Targeting the mTOR Complex by Everolimus in NRAS Mutant Neuroblastoma.
ABSTRACT: High-risk neuroblastoma remains lethal in about 50% of patients despite multimodal treatment. Recent attempts to identify molecular targets for specific therapies have shown that Neuroblastoma RAS (NRAS) is significantly mutated in a small number of patients. However, few inhibitors for the potential treatment for NRAS mutant neuroblastoma have been investigated so far. In this in-vitro study, we show that MEK inhibitors AZD6244, MEK162 and PD0325901 block cell growth in NRAS mutant neuroblastoma cell lines but not in NRAS wild-type cell lines. Several studies show that mutant NRAS leads to PI3K pathway activation and combined inhibitors of PI3K/mTOR effectively block cell growth. However, we observed the combination of MEK inhibitors with PI3K or AKT inhibitors did not show synergestic effects on cell growth. Thus, we tested single mTOR inhibitors Everolimus and AZD8055. Interestingly, Everolimus and AZD8055 alone were sufficient to block cell growth in NRAS mutant cell lines but not in wild-type cell lines. We found that Everolimus alone induced apoptosis in NRAS mutant neuroblastoma. Furthermore, the combination of mTOR and MEK inhibitors resulted in synergistic growth inhibition. Taken together, our results show that NRAS mutant neuroblastoma can be targeted by clinically available Everolimus alone or in combination with MEK inhibitors which could impact future clinical studies.
Project description:Oncogenic mutations in the Neuroblastoma Rat Sarcoma oncogene (NRAS) are frequent in melanoma, but are also found in several other cancer types, such as lung cancer, neuroblastoma and colon cancer. We designed our study to analyze changes in NRAS mutant tumor cells derived from malignancies other than melanoma. A variety of small molecule inhibitors as well as their combinations was tested in order to find beneficial inhibitory modalities in NRASQ61mutant lung cancer and neuroblastoma cell lines. Signaling changes after incubation with inhibitors were studied and compared to those found in NRAS mutant melanoma. All cell lines were most sensitive to inhibition in the MAPK pathway with the MEK inhibitor trametinib. MEK/AKT and MEK/CDK4,6 inhibitor combinations did not show any beneficial effects in vitro. However, we observed strong synergism combining MEK and PI3K/mTOR inhibitors in all cell lines. Our study provides evidence that NRAS mutant cancers share signaling similarities across different malignancies. We demonstrate that dual pathway inhibition of the MAPK and PI3K/AKT/mTOR pathway synergistically reduces cell viability in NRAS mutant cancers regardless of their tissue origin. Our results suggest that such inhibitor combinations may be a potential treatment option for non-melanoma tumors harboring activating NRAS mutations.
Project description:Activating mutations in the neuroblastoma rat sarcoma viral oncogene homolog (NRAS) gene are common genetic events in malignant melanoma being found in 15-25% of cases. NRAS is thought to activate both mitogen activated protein kinase (MAPK) and PI3K signaling in melanoma cells. We studied the influence of different components on the MAP/extracellular signal-regulated (ERK) kinase (MEK) and PI3K/mammalian target of rapamycin (mTOR)-signaling cascade in NRAS mutant melanoma cells. In general, these cells were more sensitive to MEK inhibition compared with inhibition in the PI3K/mTOR cascade. Combined targeting of MEK and PI3K was superior to MEK and mTOR1,2 inhibition in all NRAS mutant melanoma cell lines tested, suggesting that PI3K signaling is more important for cell survival in NRAS mutant melanoma when MEK is inhibited. However, targeting of PI3K/mTOR1,2 in combination with MEK inhibitors is necessary to effectively abolish growth of NRAS mutant melanoma cells in vitro and regress xenografted NRAS mutant melanoma. Furthermore, we showed that MEK and PI3K/mTOR1,2 inhibition is synergistic. Expression analysis confirms that combined MEK and PI3K/mTOR1,2 inhibition predominantly influences genes in the rat sarcoma (RAS) pathway and growth factor receptor pathways, which signal through MEK/ERK and PI3K/mTOR, respectively. Our results suggest that combined targeting of the MEK/ERK and PI3K/mTOR pathways has antitumor activity and might serve as a therapeutic option in the treatment of NRAS mutant melanoma, for which there are currently no effective therapies.
Project description:The mammalian target of rapamycin (mTOR), a vital component of signaling pathways involving PI3K/AKT, is an attractive therapeutic target in breast cancer. Everolimus, an allosteric mTOR inhibitor that inhibits the mTOR functional complex mTORC1, is approved for treatment of estrogen receptor positive (ER+) breast cancer. Other mTOR inhibitors show interesting differences in target specificities: BEZ235 and GSK2126458 are ATP competitive mTOR inhibitors targeting both PI3K and mTORC1/2; AZD8055, AZD2014 and KU-0063794 are ATP competitive mTOR inhibitors targeting both mTORC1 and mTORC2; and GDC-0941 is a pan-PI3K inhibitor. We have addressed the question of whether mTOR inhibitors may be more effective in combination than singly in inhibiting the proliferation of breast cancer cells. We selected a panel of 30 human breast cancer cell lines that included ER and PR positive, HER2 over-expressing, and "triple negative" variants, and determined whether signaling pathway utilization was related to drug-induced inhibition of proliferation. A significant correlation (p = 0.005) was found between everolimus IC50 values and p70S6K phosphorylation, but not with AKT or ERK phosphorylation, consistent with the mTOR pathway being a principal target. We then carried out combination studies with four everolimus resistant triple-negative breast cancer cell lines, and found an unexpectedly high degree of synergy between everolimus and the other inhibitors tested. The level of potentiation of everolimus inhibitory activity (measured by IC50 values) was found to be cell line-specific for all the kinase inhibitors tested. The results suggest that judicious combination of mTOR inhibitors with different modes of action could have beneficial effects in the treatment of breast cancer.
Project description:INTRODUCTION:Upregulation of PI3K/Akt/mTOR signalling in endocrine-resistant breast cancer (BC) has identified mTOR as an attractive target alongside anti-hormones to control resistance. RAD001 (everolimus/Afinitor®), an allosteric mTOR inhibitor, is proving valuable in this setting; however, some patients are inherently refractory or relapse during treatment requiring alternative strategies. Here we evaluate the potential for novel dual mTORC1/2 mTOR kinase inhibitors, exemplified by AZD8055, by comparison with RAD001 in ER?+?endocrine resistant BC cells. METHODS:In vitro models of tamoxifen (TamR) or oestrogen deprivation resistance (MCF7-X) were treated with RAD001 or AZD8055 alone or combined with anti-hormone fulvestrant. Endpoints included growth, cell proliferation (Ki67), viability and migration, with PI3K/AKT/mTOR signalling impact monitored by Western blotting. Potential ER cross-talk was investigated by immunocytochemistry and RT-PCR. RESULTS:RAD001 was a poor growth inhibitor of MCF7-derived TamR and MCF7-X cells (IC50 ?1 ?M), rapidly inhibiting mTORC1 but not mTORC2/AKT signalling. In contrast AZD8055, which rapidly inhibited both mTORC1 and mTORC2/AKT activity, was a highly effective (P?<0.001) growth inhibitor of TamR (IC50 18?nM) and MCF7-X (IC50 24 nM), and of a further T47D-derived tamoxifen resistant model T47D-tamR (IC50 19 nM). AZD8055 significantly (P?<0.05) inhibited resistant cell proliferation, increased cell death and reduced migration. Furthermore, dual treatment of TamR or MCF7-X cells with AZD8055 plus fulvestrant provided superior control of resistant growth versus either agent alone (P?<0.05). Co-treating with AZD8055 alongside tamoxifen (P?<0.01) or oestrogen deprivation (P?<0.05) also effectively inhibited endocrine responsive MCF-7 cells. Although AZD8055 inhibited oestrogen receptor (ER) ser167 phosphorylation in TamR and MCF7-X, it had no effect on ER ser118 activity or expression of several ER-regulated genes, suggesting the mTOR kinase inhibitor impact was largely ER-independent. The capacity of AZD8055 for ER-independent activity was further evidenced by growth inhibition (IC5018 and 20 nM) of two acquired fulvestrant resistant models lacking ER. CONCLUSIONS:This is the first report demonstrating dual mTORC1/2 mTOR kinase inhibitors have potential to control acquired endocrine resistant BC, even under conditions where everolimus fails. Such inhibitors may prove of particular benefit when used alongside anti-hormonal treatment as second-line therapy in endocrine resistant disease, and also potentially alongside anti-hormones during the earlier endocrine responsive phase to hinder development of resistance.
Project description:Adult T-cell leukemia (ATL) has a poor prognosis as a result of severe immunosuppression and rapid tumor progression with resistance to conventional chemotherapy. Recent integrated-genome analysis has revealed mutations in many genes involved in the T-cell signaling pathway, suggesting that the aberration of this pathway is an important factor in ATL pathogenesis and ATL-cell proliferation. We screened a siRNA library to examine signaling-pathway functionality and found that the PI3K/Akt/mTOR pathway is critical to ATL-cell proliferation. We therefore investigated the effect of mammalian target of rapamycin (mTOR) inhibitors, including the dual inhibitors PP242 and AZD8055 and the mTORC1 inhibitors rapamycin and everolimus, on human T-cell leukemia virus type 1 (HTLV-1)-infected-cell and ATL-cell lines. Both dual inhibitors inhibited the proliferation of all tested cell lines by inducing G1-phase cell-cycle arrest and subsequent cell apoptosis, whereas the effects of the 2 mTORC1 inhibitors were limited, as they did not induce cell apoptosis. In the ATL-cell lines and in the primary ATL samples, both dual inhibitors inhibited phosphorylation of AKT at serine-473, a target of mTORC2, as well as that of S6K, whereas the mTORC1 inhibitors only inhibited mTORC1. Furthermore, AZD8055 more significantly inhibited the in vivo growth of the ATL-cell xenografts than did everolimus. These results indicate that the PI3K/mTOR pathway is critical to ATL-cell proliferation and might thus be a new therapeutic target in ATL.
Project description:The PI3K/AKT/mTOR pathway plays a crucial role in the development of leiomyosarcomas (LMSs). In this study, we tested the efficacy of dual PI3K/mTOR (BEZ235), PI3K (BKM120) and mTOR (everolimus) inhibitors in three human LMS cell lines. In vitro and in vivo studies using LMS cell lines showed that BEZ235 has a significantly higher anti-tumor effect than either BKM120 or everolimus, resulting in a greater reduction in tumor growth and more pronounced inhibitory effects on mitotic activity and PI3K/AKT/mTOR signaling. Strikingly, BEZ235 but neither BKM120 nor everolimus markedly enhanced the ERK pathway. This effect was reproduced by the combination of BKM120 and everolimus, suggesting the involvement of mTORC2 via a PI3K-independent mechanism. Silencing of RICTOR in LMS cells confirmed the role of mTORC2 in the regulation of ERK activity. Combined treatment with BEZ235 and GSK1120212, a potent MEK inhibitor, resulted in synergistic growth inhibition and apoptosis induction in vitro and in vivo. These findings document for the first time that dual PI3K/mTOR inhibition in leiomyosarcomas suppress a negative feedback loop mediated by mTORC2, leading to enhanced ERK pathway activity. Thus, combining a dual PI3K/mTOR inhibitor with MEK inhibitors may be a relevant approach to increase anti-tumor activity and prevent drug resistance in patients with LMS.
Project description:Activating mutations of the NRAS (neuroblastoma rat sarcoma viral oncogene) protein kinase, present in many cancers, induce a constitutive activation of both the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) signal transduction pathway and the PI(3)K-AKT-mTOR, pathway. This in turn regulates the formation of the eIF4F eukaryotic translation initiation complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, which binds to the 7-methylguanylate cap (m(7)G) at the 5' end of messenger RNAs. Small molecules targeting MEK (MEKi: MEK inhibitors) have demonstrated activity in NRAS-mutant cell lines and tumors, but resistance sets in most cases within months of treatment. Using proximity ligation assays, that allows visualization of the binding of eIF4E to the scaffold protein eIF4G, generating the active eIF4F complex, we have found that resistance to MEKi is associated with the persistent formation of the eIF4F complex in MEKi-treated NRAS-mutant cell lines. Furthermore, inhibiting the eIF4A component of the eIF4F complex, with a small molecule of the flavagline/rocaglate family, synergizes with inhibiting MEK to kill NRAS-mutant cancer cell lines.
Project description:Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.
Project description:Attempts to directly block the mutant neuroblastoma rat sarcoma oncogene (NRAS) protein, a driving mutation in many cancer types, have been unsuccessful. Current treatments focus on inhibition of different components of NRAS' two main downstream cascades: PI3K/AKT/mTOR and MAPK. Here we test a novel dual therapy combination of metformin and trametinib on a panel of 16 NRAS mutant cell lines, including melanoma cells, melanoma cells with acquired trametinib resistance, lung cancer and neuroblastoma cells. We show that both of the main downstream cascades of NRAS can be blocked by this combination: metformin indirectly inhibits the PI3K/AKT/mTOR pathway and trametinib directly impedes the MAPK pathway. This dual therapy synergistically reduced cell viability in vitro and xenograft tumor growth in vivo. We conclude that metformin and trametinib combinations are effective in preclinical models and may be a possible option for treatment of NRAS mutant cancers.
Project description:In an effort to understand the mechanisms of acquired resistance to BRAF inhibitors, we isolated clones that acquired resistance to the BRAF inhibitor GSK2118436 derived from the A375 BRAF V600E mutant melanoma cell line. This resistance clones acquired mutations in NRAS and MEK1. One clones, 16R6-4, acquired two mutations in NRAS – Q61K and A146T. Proliferation and western blot analyses demonstrated that these clones were insensitive to single agent GSK2118436 or GSK1120212 (an allosteric MEK inhibitor) but were sensitive to the combination of GSK2118436 and GSK1120212. To further characterize this combination, global transcriptomic analysis was performed in A375 and 16R6-4 after 24 hour treatment with GSK2118436, GSK1120212 or the combination of GSK2118436 and GSK1120212. This data set was published in Molecular Cancer Therapeutics with the title “Combined inhibition of BRAF and MEK, BRAF and PI3K/mTOR, or MEK and PI3K/mTOR overcomes acquired resistance to the BRAF inhibitor GSK2118436, mediated by NRAS or MEK mutations” by Greger, J.G., et.al. A375 and 16R6-4 (an A375 derived GSK2118436 resistance clone) were treated for 24 hours with 0.1 micromolar GSK2118436, 1 micromolar GSK2118436, 0.01 micromolar GSK1120212, 0.1 micromolar GSK2118436 + 0.01 micromolar GSK1120212, or 1 micromolar GSK2118436 + 0.01 micromolar GSK1120212.