Purpose: Inhibiting mammalian target of rapamycin (mTOR) signaling in acute myelogenous leukemia (AML) blasts and leukemic stem cells may enhance their sensitivity to cytotoxic agents. We sought to determine the safety and describe the toxicity of this approach by adding the mTOR inhibitor, sirolimus (rapamycin), to intensive AML induction chemotherapy.

Experimental Design: We performed a phase I dose escalation study of sirolimus with the chemotherapy regimen MEC (mitoxantrone, etoposide, and cytarabine) in patients with relapsed, refractory, or untreated secondary AML.

Results: Twenty-nine subjects received sirolimus and MEC across five dose levels. Dose-limiting toxicities were irreversible marrow aplasia and multiorgan failure. The maximum tolerated dose (MTD) of sirolimus was determined to be a 12 mg loading dose on day 1 followed by 4 mg/d on days 2 to 7, concurrent with MEC chemotherapy. Complete or partial remissions occurred in 6 (22%) of the 27 subjects who completed chemotherapy, including 3 (25%) of the 12 subjects treated at the MTD. At the MTD, measured rapamycin trough levels were within the therapeutic range for solid organ transplantation. However, direct measurement of the mTOR target p70 S6 kinase phosphorylation in marrow blasts from these subjects only showed definite target inhibition in one of five evaluable samples.

Conclusions: Sirolimus and MEC is an active and feasible regimen. However, as administered in this study, the synergy between MEC and sirolimus was not confirmed. Future studies are planned with different schedules to clarify the clinical and biochemical effects of sirolimus in AML and to determine whether target inhibition predicts chemotherapy response. (Clin Cancer Res 2009;15(21):6732–9)

Translational Relevance

Signaling through the phosphatidylinositol-3′-kinase pathway and its downstream effectors, AKT and mammalian target of rapamycin (mTOR), is abnormally activated in acute myelogenous leukemia (AML) and hypothesized to contribute to chemotherapy resistance. Preclinical data suggest that inhibiting mTOR with rapamycins enhances AML cells' response to chemotherapy and impairs leukemic stem cell function. Here, we describe the first clinical trial combining a mTOR inhibitor with dose-intensive AML chemotherapy. We performed a phase I dose escalation of sirolimus (rapamycin) in combination with fixed doses of a multiagent induction chemotherapy regimen. This approach produced serum rapamycin concentrations capable of inhibiting mTOR signaling in leukemic blasts and was safe and worthy of future efficacy studies. Interestingly, many subjects' leukemia samples showed biochemical rapamycin resistance despite trough concentrations that would be adequate for transplant immunosuppression. This finding has implications for the clinical development of mTOR inhibitors in hematologic and solid tumors, and requires future study.

Although intensive combination chemotherapy is effective in temporizing acute myelogenous leukemia (AML) in most treated patients, the majority of those who achieve complete remission (CR) subsequently relapse and ultimately die of their disease (13). More than 30 years of combination chemotherapy clinical trials have failed to substantially improve survival in AML, leading to efforts to develop targeted therapeutics for this disease. We and others have concentrated on identifying agents or signaling inhibitors that target AML cells and AML stem cells without additional toxicity to normal hematopoietic cells. Some of these agents seem to be cytotoxic by themselves (49), whereas others alter the response of AML cells to chemotherapy (10, 11). The most extensively studied drugs in this class are the FLT3 inhibitors, for which clinical trials are ongoing (1216). Use of FLT3 inhibitors has been studied primarily in patients with FLT3 mutations, which are known to confer a poor prognosis. Studies that combined other signaling inhibitors with chemotherapy in AML have only rarely been previously reported (12, 15, 17, 18).

Phosphatidylinositol-3′-kinase (PI3K) is a potential novel therapeutic target in AML. The role of PI3K signaling in leukemia has been studied in both murine models and primary human cells. Yilmaz et al. studied a murine leukemia model that conditionally induces activated PI3K signaling in hematopoietic cells (19). This model rapidly generated transplantable acute myeloid and lymphoid leukemias. Treatment of these mice with the mammalian target of rapamycin (mTOR) inhibitor rapamycin (sirolimus) eradicated the leukemic phenotype, including loss of serial transplant capacity. We and others have also shown that PI3K and mTOR are activated in primary human AML cells and AML stem cells (5, 2023). Inhibition of PI3K signaling with LY294002 or prolonged exposure to rapamycin is cytotoxic to primary AML samples in vitro (5, 20, 22, 24). Importantly, we also showed that rapamycin dramatically enhances in vitro response to etoposide and abrogates engraftment of primary AML samples in immunodeficient mice (24). Taken together, these data show that mTOR inhibition enhances chemotherapy response and disrupts the survival of AML stem cells.

We therefore sought to test the feasibility of targeting mTOR in AML therapy by combining sirolimus with chemotherapy for AML. Prior studies have shown that mTOR inhibitors had modest antileukemic activity as single agents but very limited pharmacodynamic studies have been published to correlate any clinical responses with actual disruption of mTOR signaling in leukemic blasts (22, 2527). Sirolimus or other mTOR inhibitors have not previously been combined with induction chemotherapy for acute leukemia and the toxicity of this combination is unknown.

Based on our preclinical data, we chose to combine sirolimus with the etoposide-containing induction regimen MEC (mitoxantrone, etoposide, and cytarabine). MEC has primarily been tested in patients with relapsed and refractory AML (2830), and shows activity and toxicity comparable to other high-dose cytarabine-containing regimens (3133). Reversible mucositis, transaminase elevation, diarrhea, and neutropenic infection are the most commonly reported toxicities of this treatment. Sirolimus has been used for solid organ and bone marrow transplantation for more than a decade. Its toxicity profile has been extensively studied in the context of prolonged administration. In this setting, like other immunosuppressant medications, infections are the most common serious side effects. Other notable sirolimus toxicities include headache, acneiform rash, stomatitis, hyperlipidemia, mild thrombocytopenia, and rarely, thrombotic microangiopathy (3436).

We did a phase I dose escalation trial of sirolimus in combination with MEC to determine the toxicity profile of this combination as well as the maximally tolerated dose. We also examined rapamycin levels during therapy and studied the regimen's biochemical effects on mTOR signaling in leukemic blasts.

Subject selection

Subjects were recruited from the clinical practices of the University of Pennsylvania between May 2004 and January 2007. Eligible subjects were ages 18 to 65 with non-M3 AML that either had relapsed following prior chemotherapy or was refractory to induction chemotherapy. Primary refractory leukemia was defined as persistent disease (>5% marrow blasts) following two induction cycles or recurrent leukemia following a single induction cycle that had resulted in a nadir marrow biopsy with no leukemia. Patients with untreated secondary leukemia—defined as AML arising out of an antecedent hematologic disorder or following chemotherapy or radiation therapy—were also eligible. Patients with accelerated or myeloid blast phase chronic myelogenous leukemia could participate provided they had progressive disease despite treatment with BCR-ABL kinase inhibitors. Pathology review at the Hospital of the University of Pennsylvania was done to confirm diagnoses in all cases.

All subjects were required to have an Eastern Cooperative Oncology Group performance status of 0 to 1 and resolution of prior treatment-related toxicities. Required baseline organ function studies specified an ejection fraction >45%, creatinine ≤2.0 mg/dL, total bilirubin ≤1.5 mg/dL, hepatic transaminases ≤3× upper level of normal, no uncontrolled infections, and no unstable baseline comorbidities that would jeopardize toxicity assessment. Due to significant drug-drug interactions between sirolimus and systemic imidazole or triazole antifungals, these medications were prohibited for 1 wk prior to enrollment.

The research was approved by the Institutional Review Boards of the University of Pennsylvania and the study was monitored by the Clinical Trials Safety Review and Monitoring Committee of the Abramson Cancer Center of the University of Pennsylvania. Written informed consent was obtained from all subjects according to the Declaration of Helsinki.

Treatment schema

The treatment regimen is shown in Fig. 1 and consisted of a loading dose of oral sirolimus on day 1 followed 24 h later by a daily dose of sirolimus administered on days 2 to 7. Twelve hours after the sirolimus loading dose, subjects began i.v. MEC chemotherapy (mitoxantrone 8 mg/m2/d on days 1-5, etoposide 100 mg/m2/d on days 1-5, and cytarabine 1,000 mg/m2/d on days 1-5). In the absence of dose-limiting toxicity (DLT), the sirolimus loading and daily doses were to be escalated over five dose levels as described below in toxicity assessment. The loading dose remained thrice the daily dose for all treatment levels and ranged from 3 to 15 mg and 1 to 5 mg, respectively. Sirolimus doses were not adjusted for weight or altered based on drug levels. Each intravenous chemotherapy drug was administered over 1 h and actual body weight was used for chemotherapy calculations.

Fig. 1.

Treatment schema: a sirolimus (S) loading dose was administered on day 1. Twenty-four hours after the loading dose, sirolimus was administered once daily for six doses at one third the loading dose. MEC (mitoxantrone, 8 mg/m2/d; etoposide, 100 mg/m2/d; cytarabine, 1,000 mg/m2/d) chemotherapy was administered once daily for 5 d starting 12 h after the sirolimus loading dose. Marrow aspirates to measure mTOR inhibition in leukemic blasts were obtained prior to study therapy and on day 3. Rapamycin levels were measured as troughs just prior to sirolimus doses on days 3 and 6.

Fig. 1.

Treatment schema: a sirolimus (S) loading dose was administered on day 1. Twenty-four hours after the loading dose, sirolimus was administered once daily for six doses at one third the loading dose. MEC (mitoxantrone, 8 mg/m2/d; etoposide, 100 mg/m2/d; cytarabine, 1,000 mg/m2/d) chemotherapy was administered once daily for 5 d starting 12 h after the sirolimus loading dose. Marrow aspirates to measure mTOR inhibition in leukemic blasts were obtained prior to study therapy and on day 3. Rapamycin levels were measured as troughs just prior to sirolimus doses on days 3 and 6.

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Supportive care

Subjects were treated on the hematologic malignancies service and central venous catheters were used in all subjects. A low bacteria diet was provided during periods of neutropenia. All subjects received aggressive intravenous hydration and oral allopurinol 300 mg/d during chemotherapy or until signs and symptoms of tumor lysis syndrome had resolved. Dexamethasone eye drops were provided during cytarabine administration as keratitis prophylaxis. Cerebellar function was tested prior to each cytarabine dose. Subjects with positive herpes simplex virus (HSV) 1 or 2 antibody titers received oral acyclovir prophylaxis. Prophylactic antibiotics and antifungals were not routinely given. Neutropenic fevers were treated as per institutional guidelines. Azole antifungals (e.g., voriconazole) were permitted only after completion of all sirolimus doses. RBCs were transfused for symptomatic anemia or a hemoglobin level <8 gm/dL. Single donor platelets were transfused for bleeding, disseminated intravascular coagulation, or asymptomatic platelet counts <10,000/μL. All blood products were leukofiltered and irradiated.

Toxicity assessment

Toxicity was assessed using the Common Toxicity Criteria for Adverse Events version 3.0. Any grade 3 or greater nonhematologic toxicity thought possibly, probably, or definitely related to sirolimus was considered a DLT. Marrow aplasia (<10% cellularity with <10% blasts) that did not resolve for at least 4 weeks was considered hematologic DLT. The following toxicities were excluded as dose-limiting: grade 3 nausea and vomiting responsive to medications, grade 3 neutropenic fevers or infections, grade 3 tumor lysis syndrome, grade 3 or 4 metabolic derangements attributed to antifungal medications or tumor lysis that corrected with intravenous or oral supplementation, grade 3 stomatitis that resolved within 7 d of discontinuation of medications, grade 3 or 4 hypercholesterolemia, grade 3 or 4 hyperbilirubinemia or transaminitis that resolved to below grade 2 within 14 d, and anemia, neutropenia, or thrombocytopenia, even if requiring transfusion.

A standard phase I dose escalation scheme was used. Subjects were to be enrolled in cohorts of three to each of the five planned sirolimus dose levels. Dose escalation did not occur until 28 d following the final subject's enrollment on any dose level. In the case of a DLT, up to three additional subjects were added to this level. Dose escalation proceeded as long as no more than one subject per level experienced a DLT. If two or more subjects at a treatment level experienced a DLT, the maximum tolerated dose (MTD) was considered to have been exceeded and the dose of sirolimus was de-escalated by one level. This dose level was expanded to a target of 6 to 12 subjects to gather additional toxicity data.

Response criteria

CR was defined as a marrow aspirate (or core biopsy specimen, if hemodilute) containing <5% blasts, an absolute neutrophil count of >1,000/μL, an untransfused platelet count of >100,000/μL, and no evidence of circulating or extramedullary leukemic blasts. CR without platelet recovery met all criteria for CR except the platelet count failed to exceed 100,000/μL. A partial remission met all criteria for CR, except that the marrow blast percentage was between 5% and 25%. Progressive disease was defined as an increase of at least 25% in the absolute number of leukemic cells in peripheral blood or bone marrow/aspirate, the development of extramedullary disease, or other evidence of increased tumor burden.

Serum rapamycin levels and pharmacodynamic assessment

Rapamycin troughs were drawn prior to sirolimus doses on days 3 and 6, and assessed by high-pressure liquid chromatography in the clinical laboratories of the Hospital of the University of Pennsylvania. A baseline marrow aspirate was collected within 14 d prior to initiation of therapy. A repeat marrow aspirate was obtained on day 3 for pharmacodynamic monitoring. Mononuclear cells from marrow aspirates were separated by Ficoll/Hypaque density centrifugation and stored as viable cells in serum with 10% DMSO in a liquid nitrogen freezer. For pharmacodynamic analysis, samples were rapidly thawed and washed with PBS to remove DMSO. All subsequent steps were carried out on ice. Cells were pelleted by centrifugation, washed once in cold PBS and immediately lysed in a buffer containing 1% Triton X-100, 150 mmol/L of NaCl, 50 mmol/L of Tris [tris(hydroxymethyl)aminomethane]-HCl with protease and phosphatase inhibitors (10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mg/mL pepstatin, 5 mmol/L EDTA, 5 mmol/L sodium orthovanadate, 50 mmol/L NaF, 50 mmol/L Na-PPi, and 150 μmol/L phenylmethylsulfonyl fluoride). Total cell lysates (100 μg by Bradford method) per sample were loaded on a SDS-PAGE gel and separated by electrophoresis. Proteins were blotted onto nitrocellulose using either wet or semi-dry transfer apparatus (Bio-Rad). Blots were blocked in 5% dried milk solution diluted in TBST (TBS/0.1% Tween 20), incubated with the indicated antibody, and bound antibody detected by hybridization with a horseradish peroxidase–conjugated secondary antibody and enhanced chemiluminescence detection (Amersham Biosciences). Blots were stripped with 2% SDS at 50°C for 30 min and re-probed as needed. In a subset of immunoblots, blocking occurred in 5% bovine serum albumin or 5% Bailey's Irish Cream diluted in PBS, followed by incubation with the indicated antibody plus 3% to 5% dried milk solution diluted in TBST, and subsequently probed with fluorescent secondary antibodies diluted in TBST or TBST with 3% dry nonfat milk. Blots were washed extensively in TBST to reduce autofluorescence from nonspecifically bound milk, and finally in PBS to diminish autofluorescence from Tween 20. Fluorescent secondary antibody was detected and quantified using a Li-COR Odyssey scanner and associated software (version 3.0). Primary antibodies used included phosphorylated p70 S6 kinase (T389), phosphorylated and total ribosomal S6 kinase (S235/236), phosphorylated and total 4EBP1 (T70) from Cell Signaling Technology, and total p70 S6 kinase from Upstate. Horseradish peroxidase–conjugated secondary antibodies were purchased from Amersham and species-specific fluorescent antibodies (Alexa 680 or IRDye 800) from Molecular Probes and Rockland Immunochemicals.

Subject demographics

Twenty-nine subjects were treated. The pretreatment characteristics of the enrolled subjects are summarized in Table 1. All subjects had AML. Twenty-six (90%) subjects had relapsed or refractory disease after prior therapy and three (10%) had previously untreated AML arising out of prior MDS. The majority of subjects (n = 20, 70%) had previously received at least one cycle of high-dose cytarabine (≥1 g/m2/dose) and four subjects (14%) had had a prior myeloablative stem cell transplantation. Of the 18 subjects in first relapse, only 7 (24% of entire cohort) had had a first remission duration of >12 months. Cytogenetic data was informative for all but three subjects. Two subjects had abnormal karyotypes of unknown prognostic significance, two subjects had inv(16) karyotypes, and all other subjects had intermediate to poor risk karyotypes using Southwest Oncology Group/Eastern Cooperative Oncology Group criteria (37).

Table 1.

Baseline characteristics (n = 29)

Age (median) 58 (range, 22-65) 
Sex 
    Male 16 (55%) 
    Female 13 (45%) 
Disease status 
    Primary refractory 6 (21%) 
    First relapse 18 (62%) 
        First CR <6 mo 6 (21%) 
        First CR 6-12 mo 5 (17%) 
        First CR >12 mo 7 (24%) 
    >First relapse 2 (7%) 
    Secondary AML 3 (10%) 
Karyotype 
    Favorable 2 (7%) 
    Intermediate 11 (38%) 
    Unfavorable 11 (38%) 
    Unknown/unsuccessful 5 (17%) 
Prior therapy 
    High-dose cytarabine (≥1 g/m2/dose) 20 (70%) 
    Myeloablative stem cell transplant 4 (14%) 
        Allogeneic 3 (10%) 
        Autologous 1 (3%) 
Age (median) 58 (range, 22-65) 
Sex 
    Male 16 (55%) 
    Female 13 (45%) 
Disease status 
    Primary refractory 6 (21%) 
    First relapse 18 (62%) 
        First CR <6 mo 6 (21%) 
        First CR 6-12 mo 5 (17%) 
        First CR >12 mo 7 (24%) 
    >First relapse 2 (7%) 
    Secondary AML 3 (10%) 
Karyotype 
    Favorable 2 (7%) 
    Intermediate 11 (38%) 
    Unfavorable 11 (38%) 
    Unknown/unsuccessful 5 (17%) 
Prior therapy 
    High-dose cytarabine (≥1 g/m2/dose) 20 (70%) 
    Myeloablative stem cell transplant 4 (14%) 
        Allogeneic 3 (10%) 
        Autologous 1 (3%) 

Toxicities

Grades 3 and 4 adverse events and DLT are summarized in Table 2. Severe myelosuppression and neutropenic fevers, typical of induction regimens, occurred in all subjects. Documented grades 3 or 4 infections occurred in 15 (52%) subjects and no opportunistic organisms (e.g., Pneumocystis jiroveci) were seen. Three infection-related deaths occurred in the study (10%; further details are provided below). No other causes of death were observed. Diarrhea was commonly observed (grade 3 in 7 of 29), and grade 3 oral stomatitis was noted in two subjects. No grade 4 mucosal or intestinal toxicity was seen. Transient hyperbilirubinemia or transaminase elevations were commonly noted and were generally mild and not associated with synthetic dysfunction or encephalopathy.

Table 2.

Toxicity of sirolimus MEC (adverse events grade 3 or greater, regardless of attribution to sirolimus)

ToxicityGrade 3Grade 4Grade 5Total (%)
Neutropenic fever 29   29 (100%) 
Bacteremia 15   15 (52) 
Diarrhea   7 (24) 
Transaminase/bilirubin elevation  4 (14) 
Pneumonia  3 (10) 
Metabolic (hyperglycemia, hypokalemia, hypocalcemia)   2 (7) 
Mucositis   2 (7) 
Sepsis/multiorgan failure   1 (3) 
Anorexia   1 (3) 
Tachypnea   1 (3) 
Tachycardia   1 (3) 
Aplasia   1 (3) 
Arterial thrombosis   1 (3) 
Ataxia   1 (3) 
ToxicityGrade 3Grade 4Grade 5Total (%)
Neutropenic fever 29   29 (100%) 
Bacteremia 15   15 (52) 
Diarrhea   7 (24) 
Transaminase/bilirubin elevation  4 (14) 
Pneumonia  3 (10) 
Metabolic (hyperglycemia, hypokalemia, hypocalcemia)   2 (7) 
Mucositis   2 (7) 
Sepsis/multiorgan failure   1 (3) 
Anorexia   1 (3) 
Tachypnea   1 (3) 
Tachycardia   1 (3) 
Aplasia   1 (3) 
Arterial thrombosis   1 (3) 
Ataxia   1 (3) 

The study's dose escalation plan, measured rapamycin levels, and observed DLTs are summarized in Table 3. For reference, steady state rapamycin troughs of 4 to 12 μg/L were targeted during solid organ transplantation. DLTs were seen in four subjects. At dose level 2, one subject experienced reversible grade 3 cerebellar toxicity, prompting dose expansion to enroll three more subjects at this level prior to escalation. After additional review of medical history, this subject reported cerebellar toxicity with prior high-dose Ara-C. This had not been disclosed at study entry and would have precluded participation. At dose level 3, one subject experienced grade 3 hepatic transaminase elevation, an expected toxicity from etoposide and/or cytarabine. According to the protocols, at the time of this subject's enrollment, this toxicity necessitated chemotherapy interruption and dose expansion at this level to a total of six subjects. Following this toxicity, a protocol amendment was made to reflect the known transient hepatotoxicity of the MEC regimen. Subsequently, only transaminase elevations that did not resolve within 14 days were considered dose-limiting. At dose level 5, one subject developed multiorgan dysfunction characterized by pneumonia, cardiomyopathy, respiratory failure, and culture-negative sepsis. The contribution of sirolimus to her cardiomyopathy and respiratory failure could not be excluded and this toxicity was considered dose-limiting. A second subject on dose level 5 developed prolonged marrow aplasia. Repeated marrow biopsies up to day 84 showed no evidence of leukemic involvement and persistent cellularity <10%. The subject experienced recurrent Gram-negative infections that ultimately were fatal on day 120. Of note, persistent cytopenias were seen in seven other subjects (24%), and were attributable in all these cases to recurrent marrow involvement by AML.

Table 3.

Dose escalation, rapamycin levels, and DLT of sirolimus MEC

Dose levelSirolimus loading dose (mg)Sirolimus daily dose (mg)Subjects treatedRapamycin trough (μg/L), day 3 (n)Rapamycin trough (μg/L), day 6 (n)DLTOutcome of DLT
1.9 (3) 1.6 (2)   
3.1 (4) 2.6 (4) Grade 3 cerebellar ataxia Resolved 
3.9 (6) 4.1 (5) Grade 3 transaminase elevation Resolved 
12 12 6.4 (10) 5.0 (9)   
15 7.2 (2) 5.1 (2) Grade 4 sepsis/multiorgan failure Death 
      Aplasia Death 
Dose levelSirolimus loading dose (mg)Sirolimus daily dose (mg)Subjects treatedRapamycin trough (μg/L), day 3 (n)Rapamycin trough (μg/L), day 6 (n)DLTOutcome of DLT
1.9 (3) 1.6 (2)   
3.1 (4) 2.6 (4) Grade 3 cerebellar ataxia Resolved 
3.9 (6) 4.1 (5) Grade 3 transaminase elevation Resolved 
12 12 6.4 (10) 5.0 (9)   
15 7.2 (2) 5.1 (2) Grade 4 sepsis/multiorgan failure Death 
      Aplasia Death 

The fourth sirolimus dose level (12 mg load followed by 4 mg dose on days 2-7) was therefore determined to be the MTD. This level was expanded to treat a total of 12 subjects. The toxicities identified at this dose level were similar to those identified at lower sirolimus doses.

Clinical outcomes

Two subjects did not complete all chemotherapy due to toxicity (cerebellar ataxia and hepatic transaminase elevation). Although day 14 marrow studies were not specified by protocol, they were commonly done at the treating physician's discretion. Three subjects received additional chemotherapy off protocol based on persistent leukemia at nadir. The median time to absolute neutrophil count recovery (>500 cells/μL) was 32 days.

Six subjects responded to sirolimus MEC (22% of those who received all chemotherapy doses; summarized in Table 4). Four subjects, all in first relapse with prior a CR duration of >6 months, achieved CR. Two subjects achieved a partial remission. Of those achieving a CR to sirolimus MEC, three subjects went on to subsequent stem cell transplant and remain alive in remission. One subject declined transplantation following sirolimus MEC and experienced an unmaintained 16-month remission prior to eventual disease progression. Clinical responses occurred across all dose levels and no clear dose response to sirolimus was noted. Of the 12 subjects treated at the MTD, two CRs and one partial remission (PR) were achieved (overall response rate was 25% at the MTD).

Table 4.

Characteristics of subjects who attained clinical responses to sirolimus MEC

Subject no.Dose levelAgeTreatment status at enrollmentKaryotypeClinical responseSubsequent therapyOutcome
53 First relapse, prior CR >6 mo 46, XY CR Autologous SCT Treated for relapse at 9 mo, alive in third remission 
63 First relapse, refractory to initial salvage 46, XY, inv(16)(p13;q22) CR None Treated for relapse at 16 mo, deceased 
58 First relapse, prior to CR >6 mo 46, XX PR None Treated for progressive AML at 2 mo, deceased 
17 39 First relapse, prior to CR >6 mo 46, XY, t(6;11)(q27;q23) CR Allogeneic SCT Alive in continuous remission 
18 61 First relapse, prior to CR >6 mo 47, XY, t(3;22)(p25;q13), t(12;16)(q?13;p?11.2),+22* CR Allogeneic SCT Alive in continuous remission 
27 37 Primary refractory 46, XY PR None Treated for progressive AML at 1 mo, deceased 
Subject no.Dose levelAgeTreatment status at enrollmentKaryotypeClinical responseSubsequent therapyOutcome
53 First relapse, prior CR >6 mo 46, XY CR Autologous SCT Treated for relapse at 9 mo, alive in third remission 
63 First relapse, refractory to initial salvage 46, XY, inv(16)(p13;q22) CR None Treated for relapse at 16 mo, deceased 
58 First relapse, prior to CR >6 mo 46, XX PR None Treated for progressive AML at 2 mo, deceased 
17 39 First relapse, prior to CR >6 mo 46, XY, t(6;11)(q27;q23) CR Allogeneic SCT Alive in continuous remission 
18 61 First relapse, prior to CR >6 mo 47, XY, t(3;22)(p25;q13), t(12;16)(q?13;p?11.2),+22* CR Allogeneic SCT Alive in continuous remission 
27 37 Primary refractory 46, XY PR None Treated for progressive AML at 1 mo, deceased 

Abbreviation: SCT, stem cell transplant.

*Precise translocation breakpoints could not be defined for t(12;16).

Rapamycin drug levels and evidence of mTOR signal disruption

Rapamycin levels were measured and showed dose-dependent increases (Table 3). Similar levels were seen on days 3 and 6 measurements. At the MTD, the mean rapamycin levels at day 3 and 6 were 6.4 and 5.0 μg/L, respectively. The laboratory's therapeutic rapamycin level reference range for transplant immunosuppression was 4 to 12 μg/L. One subject did not report his use of voriconazole just prior to study entry and developed markedly elevated rapamycin levels (day 3 = 21.9 μg/L; day 6 = 31.2 μg/L). This subject was excluded from drug level analysis due to this protocol violation. Of note, this subject neither experienced prolonged myelosuppression nor other unusual toxicities. He did not clinically respond to sirolimus MEC.

Paired baseline and day 3 pharmacodynamic assessments of mTOR signaling were evaluable in 12 subjects. Paired samples could not be obtained in 11 subjects, and in 6 subjects, samples were obtained but were technically not evaluable. Unevaluable samples occurred due to low cell and protein yields from postchemotherapy (day 3) samples, target protein degradation, or insufficient signal from p-70 S6K antibody for reliable controls.

All evaluable baseline samples showed p70 S6 kinase, S6 ribosomal protein, or 4EBP1 phosphorylation, consistent with mTOR activation (data not shown). Five of 12 subjects with evaluable samples were considered to have definite (n = 2) or possible (n = 3) target inhibition. Inhibition of mTOR, as shown by abrogation of p70 S6K, S6 ribosomal protein, or 4EBP1 phosphorylation occurred across all dose levels. Among subjects treated at or above the MTD, five had evaluable paired samples (Fig. 2). Considering these five samples from dose levels 4 and 5, only one (subject 17) showed definite evidence of mTOR inhibition. No association between serum rapamycin level and biochemical response was noted to suggest a critical drug level above which mTOR consistently occurred.

Fig. 2.

mTOR inhibition in leukemic blasts at or above the MTD. Among subjects treated at or above the MTD (dose level 4), paired marrow aspirates were evaluable for signaling end points in five subjects. Western blots for phosphorylated and total p70 S6 kinase, a surrogate marker of mTOR activation, are shown for these subjects at baseline (pre) and day 3 (post). Each subject's day 3 rapamycin level and clinical response to the sirolimus MEC are shown. All subjects depicted were treated on dose level 4, except subject 20, who was treated on dose level 5. As a control, untreated lysate from a myeloid cell line (U937) is shown and compared with the same cell line following 4 h of in vitro treatment with LY294002 (25 μmol/L), an inhibitor of PI3K and mTOR that disrupts signaling through downstream targets, including p70 S6K. The anti-phosphorylated p70 antibody recognizes two subunits of the S6 kinase, p70 (lower arrow) and p85 (upper arrow). Only p70 phosphorylation is altered by the mTOR activation state, as seen in the U937 control lanes. Some samples show two p70 bands, representing alternate phosphorylation. Subject 17 shows inhibition of mTOR, as shown by abrogated phosphorylated p70 S6 kinase signal on day 3 samples compared with baseline. None of the other four samples were considered to have a definite biochemical response. NR, no remission.

Fig. 2.

mTOR inhibition in leukemic blasts at or above the MTD. Among subjects treated at or above the MTD (dose level 4), paired marrow aspirates were evaluable for signaling end points in five subjects. Western blots for phosphorylated and total p70 S6 kinase, a surrogate marker of mTOR activation, are shown for these subjects at baseline (pre) and day 3 (post). Each subject's day 3 rapamycin level and clinical response to the sirolimus MEC are shown. All subjects depicted were treated on dose level 4, except subject 20, who was treated on dose level 5. As a control, untreated lysate from a myeloid cell line (U937) is shown and compared with the same cell line following 4 h of in vitro treatment with LY294002 (25 μmol/L), an inhibitor of PI3K and mTOR that disrupts signaling through downstream targets, including p70 S6K. The anti-phosphorylated p70 antibody recognizes two subunits of the S6 kinase, p70 (lower arrow) and p85 (upper arrow). Only p70 phosphorylation is altered by the mTOR activation state, as seen in the U937 control lanes. Some samples show two p70 bands, representing alternate phosphorylation. Subject 17 shows inhibition of mTOR, as shown by abrogated phosphorylated p70 S6 kinase signal on day 3 samples compared with baseline. None of the other four samples were considered to have a definite biochemical response. NR, no remission.

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Considering subjects with clinical responses to sirolimus MEC, three subjects with a CR or PR to sirolimus MEC had evaluable paired samples. One subject at dose level 4 achieved a CR and showed definite target inhibition (Fig. 2, subject 17), one subject on dose level 1 achieved a CR and showed possible inhibition (data not shown), and one subject on dose level 4 achieved a PR but did not show phosphorylated p70 target inhibition (Fig. 2, subject 27). Among the remaining eight subjects who did not respond to sirolimus MEC but did have evaluable samples, one subject showed definite p70 S6K inhibition and two subjects showed possible inhibition. The remaining five subjects who did not respond clinically to the regimen had no significant change in downstream mTOR target phosphorylation.

This trial represents the first study to combine mTOR inhibitors with induction chemotherapy for acute leukemia. Our study shows the feasibility of combining agents that target signal transduction in leukemic stem cells with highly myelosuppressive cytotoxic chemotherapy.

Of the toxicities identified with this regimen, prolonged marrow aplasia in the absence of leukemic relapse was unexpected and occurred in one subject at dose level 5. This finding raised the concern that sirolimus plus MEC was irreversibly toxic to normal as well as leukemic stem cells, despite preclinical data to the contrary. Of note, our preclinical data was completed with rapamycin and etoposide only, and did not examine all chemotherapy agents involved in this trial. One subject that experienced a hematologic DLT was a 48-year-old man treated at first relapse. He did not have a history of heavy pretreatment, antecedent MDS, extensive marrow fibrosis, or aplastic anemia that might have predicted this toxicity. Rapamycin levels for this subject were 5.3 and 3.1 μg/L, which were not notably different from that seen in subjects on dose level 4. Although prolonged aplasia is rare from induction chemotherapy, it has been reported in populations similar to that studied here, including regimens that combine signal transduction inhibitors and induction chemotherapy (38, 39). We therefore considered aplasia to be possibly related to sirolimus.

The addition of sirolimus to MEC chemotherapy did not obviously increase nonhematologic toxicity above that seen historically with MEC alone. Abnormal liver transaminases and cerebellar toxicity necessitated dose expansion on this study, yet both toxicities are well described with etoposide or cytarabine in the absence of other agents. Although the two subjects with fatal complications of sirolimus MEC therapy at dose level 5 suggested additional toxicity from the mTOR inhibitor when added to chemotherapy, these toxicities were more likely multifactorial. The case of prolonged aplasia is considered above. In the case of multisystem organ dysfunction, the subject had a history of prior allogeneic stem cell transplant and multiple prior anthracycline-based regimens. This subject's extensive prior therapy likely contributed to her impaired ability to withstand infectious complications of induction, despite her young age (31) and adequate baseline organ studies at enrollment. In summary, it was not clear that sirolimus increased the hematologic or nonhematologic toxicity of the MEC regimen.

The integration of signal transduction inhibitors into chemotherapy regimens for AML and other cancers is a promising area of clinical development, but is also one in which there has been little detailed analysis of pharmacodynamic end points. As such, few data exist to guide the use of these molecularly targeted approaches. Preclinical data suggested that mTOR inhibition should dramatically enhance the effects of etoposide-based chemotherapy. Although not statistically powered to show comparative efficacy, it is notable that the response rate on this clinical trial at the MTD is similar to published reports using MEC chemotherapy alone in relapsed and refractory patient populations. Our dose 4 expansion cohort provides a preliminary efficacy assessment, but this response rate fails to show the strong synergy of rapamycin and etoposide predicted by our in vitro studies (5). The lack of mTOR inhibition in the majority of evaluable samples at or above dose level 4 suggests one reason why this may have occurred.

Pharmacodynamic measurement was critical to confirm or refute our hypothesis that mTOR inhibition in leukemic blasts would predict clinical response. Unfortunately, an adequate amount of sample to assess biochemical response could not be obtained in many subjects. We suspect that low cell and protein yield on day 3 samples was due to apoptosis from chemotherapy, a finding that was not directly measured here. If true, the results of our pharmacodynamic studies may represent sampling of the residual, apoptosis-resistant population. In this context, the presence of mTOR activation in these cells is actually not surprising, as data suggest constitutive mTOR activation provides a strong antiapoptotic stimulus.

Other considerations for the lack of mTOR inhibition include at least two explanations. It is possible that this finding is a technical artifact of the methodology. We have assayed mTOR on the total mononuclear cell population, and not specifically in leukemic cells. New approaches using intracellular flow cytometry may provide a more leukemia-specific assessment of pharmacodynamic effects (40). Alternatively, in vivo effects may be lost when primary cells are studied in vitro, leading to impaired sensitivity to detect rapamycin responsiveness in AML blasts. Further studies will be needed to test this hypothesis.

In summary, targeting oncogenic signaling in AML stem cells through the mTOR inhibitor rapamycin remains a promising, but challenging goal. We show that combining sirolimus and etoposide-based induction chemotherapy for AML is feasible. However, using the doses and schedule tested, we did not show synergy between sirolimus and chemotherapy. A possible reason for this finding is the inability to indicate mTOR inhibition in tumor cells that were left after 3 days of chemotherapy. We have recently begun testing the sequential administration of sirolimus alone followed by combined sirolimus and MEC. Preliminary analysis of these trials shows evaluable pharmacodynamic results in all samples, and inhibition of mTOR more frequently than presented here. Other inhibitors of the PI3K pathway (e.g., rapamcyin analogues or direct inhibitors of PI3K, AKT, or mTOR kinases) combined with other chemotherapeutic agents, may also ultimately prove more efficacious than rapamycin as used in the current study. We finally conclude that careful pharmacodynamic analysis in tumor cells is critical for the evaluation and integration of signal transduction inhibitors with chemotherapy regimens for molecularly targeted cancer therapy.

S. Luger owns stock in Wyeth Pharmaceuticals.

1
Goldstone
AH
,
Burnett
AK
,
Wheatley
K
,
Smith
AG
,
Hutchinson
RM
,
Clark
RE
. 
Attempts to improve treatment outcomes in acute myeloid leukemia (AML) in older patients: the results of the United Kingdom Medical Research Council AML11 trial
.
Blood
2001
;
98
:
1302
11
.
2
Mayer
RJ
,
Davis
RB
,
Schiffer
CA
, et al
. 
Intensive postremission chemotherapy in adults with acute myeloid leukemia. Cancer and Leukemia Group B
.
N Engl J Med
1994
;
331
:
896
903
.
3
Bennett
JM
,
Young
ML
,
Andersen
JW
, et al
. 
Long-term survival in acute myeloid leukemia: the Eastern Cooperative Oncology Group experience
.
Cancer
1997
;
80
:
2205
9
.
4
Guzman
ML
,
Rossi
RM
,
Karnischky
L
, et al
. 
The sesquiterpene lactone parthenolide induces apoptosis of human acute myelogenous leukemia stem and progenitor cells
.
Blood
2005
;
105
:
4163
9
.
5
Xu
Q
,
Simpson
SE
,
Scialla
TJ
,
Bagg
A
,
Carroll
M
. 
Survival of acute myeloid leukemia cells requires PI3 kinase activation
.
Blood
2003
;
102
:
972
80
.
6
Liesveld
JL
,
Abboud
CN
,
Lu
C
, et al
. 
Flavonoid effects on normal and leukemic cells
.
Leuk Res
2003
;
27
:
517
27
.
7
Shao
RG
,
Shimizu
T
,
Pommier
Y
. 
7-Hydroxystaurosporine (UCN-01) induces apoptosis in human colon carcinoma and leukemia cells independently of p53
.
Exp Cell Res
1997
;
234
:
388
97
.
8
Liesveld
JL
,
Lancet
JE
,
Rosell
KE
, et al
. 
Effects of the farnesyl transferase inhibitor R115777 on normal and leukemic hematopoiesis
.
Leukemia
2003
;
17
:
1806
12
.
9
Levis
M
,
Allebach
J
,
Tse
KF
, et al
. 
A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo
.
Blood
2002
;
99
:
3885
91
.
10
Holstein
SA
,
Hohl
RJ
. 
Interaction of cytosine arabinoside and lovastatin in human leukemia cells
.
Leuk Res
2001
;
25
:
651
60
.
11
Guzman
ML
,
Swiderski
CF
,
Howard
DS
, et al
. 
Preferential induction of apoptosis for primary human leukemic stem cells
.
Proc Natl Acad Sci U S A
2002
;
99
:
16220
5
.
12
Perl
AE
,
Carroll
M
. 
Exploiting signal transduction pathways in acute myelogenous leukemia
.
Curr Treat Options Oncol
2007
;
8
:
265
76
.
13
Pratz
KW
,
Cortes
J
,
Roboz
GJ
, et al
. 
A pharmacodynamic study of the FLT3 inhibitor KW-2449 yields insight into the basis for clinical response
.
Blood
2008
;
113
:
3938
46
.
14
Cortes
JE
,
Ghirdaladze
D
,
Foran
JM
, et al
. 
Phase 1 AML study of AC220, a potent and selective second generation FLT3 receptor tyrosine kinase inhibitor
.
ASH Annual Meeting Abstracts
2008
;
112
:
767
.
15
Levis
M
,
Smith
B
,
Beran
M
, et al
. 
A randomized, open-label study of lestaurtinib (CEP-701), an oral FLT3 inhibitor, administered in sequence with chemotherapy in patients with relapsed AML harboring FLT3 activating mutations: clinical response correlates with successful FLT3 inhibition
.
Blood
2005
;
106
:
121a
.
16
Ravandi
F
,
Cortes
J
,
Faderl
S
, et al
. 
Combination of sorafenib, idarubicin, and cytarabine has a high response rate in patients with newly diagnosed acute myeloid leukemia (AML) younger than 65 years
.
ASH Annual Meeting Abstracts
2008
;
112
:
768
.
17
Karp
JE
,
Smith
BD
,
Levis
MJ
, et al
. 
Sequential flavopiridol, cytosine arabinoside, and mitoxantrone: a phase II trial in adults with poor-risk acute myelogenous leukemia
.
Clin Cancer Res
2007
;
13
:
4467
73
.
18
Karp
JE
,
Feldman
EJ
,
Morris
L
, et al
. 
Active oral regimen for elderly adults with newly diagnosed acute myelogenous leukemia (AML): phase I trial of oral tipifarnib (T) combined with oral etoposide (E) for adults > = age 70 who are not candidates for traditional cytotoxic chemotherapy (TCC)
.
ASH Annual Meeting Abstracts
2006
;
108
:
426
.
19
Yilmaz
OH
,
Valdez
R
,
Theisen
BK
, et al
. 
Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells
.
Nature
2006
;
441
:
475
82
.
20
Zhao
S
,
Konopleva
M
,
Cabreira-Hansen
M
, et al
. 
Inhibition of phosphatidylinositol 3-kinase dephosphorylates BAD and promotes apoptosis in myeloid leukemias
.
Leukemia
2004
;
18
:
267
75
.
21
Bardet
V
,
Tamburini
J
,
Ifrah
N
, et al
. 
Single cell analysis of phosphoinositide 3-kinase/Akt and ERK activation in acute myeloid leukemia by flow cytometry
.
Haematologica
2006
;
91
:
757
64
.
22
Recher
C
,
Beyne-Rauzy
O
,
Demur
C
, et al
. 
Antileukemic activity of rapamycin in acute myeloid leukemia
.
Blood
2005
;
105
:
2527
34
.
23
Kornblau
SM
,
Womble
M
,
Qiu
YH
, et al
. 
Simultaneous activation of multiple signal transduction pathways confers poor prognosis in acute myelogenous leukemia
.
Blood
2006
;
108
:
2358
65
.
24
Xu
Q
,
Thompson
JE
,
Carroll
M
. 
mTOR regulates cell survival after etoposide treatment in primary AML cells
.
Blood
2005
;
106
:
4261
8
.
25
Yee
KW
,
Zeng
Z
,
Konopleva
M
, et al
. 
Phase I/II study of the mammalian target of rapamycin inhibitor everolimus (RAD001) in patients with relapsed or refractory hematologic malignancies
.
Clin Cancer Res
2006
;
12
:
5165
73
.
26
Yee
KWL
,
Garcia-Manero
G
,
Thomas
D
, et al
. 
A phase II study of temsirolimus (CCI-779) in patients with advanced leukemias
.
Blood-ASH Annual Meeting Abstracts
2004
;
104
:
4523a
.
27
Zeng
Z
,
Sarbassov
DD
,
Samudio
IJ
, et al
. 
Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML
.
Blood
2006
;109:3509–12.
28
Tallman
MS
,
Lee
S
,
Sikic
BI
, et al
. 
Mitoxantrone, etoposide, and cytarabine plus cyclosporine for patients with relapsed or refractory acute myeloid leukemia: an Eastern Cooperative Oncology Group pilot study
.
Cancer
1999
;
85
:
358
67
.
29
Greenberg
PL
,
Lee
SJ
,
Advani
R
, et al
. 
Mitoxantrone, etoposide, and cytarabine with or without valspodar in patients with relapsed or refractory acute myeloid leukemia and high-risk myelodysplastic syndrome: a phase III trial (E2995)
.
J Clin Oncol
2004
;
22
:
1078
86
.
30
Spadea
A
,
Petti
MC
,
Fazi
P
, et al
. 
Mitoxantrone, etoposide and intermediate-dose Ara-C (MEC): an effective regimen for poor risk acute myeloid leukemia
.
Leukemia
1993
;
7
:
549
52
.
31
Karanes
C
,
Kopecky
KJ
,
Head
DR
, et al
. 
A phase III comparison of high dose ARA-C (HIDAC) versus HIDAC plus mitoxantrone in the treatment of first relapsed or refractory acute myeloid leukemia Southwest Oncology Group Study
.
Leuk Res
1999
;
23
:
787
94
.
32
Leopold
LH
,
Willemze
R
. 
The treatment of acute myeloid leukemia in first relapse: a comprehensive review of the literature
.
Leuk Lymphoma
2002
;
43
:
1715
27
.
33
Estey
E
,
Plunkett
W
,
Gandhi
V
,
Rios
MB
,
Kantarjian
H
,
Keating
MJ
. 
Fludarabine and arabinosylcytosine therapy of refractory and relapsed acute myelogenous leukemia
.
Leuk Lymphoma
1993
;
9
:
343
50
.
34
Saunders
RN
,
Metcalfe
MS
,
Nicholson
ML
. 
Rapamycin in transplantation: a review of the evidence
.
Kidney Int
2001
;
59
:
3
16
.
35
MacDonald
AS
. 
A worldwide, phase III, randomized, controlled, safety and efficacy study of a sirolimus/cyclosporine regimen for prevention of acute rejection in recipients of primary mismatched renal allografts
.
Transplantation
2001
;
71
:
271
80
.
36
Langer
RM
,
Van Buren
CT
,
Katz
SM
,
Kahan
BD
. 
De novo hemolytic uremic syndrome after kidney transplantation in patients treated with cyclosporine-sirolimus combination
.
Transplantation
2002
;
73
:
756
60
.
37
Slovak
ML
,
Kopecky
KJ
,
Cassileth
PA
, et al
. 
Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern Cooperative Oncology Group Study
.
Blood
2000
;
96
:
4075
83
.
38
Karp
JE
,
Passaniti
A
,
Gojo
I
, et al
. 
Phase I and pharmacokinetic study of flavopiridol followed by 1-β-d-arabinofuranosylcytosine and mitoxantrone in relapsed and refractory adult acute leukemias
.
Clin Cancer Res
2005
;
11
:
8403
12
.
39
Karp
JE
,
Ricklis
RM
,
Balakrishnan
K
, et al
. 
A phase 1 clinical-laboratory study of clofarabine followed by cyclophosphamide for adults with refractory acute leukemias
.
Blood
2007
;
110
:
1762
9
.
40
Chow
S
,
Minden
MD
,
Hedley
DW
. 
Constitutive phosphorylation of the S6 ribosomal protein via mTOR and ERK signaling in the peripheral blasts of acute leukemia patients
.
Exp Hematol
2006
;
34
:
1183
91
.

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