RAD001 (Everolimus) delays tumor onset and progression in a transgenic mouse model of ovarian cancer

The mammalian target of rapamycin (mTOR) is thought to play a critical role in regulating cell growth, cell cycle progression, and tumorigenesis. Because the AKT-mTOR pathway is frequently hyperactivated in ovarian cancer, we hypothesized that the mTOR inhibitor RAD001 (Everolimus) would inhibit ovarian tumorigenesis in transgenic mice that spontaneously develop ovarian carcinomas. We used TgMISIIR-TAg transgenic mice, which develop bilateral ovarian serous adenocarcinomas accompanied by ascites and peritoneal dissemination. Fifty-eight female TgMISIIR-TAg mice were treated with 5 mg/kg RAD001 or placebo twice weekly from 5 to 20 weeks of age. To monitor tumor development, mice were examined biweekly using magnetic resonance microimaging. In vivo effects of RAD001 on Akt-mTOR signaling, tumor cell proliferation, and blood vessel area were analyzed by immunohistochemistry and Western blot analysis. RAD001 treatment markedly delayed tumor development. Tumor burden was reduced by approximately 84%. In addition, ascites formation, together with peritoneal dissemination, was detected in only 21% of RAD001-treated mice compared with 74% in placebo-treated animals. Approximately 30% of RAD001-treated mice developed early ovarian carcinoma confined within the ovary, whereas all placebo-treated mice developed advanced ovarian carcinoma. Treatment with RAD001 diminished the expression of vascular endothelial growth factor in tumor-derived cell lines and inhibited angiogenesis in vivo. RAD001 also attenuated the expression of matrix metalloproteinase-2 and inhibited the invasiveness of tumor-derived cells. Taken together, these preclinical findings suggest that mTOR inhibition, alone or in combination with other molecularly targeted drugs, could represent a promising chemopreventive strategy in women at high familial risk of ovarian cancer.     

Antiangiogenic therapy with mammalian target of rapamycin inhibitor RAD001 (Everolimus) increases radiosensitivity in solid cancer.

PURPOSE: Radiotherapy exerts direct antivascular effects in tumors and also induces a proangiogenic stress response in tumor cells via the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Therefore, the combination of radiotherapy and antiangiogenic therapy with mTOR inhibitor RAD001 (Everolimus) might exert additive/synergistic effects on tumor growth. EXPERIMENTAL DESIGN: Effects of radiation combined with mTOR inhibitor RAD001 were studied on proliferation of murine colon cancer CT-26, human pancreatic cancer L3.6pl, and human umbilical vascular endothelial cells in vitro. In vivo tumor growth of subcutaneous colon cancer CT 26 and orthotopic pancreatic cancer L3.6pl was assessed after fractionated radiotherapy (5 x 2 or 5 x 4 Gy) with or without the addition of the mTOR inhibitor RAD001. RAD001 (1.5 mg/kg/d) was administered until the end of experiments beginning before or after radiotherapy. RESULTS: A single dose of 2 Gy reduced in vitro proliferation of L3.6pl (-16%), CT-26 (-70%), and human umbilical vascular endothelial cells (HUVEC; -72%). The mTOR inhibitor RAD001 (10 ng/mL) suppressed proliferation of HUVEC (-83%), L3.6pl (-8%), and CT-26 (-82%). Combination of even low concentrations of 0.01 ng/mL RAD001 and 0.25 Gy radiation significantly reduced proliferation of HUVECs (-57%), whereas additive effects of RAD001 and radiation on tumor cells were seen only at the highest concentrations tested. In vivo, RAD001 introduced before radiotherapy (5 x 2 Gy) improved tumor growth control in mice (L3.6pl: 326 mm(3) versus 1144 mm(3); CT-26: 210 mm(3) versus 636 mm(3); P < 0.05 versus control). RAD001 turned out to possess a dose-modifying effect on radiotherapy. CONCLUSION: Endothelial cells seem to be most sensitive to combination of mTOR inhibition and radiotherapy. Additive tumor growth delay using the mTOR inhibitor RAD001 and radiotherapy in vivo therefore might rely on combined antiangiogenic and antivascular effects.     

Everolimus (RAD001) in the treatment of advanced renal cell carcinoma: biology and pathways

Everolimus (RAD001) is an orally administered inhibitor of the mammalian target of rapamycin (mTOR), an intracellular serine/threonine kinase that regulates protein synthesis and cell growth, proliferation and survival. Dysfunction of mTOR has been implicated in a number of human illnesses including cancer and everolimus is used for a variety of therapeutic indications and is under evaluation in clinical trials for the treatment of cancer. Two phase I studies evaluating the dosing, toxicity, pharmacokinetics, pharmacodynamics and potential biomarkers of everolimus in advanced cancer have been reported. Daily doses of 10 mg and weekly doses of 50 mg of everolimus appear to inhibit relevant therapeutic targets in both tumour tissue and in skin but maximum-tolerated doses of everolimus were not determined formally in these studies. A phase III study of everolimus at 10 mg daily in the treatment of patients with advanced renal cell carcinoma who had failed prior treatment with sorafenib or sunitinib has also been reported. In this study everolimus was generally well tolerated, causing rash, stomatitis and fatigue in approximately a third of patients which generally were not severe. Hyperglycaemia, hypertriglyceridaemia and hypercholesterolaemia were reported in approximately two-thirds of patients but again were easy to manage and mainly of mild or moderate severity. Severe infections and non-infectious pneumonitis were reported in less than 5% of study participants but generally responded to standard therapies. Further work is necessary to define mechanisms of activity and toxicity of everolimus in the treatment of advanced renal cell carcinoma.     

Everolimus (RAD001) in the Treatment of Advanced Renal Cell Carcinoma: A Review

Historically, there have been few treatment options for patients with advanced renal cell carcinoma (RCC) besides immunotherapy with interleukin‐2 and interferon (IFN)‐α. Targeted therapies have improved clinical outcomes over the past several years. These include the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors sunitinib and sorafenib, which inhibit angiogenic signaling in endothelial cells and vascular pericytes predominantly through VEGFR and platelet‐derived growth factor receptor β. Also included is the anti‐VEGF monoclonal antibody bevacizumab used in combination with IFN‐α. These agents mediate their antitumor effects by interfering with the VEGF signaling pathway, thereby inhibiting angiogenesis and causing tumor shrinkage. However, ultimately, most patients develop resistance and experience disease progression during VEGF/VEGFR‐targeted therapy, and until the recent approval of the mammalian target of rapamycin (mTOR) inhibitor everolimus (RAD001), there were no agents available with proven activity in this setting. This review describes the clinical development of everolimus in advanced RCC and the rationale for the use of mTOR inhibitors after failure of VEGF/VEGFR inhibitors.     

Everolimus (RAD001) in the treatment of advanced renal cell carcinoma: biology and pathways

Everolimus (RAD001) is an orally administered inhibitor of the mammalian target of rapamycin (mTOR), an intracellular serine/threonine kinase that regulates protein synthesis and cell growth, proliferation and survival. Dysfunction of mTOR has been implicated in a number of human illnesses including cancer and everolimus is used for a variety of therapeutic indications and is under evaluation in clinical trials for the treatment of cancer. Two phase I studies evaluating the dosing, toxicity, pharmacokinetics, pharmacodynamics and potential biomarkers of everolimus in advanced cancer have been reported. Daily doses of 10 mg and weekly doses of 50 mg of everolimus appear to inhibit relevant therapeutic targets in both tumour tissue and in skin but maximum-tolerated doses of everolimus were not determined formally in these studies. A phase III study of everolimus at 10 mg daily in the treatment of patients with advanced renal cell carcinoma who had failed prior treatment with sorafenib or sunitinib has also been reported. In this study everolimus was generally well tolerated, causing rash, stomatitis and fatigue in approximately a third of patients which generally were not severe. Hyperglycaemia, hypertriglyceridaemia and hypercholesterolaemia were reported in approximately two-thirds of patients but again were easy to manage and mainly of mild or moderate severity. Severe infections and non-infectious pneumonitis were reported in less than 5% of study participants but generally responded to standard therapies. Further work is necessary to define mechanisms of activity and toxicity of everolimus in the treatment of advanced renal cell carcinoma.     

The combination of RAD001 and NVP-BKM120 synergistically inhibits the growth of lung cancer in vitro and in vivo

This study focuses on determining whether the combination of NYP-BKM120 (BKM120) and RAD001 exerts enhanced therapeutic effect against lung cancer. The combination of BKM120 and RAD001 exerted synergistic inhibitory effects on the growth of lung cancer cells both in culture and in mouse xenograft model. This combination abrogated RAD001-induced Akt phosphorylation and exerted enhanced suppressive effect on 4EBP1 phosphorylation. Collectively, we suggest that the combination of RAD001 and BKM120 may be an effective regimen for treatment of lung cancer, hence warranting further evaluation of the combination in the clinic.     

PTEN loss does not predict for response to RAD001 (Everolimus) in a glioblastoma orthotopic xenograft test panel.

PURPOSE: Hyperactivation of the phosphatidylinositol 3-kinase/Akt signaling through disruption of PTEN function is common in glioblastoma multiforme, and these genetic changes are predicted to enhance sensitivity to mammalian target of rapamycin (mTOR) inhibitors such as RAD001 (everolimus). EXPERIMENTAL DESIGN: To test whether PTEN loss could be used as a predictive marker for mTOR inhibitor sensitivity, the response of 17 serially transplantable glioblastoma multiforme xenografts was evaluated in an orthotopic therapy evaluation model. Of these 17 xenograft lines, 7 have either genomic deletion or mutation of PTEN. RESULTS: Consistent with activation of Akt signaling, there was a good correlation between loss of PTEN function and elevated levels of Akt phosphorylation. However, of the 7 lines with disrupted PTEN function, only 1 tumor line (GBM10) was significantly sensitive to RAD001 therapy (25% prolongation in median survival), whereas 1 of 10 xenograft lines with wild-type PTEN was significantly sensitive to RAD001 (GS22; 34% prolongation in survival). Relative to placebo, 5 days of RAD001 treatment was associated with a marked 66% reduction in the MIB1 proliferation index in the sensitive GBM10 line (deleted PTEN) compared with a 25% and 7% reduction in MIB1 labeling index in the insensitive GBM14 (mutant PTEN) and GBM15 (wild-type PTEN) lines, respectively. Consistent with a cytostatic antitumor effect, bioluminescent imaging of luciferase-transduced intracranial GBM10 xenografts showed slowed tumor growth without significant tumor regression during RAD001 therapy. CONCLUSION: These data suggest that loss of PTEN function is insufficient to adequately predict responsiveness to mTOR inhibitors in glioblastoma multiforme.     

RAD001 (everolimus) improves the efficacy of replicating adenoviruses that target colon cancer

Selectively replicating adenoviruses have the potential to cure cancer but have shown little efficacy in clinical trials. We have tested the ability of the mTOR kinase inhibitor RAD001 (everolimus) to enhance the response of xenografts to an oncolytic adenovirus. The virus has Tcf sites inserted in the early viral promoters and replicates selectively in cells with activation of the Wnt signaling pathway. To enhance tumor cell infection, an integrin targeting peptide (CDCRGDCFC) was inserted into the fiber gene of the virus. RAD001 combines three useful properties: it inhibits tumor cell growth directly, blocks angiogenesis, and suppresses the immune response. RAD001 does not block viral protein expression, DNA replication, or cytopathic effect in tumor cells in vitro. After 6 weeks of daily RAD001 treatment, ongoing viral DNA replication could be detected in tumor xenografts, showing that RAD001 does not inhibit virus replication in vivo. I.v. injection of virus alone produced a small delay in xenograft growth, whereas combination therapy substantially prolonged the survival of the mice. We suggest that collapsing the tumor vasculature after the initial infection traps the virus and facilitates local spread within the tumor. Unlike conventional drugs, which require continued access to the tumor through the vascular system, oncolytic viruses are in principle less sensitive to late reductions in perfusion because they are produced locally within the tumor.     

Comparative pharmacokinetics of RAD001 (everolimus) in normal and tumor-bearing rodents

Purpose

Comparative pharmacokinetic (PK) analysis of the mTOR inhibitor RAD001 (everolimus) in rats and mice.

Methods

Blood cell partitioning, plasma protein binding and PK parameters of RAD001 in blood and tissues (including brain) of both mice and rats were determined. PK modeling predicted plasma/blood and tumor levels from a variety of regimens and these were compared with the known human PK profile. DCE-MRI was used to compare tumor vascularity between mice and rats. Estimation of IC50 values in vitro and ED50 values in vivo were used to provide an indication of anti-tumor activity.

Results

The PK properties of RAD001 differed between mice and rats, including erythrocyte partitioning, plasma protein binding, plasma/blood t _1/2, oral bioavailability, volume of distribution, tissue/tumor penetration and elimination. Modeling of tumor and blood/plasma PK suggested that in mice, multiple daily administrations result in a ~2-fold increase in tumor levels of RAD001 at steady state, whereas in rats, a ~7.9-fold increase would occur. Weekly high-dose regimens were predicted not to facilitate tumor accumulation in either species. Total tumor levels of RAD001 were four- to eight-fold greater in rats than in mice. Rat tumors had a >2-fold greater plasma content and permeability compared to mouse tumors, which could contribute to differences in tumor drug uptake. Maximal antitumor effects (T/C of 0.04–0.35) were observed in both species after daily administration with similar C _max and AUC values of unbound (free) RAD001. These free levels of RAD001 are exceeded in serum from cancer patients receiving clinically beneficial daily regimens. In rodents, brain penetration of RAD001 was poor, but was dose-dependent and showed over-proportional uptake in rats with a longer t _1/2 compared to the systemic circulation.

Conclusions

The PK of RAD001 differed between mice and rats, with rats having a PK profile closer to that of humans. High intermittent doses of RAD001 may be more appropriate for treatment of brain tumors.     

Zoledronic acid potentiates mTOR inhibition and abolishes the resistance of osteosarcoma cells to RAD001 (Everolimus): pivotal role of the prenylation process

Despite recent improvements in therapeutic management of osteosarcoma, ongoing challenges in improving the response to chemotherapy warrants new strategies still needed to improve overall patient survival. In this study, we investigated in vivo the effects of RAD001 (Everolimus), a new orally available mTOR inhibitor, on the growth of human and mouse osteosarcoma cells either alone or in combination with zoledronate (ZOL), an anti-osteoporotic drug used to treat bone metastases. RAD001 inhibited osteosarcoma cell proliferation in a dose- and time-dependent manner with no modification of cell-cycle distribution. Combination with ZOL augmented this inhibition of cell proliferation, decreasing PI3K/mTOR signaling compared with single treatments. Notably, in contrast to RAD001, ZOL downregulated isoprenylated membrane-bound Ras concomitantly with an increase of nonisoprenylated cytosolic Ras in sensitive and resistant osteosarcoma cell lines to both drugs. Moreover, ZOL and RAD001 synergized to decrease Ras isoprenylation and GTP-bound Ras levels. Further, the drug combination reduced tumor development in two murine models of osteoblastic or osteolytic osteosarcoma. We found that ZOL could reverse RAD001 resistance in osteosarcoma, limiting osteosarcoma cell growth in combination with RAD001. Our findings rationalize further study of the applications of mTOR and mevalonate pathway inhibitors that can limit protein prenylation pathways.     

RAD001 shows activity against gastric cancer cells and overcomes 5-FU resistance by downregulating thymidylate synthase

We evaluated RAD001, an inhibitor of the mammalian target of rapamycin (mTOR) in human gastric cancer cell lines and determined the molecular mechanisms. RAD001 has marked growth inhibitory activity against the SNU-1 and SNU-216 cells. It inhibited phosphorylation of mTOR and S6 K, and induced G1 cell cycle arrest. Synergistic growth-inhibitory effects in combination with 5-fluorouracil (5-FU) was identified. Furthermore, RAD001 conferred sensitivity to 5-FU-resistant cell lines by downregulating thymidylate synthase (TS). In conclusion, RAD001 showed growth inhibitory activity against gastric cancer cells and acted synergistically with cytotoxic agents such as 5-FU by downregulating TS.     

The mTOR pathway inhibitor RAD001 (everolimus) is highly efficacious in tamoxifen-sensitive and -resistant breast cancer xenografts

The rapamycin derivative RAD001 (everolimus) is presently in clinical trials. Preclinical studies have suggested preferential activity in antiestrogen resistant breast cancer cells. We investigated the response of breast cancer xenografts with different tamoxifen (TAM) sensitivity towards RAD001 and analyzed the regulatory machinery as well as the cross-talk between different signaling pathways. The ERα-positive and TAM-sensitive patient-derived breast carcinoma model 3366, its TAM-resistant counterpart 3366/TAM and 4049, a breast cancer with inherent TAM-resistance, were transplanted to immunodeficient nude mice and treated with RAD001 or TAM or the combination of both compounds. Shock frozen tumors were prepared for Western Blot and immunohistochemical analysis to semi-quantitatively evaluate the expression of the ERα and the ERα regulated IGF-IR as well as PTEN, pAkt, mTOR, (phospho)-p70^S6K, (phospho)-4E-BP1 and cyclin D1. RAD001 significantly inhibited the growth of tamoxifen responding and non-responding xenografts. The highest efficacy was found for the combined treatment with TAM and RAD001. RAD001 modified the protein expression of mTOR and its downstream molecule 4E-BP1 as well as the level of PTEN and ERα, but independent of the tumors sensitivity towards TAM. The protein kinase Akt was found in the active phosphorylated form (pAkt) only in TAM-resistant xenografts, but not detectable in the TAM-responding 3366 line. All treatment modalities down-regulated pAkt expression in the TAM-resistant tumors. p70^S6K and IGF-IR proteins were not significantly influenced by RAD001 treatment. Our findings document the linkage between different growth-controlling pathways. Due to its capability to be active in a TAM-resistant in vivo model, RAD001 could potentially serve as a promising second-line therapy in breast cancer.     

A phase 1 study of weekly everolimus (RAD001) in combination with docetaxel in patients with metastatic breast cancer

BACKGROUND:

Inhibition of mammalian target of rapamycin with everolimus may improve the efficacy of taxanes. Everolimus and docetaxel are both metabolized by CYP3A4, which could result in a pharmacokinetic (PK) interaction.

METHODS:

Fifteen patients with metastatic breast cancer were treated with docetaxel (doses of 40‐75 mg/m² intravenously on day 1 of a 21‐day cycle) in combination with everolimus (doses ranging from 20 to 50 mg orally on days 1 and 8 of a 21‐day cycle) in a phase 1 trial using the continuous reassessment method to determine maximum tolerated dose. The first 2 patients developed a dose‐limiting toxicity (neutropenic infection), prompting a mandatory dose reduction and PK evaluation of both everolimus and docetaxel for patients enrolled in subsequent dosing cohorts.

RESULTS:

Fifteen patients were treated. Dose‐limiting toxicity included grade 3 mucositis (n = 1), prolonged grade 4 neutropenia (n = 1), and grade 3 infection/febrile neutropenia (n = 3). Day 8 of everolimus was commonly held for neutropenia despite a dose reduction in docetaxel to 40 mg/m². Eleven patients underwent complete PK evaluation for everolimus, and 9 patients underwent complete PK evaluation for both everolimus and docetaxel. Widely variable changes in clearance were seen for both drugs, and the study was terminated because of lack of efficacy and concerns regarding toxicity seen with the combination.

CONCLUSIONS:

Weekly everolimus in combination with docetaxel every 3 weeks was associated with excessive neutropenia and variable clearance of both drugs, making combination therapy unpredictable, even at low doses of both drugs. Cancer 2012. © 2011 American Cancer Society.