Clinical Trials Of mTOR Inhibitors Research Articles

Abstract 427: Newer generation mTOR inhibition represents effective therapeutic strategy for BRAF-mutant melanoma

Abstract Despite the advent of novel therapies, the five year survival rate for Stage IV melanoma remains at only 30%. This highlights the critical need for new therapeutics to treat this refractory disease. Aberrant activation of the PI3K-AKT pathway is common in melanoma, but efforts to drug this pathway have proven largely ineffective in clinical trials. In our study, we observed that pharmacological inhibition of AKT was ineffective whereas genetic silencing of all three AKT paralogs significantly abrogated melanoma cell growth and led to apoptosis through effects on mTORC signaling. This phenotype could be rescued by overexpression of AKT but was dependent on kinase activity. Interestingly, expression of the serine/threonine kinase SGK1 was increased following genetic suppression of AKT but only expression of activated SGK1 could rescue the lethal effect of AKT knockdown. SGK1 also increases tumor growth and decreases survival in a BRAFV600E-driven mouse model of melanoma. Despite our results suggesting that key proliferation of melanoma cells is through effects on mTOR, phase II clinical trials of mTOR inhibitors have not shown clinical advantage. This may be due to multiple reasons: firstly, mTOR inhibitors, such as rapamycin, function by destabilization of the mTORC1-Raptor complex while leaving the mTORC2-Rictor complex, intact. Rictor enables mTORC2 to directly phosphorylate Ser473, and facilitates Thr308 phosphorylation by PDK1. As both AKT and SGK are phosphorylated by mTORC2 and PDK1 to facilitate downstream signaling through mTORC1, residual activity of mTOR incompletely suppressed by rapamycin may still be sufficient to drive melanoma progression. Thus, we evaluated second and third generation mTORC inhibitors, including a dual PI3K/mTOR inhibitor, that target both mTORC1 and 2 complexes and lead to sustained suppression of PI3K>AKT signaling. The dual PI3K/mTORC inhibitor, Paxalisib, significantly reduced cell proliferation greater than combination AKT/SGK inhibition and resulted in increased overall survival in a BRAF-driven immunocompetent mouse model of melanoma (p=0.0003 vs vehicle). These results allow insight into compensatory signaling networks upon AKT inhibition and suggest that dual targeting of PI3K and both mTOR complexes may represent an effective and tolerable therapeutic strategy in this disease that could further be combined with standard of care targeted therapy. Citation Format: Gennie L. Parkman, Tursun Turapov, David Kircher, William Burnett, Christopher Stehn, Kayla O'Toole, Katie Culver, Ashley Chadwick, Riley Elmer, Ryan Flaherty, Mona Foth, Karly Stanley, Robert Andtbacka, David Lum, Robert Judson-Torres, Martin McMahon, Sheri Holmen. Newer generation mTOR inhibition represents effective therapeutic strategy for BRAF-mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 427.

Clinical trials of mTOR inhibitors to boost immunity to viral infection in older adults

Respiratory tract infections (RTIs), mainly caused by viruses, are a major cause of morbidity and mortality in older people (aged >65 years).1 During the COVID-19 pandemic, increasing age has proved to be the major risk factor for serious illness and death from infection with SARS-CoV-2. The increased susceptibility of older people to viral RTIs is at least partly driven by age-related decline in immune function, characterised by dysregulation of both innate and adaptive immune responses. In particular, attenuated type I interferon (IFN) response, the first line of defence against viruses, might play a major role.

The mTOR pathway in treatment of epilepsy: a clinical update.

Nearly a third of patients with epilepsy have seizures refractory to current medical therapies. In the search for novel drug targets, the mTOR pathway has emerged as key in the regulation of neuronal function, growth and survival, and other cellular processes related to epileptogenesis. Hyperactivation of the mTOR pathway has been implicated in tuberous sclerosis complex and other 'mTORopathies', clinical syndromes associated with cortical developmental malformations and drug-resistant epilepsy. Recently published clinical trials of mTOR inhibitors in tuberous sclerosis complex have shown that these drugs are effective at decreasing seizure frequency. Future studies may establish whether mTOR inhibitors can provide effective treatment for patients with diverse genetic and acquired epilepsies, including preventative, disease-modifying therapies.

MTOR pathway: A current, up-to-date mini-review (Review).

Mammalian target of rapamycin (mTOR) is a protein serine/threonine kinase that was initially identified as the cellular target of rapamycin. This kinase regulates cell growth, proliferation, motility and survival, as well as the gene transcription and protein synthesis that are activated in response to hormones, growth factors and nutrients. Results from preclinical studies have indicated that factors antagonizing the mTOR pathway exert an antitumor effect on lung cancer. Furthermore, primary clinical trials of mTOR inhibitors have demonstrated that the inhibitors may be effective against lung carcinoma. The present study explores the association between mTOR and lung carcinogenesis and describes the clinical trials of mTOR inhibitors.

MTOR inhibitors and dyslipidemia in transplant recipients: a cause for concern?

Post-transplant dyslipidemia is exacerbated by mammalian target of rapamycin (mTOR) inhibitors. Early clinical trials of mTOR inhibitors used fixed dosing with no concomitant reduction in calcineurin inhibitor (CNI) exposure, leading to concerns when consistent and marked dyslipidemia was observed. With use of modern concentration-controlled mTOR inhibitor regimens within CNI-free or reduced-exposure CNI regimens, however, the dyslipidemic effect persists but is less pronounced. Typically, total cholesterol levels are at the upper end of normal, or indicate borderline risk, in kidney and liver transplant recipients, and are lower in heart transplant patients under near-universal statin therapy. Of note, it is possible that mTOR inhibitors may offer a cardioprotective effect. Experimental evidence for delayed progression of atherosclerosis is consistent with evidence from heart transplantation that coronary artery intimal thickening and the incidence of cardiac allograft vasculopathy are reduced with everolimus versus cyclosporine therapy. Preliminary data also indicate that mTOR inhibitors may improve arterial stiffness, a predictor of cardiovascular events, and may reduce ventricular remodeling and decrease left ventricular mass through an anti-fibrotic effect. Post-transplant dyslipidemia under mTOR inhibitor therapy should be monitored and managed closely, but unless unresponsive to therapy should not be regarded as a barrier to its use.

Nursing Implications for the Lifelong Management of Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is a genetic disorder that can affect multiple organ systems, including the brain, heart, skin, kidney, and lung, by formation of benign hamartomas. It can be associated with autism, epilepsy, and other neurocognitive and behavioral disabilities. The incidence of TSC is approximately 1 in 6,000 live births, but it may be underdiagnosed. Mutations to either the TSC1 (coding for hamartin) or TSC2 (coding for tuberin) genes are present in 85% of patients with TSC. The TSC1/TSC2 protein dimer complex is a crucial inhibitory element in the mammalian target of rapamycin (mTOR) complex 1 pathway that regulates cell growth and proliferation. The manifestations of TSC usually require management over the entire life of the patient. Until recently, there were few options, other than surgical removal, for treating the symptoms of TSC related to growth of hamartomas. Increased understanding of the genetic cause of the disease and the underlying dysregulation of the mTOR pathway has led to clinical trials of mTOR inhibitors including sirolimus and everolimus. This article will review the various manifestations of TSC and describe treatment strategies, recommendations for surveillance, and use of mTOR inhibitors in their management.

Pulmonary complications with the use of mTOR inhibitors in targeted cancer therapy: a systematic review and meta-analysis

Mammalian target of rapamycin (mTOR) inhibitors have gained regulatory approval for use in several cancer types. Pulmonary adverse events associated with mTOR inhibitors are well recognized but their frequency has varied considerably among trials. PubMed and ASCO abstracts were searched to identify clinical trials of mTOR inhibitors in solid tumors. Twenty-two eligible trials on which 4,242 patients were treated met the criteria for inclusion in this systematic review and meta-analysis. Adverse event data were extracted and used to determine the incidence rate and incidence rate ratio for pneumonitis, dyspnea, and cough. The incidence rate of any grade pneumonitis in patients with solid tumors treated with mTOR inhibitors was 0.11 (95% confidence interval (CI), 0.06-0.17) per patient, while the incidence of grade 3-4 pneumonitis was 0.03 (95% CI, 0.01-0.04) per patient. The incidence rate ratio (IRR) of any grade pneumonitis with mTOR inhibitors relative to controls was 19.0 (95% CI, 6.5-55.4), and for grade 3-4 pneumonitis was 8.0 (95% CI, 2.6-24.1). The incidence rate for any grade and grade 3-4 cough was 0.23 (95% CI, 0.20-0.27) per patient and 0.01 (95% CI, 0.00-0.01) per patient, respectively. The incidence rate for any grade and grade 3-4 dyspnea was 0.15 (95% CI, 0.10-0.21) per patient and 0.03 (95% CI, 0.02-0.04) per patient, respectively. Compared to control, treatment with mTOR inhibitors were associated with a significant increase in any grade cough [IRR = 1.9 (95% CI, 1.6-2.4)] and grade 3-4 dyspnea [IRR = 2.0 (95% CI, 1.2-3.3)]. This study provides an estimation of the risk of pulmonary adverse events in solid tumor patients treated with mTOR inhibitors. While pulmonary adverse events are relatively common with mTOR inhibitors, most are low grade and asymptomatic.

MTOR inhibitors control the growth of EGFR mutant lung cancer even after acquiring resistance by HGF.

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, is a critical problem in the treatment of EGFR mutant lung cancer. Several mechanisms, including bypass signaling by hepatocyte growth factor (HGF)-triggered Met activation, are implicated as mediators of resistance. The mammalian target of rapamycin (mTOR), is a downstream conduit of EGFR and MET signaling, and is thus considered a therapeutically attractive target in the treatment of various types of cancers. The purpose of this study was to examine whether 2 clinically approved mTOR inhibitors, temsirolimus and everolimus, overcome HGF-dependent resistance to EGFR-TKIs in EGFR mutant lung cancer cells. Both temsirolimus and everolimus inhibited the phosphorylation of p70S6K and 4E-BP1, which are downstream targets of the mTOR pathway, and reduced the viability of EGFR mutant lung cancer cells, PC-9, and HCC827, even in the presence of HGF in vitro. In a xenograft model, temsirolimus suppressed the growth of PC-9 cells overexpressing the HGF-gene; this was associated with suppression of the mTOR signaling pathway and tumor angiogenesis. In contrast, erlotinib did not suppress this signaling pathway or tumor growth. Multiple mechanisms, including the inhibition of vascular endothelial growth factor production by tumor cells and suppression of endothelial cell viability, contribute to the anti-angiogenic effect of temsirolimus. These findings indicate that mTOR inhibitors may be useful for controlling HGF-triggered EGFR-TKI resistance in EGFR mutant lung cancer, and they provide the rationale for clinical trials of mTOR inhibitors in patients stratified by EGFR mutation and HGF expression status.

Pulmonary complications with the use of mTOR inhibitors in targeted cancer therapy: A systematic review and meta-analysis.

362 Background: mTOR inhibitors are approved in several malignancies including renal cell carcinoma (RCC). While pulmonary toxicities are a recognized adverse effect associated with this drug class, the frequency and risk of these side effects have not been well characterized. Methods: Clinical trials of mTOR inhibitors in solid tumors were identified through a search of PubMed and ASCO abstracts. Prospective studies of temsirolimus, everolimus, and ridaforolimus in solid tumors were evaluated for inclusion. 22 eligible phase II and phase III trials that included 4,242 patients were identified and included in a systematic review and meta-analysis. Adverse event data was extracted for pulmonary complications including pneumonitis, dyspnea, and cough. The incidence rate and the incidence rate ratios were determined for these pulmonary adverse events. Results: Based on our analysis of the 20 trials that reported pneumonitis, the incidence rate of any grade pneumonitis in patients with solid tumors treated with mTOR inhibitors is 0.11 (95% CI, 0.06-0.17). The incidence rate of grade 3 or 4 pneumonitis is 0.03 (95% CI, 0.01-0.04). The incidence rate ratio of any grade pneumonitis with mTOR inhibitors relative to controls is 18.9 (95% CI, 6.5-55.1), and the incidence rate ratio for the development of grade 3 or 4 pneumonitis is 7.9 (95% CI, 2.6-24.0). The incidence rates of any grade cough and dyspnea were found to be 0.23 (95% CI, 0.20-0.27) and 0.15 (95% CI, 0.10-0.21), respectively. The incidence rates of grade 3 or 4 cough and dyspnea are found to be 0.01 (95% CI, 0.00-0.01) and 0.03 (95% CI, 0.02-0.04), respectively. There was a statistically significant, but modest increase in risk of developing any grade cough (incidence rate ratio of 1.9 [95% CI, 1.6-2.4]) and grade 3 or 4 dyspnea (incidence rate ratio of 2.0 [95% CI, 1.2-3.3]) with mTOR inhibitors relative to controls. Conclusions: This study confirms that mTOR inhibitors are associated with pulmonary adverse events and provides a quantitative estimation of the risk of these adverse events in solid tumor patients treated with these drugs. The majority of pulmonary adverse events are low grade.

1162P - Phase 1 Study of Everolimus Combined with Lutetium-177 Octreotate Radiopeptide Therapy of Advanced Low-Grade Neuroendocrine Tumours

ABSTRACT Background Recent clinical trials of mTOR inhibitors have shown benefit from everolimus treatment of advanced low-grade neuroendocrine tumours (NETs). Targeting of NET somatostatin receptors with radiopeptides is another effective therapeutic option. This phase I study investigates the safety of combining everolimus with lutetium-177 octreotate. Methods Patients with biopsy proven low-grade NETs (Ki 67 index Results Eleven patients have been treated, 4 patients at the 5 mg everolimus dose level and 3 each at the 7.5 mg and 10 mg levels. Full dose lutetium-177 octreotate for 4 cycles was possible in 6 of 7 patients in cohorts 1 and 2, whilst only 1 of 3 patients completed all cycles at the 10 mg dose level. Patients at 5 mg and 7.5 mg doses of everolimus received 85% of the total planned dosage over 24 weeks. All patients required dose reduction or complete cessation of everolimus at the 10 mg level because of unacceptable toxicities. The commonest reasons for dose reductions in cohorts 1 and 2 were grade 2 or 3 neutropenia and thrombocytopenia. Unexpected reversible grade 2 nephrotoxicity caused significant reductions in creatinine clearance levels of 40-50% baseline in 2 of 3 patients in the 10 mg cohort, not seen in the lower dose levels. Tumour responses have been observed at each dose level. Conclusion Everolimus can be combined safely with lutetium-177 octreotate to treat low-grade NETs. Toxicities were observed at all dosage levels of everolimus but appear manageable and reversible. The maximum tolerated dose (MTD) of everolimus was 7.5 mg daily in combination with 7.8 GBq lutetium-177 octreotate in a 4 cycle course over 24 weeks. Disclosure P. Claringbold: Dr Claringbold has acted on an advisory panel and received financial support from Novartis Pharmaceuticals. J.H. Turner: Dr Turner has acted on an advisory panel and received financial support from Novartis Pharmaceuticals.

Non-clear cell renal cell carcinoma: does the mammalian target of rapamycin represent a rational therapeutic target?

Non-clear cell renal cell carcinomas (nccRCCs) comprise a heterogenous and poorly characterized group of tumor types for which few treatments have been approved. Although targeted therapies have become the cornerstones of systemic treatment for metastatic renal cell carcinoma, patients with nccRCC have been excluded from many pivotal clinical trials. As such, robust clinical evidence supporting the use of these agents in patients with nccRCC is lacking. Here, we review the disparate nccRCC subtypes, the criteria for diagnosis, and the prognoses associated with each subtype, in addition to evaluating the potential use of mammalian target of rapamycin (mTOR) inhibitors in treating patients with nccRCC. Both genetic analyses and preclinical research indicate a central role for mTOR in nccRCC; a therapy that targets this ubiquitous regulator of cellular signaling could prove efficacious across various tumor subtypes. Results from recent studies exploring targeted therapies as both monotherapy and combination therapy have provided early indications of efficacy in patients with nccRCC. Exploratory analyses support further research with the mTOR inhibitors everolimus and temsirolimus in patients with nccRCC. Current clinical practice guidelines support the use of mTOR inhibitors in patients with nccRCC; however, these recommendations are based on low levels of evidence. Further results from randomized, controlled clinical trials are needed to determine the optimal choice of therapy for patients with nccRCC. Results from ongoing clinical trials of mTOR inhibitors and other agents in nccRCC, as well as their impact on the nccRCC treatment paradigm, are eagerly awaited.

Abstract LB-201: Prostate cancer stem cells display low mTOR activity and resistance to mTOR inhibitors in hypoxia

Abstract Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSCs), were recently identified and characterized in prostate cancer (PCa). We hypothesized that one of the frequently altered molecular signaling pathways in PCa, the PI3K/AKT pathway, and mammalian target of rapamycin (mTOR) in particular, play a role in PCa stem cell maintenance. In addition, hypoxia is known to be involved in cancer stem cell maintenance and to have a regulating effect on mTOR signaling. In our study, we evaluated the effects of mTOR inhibitors on proliferation of human as well as murine CSCs in hypoxic conditions in vitro. We isolated CSC like subpopulations from the androgen independent human prostate cancer cell line DU145 using fluorescence activated cell sorting according to their expression of the stem cell marker CD44. Further, we sorted the murine prostate cancer cell line TRAMP-C1 based on the expression of the stem cell markers Sca-1 and CD49f. We used sphere formation assays to confirm the stem and progenitor cell properties of the sorted subpopulations. Next, we treated the CSC and non-CSC cell populations with mTOR inhibitors rapamycin and everolimus under normoxic (20% O2) and hypoxic (3% O2) conditions in order to determine their viability at time points up to 72 hours. Our results suggest that CSC like prostate cancer cells with elevated expression of stem cell markers, such as CD44, Sca-1 or CD49f, are more resistant to mTOR inhibitors when compared to their non-CSC counterparts. Most interestingly, while the non-CSC subpopulation of prostate cancer cells displays increased sensitivity to mTOR inhibitors under hypoxic conditions, the viability of CSCs remains essentially unaffected. Immunoblotting reveals lower mTOR activity in prostate cancer stem cells compared to non-CSCs. Hypoxia in this context leads, as expected, to a decrease in mTOR activity in both subpopulations, though this effect is significantly pronounced in CSCs like subpopulations of both cancer cell lines in our study. At the same time, we do not observe alterations in signaling through other PI3K downstream effectors, such as AKT. We propose that prostate cancer stem cells might be resistant against small molecule inhibitors of mTOR and that inhibitors targeting multiple kinases along the PI3K/AKT/mTOR axis would be more effective in this setting. We also hypothesize that intratumoral hypoxia might contribute to the mTOR resistance of prostate cancer stem cells by inhibiting mTOR signaling and promoting cell growth through other, PI3K dependent pathways. These findings could be helpful to assess the impact of mTOR targeted therapy in prostate cancer, notably in light of ongoing clinical trials of mTOR inhibitors in this entity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-201. doi:1538-7445.AM2012-LB-201

Single-Cell Pharmacodynamic Monitoring of S6 Ribosomal Protein in AML Blasts During a Clinical Trial Combining the mTOR Inhibitor Sirolimus with Mitoxantrone, Etoposide, and Cytarabine Chemotherapy.

Abstract 413The mammalian target of rapamycin (mTOR) is an emerging molecular target in cancer therapy, however the relationship between target activation in individual tumors as well as inhibition of target and clinical response is poorly defined. This is in large part due to the difficulty of real-time monitoring of mTOR activation in patient samples. mTOR is activated in acute myelogneous leukemia (AML) cells and the mTOR inhibitor rapamycin enhances cytotoxic effects of chemotherapy in vitro and in vivo. We therefore developed a new application of a recently-developed, whole blood fixation/permeabilization technique for intracellular flow cytometry (Chow, et al. Cytometry A 2005). Using this approach, we sought to serially monitor S6 ribosomal protein (S6) phosphorylation in peripheral blood leukemic blasts during clinical trials of mTOR inhibitors. S6 is a known target of mTOR and its phosphorylation is a surrogate marker for mTOR kinase activation. We applied this methodology during a recent pilot trial in which an oral mTOR inhibitor, sirolimus (rapamycin), was administered in sequence with intensive combination chemotherapy (mitoxantrone, etoposide, and cytarabine, or MEC) in patients with relapsed, refractory, or secondary AML. The whole blood fixation process sufficiently preserved surface epitopes and light scatter properties for immunophenotyping, allowing specific signaling analysis of leukemic blasts as well as non-malignant cell populations. Importantly, even leukopenic trial samples containing as few as 20% blasts provided robust signaling data in malignant cells. S6 phosphorylation was readily apparent in leukemic blasts prior to therapy and, consistent with prior reports, occurred only in a subset of blasts. Exposing aliquots of pre-treatment whole blood samples to increasing concentrations of rapamycin ex vivo determined that leukemic blasts from most samples showed inhibition of S6 phosphorylation at clinically achievable concentrations (between 10-20 nM). Notably, some subjects' leukemic blasts showed no inhibition to >50 nM rapamycin, which far exceeded trough concentrations measured on our studies. To examine rapamycin's in vivo biochemical effects, we performed a paired analysis of clinical samples drawn at study entry and after 72 hours of oral sirolimus. 10 subjects provided paired samples, of which 2 did not show baseline S6 phosphorylation, 6 showed baseline S6 phosphorylation that inhibited during therapy, and 2 showed baseline S6 phosphorylation but no inhibition. Trough rapamycin levels were similar among rapamycin responsive and resistant subjects. Considering the 6 subjects with in vivo mTOR inhibition, 3 subjects achieved complete or partial remissions from the regimen. Neither subject with in vivo rapamycin resistance had a clinical response. Overall, we conclude that effective inhibition of mTOR signaling in AML blasts occurs in the majority of subjects during sirolimus treatment at the dose studied. However, cell-intrinsic rapamycin resistance occurs in a minority of patients and requires further study to clarify its mechanism and effects upon concurrent chemotherapy response. These data demonstrate the feasibility of real-time, intra-tumoral pharmacodynamic monitoring of S6 phosphorylation by flow cytometry during clinical trials combining intensive chemotherapy and signal transduction inhibitors for leukemia. Our approach greatly clarifies pharmacokinetic/pharmacodynamic relationships and has broad application to pre-clinical and clinical testing of drugs whose direct or downstream effects disrupt PI3K/AKT/mTOR signaling. Such compounds include inhibitors of FLT3, c-KIT, BCR-ABL, JAK2, and ras/raf/MAPK. Multicenter/cooperative group phase II testing of sirolimus plus MEC in AML has been initiated to establish the regimen's response rate and test the extent to which our pharmacodynamic studies predict clinical response. Disclosures:Off Label Use: The use of sirolimus in the therapy of AML is investigational and off-label. Carroll:Cephalon consultancy: Consultancy; Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceutical: Research Funding.

Histone Deacetylase Inhibition with LBH589 Inhibits the Rapamycin Insensitive Rictor-mTOR (mTORC2) Complex and Translation Initiation Factor eIF4E Activation in Diffuse Large B-Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma and the treatment of these patients has improved with rituximab-based chemo-immunotherapy. However, approximately 40% of patients die of their disease and new agents with novel mechanisms of action are needed for this disease. The mammalian target of rapamycin (mTOR) has emerged as an important therapeutic target in cancer cells and rapamycin and its derivatives, which specifically inhibit mTOR, are now being actively evaluated. Recent clinical trials of mTOR inhibitors have demonstrated that a fraction of patients (35%) with relapsed DLBCL respond to single-agent temsirolimus (Smith S et al J Clin Oncol 26; May 20 Supplement abstract 8514) or everolimus (Reeder et al 2007 ASH Annual Meeting Abstracts 110 (11) abstract 121) in relapsed DLBCL, however most patients are resistant to this therapy. The mechanism of this resistance remains a subject of major therapeutic significance. Herein, we report that cells from DLBCL lines (DHL-6, Ly7 and Ly3) and primary tissues from DLBCL patients (n=10) display persistent activation of the mTOR pathway as determined by phosphorylation of mTOR targets S6 ribosomal protein (S6rp) and 4E-binding protein 1 (4E-BP1). Treatment of human DLBCL cells with various doses of rapamycin predictably demonstrated a decrease in proliferation but less than 10% reduction in overall cell survival. However, rapamycin suppressed the phosphorylation of S6rp and 4E-BP1, indicating an inhibition of raptor-mTOR (mTORC1) signaling. Paradoxically, rapamycin also concurrently increased, through a negative feedback mechanism, the phosphorylation of Akt that may contribute to drug resistance. Interestingly we found that rapamycin treatment also increased the phosphorylation of eIF4E, a survival protein downstream of mTOR, which may also be responsible for resistance of rapamycin along with Akt. To determine whether the observations found in vitro are clinically relevant, we obtained peripheral blood samples from patients with aggressive lymphoma treated with the rapamycin derivative everolimus. The levels of phosphorylated Akt and eIF4E were increased in 3 of 3 patient samples at 48 hrs and after 1 cycle compared to untreated control. Our observations therefore suggest that rapamycin derivatives potently activate Akt and eIF4E activity via activation of the mTORC2 assembly, in addition to its well-characterized ability to suppress the mTORC1 pathway. Treatment of DLBCL cells with histone deacetylase (HDAC) inhibitor LBH589 induced growth inhibition of DLBCL cells at nanomolar concentrations in a dose dependent manner in association with hyperacetylation of histones H3 and H4. LBH alone was able to inhibit the phosphorylation of S6rp and 4E-BP1, while combined treatment with rapamycin inhibited the phosphorylation of S6rp and 4EBP1 to a greater extent than either agent alone. Surprisingly LBH inhibited constitutive as well as rapamycin-induced activation of Akt and eIF4E in a time and dose dependent manner. Our co-immunoprecipitation data suggest that LBH alone was able to alter the level of intact mTORC2 by reducing the amounts of rictor bound to mTOR, which is further decreased when combined with rapamycin. In support of this model, rapamycin combined with LBH exhibited enhanced synergistic inhibitory effects on survival and proliferation of DLBCL cells. The mTOR pathway is also considered to control the translation of specific mRNA species, some of which are involved in cell cycle control and angiogenesis (e.g. cyclin D1, c-Myc and HIF-1a). LBH alone down-regulated expression of c-Myc and HIF-1a, while have no effect on cyclin D1 expression. Combination of LBH with rapamycin further decreased expression of c-Myc and HIF-1a. Overall these results indicate that pharmacological inhibition of the mTOR pathway by rapamycin and LBH interferes with essential survival and proliferating pathways in DLBCL cells. We propose that the rapamycin-induced functional blockade of Akt and eIF4E is inhibited by LBH, and the combination increased the anti tumor activity of rapamycin. In summary, our data provide a mechanistic basis for enhancing mTORtargeted cancer therapy by combining an mTOR inhibitor with a HDAC inhibitor such as LBH. A phase I/II trial of this combination is planned.

Multicenter trial of everolimus in pediatric renal transplant recipients: Results at three year

There are few prospective clinical trials of mTOR inhibitors (or proliferation signal inhibitors) combined with CNI inhibitors in de novo pediatric renal transplantation. Results reported here are from a multicenter, open-label study in de novo pediatric renal transplant patients (<or=16 yr), in which patients received everolimus with cyclosporine and corticosteroids for one yr, then entered an extension study for a further two yr. Nineteen patients completed the one-yr study, of whom three discontinued study medication. Fifteen of the remaining 16 patients entered the extension study, eight of whom were aged <10 yr (Group 1) and seven were aged 10-16 yr (Group 2). Mean daily dose of everolimus during the first 36 months was 1.53 mg/m(2) BSA. Biopsy-proven acute rejection occurred in three patients in Group 2 and in one patient in Group 1. Biopsy-proven chronic allograft rejection was reported in four patients (two in each age group). Graft survival at one yr was 100%; one patient in Group 2 lost their graft subsequently during the extension. For patients entering the extension, patient survival at three yr was 100%. There were three cases of viral infection, including one case of cytomegalovirus infection. At three yr, mean total cholesterol was 5.5 +/- 0.8 mm/L (213 +/- 31 mg/dL) and four patients received statin therapy. Mean serum creatinine at 36 months was 96 +/- 36 microm/L (1.1 +/- 0.4 mg/dL). This is the first long-term prospective study to demonstrate that a regimen of everolimus, cyclosporine, and corticosteroids provides good efficacy, tolerability, and safety in de novo pediatric renal transplant patients.