Purpose: To determine the MTD, dose-limiting toxicities (DLT), pharmacokinetics, and biologic effects of cixutumumab administered in combination with temsirolimus to children with refractory solid tumors.

Experimental Design: Cixutumumab and temsirolimus were administered intravenously once every 7 days in 28-day cycles. Pharmacokinetic and biology studies, including assessment of mTOR downstream targets in peripheral blood mononuclear cells, were performed during the first cycle.

Results: Thirty-nine patients, median age 11.8 years (range, 1–21.5), with recurrent solid or central nervous system tumors were enrolled, of whom 33 were fully assessable for toxicity. There were four dose levels, which included two dose reductions and a subsequent intermediated dose escalation: (i) IMC-A12 6 mg/kg, temsirolimus 15 mg/m2; (ii) IMC-A12 6 mg/kg, temsirolimus 10 mg/m2; (iii) IMC-A12 4 mg/kg, temsirolimus 8 mg/m2; and (iv) IMC-A12 6 mg/kg, temsirolimus 8 mg/m2. Mucositis was the predominant DLT. Other DLTs included hypercholesterolemia, fatigue, thrombocytopenia, and increased alanine aminotransferase. Target inhibition (decreased S6K1 and PAkt) in peripheral blood mononuclear cells was noted at all dose levels. Marked interpatient variability in temsirolimus pharmacokinetic parameters was noted. At 8 mg/m2, the median temsirolimus AUC was 2,946 ng • h/mL (range, 937–5,536) with a median sirolimus AUC of 767 ng • h/mL (range, 245–3,675).

Conclusions: The recommended pediatric phase II doses for the combination of cixutumumab and temsirolimus are 6 mg/kg and 8 mg/m2, respectively. Clin Cancer Res; 21(7); 1558–65. ©2014 AACR.

Translational Relevance

Deregulated insulin-like growth factor signaling through the type 1 receptor is common to many childhood solid tumors. Similarly, the cell signaling pathways that activate the mTOR are altered in many pediatric cancers. IGFIR inhibition should prevent rapamycin-induced Akt activation, hence abrogating at least one survival pathway and synergizing with mTOR inhibitors. This first pediatric phase I combination study established the recommended pediatric phase II doses for cixutumumab and temsirolimus at 6 mg/kg and 8 mg/m2, respectively, with mucositis as the dose-limiting toxicity. Target inhibition (decreased p-S6K1 and p-Akt) in peripheral blood mononuclear cells was noted at all dose levels. One partial response was reported in a patient with medulloblastoma; prolonged stable disease was noted in 3 patients (median, 12 cycles). These results lay the groundwork for additional combination studies with IGFR and mTOR inhibitors. A phase II study of this combination has recently been completed by the Children's Oncology Group (COG).

Deregulated insulin-like growth factor signaling through the type 1 receptor is common to many childhood solid tumors (1), including Ewing sarcoma (2), rhabdomyosarcoma (3), osteosarcoma (4, 5), neuroblastoma (6–8), brain tumors (9–13), Wilms tumor (14), and hepatoblastoma (15). Similarly, the cell signaling pathways that activate the mTOR are altered in many pediatric cancers, making them susceptible to mTOR inhibition by sirolimus and its analogues (16–18). Although we have previously shown preclinical activity of mTOR inhibitors in pediatric cancers (17), monotherapy with these agents has had limited clinical activity (16, 19). This may, in part, be explained by a feedback loop in which inhibition of mTOR signaling leads to upregulation of IGFIR signaling through loss of S6K1 phosphorylation, leading to activation of PI3K and Akt (20–22). Indeed, Akt activation has been demonstrated in cancer cell lines and in patients treated with mTOR inhibitors (23). IGFIR inhibition should prevent rapamycin-induced Akt activation, hence abrogating at least one survival pathway and synergizing with mTOR inhibitors (20, 21, 24).

Cixutumumab (IMC-A12) is a fully recombinant IgG1 monoclonal antibody that specifically targets the human IGFIR1 with high affinity. It acts as an antagonist of IGFI and IGFII ligand binding and signaling. It also blocks ligand binding to IGFIR and inhibits downstream signaling of the two major insulin-like growth factor pathways: MAPK and PI3K/AKT. Cixutumumab has potent dose-dependent in vitro and in vivo antitumor activity in a variety of cell lines and xenografts.

Temsirolimus is a small-molecule inhibitor of mTOR. Like sirolimus and everolimus, temsirolimus forms a gain-of-function complex with FK506-binding protein 12 (FKBP12) that binds and inhibits mTOR, leading to antiproliferative effects, including G1-phase cell-cycle arrest (25), and apoptosis. The primary downstream targets of mTOR include eIF4E-binding protein (4E-BP1; refs. 26, 27) and p70S6 kinase, important in the translation regulation of mRNA encoding proteins involved in G1 phase progression. mTOR inhibitors have potent in vitro activity against many human cancer cell lines and xenograft models. The Pediatric Preclinical Testing Program (17, 28) and others have reported preclinical single agent and synergistic combination activity of these agents in many solid tumors (24, 29, 30).

We report the results of a phase I trial of cixutumumab in combination with temsirolimus in children with recurrent or refractory solid tumors. The primary objectives were to estimate the MTD, determine dose-limiting toxicities (DLT), and characterize the pharmacokinetics of IMC-A12 and temsirolimus administered once weekly in combination, to children with refractory solid tumors. The secondary objectives were to assess the biologic activity of temsirolimus by measuring levels of phospho-S6Ser235/236, phospho-AKTSer473, and phospho-4EBP1Ser65 in peripheral blood mononuclear cells (PBMC).

Patient eligibility

Patients >12 months and <22 years with measurable or evaluable solid tumors refractory to therapy were eligible. Histologic verification of malignancy was required except for patients with intrinsic brainstem glioma. Other eligibility criteria included Lansky or Karnofsky score ≥50; recovery from the acute toxic effects of prior therapy; ≥3 months since total body irradiation, craniospinal or hemi-pelvic radiation, and ≥2 months since a stem cell transplant; adequate bone marrow function [peripheral absolute neutrophil count (ANC) ≥ 1,000/μL, platelets ≥ 100,000/μL (transfusion independent), hemoglobin ≥ 8.0 g/dL]; adequate renal function (age-adjusted normal serum creatinine or a glomerular filtration rate ≥ 70 mL/min/1.73 m2); adequate liver function [total bilirubin ≤ 1.5× institutional upper limit of normal for age, SGPT (alanine aminotransferase, ALT) ≤ 5× institutional upper limit of normal for age and albumin ≥ 2 g/dL]; prothrombin time and international normalized ratio < 1.2× upper limit of normal. Patients receiving corticosteroids had to be on a stable or decreasing dose for ≥7 days before study enrollment.

Patients were excluded if they had known bone marrow involvement; had received prior temsirolimus or monoclonal antibody therapy targeting IGFIR; were pregnant or lactating; had an uncontrolled infection; were receiving enzyme inducing anticonvulsants (EIACD), insulin, growth hormone therapy, or any of the following CYP3A4 inducers or inhibitors: erythromycin, clarithromycin, ketoconazole, azithromycin, itraconazole, grapefruit juice, or St. John's wort or other noncytotoxic anticancer agents. Also excluded were patients with a history of allergic reactions attributed to compounds of similar chemical or biologic composition to cixutumumab or temsirolimus, or patients who had undergone major surgery within 6 weeks before study enrollment. The Institutional Review Boards of participating institutions approved the protocol. Informed consent and assent, as appropriate, were obtained according to local institutional guidelines.

Drug administration

Cixutumumab [5 mg/mL (250 mg) or 10 mg/mL (500 mg) vials] and temsirolimus (25 mg/mL), supplied by the Cancer Therapy Evaluation Program (NCI, Bethesda, MD), were administered weekly as a 1 hour and 30 minute infusion, respectively, in 28-day cycles. In the absence of disease progression, patients could receive a total of 25 cycles of therapy.

The starting dose of cixutumumab was 6 mg/kg (one dose level below the recommended phase II dose of 9 mg/kg in the pediatric phase I study; ref. 31) and of temsirolimus was 15 mg/m2, with planned dose escalations to a maximum of 9 mg/kg for cixutumumab and 35 mg/m2 for temsirolimus. The starting dose of temsirolimus was based on published studies in adult combination trials (32). Because of toxicity, the study was amended to incorporate several dose de-escalations as shown in Table 1.

Table 1.

Dose-limiting toxicity

Dose levelIMC-A12 (mg/kg)Temsirolimus (mg/m2)Number of patients enteredNumber of patients evaluableNumber of patients with DLTType of DLT (n)
15 15 12 Platelets (1), fatigue (1), mucositis (1), ALT (1), hypercholesterolemia (1) 
10 Mucositis/stomatitis (2) 
−1  
Intermediate Mucositis (1) 
Expanded PK cohort  
Dose levelIMC-A12 (mg/kg)Temsirolimus (mg/m2)Number of patients enteredNumber of patients evaluableNumber of patients with DLTType of DLT (n)
15 15 12 Platelets (1), fatigue (1), mucositis (1), ALT (1), hypercholesterolemia (1) 
10 Mucositis/stomatitis (2) 
−1  
Intermediate Mucositis (1) 
Expanded PK cohort  

Abbreviation: PK, pharmacokinetic.

Trial design

Toxicities were graded according to the Common Terminology Criteria for Adverse Events (CTCAE) v3.0. (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf). Hematologic DLT was defined as grade 4 neutropenia for >7 days, or before drug administration on days 8, 15, or 21; platelet count <20,000/mm3 on 2 separate days or requiring platelet transfusions on 2 separate days within a 7-day period, or myelosuppression that caused a delay of ≥14 days between treatment cycles. Nonhematologic DLT was defined as grade 3 or 4 nonhematologic toxicity with the specific exclusion of grade 3 nausea and vomiting of <3 days duration, grade 3 transaminase elevations that returned to grade ≤ 1 or baseline before the time for the next treatment cycle, grade 3 fever or infection, nonhematologic toxicity that caused a delay of ≥14 days between treatment cycles.

Any patient who experienced DLT at any time during protocol therapy was considered fully evaluable for toxicity. Patients without DLT who received at least 85% of the prescribed dose during the first 28-day cycle were considered evaluable for toxicity as well as response. Dose escalation proceeded using a modified 3 + 3 cohort design in which 3 patients were initially studied at each dose level. If none of these 3 patients experience DLT, the dose was escalated to the next higher level. If one of three patients experienced DLT, then up to three more patients were accrued at the same dose level. If none of these three additional patients experienced DLT, then the dose was escalated. If one or more of these three additional patients experience DLT, the MTD was exceeded, unless one of the DLTs did not appear to be related to dose or the DLTs were of different classes and the toxicities were readily reversible. In that circumstance, the cohort could be expanded to 12. The MTD was the maximum dose at which fewer than one third of patients experienced DLT. Once the MTD for the combination was defined, up to 6 patients with recurrent or refractory solid tumors were to be enrolled to acquire additional pharmacokinetic data in patients <12 years.

Response evaluation

Disease evaluations were obtained at baseline, at the end of cycle 1, and after every other cycle. Tumor response was reported using the RECIST (33).

Pharmacokinetics studies

Blood samples (5 mL) for pharmacokinetic analysis of cixutumumab were obtained before administration on days 1, 8, 15, 22, and 28 of cycle 1, and days 15 and 28 of cycle 2. In patients who agreed to have additional pharmacokinetic sampling, 3 mL of blood were collected at the end of the day 1 infusion, and 1, 3, 6, and 24 (±2) hours, and 4 days (±1 day) after the first dose. All blood samples were collected at a site distant from the infusion, in tubes without anticoagulant, allowed to clot at room temperature, centrifuged at 1,500 rpm for 15 minutes, and stored at less than −20°C. A validated ELISA was used to quantify levels of cixutumumab as previously described (31).

Blood samples (1 mL) for pharmacokinetic analysis of temsirolimus were obtained before temsirolimus administration on days 1 and 28 of cycle 1, and days 15 and 28 of cycle 2. In patients who agreed to have additional pharmacokinetic sampling, blood was collected prior at the end and at 15 minutes, 30 minutes, 1, 3, 6, and 24 (±2) hours after the infusion on day 1. All blood samples were collected at a site distant from the infusion.

Temsirolimus and sirolimus concentrations were determined using validated isotope-dilution high-performance liquid chromatography–tandem mass spectrometry assays based on previously described principles (34).

The maximum plasma concentration (Cmax) and time to maximum plasma concentration (Tmax) were determined by visual inspection of the plasma concentration-time profiles for subjects participating in the extended pharmacokinetic sampling. Temsirolimus clearance (CL), steady-state volume of distribution (Vdss), half-life (t1/2), and area under the plasma concentration versus time curve (AUC0–24 and AUC0–∞) were analyzed using standard noncompartmental methods (NCA; Phoenix WinNonlin, Pharsight Corp). Sirolimus pharmacokinetic parameter estimates for t1/2 and AUC0–∞ were generated using model-based Bayesian analysis (MW/Pharm; Version 3.60, Mediware; ref. 35).

Biologic assays

Whole blood (4 mL) was collected in BD Vacutainer CPT sodium citrate tubes before administration of protocol therapy on days 1, 8, and 28 of cycle 1 in consenting patients. Within 1 hour of acquisition, PBMC lysate was prepared as described (36), snap-frozen in liquid nitrogen, and stored at −80ºC until phosphoprotein analysis. Phosphorylated and total S6K1, Akt, and 4E-BP1 detection and quantification were performed via Western blot analysis followed by densitometric analysis with ImageQuant 5.0 (Molecular Dynamics). The ratio of phosphorylated protein to total (phosphorylated and unphosphorylated) protein in each patient sample was then calculated.

Of 39 patients enrolled, all were eligible, six were not fully assessable for toxicity: two never received protocol therapy because of clinical deterioration, two experienced progressive disease during cycle 1, and two withdrew consent for non-DLTs [grade 2 ALT/AST (aspartate aminotransferase; n = 1) and grade 2 mucositis (n = 1)]. Table 2 summarizes the characteristics of the eligible patients. Patients received a median of 2 cycles (range, 1–20) of therapy.

Table 2.

Characteristics of eligible patients (n = 39)

CharacteristicNumber
Age (years)  
 Median (range) 11.8 (1.0–21.5) 
Male:female 20 (51%):19 (49%) 
Diagnosis  
 Rhabdomyosarcoma 
 Osteosarcoma 
 Ewing sarcoma 
 Malignant glioma (including GBM) 
 Medulloblastoma 
 Wilms tumor 
 Desmoplastic small round cell tumor 
 Astrocytoma NOS 
 Choroid plexus papilloma 
 Neuroblastoma 
 Neurilemmoma 
 Ganglioneuroblastoma 
 Gastrointestinal stromal tumor 
 Hepatocellular carcinoma 
 Atypical teratoid rhabdoid tumor 
 Epithelioid sarcoma 
Prior therapy  
 Chemotherapy regimens: median (range) 2 (0–7) 
 Prior radiotherapy 28 
CharacteristicNumber
Age (years)  
 Median (range) 11.8 (1.0–21.5) 
Male:female 20 (51%):19 (49%) 
Diagnosis  
 Rhabdomyosarcoma 
 Osteosarcoma 
 Ewing sarcoma 
 Malignant glioma (including GBM) 
 Medulloblastoma 
 Wilms tumor 
 Desmoplastic small round cell tumor 
 Astrocytoma NOS 
 Choroid plexus papilloma 
 Neuroblastoma 
 Neurilemmoma 
 Ganglioneuroblastoma 
 Gastrointestinal stromal tumor 
 Hepatocellular carcinoma 
 Atypical teratoid rhabdoid tumor 
 Epithelioid sarcoma 
Prior therapy  
 Chemotherapy regimens: median (range) 2 (0–7) 
 Prior radiotherapy 28 

Abbreviations: GBM, glioblastoma multiforme; NOS, not otherwise specified.

Toxicity

Table 1 summarizes the DLTs across all dose levels. At the starting dose [cixutumumab (6 mg/kg); temsirolimus (15 mg/m2)], one patient in the initial cohort of three had dose-limiting hypercholesterolemia (grade 3). The cohort was expanded to include three more evaluable patients, one of whom had dose-limiting mucositis (grade 3). Because the observed DLTs observed were readily reversible, of different classes and unrelated to dose, the statistical design allowed enrollment of up to six additional patients at this dose level. Three different DLTs were observed among this expanded cohort of 6 (grade 3 ALT, fatigue, and prolonged thrombocytopenia >14 days), resulting in a dose de-escalation. At dose level 0 [cixutumumab (6 mg/kg); temsirolimus (10 mg/m2)], 1 of 3 patients experienced grade 3 dose-limiting mucositis. Three additional patients were enrolled, one of whom experienced grade 3 dose-limiting mucositis. As a result, the protocol was amended to incorporate dose level −1 in which the doses of both agents were further decreased [cixutumumab (4 mg/kg) and temsirolimus (8 mg/m2)]. None of the patients at dose level −1 experienced a DLT. An intermediate dose level [cixutumumab (6 mg/kg) and temsirolimus (8 mg/m2)] was subsequently added and one of six patients experienced grade 3 mucositis; none of 6 patients experienced a DLT among an expanded cohort for children <12 years.

Table 3 summarizes all non-DLTs at least possibly attributable to cixutumumab or temsirolimus in the 33 patients fully assessable for toxicity.

Table 3.

Non-DLTs related to protocol therapy observed in evaluable patients (n = 33)

Maximum grade of toxicity across cycle 1 (total, 33 cycles)Maximum grade of toxicity across cycles 2 to 20 (total, 77 + 22 follow-up cycles)
Toxicity typeGrade 1Grade 2Grade 3Grade 4Grade 1Grade 2Grade 3Grade 4
Hematologic 
 Anemia 11    
 Lymphocyte count decreased 11  
 Neutrophil count decreased  
 Platelet count decreased 17   
 White blood cell decreased   
Nonhematologica 
 ALT increased    
 AST increased     
 Cholesterol high 13     
 Constipation       
 Creatinine increased       
 Fatigue      
 Headache      
 Hyperglycemia 15     
 Hypermagnesemia       
 Hypertriglyceridemia 14     
 Hypocalcemia      
 Hypokalemia       
 Hyponatremia       
 Hypophosphatemia   
 Mucositis oral    
 Nausea    
 Proteinuria      
 Rash acneiform       
 Weight loss     
Maximum grade of toxicity across cycle 1 (total, 33 cycles)Maximum grade of toxicity across cycles 2 to 20 (total, 77 + 22 follow-up cycles)
Toxicity typeGrade 1Grade 2Grade 3Grade 4Grade 1Grade 2Grade 3Grade 4
Hematologic 
 Anemia 11    
 Lymphocyte count decreased 11  
 Neutrophil count decreased  
 Platelet count decreased 17   
 White blood cell decreased   
Nonhematologica 
 ALT increased    
 AST increased     
 Cholesterol high 13     
 Constipation       
 Creatinine increased       
 Fatigue      
 Headache      
 Hyperglycemia 15     
 Hypermagnesemia       
 Hypertriglyceridemia 14     
 Hypocalcemia      
 Hypokalemia       
 Hyponatremia       
 Hypophosphatemia   
 Mucositis oral    
 Nausea    
 Proteinuria      
 Rash acneiform       
 Weight loss     

aNonhematologic toxicities are those that occurred in >10% of patients as determined in the first cycle of therapy.

Responses

One partial response, confirmed on central review, was observed following cycle 1 in a patient with medulloblastoma enrolled at dose level 1. Unfortunately, tumor progression was noted on surveillance imaging studies performed after cycle 4. Stable disease for ≥3 cycles was observed in 3 patients (median, 12 cycles; range, 11–20) with epithelioid sarcoma, neuroblastoma, and alveolar rhabdomyosarcoma, respectively.

Pharmacokinetics

Cixutumumab single-dose pharmacokinetics were studied in 19 patients, and steady-state pharmacokinetics were studied in 36 patients who received the combination of cixutumumab with temsirolimus. There were no changes in cixutumumab clearance as the temsirolimus dose was increased (Table 4). The median weight-adjusted clearance and half-life values for all patients were 0.17 mL/hr/kg (range, 0.07–0.43 mL/hr/kg) and 122 hours (range, 64–446 hours), respectively. Cixutumumab serum trough concentrations (Ctrough) exhibited marked interpatient variability, increased throughout the first cycle of treatment (Supplementary Fig. S1), and a median Ctrough of approximately 200 μg/mL was achieved on day 28 following a 6 mg/kg dose (Supplementary Table S1). The median cixutumumab Ctrough at the recommended phase II dose was 197 μg/mL (range, 60–544).

Table 4.

Summary of cycle 1 IMC-A12 pharmacokinetic parameters

6 mg/kg6 mg/kg6 mg/kg
IMC-A12 dose8 mg/m210 mg/m215 mg/m2
Temsirolimus(n = 8)(n = 3)(n = 4)
doseMedianRangeMedianRangeMedianRange
Cmax (μg/mL) 281 239–355 277 219–390 230 161–464 
Tmax (h) 2.0 1.0–7.0 1.1 1.1–2.2 2.1 1.1–7.0 
t1/2 (h) 98 64–161 122 88–133 130 98–446 
AUC0–7d (h • μg/L) 20.6 11.6–29.7 25.6 20.6–28.7 26.4 14.8–34.0 
CL (mL/h) 8.3 1.1–15.2 9.9 2.9–14.5 7.0 3.1–14.3 
CL (mL/h/kg) 0.19 0.12–0.43 0.17 0.12–0.18 0.13 0.07–0.24 
Vdss (mL) 1050 150–2,390 1750 470–1,840 1860 590–2,400 
Vdss (mL/kg) 25.8 20.8–40.3 22.7 20.7–29.4 31.1 18.6–42.3 
6 mg/kg6 mg/kg6 mg/kg
IMC-A12 dose8 mg/m210 mg/m215 mg/m2
Temsirolimus(n = 8)(n = 3)(n = 4)
doseMedianRangeMedianRangeMedianRange
Cmax (μg/mL) 281 239–355 277 219–390 230 161–464 
Tmax (h) 2.0 1.0–7.0 1.1 1.1–2.2 2.1 1.1–7.0 
t1/2 (h) 98 64–161 122 88–133 130 98–446 
AUC0–7d (h • μg/L) 20.6 11.6–29.7 25.6 20.6–28.7 26.4 14.8–34.0 
CL (mL/h) 8.3 1.1–15.2 9.9 2.9–14.5 7.0 3.1–14.3 
CL (mL/h/kg) 0.19 0.12–0.43 0.17 0.12–0.18 0.13 0.07–0.24 
Vdss (mL) 1050 150–2,390 1750 470–1,840 1860 590–2,400 
Vdss (mL/kg) 25.8 20.8–40.3 22.7 20.7–29.4 31.1 18.6–42.3 

Results of the pharmacokinetic data (n = 19) following the first dose of temsirolimus at 8, 10, and 15 mg/m2/dose are summarized in Table 5. There was marked interpatient and interdose group variability. Sirolimus, the metabolite of temsirolimus, exposure did not appreciably increase with dose. The steady-state volume of distribution (Vss) was large and increased with dose, ranging from 46.3 L (after 8 mg/m2 dose) to 74.9 L (after 15 mg/m2 dose). Similarly, temsirolimus clearance from whole blood increased with increasing dose, from 2.3 L/h after an 8 mg/m2 dose to 6.7 L/h after a 15 mg/m2 dose.

Table 5.

Summary of cycle 1 pharmacokinetic data for temsirolimus and its metabolite

Temsirolimus8 mg/m2(n = 11)10 mg/m2(n = 3)15 mg/m2(n = 5)
doseMedianRangeMedianRangeMedianRange
Temsirolimus 
Cmax (ng/mL) 509 187 2,010 606 283 675 660 615 1,310 
Tmax (h) 0.8 0.5 1.2 0.7 0.6 1.3 0.6 0.5 0.7 
 CL (L/h) 2.3 1.1 8.2 5.5 1.5 11.4 6.7 5.5 24.7 
Vdss (L) 46.3 6.2 124.5 50.4 40.5 68.1 74.9 18.4 99.6 
t1/2 (h) 10.3 5.3 45.9 10.5 3.8 18.4 10.3 7.3 12.9 
 AUC0–24h (h • ng/mL) 2,022 698 5,194 2,654 1,650 2,975 2,784 973 4,028 
 AUCinf (h • ng/mL) 2,946 937 5,536 3,646 1,666 4,820 3,274 973 5,293 
Sirolimus 
Cmax (ng/mL) 20.3 3.4 83.2 21.3 10.2 28.1 32.3 21.4 38.5 
Tmax (h) 1.6 1.0 6.5 1.7 1.3 1.7 1.0 0.8 1.2 
t1/2 (h) 51.0 25.4 126.8 68.9 60.8 71.0 70.9 15.3 123.5 
 AUC0–24h (h • ng/mL) 273 77 1,252 285 160 353 579 220 661 
 AUCinf (h • ng/mL)a 767 245 3,675 1,268 551 3,210 2,870 1,850 5,563 
 Sum AUC_T+S (h • ng/mL) 5,750 1,182 8,932 4,914 4,876 5,371 6,446 2,823 8,464 
 Ratio Sir/Tem 0.3 0.1 1.7 0.3 0.1 1.9 1.0 0.5 1.9 
Temsirolimus8 mg/m2(n = 11)10 mg/m2(n = 3)15 mg/m2(n = 5)
doseMedianRangeMedianRangeMedianRange
Temsirolimus 
Cmax (ng/mL) 509 187 2,010 606 283 675 660 615 1,310 
Tmax (h) 0.8 0.5 1.2 0.7 0.6 1.3 0.6 0.5 0.7 
 CL (L/h) 2.3 1.1 8.2 5.5 1.5 11.4 6.7 5.5 24.7 
Vdss (L) 46.3 6.2 124.5 50.4 40.5 68.1 74.9 18.4 99.6 
t1/2 (h) 10.3 5.3 45.9 10.5 3.8 18.4 10.3 7.3 12.9 
 AUC0–24h (h • ng/mL) 2,022 698 5,194 2,654 1,650 2,975 2,784 973 4,028 
 AUCinf (h • ng/mL) 2,946 937 5,536 3,646 1,666 4,820 3,274 973 5,293 
Sirolimus 
Cmax (ng/mL) 20.3 3.4 83.2 21.3 10.2 28.1 32.3 21.4 38.5 
Tmax (h) 1.6 1.0 6.5 1.7 1.3 1.7 1.0 0.8 1.2 
t1/2 (h) 51.0 25.4 126.8 68.9 60.8 71.0 70.9 15.3 123.5 
 AUC0–24h (h • ng/mL) 273 77 1,252 285 160 353 579 220 661 
 AUCinf (h • ng/mL)a 767 245 3,675 1,268 551 3,210 2,870 1,850 5,563 
 Sum AUC_T+S (h • ng/mL) 5,750 1,182 8,932 4,914 4,876 5,371 6,446 2,823 8,464 
 Ratio Sir/Tem 0.3 0.1 1.7 0.3 0.1 1.9 1.0 0.5 1.9 

aInf = from time zero to infinity (0–∞).

mTOR pathway activity

At the recommended phase II dose, a total of 23 PBMC samples were obtained from 9 consenting patients. Four patients had suitable samples from the days 1, 8, and 28 timepoints, three had samples from days 1 and 8, and one had samples from days 1 and 28. Baseline variation in total S6K1 protein was observed among patients, but intrapatient levels were consistent at each time point (days 1, 8, and 28). Phospho-S6K1 (T389) was reduced at day 8 in the seven evaluable patients when compared with baseline (day 1), and in the majority of cases was further reduced at day 28; in only one patient was S6K1 phosphorylation higher at day 28 when compared with baseline. Unlike S6K1, total and phospho-4EPB1 (S65) followed expression levels at each time point, with no observed change in phosphorylation as a function of treatment on days 8 or 28. Akt phosphorylation (S473) was more variable than S6K1, showing a reduction at day 8 in five patients, and slight or no increase in two others. By day 28, Akt phosphorylation in the five evaluable patients remained variable, with evidence of an increase in three patients, and decrease in the other two, when compared with baseline. Figure 1 depicts a Western blot of representative patient PBMC samples showing inhibition of target proteins p-S6K1 T389 and p-AKT S473.

Figure 1.

Western blot of representative patient samples showing inhibition of target proteins pS6K1 T389 and pAKT S473 along with the respective total protein. Samples were taken before the first dose was administered (1) and on days 8 and 28 after treatment.

Figure 1.

Western blot of representative patient samples showing inhibition of target proteins pS6K1 T389 and pAKT S473 along with the respective total protein. Samples were taken before the first dose was administered (1) and on days 8 and 28 after treatment.

Close modal

The recommended pediatric phase II doses of the combination of cixutumumab and temsirolimus in children with refractory solid tumor are 6 mg/kg and 8 mg/m2, respectively. Similar to adults, observed DLTs included mucositis, fatigue, hypercholesterolemia, and transaminitis. However, the MTD of temsirolimus for this combination in children was significantly lower than observed in adults with advanced cancers (37), relapsed Ewing sarcoma [ref. 38; cixutumumab 6 mg/kg i.v. and temsirolimus 25-mg flat dose (14 mg/m2)], or metastatic prostate cancer [cixutumumab 6 mg/kg i.v. and temsirolimus 20-mg flat dose (11 mg/m2); ref. 39]. It was noted in the prostate cancer trial that the weekly schedule was difficult to maintain long term due to chronic toxicities. Pharmacokinetic data were not available for any of the adult trials. Interestingly, in the adult trials, grade 3 mucositis was observed in 3% to 27% of patients (37–39). In our study, 4 of 27 patients or 15% experienced grade 3 mucositis, leading to repeated dose de-escalations of the temsirolimus and cixutumumab dose.

Although the pharmacokinetics of cixutumumab (31) and temsirolimus (40) have been previously reported in single-agent pediatric studies, this is the first description of the disposition of these drugs when used in combination in children. Cixutumumab clearance, Cmax, and half-life at 6 mg/kg when combined with temsirolimus at the MTD of 8 mg/m2 were similar to those reported earlier in the pediatric phase I single-agent study of cixutumumab (31). Furthermore, cixutumumab half-life and steady-state trough concentrations of 122 hours and 197 mg/mL, respectively, at 6 mg/kg were similar to the values of 209 hours and 145 mg/mL reported for adults at that same dose (41).

Although there was large interpatient and interdose variability in pharmacokinetic parameter estimates for temsirolimus and its active metabolite sirolimus, the temsirolimus and sirolimus AUCs did not increase significantly with dose, which has previously been reported (42, 43). The steady-state volume of distribution (Vss) of temsirolimus was generally large, increased with dose, and ranged from 230 L (after a 25-mg dose) to as high as 900 L (after a 250-mg dose). The distribution of temsirolimus into red blood cells appeared to be saturable at higher doses. In addition, the clearance from whole blood increased with increasing dose, as observed with Vss, with mean values ranging from 20 L/h after a 25-mg dose to 100 L/h after a 250-mg dose (coefficient of variation, approximately 16%–27%; ref. 43). We compared the pharmacokinetic parameters in the pediatric single-agent temsirolimus phase I trial with the current study. Spunt and colleagues (40) reported no MTD for temsirolimus at dose levels ranging from 10 to 150 mg/m2. The median temsirolimus Cmax and AUC values at the MTD of 8 mg/m2 in our study were comparable with median values reported at the 25 and 75 mg/m2 dose level reported by Spunt and colleagues By contrast, the sirolimus AUC and Cmax were substantially lower in our study at similar doses, with lower sirolimus to temsirolimus ratios at all dose levels tested. The AUCs and Cmax of temsirolimus and AUCs and Cmin of sirolimus and cixutumumab observed concentrations that did not show an association with toxicity or inhibition of target. The lower MTD observed in this study is possibly pharmacodynamic in nature and related to the combination of IGFIR and m-TOR inhibition, but requires further evaluation in a larger patient cohort. As these inhibitors affect signaling pathways that regulate glucose, lipid, and energy metabolism, it is possible that children may be biologically more sensitive to these drugs, particularly in combination; however, this determination would require further evaluation in a larger patient cohort.

We demonstrated that this combination significantly inhibited the phosphorylation of S6K1, but not 4E-BP1 in PBMCs at all dose levels. Phospho-S6K1 was reduced at day 8 in the 7 patients with evaluable samples, and was further reduced in 4 of 5 patients with samples at day 28. Although S6K1 and 4E-BP1 are both downstream effectors of mTORC1, these data are consistent with reports that in some cells, rapamycin and the rapalogs are incomplete inhibitors of mTORC1, affecting S6K1 phosphorylation more strongly than 4E-BP1 (44). Similar to previous pediatric and adult reports (40, 45), the phosphorylation status of S6K1, 4E-BP1, and Akt did not correlate with objective responses or prolonged stabilization of disease, limiting their use as biomarkers. There was considerable variation in Akt phosphorylation on days 8 and 28, with either an increase or decrease in Akt phosphorylation when compared with baseline. The pAkt variability is consistent with an earlier pediatric temsirolimus study that demonstrated a lack of correlation between temsirolimus dose and phosphorylation of Akt, S6, and 4E-BP1 (40). However, in the current study, phosphorylation trends of the mTORC1 targets S6K1 and 4E-BP1 were consistently decreased or unchanged, respectively. In 5 patients, Akt S473 phosphorylation was generally decreased in response to treatment, paralleling S6K1 phosphorylation and consistent with the inhibition of mTOR complex 2 (mTORC2) observed in cells exposed to long-term treatment with rapamycin (46). This decrease may also reflect the efficacy of cixutumumab, but the contribution of IGFIR signaling to PI3K and Akt may be minor in PBMCs. In two patients, there was an inverse correlation between pS6K1 (decreased) and pAkt (increased), which may be explained by temsirolimus-mediated inhibition of the mTORC1-S6K1–negative feedback loop (47–49), leading to increased PI3K signaling to Akt. The variability of pAkt among patients does not appear to correlate with the magnitude of S6K1 inhibition, but instead may be due to a multitude of factors that are patient-specific, including variability in the makeup of the lymphocytes and monocytes in the PBMC population, and unknown changes in the status of the immune system following prolonged treatment with temsirolimus and cixutumumab.

This study demonstrates that the combination of cixutumumab and temsirolimus can be safely given to children with recurrent solid tumors, albeit at lower doses than in adult combination studies. One patient with a medulloblastoma had a confirmed partial response and 3 patients experienced prolonged disease stabilization for ≥3 cycles. Target inhibition was noted in PBMCs at all dose levels as evidenced by a decrease in phosphor-S6K1 and pAKT. A phase II study of this combination has recently been completed in children with recurrent tumors in the Children's Oncology Group.

D.A. Krueger reports receiving a commercial research grant from, is a consultant/advisory board member for, and reports receiving speakers bureau honoraria from Novartis. No potential conflicts of interest were disclosed by the other authors.

Conception and design: M. Fouladi, J.P. Perentesis, P.J. Houghton, L.A. Doyle, H. Chen, B. Weigel, S.M. Blaney

Development of methodology: M. Fouladi, A.A. Vinks, G. Thomas, P.J. Houghton

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Fouladi, L.M. Wagner, C.A. Mercer, D.A. Krueger, P.J. Houghton, B. Weigel

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Fouladi, A.A. Vinks, J.M. Reid, C. Ahern, C.A. Mercer, D.A. Krueger, S.M. Blaney

Writing, review, and/or revision of the manuscript: M. Fouladi, J.P. Perentesis, L.M. Wagner, J.M. Reid, C. Ahern, C.A. Mercer, D.A. Krueger, P.J. Houghton, L.A. Doyle, B. Weigel, S.M. Blaney

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Fouladi, H. Chen

Study supervision: M. Fouladi, J.P. Perentesis, L.A. Doyle, B. Weigel

Others (population pharmacokinetic modeling): A.A. Vinks

The authors thank Melissa Asman for excellent technical assistance as well as Biljana Georgievska, Thalia Beeles, and Mark Ingle of the COG Phase 1/Pilot Consortium Coordinating Center for outstanding administrative and data management support throughout the development and conduct of this trial.

This work was supported by NCI grant nos. U01 CA97452 and UM1 CA097452-11.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Kim
SY
,
Toretsky
JA
,
Scher
D
,
Helman
LJ
. 
The role of IGF-1R in pediatric malignancies
.
Oncologist
2009
;
14
:
83
91
.
2.
Scotlandi
K
,
Benini
S
,
Sarti
M
,
Serra
M
,
Lollini
PL
,
Maurici
D
, et al
Insulin-like growth factor I receptor-mediated circuit in Ewing's sarcoma/peripheral neuroectodermal tumor: a possible therapeutic target
.
Cancer Res
1996
;
56
:
4570
4
.
3.
Cao
L
,
Yu
Y
,
Darko
I
,
Currier
D
,
Mayeenuddin
LH
,
Wan
X
, et al
Addiction to elevated insulin-like growth factor I receptor and initial modulation of the AKT pathway define the responsiveness of rhabdomyosarcoma to the targeting antibody
.
Cancer Res
2008
;
68
:
8039
48
.
4.
Kappel
CC
,
Velez-Yanguas
MC
,
Hirschfeld
S
,
Helman
LJ
. 
Human osteosarcoma cell lines are dependent on insulin-like growth factor I for in vitro growth
.
Cancer Res
1994
;
54
:
2803
7
.
5.
Burrow
S
,
Andrulis
IL
,
Pollak
M
,
Bell
RS
. 
Expression of insulin-like growth factor receptor, IGF-1, and IGF-2 in primary and metastatic osteosarcoma
.
J Surg Oncol
1998
;
69
:
21
7
.
6.
El-Badry
OM
,
Romanus
JA
,
Helman
LJ
,
Cooper
MJ
,
Rechler
MM
,
Israel
MA
. 
Autonomous growth of a human neuroblastoma cell line is mediated by insulin-like growth factor II
.
J Clin Invest
1989
;
84
:
829
39
.
7.
El-Badry
OM
,
Helman
LJ
,
Chatten
J
,
Steinberg
SM
,
Evans
AE
,
Israel
MA
. 
Insulin-like growth factor II-mediated proliferation of human neuroblastoma
.
J Clin Invest
1991
;
87
:
648
57
.
8.
van Golen
CM
,
Schwab
TS
,
Kim
B
,
Soules
ME
,
Su Oh
S
,
Fung
K
, et al
Insulin-like growth factor-I receptor expression regulates neuroblastoma metastasis to bone
.
Cancer Res
2006
;
66
:
6570
8
.
9.
Sun
Q
,
Wei
X
,
Feng
J
,
Zhang
R
,
Shen
Q
,
Dong
J
, et al
Involvement of insulin-like growth factor-insulin receptor signal pathway in the transgenic mouse model of medulloblastoma
.
Cancer Sci
2008
;
99
:
234
40
.
10.
Del Valle
L
,
Enam
S
,
Lara
C
,
Ortiz-Hidalgo
C
,
Katsetos
CD
,
Khalili
K
. 
Detection of JC polyomavirus DNA sequences and cellular localization of T-antigen and agnoprotein in oligodendrogliomas
.
Clin Cancer Res
2002
;
8
:
3332
40
.
11.
Wang
JY
,
Del Valle
L
,
Gordon
J
,
Rubini
M
,
Romano
G
,
Croul
S
, et al
Activation of the IGF-IR system contributes to malignant growth of human and mouse medulloblastomas
.
Oncogene
2001
;
20
:
3857
68
.
12.
Del Valle
L
,
Enam
S
,
Lassak
A
,
Wang
JY
,
Croul
S
,
Khalili
K
, et al
Insulin-like growth factor I receptor activity in human medulloblastomas
.
Clin Cancer Res
2002
;
8
:
1822
30
.
13.
Hoelzinger
DB
,
Mariani
L
,
Weis
J
,
Woyke
T
,
Berens
TJ
,
McDonough
WS
, et al
Gene expression profile of glioblastoma multiforme invasive phenotype points to new therapeutic targets
.
Neoplasia
2005
;
7
:
7
16
.
14.
Gansler
T
,
Allen
KD
,
Burant
CF
,
Inabnett
T
,
Scott
A
,
Buse
MG
, et al
Detection of type 1 insulinlike growth factor (IGF) receptors in Wilms' tumors
.
Am J Pathol
1988
;
130
:
431
5
.
15.
Gray
SG
,
Eriksson
T
,
Ekstrom
C
,
Holm
S
,
von Schweinitz
D
,
Kogner
P
, et al
Altered expression of members of the IGF-axis in hepatoblastomas
.
Br J Cancer
2000
;
82
:
1561
7
.
16.
Fouladi
M
,
Laningham
F
,
Wu
J
,
O'Shaughnessy
MA
,
Molina
K
,
Broniscer
A
, et al
Phase I study of everolimus in pediatric patients with refractory solid tumors
.
J Clin Oncol
2007
;
25
:
4806
12
.
17.
Houghton
PJ
,
Morton
CL
,
Kolb
EA
,
Gorlick
R
,
Lock
R
,
Carol
H
, et al
Initial testing (stage 1) of the mTOR inhibitor rapamycin by the pediatric preclinical testing program
.
Pediatr Blood Cancer
2008
;
50
:
799
805
.
18.
Bjornsti
MA
,
Houghton
PJ
. 
The TOR pathway: a target for cancer therapy
.
Nat Rev Cancer
2004
;
4
:
335
48
.
19.
Spunt
SL
,
Grupp
SA
,
Vik
TA
. 
Phase I study of temsirolimus in pediatric patients with recurrent refractory solid tumors
.
J Clin Oncol
2011
;
29
:
2933
40
.
20.
O'Reilly
KE
,
Rojo
F
,
She
QB
,
Solit
D
,
Mills
GB
,
Smith
D
, et al
mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt
.
Cancer Res
2006
;
66
:
1500
8
.
21.
Shi
Y
,
Yan
H
,
Frost
P
,
Gera
J
,
Lichtenstein
A
. 
Mammalian target of rapamycin inhibitors activate the AKT kinase in multiple myeloma cells by up-regulating the insulin-like growth factor receptor/insulin receptor substrate-1/phosphatidylinositol 3-kinase cascade
.
Mol Cancer Ther
2005
;
4
:
1533
40
.
22.
Wan
X
,
Harkavy
B
,
Shen
N
,
Grohar
P
,
Helman
LJ
. 
Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism
.
Oncogene
2007
;
26
:
1932
40
.
23.
Cloughesy
TF
,
Yoshimoto
K
,
Nghiemphu
P
,
Brown
K
,
Dang
J
,
Zhu
S
, et al
Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma
.
PLoS Med
2008
;
5
:
e8
.
24.
Kurmasheva
RT
,
Dudkin
L
,
Billups
C
,
Debelenko
LV
,
Morton
CL
,
Houghton
PJ.
The insulin growth factor-1 receptor targeting antibody, CP-751,781, suppresses tumor-derived VEGF and synthesizes with rapamycin in models of childhood sarcoma
.
Cancer Res
2009
;
69
:
7662
71
.
25.
Brown
EJ
,
Albers
MW
,
Shin
TB
,
Ichikawa
K
,
Keith
CT
,
Lane
WS
, et al
A mammalian protein targeted by G1-arresting rapamycin-receptor complex
.
Nature
1994
;
369
:
756
8
.
26.
Lin
TA
,
Kong
X
,
Haystead
TA
,
Pause
A
,
Belsham
G
,
Sonenberg
N
, et al
PHAS-I as a link between mitogen-activated protein kinase and translation initiation
.
Science
1994
;
266
:
653
6
.
27.
Graves
LM
,
Bornfeldt
KE
,
Argast
GM
,
Krebs
EG
,
Kong
X
,
Lin
TA
, et al
cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells
.
Proc Natl Acad Sci U S A
1995
;
92
:
7222
6
.
28.
Kolb
EA
,
Gorlick
R
,
Maris
JM
,
Keir
ST
,
Morton
CL
,
Wu
J
, et al
Combination testing (Stage 2) of the Anti-IGF-1 receptor antibody IMC-A12 with rapamycin by the pediatric preclinical testing program
.
Pediatr Blood Cancer
2012
;
58
:
729
35
.
29.
Chung
J
,
Kuo
CJ
,
Crabtree
GR
,
Blenis
J
. 
Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases
.
Cell
1992
;
69
:
1227
36
.
30.
Kurihara
M
,
Tokunaga
Y
,
Tsutsumi
K
,
Kawaguchi
T
,
Shigematsu
K
,
Niwa
M
, et al
Characterization of insulin-like growth factor I and epidermal growth factor receptors in meningioma
.
J Neurosurg
1989
;
71
:
538
44
.
31.
Malempati
S
,
Weigel
B
,
Ingle
AM
,
Ahern
CH
,
Carroll
JM
,
Roberts
CT
, et al
Phase I/II trial and pharmacokinetic study of cixutumumab in pediatric patients with refractory solid tumors and Ewing sarcoma: a report from the Children's Oncology Group
.
J Clin Oncol
2012
;
30
:
256
62
.
32.
Ma
CX
,
Suman
VJ
,
Goetz
M
,
Haluska
P
,
Moynihan
T
,
Nanda
R
, et al
A phase I trial of the IGF-1R antibody Cixutumumab in combination with temsirolimus in patients with metastatic breast cancer
.
Breast Cancer Res Treat
2013
;
139
:
145
53
.
33.
Therasse
P
,
Arbuck
SG
,
Eisenhauer
EA
,
Wanders
J
,
Kaplan
RS
,
Rubinstein
L
, et al
New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada
.
J Natl Cancer Inst
2000
;
92
:
205
16
.
34.
Christians
U
,
Jacobsen
W
,
Serkova
N
,
Benet
LZ
,
Vidal
C
,
Sewing
KF
, et al
Automated, fast and sensitive quantification of drugs in blood by liquid chromatography-mass spectrometry with on-line extraction: immunosuppressants
.
J Chromatogr B Biomed Sci Appl
2000
;
748
:
41
53
.
35.
Proost
JH
,
Meijer
DK
. 
MW/Pharm, an integrated software package for drug dosage regimen calculation and therapeutic drug monitoring
.
Comput Biol Med
1992
;
22
:
155
63
.
36.
Boulay
A
,
Zumstein-Mecker
S
,
Stephan
C
,
Beuvink
I
,
Zilbermann
F
,
Haller
R
, et al
Antitumor efficacy of intermittent treatment schedules with the rapamycin derivative RAD001 correlates with prolonged inactivation of ribosomal protein S6 kinase 1 in peripheral blood mononuclear cells
.
Cancer Res
2004
;
64
:
252
61
.
37.
Naing
A
,
Kurzrock
R
,
Burger
A
,
Gupta
S
,
Lei
X
,
Busaidy
N
, et al
Phase I trial of cixutumumab combined with temsirolimus in patients with advanced cancer
.
Clin Cancer Res
2011
;
17
:
6052
60
.
38.
Naing
A
,
LoRusso
P
,
Fu
S
,
Hong
DS
,
Anderson
P
,
Benjamin
RS
, et al
Insulin growth factor-receptor (IGF-1R) antibody cixutumumab combined with the mTOR inhibitor temsirolimus in patients with refractory Ewing's sarcoma family tumors
.
Clin Cancer Res
2012
;
18
:
2625
31
.
39.
Rathkopf
DE
,
Danila
DC
,
Morris
MJ
,
Slovin
SF
,
Borwick
LS
,
Momen
L
,
Curley
T
, et al
Anti-insulin-like growth factor-1 receptor (IGF-1R) monoclonal antibody cixutumumab (cix) plus mTOR inhibitor temsirolimus (tem) in metastatic castration-resistant prostate cancer (mCRPC): Results of a phase I pilot study
.
J Clin Oncol
29
: 
2011
(
suppl; abstr e15081
).
40.
Spunt
SL
,
Grupp
SA
,
Vik
TA
,
Santana
VM
,
Greenblatt
DJ
,
Clancy
J
, et al
Phase I study of temsirolimus in pediatric patients with recurrent/refractory solid tumors
.
J Clin Oncol
2011
;
29
:
2933
40
.
41.
McKian
KP
,
Haluska
P
. 
Cixutumumab
.
Expert Opin Investig Drugs
2009
;
18
:
1025
33
.
42.
Boni
J
,
Leister
C
,
Burns
J
,
Cincotta
M
,
Hug
B
,
Moore
L
. 
Pharmacokinetic profile of temsirolimus with concomitant administration of cytochrome p450-inducing medications
.
J Clin Pharmacol
2007
;
47
:
1430
9
.
43.
Atkins
MB
,
Hidalgo
M
,
Stadler
WM
,
Logan
TF
,
Dutcher
JP
,
Hudes
GR
, et al
Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma
.
J Clin Oncol
2004
;
22
:
909
18
.
44.
Dowling
RJ
,
Topisirovic
I
,
Alain
T
,
Bidinosti
M
,
Fonseca
BD
,
Petroulakis
E
, et al
mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs
.
Science
2010
;
328
:
1172
6
.
45.
Chan
S
,
Scheulen
ME
,
Johnston
S
,
Mross
K
,
Cardoso
F
,
Dittrich
C
, et al
Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer
.
J Clin Oncol
2005
;
23
:
5314
22
.
46.
Sarbassov
DD
,
Ali
SM
,
Sengupta
S
,
Sheen
JH
,
Hsu
PP
,
Bagley
AF
, et al
Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB
.
Mol Cell
2006
;
22
:
159
68
.
47.
Zick
Y
. 
Insulin resistance: a phosphorylation-based uncoupling of insulin signaling
.
Trends Cell Biol
2001
;
11
:
437
41
.
48.
Um
SH
,
Frigerio
F
,
Watanabe
M
,
Picard
F
,
Joaquin
M
,
Sticker
M
, et al
Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity
.
Nature
2004
;
431
:
200
5
.
49.
Zhang
H
,
Bajraszewski
N
,
Wu
E
,
Wang
H
,
Moseman
AP
,
Dabora
SL
, et al
PDGFRs are critical for PI3K/Akt activation and negatively regulated by mTOR
.
J Clin Invest
2007
;
117
:
730
8
.