Mammalian Target of Rapamycin
`(mTOR) Inhibitors
`Janice P. Dutcher, MD
`
`Address
`Our Lady of Mercy Cancer Center, New York Medical College,
`600 East 233rd Street, Bronx, NY 10466, USA.
`E-mail: jpd4401@aol.com
`Current Oncology Reports 2004, 6:111–115
`Current Science Inc. ISSN 1523-3790
`Copyright © 2004 by Current Science Inc.
`
`Current efforts in anticancer drug development are target-
`ing key factors in cell-cycle regulation. Mammalian target of
`rapamycin (mTOR) is one such protein kinase that facili-
`tates cell growth by stimulating the cell to traverse the G1
`to S phase of the cell cycle. Rapamycin is the first defined
`inhibitor of mTOR, and the demonstration of its antitumor
`activity has led to great interest in this pathway as an antitu-
`mor mechanism. Analogues with better pharmacologic
`properties have been developed and have entered clinical
`trials. Human cell lines of renal cell cancer, among several
`other tumors, are sensitive to growth inhibition via this
`pathway. Ongoing clinical trials are evaluating renal cell can-
`cer and other malignancies using therapy with mTOR inhib-
`itors. These agents are more likely to induce growth
`inhibition rather than tumor regression.
`
`Introduction
`Current efforts in anticancer drug development are tar-
`geting key regulatory proteins in the cell cycle that may
`be amenable to control by pharmacologic intervention.
`An understanding of the pathways and the role of vari-
`ous positive and negative control substances has led to
`the development of a number of agents that may selec-
`tively control growth of tumor cells. Agents that affect
`certain checkpoints or control points in the cell cycle are
`of particular interest.
`Rapamycin, a natural product derived from Streptomyces
`hygroscopicus, is used as an immunosuppressive agent in
`renal transplantation, where it was observed to have antitu-
`mor as well as immunosuppressive activity [1–3]. A num-
`ber of pathways of cell regulation were elucidated in
`subsequent investigations of its mechanism of action and
`sites of action with respect to the cell cycle. Rapamycin
`blocks cellular proliferation by inhibition of cell-cycle pro-
`gression at G1 to S [4]. The activity of rapamycin as a
`potential antitumor agent has led to the development of a
`number of analogues with more favorable pharmacologic
`
`properties that are undergoing clinical investigation (Figs.
`1 and 2) [5(cid:127)]. The clinical data from these trials are sum-
`marized in this paper.
`Rapamycin binds to its immunophilin, FK binding pro-
`tein (FKBP12), and takes a different pathway than that of
`the other immunosuppressive agents used in solid organ
`transplantation (cyclosporine A, FK506). Rapamycin com-
`bined with FKBP12 interacts with mTOR and inhibits its
`activation [6(cid:127)]. Thus, rapamycin is the first identified
`mTOR inhibitor.
`The inhibition of mTOR appears to be critical to cell-
`cycle control in malignant cells, and this pathway is of
`great interest as a possible anticancer target. It is more
`sensitive to inhibition in malignant cells than in normal
`cells. As a protein kinase that regulates cell-cycle progres-
`sion from G1 to S, mTOR promotes cell growth [5(cid:127)]. In
`cells that respond to growth factors through growth factor
`receptors, the upstream regulator of mTOR, Akt, a serine–
`threonine kinase, is activated, and then mTOR is acti-
`vated. In response to activation, mTOR signals two sepa-
`rate downstream pathways, which stimulate translation
`of specific mRNAs that signal cell growth at the G1 to S
`phase of the cell cycle. The first pathway is through phos-
`phorylation of the eukaryotic translation initiation factor,
`4E binding protein-1 (4E-BP1), and the second is
`through the 40S ribosomal protein p70 S6 kinase
`[5(cid:127),7,8]. Cyclin D3 and c-myc are among the other pro-
`teins regulated in part by mTOR [8].
`An additional component regulating the mTOR path-
`way is control of the upstream regulator, Akt. Akt is acti-
`vated by phosphatidylinositol 3-kinase (PI3K), which in
`turn is controlled by PTEN (a tumor suppressor gene).
`The dysregulation of cell proliferation that occurs in
`tumors has been attributed in some cases to 1) constitu-
`tive activity of Akt, or 2) mutation or deletion of PTEN.
`In cases where either of these two events has occurred,
`there is uncontrolled activation of mTOR, stimulating
`increased cell proliferation. Therefore, it is hypothesized
`that tumors with increased mTOR activity will be highly
`sensitive to mTOR inhibition.
`This process has been investigated in human tumor cell
`lines. Cell lines with mutations of PTEN have increased
`levels of activated Akt and mTOR [6(cid:127),8,9]. Similarly, in
`tests of sensitivity to rapamycin or its analogue, CCI-779,
`the pattern of sensitivity matches that of the cell lines with
`PTEN mutations or increased activation of Akt [10].
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`Figure 1. The rapamycin-sensitive signal
`transduction pathway is shown. Rapamycin
`and CCI-779 bind to the immunophilin FK506
`binding protein 12 (FKBP-12). The rapamycin–
`FKBP12 complex blocks the kinase activity of
`the mammalian target of rapamycin (mTOR).
`The inhibition of mTOR kinase activity inhibits
`the downstream translational regulators of
`4E-BP1/PHAS and p70s6k. The inhibition of
`4E-BP1/PHAS and p70s6k decreases the
`translation of mRNA of specific proteins
`essential for cell-cycle progression from G1
`to S phase. (From Hidalgo and Rowinsky [5(cid:127)];
`with permission.)
`
`With respect to renal cell carcinoma, another pathway
`influenced by mTOR could also be contributory and may
`provide another antitumor mechanism. Sporadic renal cell
`cancer is associated with loss of function of the von Hip-
`pel–Lindau (VHL) tumor suppressor gene [11(cid:127)]. The VHL
`gene product, a protein, is important for causing destruc-
`tion via the proteosome of the oxygen-sensitive transcrip-
`tion factors, termed hypoxia-inducible factor (HIF)-1α and
`HIF-2α [12]. Both of these factors are key in stimulating
`vascular growth in tumors in the setting of hypoxia [13].
`When VHL function is lost, these factors are not degraded,
`and therefore accumulation of HIFs is increased and HIFs
`continue to function. Both HIFs have been noted to stimu-
`late increased expression of vascular endothelial growth
`factor (VEGF), platelet-derived growth factor (PDGF), and
`transforming growth factor (TGF)-α, among other growth
`factors [13]. Thus, loss of function of VHL leads to
`increases in HIF activity and therefore increases in growth
`factors key to tumor and vascular growth. This is thought
`to be a major pathway of growth and persistence of renal
`cell cancer. Recently, it was determined that mTOR activa-
`tion increases HIF-1α gene expression through mRNA
`translation and protein stabilization [14,15].
`Thus, mTOR inhibition is of general interest as an anti-
`cancer approach because it affects multiple pathways in a
`number of malignancies—through the ribosomal pathway
`and the factor 4E pathway, and in tumors with mutations of
`PTEN. However, mTOR inhibition may also be of interest in
`renal cell cancer through the potential effect on HIF-α.
`
`Preclinical Evaluation of mTOR Inhibition
`Considerable preclinical work has been done in human
`tumor cell lines and animal models of human xenografts
`to evaluate the effect of mTOR inhibition on cell growth
`and tumor growth. A striking effect was initially evaluated
`using rapamycin, the prototype agent to induce mTOR
`
`inhibition [16,17]. The rapamycin analogues, CCI-779,
`RAD001, and ap23573, are now being evaluated. CCI-779
`is an ester of rapamycin, and its active metabolite is siroli-
`mus. RAD001 is also a derivative of rapamycin, and all are
`inhibitors of mTOR with cell-line sensitivity patterns of
`inhibition similar to that of rapamycin.
`In tests of the National Cancer Institute human tumor
`cell-line panel, antitumor activity for rapamycin and its
`derivatives showed significant growth inhibition at con-
`centrations of less than 0.01 µM for breast cancer, prostate
`cancer, leukemia, melanoma, renal cell cancer, glioblas-
`toma, and pancreatic cancer [10]. In addition, some cell
`lines of virtually every tissue tested showed sensitivity. Half
`of glioblastomas have PTEN mutations, which could make
`them increasingly sensitive to mTOR inhibition. Addition-
`ally, tumors with lesions of other proteins that regulate
`progression through G1 may also be more susceptible to
`mTOR inhibition, such as deletions of p16 to glioma and
`pancreatic cancer (Wyeth, Personal communication). In
`studies of human tumors as xenografts in mice, evaluation
`of growth inhibition demonstrated prolonged time to
`tumor growth in a similar spectrum of diseases. Growth
`inhibition rather than tumor regression appeared to be the
`most consistent outcome, and compared with untreated
`control subjects, there was significant increase in time.
`Geoerger et al. [18] investigated the antitumor activity of
`CCI-779 in human primitive neuroectodermal tumor/
`medulloblastoma (PNET/MB) models, the most common
`form of malignant pediatric brain tumor. In cell lines and
`in xenografts, significant growth inhibition was exhibited.
`An additive effect was also seen in the xenograft when CCI-
`779 was given in combination with cisplatin, another
`active agent in PNET [18]. This carefully done study exem-
`plifies the unusual features of mTOR inhibitors: 1) A linear
`dose-response effect is not apparent; there appears to be a
`dose threshold that is effective with minimal benefit from
`incremental increases in dose; 2) intermittent dosing is
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`Mammalian Target of Rapamycin (mTOR) Inhibitors (cid:127) Dutcher
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`113
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`Figure 2. Rapamycin and CCI-779 inhibit the
`phosphorylation of 4E-BP1/PHAS, preventing
`the release of the eIF-4E and the activation of
`the eIF4F complex. (From Hidalgo and
`Rowinsky [5(cid:127)]; with permission.)
`
`effective; and 3) a 2-week period of daily dosing was supe-
`rior to 1 week, superior to one large single dose, and supe-
`rior to longer than 2 weeks of treatment. Thus, sufficient
`preclinical data demonstrating antitumor activity of rapa-
`mycin, and subsequently of its analogues, have led to the
`initiation of clinical trials to investigate the effectiveness of
`mTOR inhibition as an anticancer modality.
`
`The responses in these phase I studies, at doses that
`were tolerable, have led to development of disease-directed
`phase II trials and trials evaluating this drug in combina-
`tion with chemotherapy. Two phase I studies have been
`initiated with combinations of chemotherapy, one of CCI-
`779 plus gemcitabine, and one with 5-fluorouracil and leu-
`covorin. The results of these two studies are yet to be pre-
`sented. (Wyeth, Personal communication).
`
`Phase I Clinical
`Investigations in Cancer Patients
`The greatest amount of clinical data amassed for the ana-
`logues concerns CCI-779, for which phase I and II trials have
`been completed. Although animal data provide some guid-
`ance, with any new agent, phase I clinical trials often demon-
`strate new information. Phase I trials with CCI-779 were
`conducted with intermittent schedules to avoid prolonged
`immunosuppression, as is seen and desired with rapamycin.
`Raymond et al. [19] conducted a trial using a 30-
`minute weekly intravenous infusion. The maximally toler-
`ated dose was not reached with this study. The doses
`ranged from 7.5 to 220 mg/m2/wk. Grade 3 mucositis was
`observed. A partial response (PR) was seen in one patient
`each with renal cell cancer, breast cancer, and neuroendo-
`crine tumor. Hidalgo et al. [20] conducted a second phase I
`study, with a schedule of daily 30-minute infusion for 5
`days, repeated every 2 weeks. For heavily pretreated
`patients, the maximally tolerated dose was 15 mg/m2/d,
`and for minimally pretreated patients the maximally toler-
`ated dose was 19 mg/m2/d. The dosage ranged from 0.75
`to 19.1 mg/m2/d. Dose-limiting toxicities included grade 3
`hypocalcemia, grade 3 elevation in liver function tests, and
`grade 3 thrombocytopenia. One patient with non–small-
`cell lung cancer experienced a PR.
`Toxicities observed with some frequency included folliculi-
`tis, nail bed changes, maculopapular rash, and eczematoid
`reactions as dermatologic changes. Hematologic changes were
`primarily thrombocytopenia. Biochemical alterations included
`abnormalities of liver function tests, hypertriglyceridemia,
`hypercholesterolemia, and reversible decrements in testoster-
`one. Some patients also developed mucositis. All of these toxic-
`ities were reported as relatively mild in the phase I studies.
`
`Phase II Clinical Trial in Renal Cell Carcinoma
`In single-agent phase II studies initiated with a weekly
`schedule, CCI-779 appeared to have activity and was toler-
`ated well over a large range of doses. Because selection of a
`dose for the weekly schedule was problematic, three doses
`were used in a study of patients with metastatic renal cell
`cancer: 25 mg/m2, 75 mg/m2, and 250 mg/m2, each
`administered weekly (Submitted manuscript) [21(cid:127)]. Both
`the investigator and the patient were blinded to the dose
`level. Dose reductions were required for grade 3 or greater
`toxicity. Treatment was continued until evidence of pro-
`gression was shown or unacceptable toxicity was reached.
`Patients were premedicated with diphenhydramine to pre-
`clude allergic reactions that had been seen earlier in the
`trial. One hundred eleven patients with renal cell cancer
`who had previously received some type of systemic therapy
`(usually a cytokine) were enrolled, and 110 were treated.
`The patients were randomly assigned to dose levels and
`evenly distributed, with 36 treated at 25 mg/m2/wk, 38 at
`75 mg/m2/wk, and 36 at 250 mg/m2/wk. Sixty-two percent
`were classified as Eastern Cooperative Oncology Group
`(ECOG) performance status 1, and 38% were classified as
`ECOG performance status 0. The most common clinical
`adverse events were rash (72%), mucositis (65%), asthenia
`(39%), nausea (36%), and acne (30%). The most common
`laboratory adverse events were thrombocytopenia (24%),
`hypertriglyceridemia (24%), and anemia (23%). Most
`adverse events were grade 1 or 2. the most common grade
`3 or 4 adverse events were hyperglycemia and anemia.
`Some occurrences of nonspecific pneumonitis, the major-
`ity asymptomatic, were observed. Median time to progres-
`sion and overall survival were 5.8 months and 15 months,
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`respectively, for the entire group. Although the response
`rate (complete response [CR] + PR) was 7% for the entire
`group, the cumulative responses (CR + PR) added to minor
`responses and stable disease greater than 24 weeks com-
`prised 51% of the entire group.
`Subsequently, in renal cell cancer, a study of CCI-779
`plus interferon (IFN) alfa was initiated, based on data
`showing significant synergy in murine systems [22]. This
`phase I study attempted to escalate doses of CCI-779, given
`as a weekly intravenous infusion, and IFN, given as a sub-
`cutaneous injection three times weekly [22]. The initial
`dose of IFN was 6 MU three times weekly. IFN alone was
`given during the first week. The initial dose of CCI-779 was
`5 mg/m2/wk. CCI-779 dose escalation steps were 10 mg/
`m2, 15 mg/m2, and 25 mg/m2. Once the maximum dose
`of CCI-779 was determined, IFN was to be escalated if pos-
`sible to 9 MU three times weekly. As of November 2002, 20
`patients were entered at four different dose levels of CCI-
`779. Fifteen had remained on study for greater than 7
`months. Partial responses were reported in two patients
`and stable disease in six, and the study was continuing to
`accrue. At this early evaluation, the combination of CCI-
`779 and IFN was considered tolerable, and antitumor
`activity was observed. It is too early to determine if the
`level of activity is additive or synergistic, or if it is consis-
`tent with expectations with either drug alone.
`
`Phase II Trial in Metastatic Breast Cancer
`A study in patients with locally advanced or metastatic
`breast cancer was initiated, using two weekly dose levels of
`CCI-779, 75 mg/m2 and 250 mg/m2 [23(cid:127)]. Patients had
`been treated previously with anthracyclines (45%), taxanes
`(5%), or both (50%). One hundred nine patients were
`enrolled, and 106 were treated. Liver, lung, and bone disease
`were reported in 59%, 36%, and 42% of patients, respec-
`tively. Dose reductions were required in 31% of patients
`treated at 75 mg/m2 and in 51% of those treated at 250 mg/
`m2. Fewer grade 3 or 4 toxicities occurred among the
`patients treated at 75 mg/m2, and it is clear that prior che-
`motherapy had an impact on the level and frequency of tox-
`icities from CCI-779. Leukopenia and thrombocytopenia
`were seen in both groups. Clinical benefit was observed in
`49% of the patients, based on the report of one CR, eight
`PRs, and 43 patients who remained stable for more than 8
`weeks, including eight unconfirmed PRs. Activity was
`observed at both dose levels and was seen in patients with
`liver metastases. Further evaluation of the data is ongoing,
`and plans for combination studies are being evaluated.
`
`Phase I Trial in Primary Brain Tumors
`An additional phase I study is being conducted in patients
`with primary brain tumors, again with 50% or more
`expressing loss or mutation of PTEN [24]. Patients with
`
`primary brain tumors were treated for multiple cycles of
`eight weekly doses, and some received as many as 17
`cycles. Higher doses were administered due to the promet-
`abolic effect of corticosteroids in patients with primary
`brain tumors.
`
`Oral Formulations of mTOR Inhibitors
`An oral formulation of CCI-779 is under development and
`has been used in treatment of rheumatologic patients and
`in normal control subjects (Wyeth, Personal communica-
`tion). Its activity in cancer patients will be evaluated if the
`level of intravenous activity is deemed sufficient to con-
`tinue studies. RAD001 is an oral formulation of another
`mTOR inhibitor. A phase I study with weekly administra-
`tion of this agent has been reported, and combination
`studies are in progress (Novartis, Personal communica-
`tion) [25]. Similarly, ap23573, a third mTOR inhibitor, is
`undergoing animal studies and is expected to enter clinical
`trials in humans (ARIAD Pharmaceuticals, Personal com-
`munication) [26].
`
`Conclusions
`Inhibition of mTOR appears to be a viable approach to
`anticancer therapy. Ongoing clinical trials suggest that
`agents that inhibit this pathway mediate changes in cell
`proliferation but may not cause regression in the majority
`of patients. Combination studies are still at early stages
`and may be difficult to execute. This pathway should be
`evaluated very carefully and thoroughly because it may be
`difficult to elucidate precisely what determines a beneficial
`effect. The gestalt from treating physicians and nurses is
`that inhibition of this pathway is beneficial to patients
`with renal cell cancer in that the duration of stable disease
`is prolonged. Somehow, this observation needs to be
`quantified, and that is the difficulty. Nevertheless, mTOR
`inhibition is a promising area for clinical development.
`
`Acknowledgments
`This work was supported by the Kidney Cancer Fund of the
`Cancer Research Foundation.
`
`References and Recommended Reading
`Papers of particular interest, published recently, have been
`highlighted as:
`(cid:127)
`Of importance
`(cid:127)(cid:127) Of major importance
`
`1.
`
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