Dual inhibition of EGFR and mTOR pathways in small cell lung cancer.

BACKGROUND: In this report we investigated the combination of epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) pathway inhibition as a possible new therapeutic strategy for small cell lung cancer (SCLC). METHODS: EGFR, p-AKT, p-ERK, p-mTOR and p-p70s6K protein expressions were studied by immunohistochemistry in 107 small cell lung carcinomas and correlated with clinicopathological parameters. Cells of SCLC were treated with erlotinib+/-RAD001 and analysed for cell viability, proliferation, autophagy, and pathway regulation. RESULTS: Epidermal growth factor receptor, p-AKT, p-ERK, p-mTOR, and p-p70s6K were expressed in 37, 24, 13, 55 and 91% of the tumour specimens of all SCLC patients, respectively, and were not associated with disease-free or overall survival. The expression of EGFR was lower in neoadjuvant-treated patients (P=0.038); mTOR pathway activation was higher in the early stages of disease (P=0.048). Coexpression of EGFR/p-mTOR/p-p70s6K was observed in 28% of all patients . EGFR immunoreactivity was associated with p-ERK and p-mTOR expression (P=0.02 and P=0.0001); p-mTOR immunoreactivity was associated with p-p70s6K expression (P=0.001). Tumour cells comprised a functional EGFR, no activating mutations in exons 18-21, and resistance to RAD001 monotherapy. We found synergistic effects of erlotinib and RAD001 combination therapy on the molecular level, cell viability, proliferation and autophagy. CONCLUSIONS: The combined inhibition of EGFR/mTOR pathways could be a promising approach to treat SCLC.

Use of Ipilimumab and Pembrolizumab in Metastatic Melanoma in a Combined Heart and Kidney Transplant Recipient: A Case Report.

New therapeutic agents such as checkpoint inhibitors are promising strategies in the treatment of metastatic melanoma. Transplant recipients are generally at higher risk of malign diseases. Limited data are available for the use and safety of these agents in this population. We describe a patient who had a stable transplant function over years after a combined heart and kidney transplantation. Immunosuppressive medications included tacrolimus and azathioprine and were later switched to sirolimus and azathioprine. Metastatic melanoma was diagnosed; after detailed explanation of the potential risk, the patient was initially treated with the anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibody ipilimumab but experienced an acute kidney graft rejection and rapid progression. Rejection therapy with high-dose corticosteroids was successful, and kidney function was stabilized. Because of the urgent request of the patient for additional therapy, he received a PD-1 inhibitor. Acute kidney graft rejection resulted, with indication for acute dialysis. He developed severe candida pneumonia and died despite extensive antimicrobial therapy. Checkpoint inhibitors have become the standard in a broad entity of tumors. Organ transplant patients are at higher risk of developing malignant diseases. Limited data are available for the use of checkpoint inhibitors in this subgroup, and the use of checkpoint inhibitors is associated with a high risk of graft rejection.

FDG uptake is a surrogate marker for defining the optimal biological dose of the mTOR inhibitor everolimus in vivo.

This study aimed to test whether [(18)F]fluoro-D-glucose (FDG) uptake of tumours measured by positron emission tomography (PET) can be used as surrogate marker to define the optimal biological dose (OBD) of mTOR inhibitors in vivo. Everolimus at 0.05, 0.5, 5 and 15 mg kg(-1) per day was administered to gastric cancer xenograft-bearing mice for 23 days and FDG uptake of tumours was measured using PET from day 1 to day 8. To provide standard comparators for FDG uptake, tumour volume, S6 protein phosphorylation, Ki-67 staining and everolimus blood levels were evaluated. Everolimus blood levels increased in a dose-dependent manner but antitumour activity of everolimus reached a plateau at doses >or=5 mg kg(-1) per day (tumour volume treated vs control (T/C): 51% for 5 mg kg(-1) per day and 57% for 15 mg kg(-1) per day). Correspondingly, doses >or=5 mg kg(-1) per day led to a significant reduction in FDG uptake of tumours. Dose escalation above 5 mg kg(-1) per day did not reduce FDG uptake any further (FDG uptake T/C: 49% for 5 mg kg(-1) per day and 52% for 15 mg kg(-1) per day). Differences in S6 protein phosphorylation and Ki-67 index reflected tumour volume and changes in FDG uptake but did not reach statistical significance. In conclusion, FDG uptake might serve as a surrogate marker for dose finding studies for mTOR inhibitors in (pre)clinical trials.

Suppression of mTOR complex 2-dependent AKT phosphorylation in melanoma cells by combined treatment with rapamycin and LY294002.

BACKGROUND: Inhibition of mTOR complex 1 (mTORC1) with rapamycin leads to phosphorylation of AKT in some cancer cells, with unknown biological consequences. The role of this phosphorylation in melanoma is unknown, although preliminary clinical data indicate poor activity of rapalogues in melanoma. OBJECTIVES: We aimed at elucidating the role of AKT phosphorylation after mTORC1 inhibition in melanoma cells. METHODS: Western blotting, apoptosis assays, cell cycle analyses and viability assays were performed to analyse the effects of rapamycin and LY294002 treatment on melanoma cells. For suppression of mTOR complex 2 (mTORC2) an siRNA directed against rictor was used. RESULTS: Rapamycin showed limited effects on cell viability but resulted in strong and lasting AKT phosphorylation in melanoma cells. Combined PI3K/mTOR inhibition with LY294002 had pronounced effects on viability but also led to increased AKT phosphorylation after prolonged treatment. In contrast, combination of rapamycin plus LY294002 suppressed AKT phosphorylation. Suppression of AKT phosphorylation did not correlate with decreases in cell viability. Inhibition of mTORC2 led to reduced levels of phosphorylated AKT. CONCLUSIONS: mTORC1 inhibition with rapamycin and with LY294002 can lead to AKT phosphorylation in melanoma cells via mTORC2. Combination of rapamycin and LY294002 suppresses AKT phosphorylation but without significant effect on treatment efficacy.

The effect of RANKL and OPG on bone mineral density in pre-dialysis chronic renal failure.

AIMS: The influence of pre-dialysis chronic kidney disease (CKD) on bone is ill defined. Isolation of specific pathogenic mechanisms would improve the understanding and therapeutic options. We therefore investigated whether parathyroid dysfunction, altered vitamin D and hormonal status, or RANKL and OPG have an influence on bone mineral density (BMD) in patients with pre-dialysis renal failure. MATERIAL: 132 patients with chronic renal failure stage 1 - 5 (not yet on dialysis) were investigated in a cross sectional study. Osteoprotegerin (OPG), receptor activator of nuclear factor kB ligand (RANKL), parathyroid hormone (whole, intact and 7-84 fragment), bone markers, sex hormones, and vitamin D status were assessed together with femoral neck and trochanter z-score. Correlation and multivariate analyses were performed between the different parameters and BMD. RESULTS: In the multivariate analysis a significant association was found between the femoral neck z-score and sRANKL (B = -0.45; p < 0.001), and OPG (B = 0.20; p < 0.05). A significant negative association was also found between the trochanter z-score and sRANKL (B = -0.32; p < 0.001). No associations were found between the trochanter z-score and OPG or the sRANKL/OPG ratio. The body mass index was the only additional marker associated with both FN z-score (B = 0.20, p < 0.05) and TR z-score (B = 0.20, p < 0.05). Neither markers of osteoblast nor osteoclast activity, or intact PTH, whole PTH, the PTH 7-84 fragment or vitamin D status were related to bone mineral density. CONCLUSION: Our results demonstrate that the RANKL/RANK/OPG system is associated with bone mineral density in pre-dialysis chronic renal failure.

Effect of teriparatide on early bone loss after kidney transplantation.

Kidney transplantation is associated with bone loss and a high risk of fractures. Prophylactic treatment of bone is therefore recommended in the early posttransplant period. As a large number of transplant recipients develop adynamic renal osteodystrophy, recombinant parathyroid hormone (rPTH) could be a promising therapeutic option. In a 6-month double-blind, randomized trial, 26 kidney transplant recipients were treated with daily subcutaneous injections of 20 microg teriparatide (PTH 1-34) or placebo. Bone mineral density (BMD) of the femoral neck, lumbar spine and radial bone was measured at transplantation and after 6 months. Paired bone biopsies for histomorphometric analysis were obtained in six, and for measurement of bone matrix mineralization in five patients of each group. Serologic bone markers were measured at baseline and every 3 months. A total of 24 out of 26 patients completed the study. Femoral neck BMD was stable in the teriparatide group, but decreased significantly in the placebo group. Lumbar spine and radial BMD, histomorphometric bone volume and bone matrix mineralization status remained unchanged in both groups. Serologic bone markers were similarly reduced in both groups throughout the study. We conclude that teriparatide does not improve BMD early after kidney transplantation. Neither histological analysis nor bone markers provide evidence of improved bone turnover or mineralization.

Chlorpromazine-induced increase in dipalmitoylphosphatidylserine surface area in monolayers at room temperature.

The Langmuir technique revealed that the surface area of acidic glycerophospholipids (dipalmitoylphosphatidylserine, -glycerol, and dipalmitoylphosphatidic acid) in monolayers increased dramatically when micromolar concentrations of the antipsychotic drug chlorpromazine (CPZ) were present in the subphase. Monolayers of neutral glycerophospholipids (dipalmitoylphosphatidylcholine and -ethanolamine) did not show such a large effect with CPZ. Compared to CPZ, millimolar concentrations of the monovalent cations Li+, K+, Na+, Rb+, and Cs+ did not appear to influence the dipalmitoylphosphatidylserine monolayer, suggesting that the effect of CPZ, a monovalent cationic amphophile, was due to an interaction with the acyl chains of the lipids. In addition, the effect of CPZ was reduced by 150 mM Na+, suggesting that the sodium cations might screen the negatively charged headgroups from an electrostatic interaction with the positively charged drug molecule. Two CPZ analogs, chlorpromazine sulfoxide and CPZ with 2 carbons in the side chain, were also studied. These observations suggest that part of the biological effects of CPZ, being antipsychotic and/or side effects, may be due to CPZ's action on the acidic glycerophospholipids in nerve cell membranes.