F O C U S
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`Will mTOR inhibitors make it as cancer drugs?
`Charles L. Sawyers*
`
`Howard Hughes Medical Institute; Departments of Medicine, Molecular and Medical Pharmacology, and Urology and Jonsson Cancer
`Center, David Geffen School of Medicine at UCLA, University of California, Los Angeles, California 90095
`*Correspondence: csawyers@mednet.ucla.edu
`
`Several pharmaceutical and biotechnology companies are
`actively pursuing the clinical development of inhibitors of the
`serine/threonine kinase mammalian target of rapamycin (mTOR)
`for cancer. Rapamycin, the original natural product compound
`first shown to inhibit mTOR, is already an approved drug for pre-
`vention of allograft rejection in recipients of organ transplants
`due to its potent inhibition of T cell activation. What is the logic
`behind the use of the same agent for cancer indications? This
`focus will review the background supporting the potential utility
`of mTOR inhibitors as anticancer agents, then compare and
`contrast two different approaches for its clinical development.
`The first approach is empiric, based on the traditional phase I
`design of escalation to maximum tolerated dose in a broad
`patient population, followed by larger trials focused on those
`tumor types that demonstrate hints of activity in the phase I set-
`ting. The second approach is mechanism based, building on
`knowledge of signaling pathways that activate mTOR, where
`the dose is selected by measuring target enzyme inhibition in
`tumor cells and patient eligibility is defined by molecular profil-
`ing studies. I will speculate on potential outcomes from both
`approaches as well as my view of the eventual role that mTOR
`inhibitors may play in the cancer drug armamentarium.
`
`mTOR: A central regulator of cell growth
`Rapamycin, a bacterially derived natural product, induces G1
`arrest in various cell types at low nanomolar concentrations.
`The mechanism was cleverly deciphered through yeast genetic
`screens that identified a serine/threonine kinase named target
`of rapamycin (TOR) (Heitman et al., 1991), which is a member
`of the larger phosphatidylinositol 3-kinase (PI3K) related family
`that includes PI3K, ATM, and ATR. Rapamycin exerts its action
`by first binding to the immunophilin FK506 binding protein
`(FKBP12). The FKBP12/rapamycin complex then binds mTOR,
`preventing phosphorylation of downstream targets such as S6
`kinase (S6K) and 4EBP1 (see Abraham, 2002; Schmelzle and
`Hall, 2000; Shamji et al., 2003).
`mTOR receives a diverse set of signaling inputs. Among the
`most relevant for a discussion of cancer is mTOR activation by
`growth factors like IGF-1, which activates the PI3K/Akt signaling
`pathway. Akt directly phosphorylates a number of proteins that
`impact cell survival and proliferation (reviewed in Vivanco and
`Sawyers, 2002), but the details defining the connection to
`mTOR were unclear until recently. Now a series of biochemical
`and genetic studies have established a pathway from Akt to
`mTOR involving the tuberous sclerosis complex proteins tuberin
`and hamartin, as well as the small Ras-like GTPase Rheb.
`Tuberous sclerosis complex 2 (TSC2) is a direct substrate of Akt
`(Inoki et al., 2002; Manning et al., 2002; Potter et al., 2002)
`(Figure 1). Unphosphorylated TSC2 is bound to TSC1 in a com-
`plex that blocks mTOR activation. Akt-mediated phosphory-
`lation of TSC2 disrupts the TSC1/TSC2 complex, allowing
`unrestrained mTOR kinase activity. Rheb (Ras homolog
`
`enriched in brain) functions in this pathway downstream of
`TSC2 and upstream of mTOR (Garami et al., 2003; Saucedo et
`al., 2003; Stocker et al., 2003; Zhang et al., 2003). Interestingly,
`Rheb is highly expressed in transformed cancer cell lines and
`functions as an oncogene in fibroblast transformation models,
`but can also block transformation by Ras or B-Raf (Clark et al.,
`1997; Im et al., 2002).
`A simple linear model of this pathway (Akt-TSC1/2-Rheb-
`TOR-S6K) cannot account for all experimental findings. For
`example, PI3K can activate S6K
`independently of Akt
`and mTOR through an alternative pathway involving PDK1
`(Radimerski et al., 2002). In addition, mTOR functions in a nutri-
`ent sensor pathway independent of PI3K and Akt. mTOR is
`inhibited during conditions of nutrient deprivation, which leads
`to a slowdown in cell growth (defined as cell size or mass as
`opposed to cell proliferation). This starvation response makes
`teleological sense because mTOR plays a key regulatory role in
`protein translation through modulation of S6K and 4EBP1
`action. The recently isolated mTOR binding protein Raptor pro-
`vides a potential mechanism for how mTOR regulates down-
`stream effectors (Hara et al., 2002; Kim et al., 2002). Because
`Raptor can also bind S6K and 4EBP1, it may function as a scaf-
`fold, keeping this signaling complex primed for rapid response
`to inputs from various pathways.
`Because S6K and 4EBP1 play crucial roles in regulating
`translation, there has been much interest in defining the down-
`stream mRNA targets of mTOR. The 5′ untranslated regions of
`cyclin D1 and c-Myc mRNAs both have CAP sequences, ren-
`dering them subject to regulation by 4EBP1. Cyclin D1 and c-
`Myc are regulated in part by mTOR since the levels of both
`proteins can fall in cells exposed to rapamycin in certain con-
`texts (Hosoi et al., 1998; Muise-Helmericks et al., 1998; Takuwa
`et al., 1999). Global transcriptome analyses using polysome
`fractions are beginning to define the range of mTOR-regulated
`mRNAs (Peng et al., 2002; Rajasekhar et al., 2003; Shamji et
`al., 2000). At a first approximation, these analyses appear to
`confirm important functional roles of c-Myc and cyclin D1 in
`rapamycin-induced growth arrest (Gera et al., 2003).
`
`Rapamycin has anticancer activity
`The natural products program at the National Cancer Institute
`identified rapamycin as a potential anticancer agent in the
`1970s (Douros and Suffness, 1981). Once the biochemical TOR
`was identified and more detailed activity profiles against a panel
`of human tumor cell lines were completed, some very interest-
`ing patterns emerged. Specifically, cell lines derived from differ-
`ent cancer types were noted to undergo G1 arrest when
`exposed to 1 nM rapamycin, a concentration which closely
`matches that required for biochemical inhibition of mTOR in
`cells. Notably, several other tumor cell lines that failed to
`respond to the 1 nM dose did undergo growth arrest at significantly
`higher concentrations (?1000 nM). While these phenotypic
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`Figure 1. Signaling pathways involving mTOR
`The diagram depicts the current view of mTOR regulation through the
`PI3K/Akt pathway based on biochemical and genetic studies. See text for
`more details.
`
`screening studies revealed the broad potential for rapamycin as
`an antiproliferative agent, they did not uncover the mechanism.
`Nonetheless, the results define two groups of rapamycin-sensi-
`tive cell lines—those whose response correlates with inhibition
`of mTOR (1 nM) (group 1) and those that require significantly
`higher concentrations (group 2). Of note, the failure of the group
`2 cell lines to respond to low “dose” (1 nM) rapamycin cannot be
`explained by insufficient blockade of mTOR kinase activity
`because 1 nM rapamycin was equally effective at inhibiting S6K
`and 4EBP1 phosphorylation in both groups (Neshat et al.,
`2001). These data support the notion that mTOR is the biologi-
`cally relevant target of rapamycin for the tumor lines in group 1,
`whereas other targets are relevant in group 2.
`
`Initial clinical development of mTOR inhibitors—The
`empiric strategy
`In the absence of any histologic subtype or molecular marker
`that can distinguish between group 1, group 2, and nonrespon-
`sive cell lines, the initial clinical development of mTOR inhibitors
`in cancer has proceeded empirically. Several pharmaceutical
`companies have compounds in clinical trials for this indication.
`Among the most advanced is CCI-779 from Wyeth. CCI-779 is
`an ester of rapamycin with comparable potency and specificity
`for mTOR but with a longer half-life. Phase I and phase II clinical
`trials of the intravenous formulation have been completed and
`show promising enough results to warrant a phase III random-
`ized trial that is underway. A brief review of the rationale and
`clinical details underlying this empiric approach is warranted to
`contrast with the molecularly driven approach described sub-
`sequently (see Dancey, 2002 for a comprehensive review of the
`clinical experience with mTOR inhibitors in cancer).
`Following the traditional strategy of defining the maximum
`
`tolerated dose (MTD), Wyeth has conducted two phase I dose
`escalation studies of CCI-779 in patients with solid tumors using
`two different delivery schedules—weekly versus daily for 5 days
`every 2 weeks (Hidalgo et al., 2000; Raymond et al., 2000).
`Toxicities such as low platelet counts and fatigue were observed
`at high doses, but the drug was generally well tolerated.
`Importantly, the doses tested in these cancer trials gave peak
`plasma concentrations well above that required for inhibition of
`mTOR, but the intermittent nature of the dosing allowed troughs
`to fall below mTOR inhibition levels. Clinical responses were
`observed in several patients with advanced stage kidney can-
`cer; therefore, a phase II trial was conducted in this disease.
`Using three different doses of CCI-779 given weekly, the objec-
`tive response rate was only 5%, but there was a higher rate of
`minor responses (29%) and stable disease (40%) (Atkin et al.,
`2002). Of note, responses were observed equally across all
`doses (all of which give peak serum levels that block mTOR).
`On this basis, a phase III randomized trial has been initiated.
`
`Deconvoluting the mechanism of response in kidney
`cancer
`Now that the empiric clinical development plan of CCI-779 has
`identified renal cell carcinoma as a potential mTOR-dependent
`cancer, it is interesting to speculate on possible mechanisms.
`Two scenarios come to mind based on the critical role of angio-
`genesis in these tumors due to expression of hypoxia inducible
`factor (HIF) (reviewed in (Kaelin, 2002). The first is based on
`recent evidence that mTOR inhibitors may be antiangiogenic
`agents. PI3K, Akt, and mTOR are all critical for vascular
`endothelial growth factor (VEGF)-mediated endothelial cell pro-
`liferation, survival, and migration (Yu and Sato, 1999). In fact,
`rapamycin-coated coronary artery stents prevent restenosis in
`patients with coronary artery disease who undergo angioplasty
`(Morice et al., 2002). Preclinical studies suggest that this effect
`of mTOR inhibitors on endothelial growth may also apply to
`tumor angiogenesis since rapamycin blocked the in vivo growth
`of tumor cells that were resistant to rapamycin in vitro (Guba et
`al., 2002). A recent antiangiogenesis trial in kidney cancer (not
`involving an mTOR inhibitor) demonstrated that anti-VEGF anti-
`body was not effective in causing tumors to shrink but caused
`significant delays in the time to tumor progression (Yang et al.,
`2003). This clinical outcome is strikingly reminiscent of the
`phase II results of CCI-779 in kidney cancer and begs the ques-
`tion of whether mTOR inhibitors may work by this mechanism.
`A second scenario to explain the activity of CCI-779 in kid-
`ney cancer is based on evidence that mTOR can regulate HIF
`expression through the PI3K/Akt pathway (Hudson et al., 2002;
`Zhong et al., 2000). Therefore, mTOR inhibitors could have
`direct effects on tumor cells by reducing HIF levels as well as
`indirect effects on the endothelial cells recruited for tumor
`angiogenesis.
`
`Rethinking the empiric clinical development strategy for
`mTOR inhibitors
`The usual justification for pursuing an empiric drug develop-
`ment approach is that the molecular target of the drug is
`unknown or its disease-specific role is poorly understood. As a
`result, drug dose is chosen on the basis of what can be tolerat-
`ed without inordinate toxicity. In this way, it is assumed that any
`clinical activity will not be missed due to insufficient drug levels.
`Once dose-limiting side effects are observed, the schedule of
`drug delivery is typically modified to allow recovery from any
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`Table 1. Potential mTOR-dependent cancers
`Molecular lesion
`Clinical disease
`Upstream of mTOR
`PTEN loss
`
`PI3K/Akt activation
`
`TSC1/2 loss
`
`Downstream of mTOR
`cyclin D1 overexpression
`
`Myc overexpression
`
`HIF overexpression
`
`Glioblastoma
`Prostate cancer
`Endometrial cancer
`Othersa
`Breast cancer (Her2+)
`Chronic myeloid leukemia (Bcr-Abl)
`Ovarian cancer
`(PI3K or Akt gene amplification)
`Othersa
`Tuberous sclerosis
`
`Mantle cell lymphoma
`Breast cancer
`Burkitt(cid:146)s lymphoma
`Other Myc-driven cancers?
`Kidney cancer
`Others?
`
`aFor more complete listings, see Vivanco and Sawyers, 2002.
`
`toxicities. Hence, an intermittent regimen using doses just
`below the toxic level is the typical outcome of phase I evalua-
`tions of novel agents. Based on this approach, the early phase
`CCI-779 studies converged on a weekly dosing regimen.
`However, mTOR inhibitors represent an unusual example
`because of the broad clinical experience with rapamycin as an
`immunosuppressive agent for patients receiving organ trans-
`plants. For this indication, the dose is based on that required to
`inhibit T cell activation, which correlates with mTOR inhibition in
`blood cells. These patients take rapamycin daily at relatively low
`doses (in comparison to the weekly “cancer” dose of CCI-779)
`with minimal side effects. A recent phase I clinical report of a
`second mTOR inhibitor, RAD-001 (Novartis), measured S6K in
`blood cells to guide dose selection in cancer patients
`(O’Donnell et al., 2003).
`If mTOR is the relevant target in cancer cells, why wasn’t
`this low dose, daily schedule evaluated initially? And why not
`select patients whose tumors are more likely to be mTOR
`dependent? Obviously, the details underlying the decision-
`making processes at pharmaceutical companies are not public
`knowledge, but I will speculate on a few potential reasons. First,
`a daily dosing schedule would lead to constitutive T cell sup-
`pression, a side effect that one would prefer not to have in can-
`cer patients. My personal view is that immune suppression is of
`secondary concern in the initial stages of developing an anti-
`cancer agent and should be factored into decision making only
`after the primary goal of tumor response has been achieved.
`Second, in vitro studies have defined a second population of
`rapamycin-sensitive tumor cell lines (group 2, see above) where
`the relevant target of drug action is not mTOR (perhaps another
`PIKK family kinase?). In hopes of capturing this second group of
`tumors, one would not want to restrict clinical evaluation to daily,
`low dose mTOR inhibitor because this would only be effective
`against those tumors presumed to be mTOR dependent (group
`1). Third, it would be difficult to convince upper management to
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`support a clinical development program focused exclusively on
`mTOR-dependent cancers without any knowledge of the size of
`the target population or the tools to identify the patients. Finally,
`marketing departments would presumably be concerned about
`the confusion generated by parallel use of the same drug for
`immune suppression and cancer. Reformulation into a new
`compound with a different schedule of administration could
`address this issue (albeit, cosmetically). I reiterate that my com-
`ments in this paragraph are highly speculative, but I hope that
`they help illustrate the complex set of scientific, economic, and
`regulatory considerations that influence decisions underlying a
`clinical development path.
`
`An alternative clinical development strategy using
`molecularly selected patients
`Studies of kinase inhibitors such as imatinib (also called
`Gleevec or STI571) indicate that these drugs can have tremen-
`dous clinical activity in appropriately selected patients. This
`experience has led to the notion that certain cancers are kinase
`dependent, typically due to fusions, point mutations, or amplifi-
`cation affecting the kinase gene that is targeted by the inhibitor
`(reviewed in Sawyers, 2003). In general, these genetic events
`enhance enzymatic activity of the kinase and serve as onco-
`genic events driving the growth of the cancer. Since mTOR is a
`kinase, can a similar paradigm of clinical development be
`applied here?
`Strict application of the imatinib paradigm is unlikely since
`there is currently no evidence that the mTOR gene is mutated or
`amplified in human cancer. However, preclinical observations
`suggest that tumors with primary genetic abnormalities affect-
`ing pathways that regulate mTOR are, in fact, dependent on
`mTOR. These abnormalities
`include upregulation of
`the
`PI3K/Akt pathway, directly or by loss of the tumor suppressor
`phosphatase PTEN, as well as mTOR upregulation by TSC2
`loss. PTEN null tumors are sensitive to mTOR inhibitors in sev-
`eral different human and murine preclinical models (Grunwald
`et al., 2002; Neshat et al., 2001; Podsypanina et al., 2001; Shi et
`al., 2002). Transformation induced by oncogenic alleles of Akt,
`but not Myc or Ras, is also reversed by mTOR inhibition (Aoki et
`al., 2001). Recent studies in conditional PTEN knockout or
`transgenic Akt mouse models confirm a role for mTOR in either
`aberrant cell growth (Kwon et al., 2003) or transformation
`through the PI3K/Akt pathway (E. Holland, personal communi-
`cation; W. Sellers, personal communication). Similarly, tumors
`caused by loss of TSC2 also show enhanced sensitivity to
`rapamycin in growth assays (Kenerson et al., 2002).
`The mTOR dependency of these tumors, whether induced
`by loss of PTEN or TSC2 or by activation of PI3K or Akt, shares
`conceptual similarity to synthetic lethal relationships originally
`described in yeast. Loss of PTEN or TSC2 seems to render
`mTOR essential in tumor cells but not in surrounding normal
`cells. However, genetic studies in worms and flies make it clear
`that TOR is essential for normal development (Long et al., 2002;
`Oldham et al., 2000; Zhang et al., 2000). Because TOR is highly
`conserved, it is likely that certain normal mammalian functions,
`including T cell function, will be mTOR dependent. Nonetheless,
`the clinical experience with rapamycin as an
`immuno-
`suppressive agent indicates that mTOR inhibitors are
`well tolerated.
`An alternative group of tumors that might also be mTOR
`dependent are those that express high levels of mTOR-regulat-
`ed mRNAs, such as cyclin D1 or Myc. Because both genes are
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`also regulated by mTOR-independent mechanisms, it is difficult
`to anticipate how effective an mTOR inhibitor might be in reduc-
`ing the expression of either protein. This, as well as the mTOR
`dependency of PTEN or TSC2-deficient cancers, can be tested
`in clinical trials where patient selection is based on document-
`ing the relevant molecular pathway abnormality in tumor tissue.
`The greatest challenge in designing these clinical trials is
`identification of molecularly defined patient cohorts (Table 1).
`Traditional inclusion criteria using tumor histology and site of
`origin will fail miserably because the molecular phenotype can-
`not be discerned from the clinical phenotype (tuberous sclerosis
`may be an exception). The most appropriate tools for discerning
`these phenotypes in the context of a clinical trial have not been
`defined. Among the potential approaches are proteomic or gene
`expression profiling to recognize signatures of kinase depen-
`dency. A first iteration of this approach, through the use of
`phospho-specific antibodies against specific kinase targets or
`substrates, shows promise in initial immunohistochemical appli-
`cations (Choe et al., 2003). Limitations include the tricky perfor-
`mance characteristics of certain antibodies and the potential
`need for large numbers of optimized antibodies for comprehen-
`sive evaluation. It may also be possible to recognize kinase acti-
`vation through gene expression profiling (Allander et al., 2001;
`Shai et al., 2003). Tissue availability and tumor heterogeneity
`present additional obstacles. Nonetheless, several of these
`approaches are under evaluation in small clinical studies of
`mTOR inhibitors. One possibility is that pilot trials will identify
`biomarkers that can be incorporated more easily into large
`scale studies.
`
`Expectations
`mTOR inhibitors are now far along on the clinical development
`path as anticancer agents, but it remains unclear how the story
`will unfold. The empiric approach has uncovered a low but
`reproducible objective response rate in kidney cancer patients.
`There is the impression of a much larger rate of disease stabi-
`lization, but this must be confirmed by a randomized trial. mTOR
`inhibitors will also be combined empirically with other agents
`(like interferon in kidney cancer) in an effort to increase the
`response rate, but these trials will be conducted without molec-
`ular insight into the mechanism of response. Although we all
`hope for success, this strategy is strikingly similar to recent
`combination trials of EGFR inhibitors with chemotherapy in
`advanced stage lung cancer (reviewed in Dancey and Freidlin,
`2003). Single agent response rates with EGFR inhibitors in
`these patients are low but reproducible; the molecular basis of
`response is unknown; and four large randomized trials of EGFR
`inhibitors plus chemotherapy were all negative.
`The alternative approach of using molecular insights from
`preclinical work to select patients is just now being evaluated.
`Efforts to identify appropriate patients based on immunohisto-
`chemical staining of tumor biopsies have been convincing
`enough to launch exploratory trials, but the robustness of these
`assays in realtime clinical settings remains to be defined.
`If we assume that these assays are accurate and that con-
`tinuous daily dosing of mTOR inhibitors effectively blocks mTOR
`in tumor cells, what clinical outcomes might we expect? There
`are several issues to consider. First, mTOR inhibition in sensi-
`tive tumor cell lines typically causes G1 arrest rather than apop-
`tosis. Therefore, objective response rates may be low but
`disease stabilization could be high. Second, even though we
`have the tools to recognize tumors with loss of PTEN or activa-
`
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`
`tion of Akt, we do not know if this molecular abnormality repre-
`sents an early or late event in the history of that tumor. This
`issue could be critical because an inhibitor that blocks an initiat-
`ing oncogenic event is, presumably, more likely to induce a clin-
`ical response than one that blocks a later event involved in
`disease progression.
`No matter the outcome, it is likely that combination therapy
`will be required to fully evaluate the potential of mTOR inhibitors
`in order to maximize response rates and prevent drug resis-
`tance. To avoid mistakes of the past, we need to select combi-
`nations based on mechanistic insights into why certain patients
`respond and others do not. One possibility is an mTOR plus
`EGFR inhibitor combination, particularly in a disease like
`glioblastoma where PTEN loss and genome-based EGFR acti-
`vation can occur in the same tumor (Choe et al., 2003).
`Furthermore, perturbations in one signaling pathway may alter
`the cellular response to inhibition of another, as has been
`observed with PTEN and EGFR in laboratory models (Bianco et
`al., 2003; She et al., 2003). The good news is that we finally
`have a very nice selection of signaling pathway inhibitors, and
`we have the tools to select the patients. We just have to get to
`work and do the right clinical experiments.
`
`Acknowledgments
`
`I thank Ingo Mellinghoff and George Thomas for helpful discussions.Work in my
`lab is supported by the Howard Hughes Medical Institute, Doris Duke
`Charitable Foundation, National Cancer Institute, Leukemia and Lymphoma
`Society, Department of Defense, CaPCURE, and Accelerate Brain Cancer
`Cure.
`
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