of CCI-779.
`Because CCI-779 seems to induce cytostasis by
`preventing progression through the G 1 phase, it
`is possible that abnormalities of regulators of the
`Gl checkpoint such as pRB, p16, p27, and cyclin
`D may predict for drug efficacy. The parent com(cid:173)
`pound, rapamycin, affects the efficiency with
`which cdks are activated by alteration of the
`expression of the cyclin D subunit.4o Because p16
`inhibits the cyclin n-cdk4/6 phosphorylation of
`pRb required for progression through the G 1
`phase, loss of p16 results in unregulated cyclin
`D/cdk activity. Decreasing cyclin D might reintro(cid:173)
`duce a cdk-inhibitory effect and arrest the cell
`cycle. Defining the molecular characteristics of
`tumors that correlate with the activity or inactiv(cid:173)
`ity of agents may help to identify the groups of
`patients who may benefit from treatment. Such
`information can only be obtained by systemati(cid:173)
`cally collecting and analyzing tumor samples
`from patients. Although these studies add to the
`complexity of the trial, it is possible that molecu(cid:173)
`lar characterization of patients' tumors may be
`more predicative of drug efficacy than histology. 53
`Although our understanding of the regulation
`of translation and its effects on cell proliferation
`are incomplete, targeting translation to control
`cell proliferation is a novel therapeutic strategy
`worthy of further evaluation. Although our un(cid:173)
`derstanding of the mechanisms of action this
`class of agents is imperfect and will undoubtedly
`deepen over time, these agents have significant in
`vitro and in vivo antiproliferative activity against
`a broad range of human tumor cell lines, which
`supports the initiation of clinical trials of CCI-799
`
`West-Ward Exhibit 1016
`Dancey 2000
`Page 007
`
`

`

`74
`
`JANET E. DANCEY
`
`in cancer patients. As with other molecular-tar(cid:173)
`geted therapies, the challenge to investigators
`will be to efficiently determine what roles these
`translational regulatory pathways and this class
`of agent will play in the treatment of cancer
`patients.
`
`ACKNOWLEDGMENT
`
`I thank Robert Abraham, PhD, and Peter Houghton, PhD,
`for their insights into the mechanisms of action and antipro(cid:173)
`liferative activities of these agents and Edward Sausville, MD,
`PhD, Susan Arbuck, MD, and Howard Streicher, MD, for their
`helpful critique of the manuscript.
`
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