6382s Vol. 10, 6382s– 6387s, September 15, 2004 (Suppl.)
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`Clinical Cancer Research
`
`Mammalian Target of Rapamycin Inhibition
`
`Janice P. Dutcher
`Comprehensive Cancer Center, Our Lady of Mercy Medical Center,
`Bronx, New York
`
`threonine kinase, as a key regulator of cell cycle and of many
`intracellular functions has emerged and presented itself as a
`potential target for antitumor therapeutics.
`
`ABSTRACT
`The mammalian target of rapamycin (mTOR) is a ser-
`ine/threonine kinase that has been increasingly recognized
`as key to the regulation of cell growth and proliferation.
`mTOR either directly or indirectly regulates translation
`initiation, actin organization, tRNA synthesis, ribosome bio-
`genesis, and many other key cell maintenance functions,
`including protein degradation and transcription functions.
`Inhibition of mTOR blocks traverse of the cell cycle from
`the G1 to S phase. Preclinical data show inhibition of tumor
`growth in a number of cell lines and xenograft models.
`Clinical trials are ongoing. In metastatic renal cell cancer,
`both tumor regression and prolonged stabilization have
`been noted. mTOR inhibition appears to be a key pathway
`that may be useful in antitumor therapy. Renal cell cancer
`may be particularly susceptible through both the translation
`inhibition pathway and pathways that enhance HIF-1␣ gene
`expression, a factor believed to stimulate growth in meta-
`static renal cell cancer. Additional clinical trials that use
`agents that inhibit mTOR are ongoing.
`
`INTRODUCTION
`The identification of the mammalian target of rapamycin
`(mTOR) pathway as a potential target for anticancer therapy
`emerged from efforts to understand the activity of the immuno-
`suppressive drug rapamycin (sirolimus, Rapamune, Wyeth-Ay-
`erst Laboratories, Collegeville, PA). Rapamycin, a natural prod-
`uct derived from the soil bacteria Streptomyces hygrosopius,
`was approved for use in organ transplantation in 1999 (1– 4).
`During its preclinical evaluation, studies demonstrated potent
`antitumor activity, although initially the mechanism was un-
`known (5–7). Subsequently, Dilling et al. (8) demonstrated
`potent inhibition of growth of rhabdomyosarcoma cells by ra-
`pamycin at nanogram concentrations. Clues to the pathways
`involved were derived from this cell line, which required an
`autocrine loop involving signaling through insulin-like growth
`factor receptors (9, 10). Subsequent work by many investigators
`demonstrated that rapamycin produces cell cycle arrest, prevent-
`ing progression of dividing cells from the G1 to S phase of the
`cell cycle (11, 12). Subsequently, the role of mTOR, a serine/
`
`Presented at the First International Conference on Innovations and
`Challenges in Renal Cancer, March 19 –20, 2004, Cambridge, Massa-
`chusetts.
`Requests for reprints: Janice P. Dutcher, Our Lady of Mercy Cancer
`Center, New York Medical College, 600 East 233rd Street, Bronx, NY
`10466. Phone: 718-920-1100; Fax: 718-920-1123; E-mail: jpd4401@
`aol.com.
`©2004 American Association for Cancer Research.
`
`mTOR REGULATORY PATHWAYS
`Rapamycin does not directly inhibit mTOR but binds to its
`immunophilin, FK binding protein (FKBP12). Rapamycin plus
`FKBP12 then interact with mTOR and inhibit its function (12),
`leading to inhibition of cell growth and proliferation. The down-
`stream effects of this inhibition include inhibition of transla-
`tional pathways, with loss of phosphorylation of the eukaryotic
`translation initiation factor, 4E binding protein-1, and inhibition
`of the 40S ribosomal protein p70 S6 kinase (blocking ribosomal
`biogenesis; refs. 13–15). This effect results in a 15% to 20%
`inhibition of overall protein translation and leads to cell cycle
`arrest (12). Other cellular functions that appear to be regulated
`by mTOR and, thus, affected by its inhibition include actin
`organization, membrane traffic, protein degradation, protein ki-
`nase C signaling, and tRNA synthesis (16). There are also
`regulatory effects on synthesis of essential cell cycle proteins,
`such as cyclin D1 and c-myc (15, 17–19). Recent data suggest
`that mTOR regulates protein synthesis when cellular ATP levels
`fluctuate (20).
`In addition to the downstream activities of mTOR, which
`are affected by its inhibition, important upstream regulators of
`its activity may be altered in malignant cells. This may make
`this pathway particularly important in antitumor therapeutics.
`Both phosphatidylinositol 3⬘-kinase and Akt are upstream to
`mTOR. Akt activity is regulated by PTEN, a tumor suppressor
`gene,
`thus regulating mTOR activity. This counteracts Akt
`activation through phosphatidylinositol 3⬘-kinase. Aberrations
`in these upstream regulators, therefore, may lead to alterations
`in mTOR regulatory activity. The most striking examples of this
`are the dysregulation of phosphatidylinositol 3⬘-kinase activity
`when PTEN is mutated, deleted, or methylated (21–23). In these
`situations, this could lead to uncontrolled activity of mTOR,
`leading to uncontrolled cell proliferation.
`An additional pathway influenced by mTOR that appears
`to be particularly important in renal cell carcinoma involves the
`hypoxia-inducible factor (HIF). With loss of VHL function
`commonly seen in clear cell renal cell cancer, there is accumu-
`lation of the oxygen-sensitive transcription factors HIF-1␣ and
`HIF-2␣ (24). An increase in accumulation of these factors yields
`increased stimulation of vascular endothelial growth factor
`(VEGF), platelet-derived growth factor, and transforming
`growth factor ␣ (25). This effect is augmented by the activation
`of mTOR, which stimulates both a protein stabilization function
`and a protein translational function and, thus, increases HIF-1␣
`activity (26, 27).
`In addition, it has been determined that mutations of tuber-
`ous sclerosis complex TSC1 and -2 gene products function
`together to inhibit mTOR-mediated downstream signaling (28).
`Mutations of these genes occur in tuberous sclerosis, and their
`loss of function yields yet another pathway, which leads to
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`increased activity of mTOR and disrupts phosphatidylinositol
`3⬘-kinase-Akt signaling through down-regulation of platelet-
`derived growth factor receptor (28 –30). Loss of TSC1 or TSC2
`gene activity induces VEGF production through mTOR (30).
`Signaling through this pathway with activation of Akt and
`mTOR results in increased HIF activity and increased VEGF.
`However, in TSC2-negative cells, platelet-derived growth factor
`receptor is markedly reduced (29) TSC2 regulates VEGF
`through both mTOR-dependent and -independent pathways
`(31). TSC2 also regulates HIF. Thus, studies evaluating the
`impact of TSC1 and TSC2 mutations demonstrate the connec-
`tion of increased VEGF and activated mTOR pathways to
`angiogenesis. Thus, inhibition of mTOR, leading to an antian-
`giogenic effect, can be explained by its impact on several
`proangiogenic pathways.
`
`PRECLINICAL EVALUATION OF mTOR
`INHIBITION
`As stated previously, early work evaluating the immuno-
`suppressive activity of rapamycin also demonstrated antitumor
`activity (5, 6), but
`this was not
`initially further evaluated.
`Subsequently, studies in rhabdomyosarcoma cells suggested an
`antitumor effect, particularly in cells that required stimulation
`by insulin-like growth factor (8). Subsequent studies in multiple
`other cell lines have shown both cytostatic and cytotoxic effects
`of rapamycin (8, 15, 22, 32– 41). Of interest, during these
`evaluations, it became apparent that rapamycin not only pro-
`duced cell cycle arrest in G1 but also produced effects that led
`to cell death. Experimental findings have demonstrated induc-
`tion of programmed cell death (apoptosis) in B-cells (42, 43)
`and rhabdomyosarcoma cells (35, 36).
`In tests performed by the National Cancer Institute (NIH,
`Bethesda, MD) human tumor cell line panel, rapamycin and its
`derivatives showed significant growth inhibition in breast can-
`cer, prostate cancer, leukemia, melanoma, renal cell cancer,
`glioblastoma, and pancreatic cancer (22). It is known that half of
`glioblastomas have PTEN mutations, which could make them
`increasingly sensitive to mTOR inhibition, and this is being
`investigated clinically (21, 22, 44).
`Studies in human tumor xenografts in mice have also
`demonstrated prolonged time to tumor growth (37). In studies in
`a pediatric brain tumor model that used CCI-779, an ester of
`rapamycin, there was significant growth inhibition and an ad-
`ditive effect when CCI-779 was administered with cisplatin
`(37). This interesting evaluation demonstrated new clinical char-
`acteristics: (1) there was not a linear-dose response effect but
`more of a threshold level effect, (2) intermittent dosing was
`effective, and (3) 2 weeks of daily dosing was superior to 1
`week, one large single dose, and dosing for ⬎2 weeks.
`Additionally, renal transplantation investigators have com-
`pared the tumor-promoting effect of the immunosuppressive
`agents used in renal transplantation in a murine model of met-
`astatic human renal cell cancer (45). Of interest, and consistent
`with these preclinical data, the number of pulmonary metastases
`was reduced when the animals were exposed to rapamycin but
`increased when exposed to cyclosporine, another immunosup-
`pressive agent used in transplantation (45). In these studies,
`
`rapamycin also reduced circulating levels of VEGF-A and trans-
`forming growth factor ␤1 (45).
`Because rapamycin has poor water solubility and stability
`in solution, it is a poor candidate for parenteral administration.
`Therefore, two ester analogues of rapamycin have been devel-
`oped with improved pharmaceutical properties and cellular ef-
`fects similar to rapamycin in the cell line screening evaluations:
`CCI-779 (Wyeth-Ayerst Research, Cambridge, MA) and RAD-
`001 (everolimus, Novartis AG, Basel, Switzerland). Both of
`these agents have entered clinical trials (46 – 48). A third mTOR
`inhibitor, ap23573 (ARIAD Pharmaceuticals, Cambridge, MA;
`ref. 49), is completing preclinical trials and is scheduled for
`clinical trials in late 2004.
`
`PHASE I CLINICAL INVESTIGATION IN
`CANCER PATIENTS
`Most of the current clinical data with agents that inhibit
`mTOR come from clinical trials of CCI-779 (Wyeth-Ayerst).
`This agent has been evaluated extensively in two Phase II trials,
`one using a weekly schedule and one using a daily for 5 days
`schedule. Raymond et al. (46) studied the weekly dosing as a
`30-minute infusion at doses ranging from 7.5 to 220 mg/m2 per
`week. There were no dose-limiting toxic effects, although grade
`3 mucositis was observed. There was a partial response in one
`patient each with renal cell cancer, breast cancer, and a neu-
`roendocrine tumor. The second Phase I trial, by Hidalgo et al.
`(47) evaluated daily 30-minute infusions given for 5 days every
`2 weeks. Dose-limiting toxic effects were grade 3 thrombocy-
`topenia, grade 3 elevations of liver function tests, and grade 3
`hypocalcemia. The maximally tolerated dose was 19 mg/m2
`daily for minimally pretreated patients and 15 mg/m2 daily for
`heavily pretreated patients. A partial response was noted in one
`patient with non–small cell lung cancer.
`Other relatively frequent toxic effects that occurred with
`some frequency included dermatologic effects such as eczema-
`toid reactions, maculopapular rash, nail bed changes, and fol-
`liculitis. The hematologic effect was thrombocytopenia. Eleva-
`tions in liver
`function tests and hypertriglyceridemia and
`hypercholesterolemia were noted. There were reversible decre-
`ments in testosterone. There was occasional mucositis. All of
`these effects were described as being mild.
`
`PHASE II CLINICAL TRIAL OF CCI-779 IN
`PATIENTS WITH METASTATIC RENAL
`CELL CANCER
`A Phase II trial has been completed and reported involving
`patients treated previously with metastatic renal cell cancer (50,
`51). This study used weekly administration of one of three dose
`levels of CCI-779: 25 mg/m2, 75 mg/m2, and 250 mg/m2. The
`dose used was blinded to the treating physician and patient.
`Dose reductions were prescribed for grade 3 toxic effects. Treat-
`ment was continued until evidence of progression or unaccept-
`able toxic effects. Patients were premedicated with diphenhy-
`dramine to preclude allergic reactions that were observed early
`in the trial. There were 111 patients enrolled and 110 received
`treatment, 36 at 25 mg/m2, 38 at 75 mg/m2, and 36 at 250
`mg/m2. Ninety percent of patients had received prior therapy for
`metastatic disease (usually a cytokine), and more than half had
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`received more than one prior regimen. Sixty-five percent of
`patients were Eastern Cooperative Oncology Group perform-
`ance status 1, and 35% were Eastern Cooperative Oncology
`Group performance status 0.
`As in the Phase I trials, the most common toxic effects
`observed were maculopapular rash (76%) and mucositis (70%).
`Additionally, with prolonged therapy, patients developed asthe-
`nia (50%) and nausea (43%). Grade 3 or 4 laboratory adverse
`events were hyperglycemia (17%), hypophosphatemia (13%),
`anemia (9%), and hypertriglyceridemia (6%). There did not
`appear to be a dose-toxicity relationship, although there were
`more dose reductions at the higher dose levels. Responses were
`observed with all of the dose levels.
`The objective response rate was 7%, with one complete and
`seven partial responses. Median time to tumor progression was
`5.8 months, and median survival was 15 months. The cumula-
`tive response rate, including complete responses, partial re-
`sponses, minor response, and stable disease for ⬎24 weeks, was
`51% (51).
`In this study, patients were evaluated for prognosis, using
`the prognostic factor criteria developed by Motzer et al., (52) in
`an analysis of ⬎300 patients treated with interferon at Memorial
`Hospital, New York, NY. The five factors that were determined
`to have significant prognostic impact were performance status,
`lactate dehydrogenase, serum calcium, hemoglobin, and time
`from initial diagnosis to treatment (52). Although initially de-
`veloped for untreated patients, the factors appear to segregate
`patients in the second-line setting as well (51). In this study of
`CCI-779, survival was decidedly different based on prognostic
`group among all of the dose-level patient groups (51).
`into the
`Eighty-seven percent of CCI-779 patients fell
`intermediate or poor prognosis categories for metastatic renal
`cell cancer, with ⬍10% in the good prognosis group. Median
`survival for these CCI-779 –treated patients in the intermediate
`or poor groups appeared to be 1.6- to 1.7-fold longer than those
`in the original study by Motzer et al. (52) of interferon-treated
`patients when compared with prognostic group by prognostic
`group (of first-line patients). Thus, mTOR inhibition may be of
`particular value in the poorer prognosis patients. This will be
`additionally tested in a randomized Phase III trial.
`Preclinical data have suggested synergy of CCI-779 admin-
`istered in combination with interferon ␣, which has led to the
`conduct of a Phase I/II clinical trial of this combination in renal
`cell cancer (53, 54). The study design began with an initial dose
`of interferon at 6 million units three times per week and of
`CCI-779 at 5 mg/m2 weekly. In the initial Phase I component of
`the study, the dose of CCI-779 was escalated, and interferon
`␣was kept at 6 million units three times per week. Doses of
`CCI-779 ranged from 5 to 25 mg/m2. A small cohort was also
`treated at 15 mg/m2 and 9 million units of interferon ␣. The
`Phase II component of the trial has extended the number of
`patients treated at 15 mg/m2 of CCI-779 and 6 million units of
`interferon ␣. As of December 2003, 71 patients have been
`entered, and several continue with treatment. The median time
`undergoing treatment for all of the cohorts is 7 months, and
`more than half have continued treatment for ⬎6 months. Re-
`sponses have been confirmed, and evaluation is continuing. The
`combination appears to show favorable activity and safety. A
`Phase III study has been initiated in which the combination of
`
`CCI-779 and interferon ␣ is compared with either agent alone as
`first-line therapy in patients with a poor prognosis for metastatic
`renal cell cancer. An additional randomized study is planned for
`patients who have had prior therapy.
`
`OTHER AGENTS
`The other agents that inhibit mTOR are undergoing early
`phases of evaluation. RAD001 is an oral formulation and is
`currently in Phase I clinical trials (48). The agent ap23573 is
`currently in preclinical evaluation, with human clinical trials
`planned (49).
`
`DISCUSSION
`Preclinical data demonstrate the importance of mTOR as a
`regulator of cell growth and proliferation. Additional evaluation
`of malignant cells demonstrates that the constitutive activity of
`mTOR leads to unregulated growth. Human malignancies have
`been demonstrated to have a constitutive activation of mTOR or
`its upstream regulators, leading to enhanced mTOR activity.
`Now clinical data are beginning to demonstrate that this path-
`way is a viable target for antitumor therapeutics, and the out-
`come demonstrated in the Phase II trial of CCI-779 in metastatic
`renal cell cancer is promising. The ability to evaluate and
`interpret prolonged stable disease in metastatic renal cell cancer
`trials continued to be problematic but appears to be a real
`outcome and will need to be carefully quantitated in future
`randomized trials with many of the agents currently undergoing
`evaluation, including the inhibitors of mTOR.
`As noted in the early in vitro work by Dilling et al. (8), the
`rhabdomyosarcoma cell line that they studied is regulated by an
`autocrine loop involving secretion of type 2 insulin-like growth
`factor, and signaling is through the insulin-like growth factor
`receptor. In previous studies (8, 12), the cells most sensitive to
`rapamycin were dependent on this autocrine pathway. Others
`have reported the interaction of rapamycin with control of
`glucose and lipids and the role of mTOR in the insulin-signaling
`pathway (55, 56). In view of the incidence of hyperglycemia and
`hypertriglyceridemia observed during treatment with CCI-779,
`it is possible that these biochemical parameters may correlate
`with the degree of mTOR inhibition (51). This will be evaluated
`by additionally investigating the clinical outcome in the current
`trials with CCI-779 and the levels of glucose and lipids. This
`will need additional confirmation by observations during trials
`of the other mTOR inhibitors, as to whether these clinical effects
`indeed do reflect a pharmacodynamic surrogate for mTOR
`inhibition.
`The inhibition of mTOR appears to be a promising targeted
`approach to antitumor therapy. Additional evaluation of these
`agents for schedule, route of administration, and combination
`therapy approaches is clearly warranted. Renal cell cancer ap-
`pears to be sensitive to this therapeutic approach, and it will be
`of great interest to determine whether combinations of mTOR
`inhibitors with other agents will enhance the therapeutic effi-
`cacy in this difficult disease. It will also be important to evaluate
`the biochemical parameters that might predict favorable outcome.
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`I think these are the right
`
`OPEN DISCUSSION
`Dr. Robert J. Motzer: Can you comment on the lung
`toxicity or the pulmonary infiltrates that are associated with the
`drug?
`Dr. Janice P. Dutcher: There have been some asymp-
`tomatic patients, but we did not see them at all in our group.
`Dr. Michael B. Atkins:
`In the paper that was published
`in JCO, there were 6 patients out of approximately 100 who had
`pulmonary infiltrates (J Clin Oncol 2004;22:909 –18). Some of
`these individuals had associated symptoms. All had their treat-
`ment held, 2 were not restarted probably because their disease
`progressed, 4 were restarted when their symptoms got better.
`Two did not have a recurrence of the problem, and 2 had a
`recurrence of the pulmonary infiltrates. Five of 6 happened at
`the 75-mg dose level, 1 at the 250-mg level, and none at the
`25-mg dose level. It is hard to say whether any of these side
`effects were dose dependent.
`Dr. Robert A. Figlin: My understanding of signal trans-
`duction inhibition with CCI-779 is that administered once a
`week it will not inhibit the target for an extended period. Do we
`have any readout from laboratory biology that can help us
`understand whether once a week, which is very convenient for
`the patient but might not be good at impacting the tumor, is a
`better strategy? Is that born out by laboratory models? How do
`we need to be targeting these pathways via pharmacological
`interventions?
`Dr. William G. Kaelin, Jr.:
`questions.
`Dr. Atkins: There were a lot of patients in this study who
`had minor responses. I think it was 26% of the patients who had
`more than 25% tumor regression without satisfying the criteria
`for partial response. A lot of the patients who were benefiting
`were patients who we thought would never have benefited from
`immunotherapy. There were people with hypercalcemia, fa-
`tigue, a performance status of 2, and multiple prior treatments.
`We were seeing the disease course change in those patients, so
`it suggested to us that maybe there was a different patient
`population that was responding to CCI-779 than would typically
`respond to immunotherapy. What would be the appropriate
`targets to measure in an mTOR inhibition trial and when would
`be the appropriate time to measure them?
`Dr. Figlin: That’s in fact what our trial will be looking at
`in patients who are undergoing cytoreductive nephrectomy with
`metastatic disease. We will be looking at inhibition of all of the
`targets in the mTOR pathway.
`Dr. Walter M. Stadler: There is a lot of publicity about
`these drugs being more active in the PTEN-deficient tumors.
`Has any of that been born out clinically?
`Dr. Figlin: The mTOR pathway appears to have impor-
`tance not just inside the tumor cell but also in terms of the
`angiogenesis. There may be some synergy in terms of that
`pathway being important in multiple places.
`Dr. Atkins: What about the connection between mTOR
`inhibition and HIF? Is that a direct effect or is that a more
`general effect on protein synthesis or on molecules that are
`turning over quicker?
`Dr. Kaelin: That is still being worked out, and I certainly
`don’t want to oversell the HIF connection. There clearly is a HIF
`
`connection, but mTOR is a fairly important enzyme that does
`other things. An emerging theme that is coming out of at least
`preclinical studies with various targets and cancer is this notion
`that cancer cells, after mutating a particular pathway, become
`addicted to that pathway and hypersensitive to inhibitors of that
`pathway. This happens to be one of those scenarios that have
`been described for some other targets as well.
`Dr. Michael S. Gordon: Those have been the settings
`where the greatest results, positive results, have been shown.
`Dr. Kaelin: Preclinically, in retrospect, there was evi-
`dence that leukemic cells become addicted to BCR-ABL sig-
`naling and become hypersensitive to an inhibitor. In some of
`these models, for example, after you introduce BCR-ABL into
`cells, they become hypersensitive and they will undergo apo-
`ptosis after acute interruption of that signal, which might favor
`intermittent dosing to allow normal cells to recover. If I am not
`mistaken, rapamycin is primarily antiproliferative, meaning cy-
`tostatic. There is an influence of PTEN status on responsiveness,
`but still what you see is a cytostatic effect. In that scenario, you
`could make the case for a continuous exposure.
`Dr. Daniel J. George:
`It sounds like there is still a huge
`dose range that people are struggling with. Obviously, there is
`toxicity, but does that toxicity vary? Should we be using a dose
`lower than 25 mg?
`Dr. Dutcher: The sense was that the toxicity was clearly
`manageable at all dose levels, and the dose that is in the Phase
`III trial is the 25-mg dose. There are some that would argue that
`you should stay at the 250 mg, because it would allow dose
`reductions and there was a slight difference in survival.
`Dr. Atkins: When we looked at all of the efficacy pa-
`rameters of response, time to progression, and survival, there
`was no clearcut difference between doses, indicating that even
`the 25-mg dose was probably high enough to hit the target.
`Although the survival was longer in the 250-mg patients, the
`time to progression was less, so there may have been patient
`selection that accounted for their prolonged survival. It is inter-
`esting that in the more recent studies with CCI-779 and inter-
`feron, we have seen several patients who have had significant
`hypertension as well. It was not reported in the single-agent
`Phase II study, because it probably did not get to exceed the
`10% to 15% range. Now we are seeing patients who have had
`hypertension because they have been on treatment for a while,
`which may be an indication that CCI-779 is hitting VEGF or
`HIF.
`
`Dr. Gordon:
`Is the rash an EGFR-type rash? Is it the
`same as we would see with gefitinib?
`Dr. Dutcher:
`It is not as severe, but it is similar. It
`usually did not last through the whole treatment.
`Dr. Atkins: A lot of patients got steroids to control the
`rash, which could be a potential problem. It is sort of strange to
`think about combining this with interferon even though there
`was synergy, but it is clearly an immunosuppressive agent if you
`give it frequently enough.
`Dr. Dutcher: Some of the people got antibiotics for the
`nail bed problems.
`Dr. Gordon: But that, again, is something that we have
`been seeing with the targeted therapies.
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