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Novel pharmacodynamic biomarkers for MYCN protein and PI3K/AKT/mTOR pathway signaling in children with neuroblastoma.

ABSTRACT: There is an urgent need for improved therapies for children with high-risk neuroblastoma where survival rates remain low. MYCN amplification is the most common genomic change associated with aggressive neuroblastoma and drugs targeting PI3K/AKT/mTOR, to activate MYCN oncoprotein degradation, are entering clinical evaluation. Our aim was to develop and validate pharmacodynamic (PD) biomarkers to evaluate both proof of mechanism and proof of concept for drugs that block PI3K/AKT/mTOR pathway activity in children with neuroblastoma. We have addressed the issue of limited access to tumor biopsies for quantitative detection of protein biomarkers by optimizing a three-color fluorescence activated cell sorting (FACS) method to purify CD45-/GD2+/CD56+ neuroblastoma cells from bone marrow. We then developed a novel quantitative measurement of MYCN protein in these isolated neuroblastoma cells, providing the potential to demonstrate proof of concept for drugs that inhibit PI3K/AKT/mTOR signaling in this disease. In addition we have established quantitative detection of three biomarkers for AKT pathway activity (phosphorylated and total AKT, GSK3? and P70S6K) in surrogate platelet-rich plasma (PRP) from pediatric patients. Together our new approach to neuroblastoma cell isolation for protein detection and suite of PD assays provides for the first time the opportunity for robust, quantitative measurement of protein-based PD biomarkers in this pediatric patient population. These will be ideal tools to support clinical evaluation of PI3K/AKT/mTOR pathway drugs and their ability to target MYCN oncoprotein in upcoming clinical trials in neuroblastoma.

SUBMITTER: Smith JR

PROVIDER: S-EPMC5423144 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Novel pharmacodynamic biomarkers for MYCN protein and PI3K/AKT/mTOR pathway signaling in children with neuroblastoma.

Smith Jennifer R JR Moreno Lucas L Heaton Simon P SP Chesler Louis L Pearson Andrew D J AD Garrett Michelle D MD

Molecular oncology 20151119 4

There is an urgent need for improved therapies for children with high-risk neuroblastoma where survival rates remain low. MYCN amplification is the most common genomic change associated with aggressive neuroblastoma and drugs targeting PI3K/AKT/mTOR, to activate MYCN oncoprotein degradation, are entering clinical evaluation. Our aim was to develop and validate pharmacodynamic (PD) biomarkers to evaluate both proof of mechanism and proof of concept for drugs that block PI3K/AKT/mTOR pathway activ ...[more]

PMID: 26686971

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Project description:Breaking resistance to chemotherapy is a major goal of combination therapy in many tumors, including advanced neuroblastoma. We recently demonstrated that increased activity of the PI3K/Akt network is associated with poor prognosis, thus providing an ideal target for chemosensitization. Here we show that targeted therapy using the PI3K/mTOR inhibitor NVP-BEZ235 significantly enhances doxorubicin-induced apoptosis in neuroblastoma cells. Importantly, this increase in apoptosis was dependent on scheduling: while pretreatment with the inhibitor reduced doxorubicin-induced apoptosis, the sensitizing effect in co-treatment could further be increased by delayed addition of the inhibitor post chemotherapy. Desensitization for doxorubicin-induced apoptosis seemed to be mediated by a combination of cell cycle-arrest and autophagy induction, whereas sensitization was found to occur at the level of mitochondria within one hour of NVP-BEZ235 posttreatment, leading to a rapid loss of mitochondrial membrane potential with subsequent cytochrome c release and caspase-3 activation. Within the relevant time span we observed marked alterations in a ?30 kDa protein associated with mitochondrial proteins and identified it as VDAC1/Porin protein, an integral part of the mitochondrial permeability transition pore complex. VDAC1 is negatively regulated by the PI3K/Akt pathway via GSK3? and inhibition of GSK3?, which is activated when Akt is blocked, ablated the sensitizing effect of NVP-BEZ235 posttreatment. Our findings show that cancer cells can be sensitized for chemotherapy induced cell death - at least in part - by NVP-BEZ235-mediated modulation of VDAC1. More generally, we show data that suggest that sequential dosing, in particular when multiple inhibitors of a single pathway are used in the optimal sequence, has important implications for the general design of combination therapies involving molecular targeted approaches towards the PI3K/Akt/mTOR signaling network.

Project description:The generation of excessive mitochondrial reactive oxygen species (mtROS) is associated with glutamate-stimulated neurotoxicity and pathogenesis of Alzheimer's disease (AD). Impaired mitochondrial function is accompanied with oxidative stress that is a significant contributor to initiate autophagy, but the underlying mechanisms are not fully understood. The present study aimed to investigate the neuroprotective effects of Mito-Tempo on glutamate-induced neuroblastoma SH-SY5Y cell toxicity. SH-SY5Y cells were treated with 100 μM glutamate in the presence or absence of 50 and 100 μM Mito-Tempo for 24 h. Changes in cell viability were measured by MTT assay. Cytotoxicity and intracellular ROS accumulation were also evaluated using lactate dehydrogenase (LDH) activity assay and 2,7-dichlorofluorescein diacetate (DCFDA) Reactive Oxygen Species Assay kit, respectively. Mitochondrial membrane potential was analyzed by tetraethylbenzimidazoly-lcarbocyanine iodide (JC-1) staining. Expression of PI3K/AKT/mTOR pathway and autophagy markers, including LC3 (LC3-I/-II) and p62 (SQSTM1) were performed using Western blot analysis. Our results demonstrated that glutamate-exposed cells significantly increased cellular oxidative stress by enhancing ROS production. Glutamate treatment also increased LDH release follows the loss of mitochondrial membrane potential, caused cell viability loss. Treatment with Mito-Tempo not only attenuated the generation of ROS and improved mitochondrial membrane potential but also reduced the neurotoxicity of glutamate in a concentration-dependent manner, which leads to increased cell viability and decreased LDH release. Mito-Tempo has a greater protective effect by enhancing superoxide dismutase (SOD) activity and PI3K/AKT/mTOR phosphorylation. Moreover, Mito-Tempo treatment altered the autophagy process resulting in the decline in the ratio of the autophagy markers LC3-I/-II and p62 (SQSTM1). We propose that Mito-Tempo can improve neuronal properties against glutamate cytotoxicity through its direct free radical scavenging activity and inhibit excessive autophagy signaling pathway, therefore, allow for further studies to investigate the therapeutic potentials of Mito-Tempo in animal disease models and human.

Project description:BackgroundNeuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB.MethodsUsing small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses.ResultsCo-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy.ConclusionsTogether, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.

Project description:The phosphatidylinositiol 3-kinase (PI3K), AKT, mammalian target of rapamycin (mTOR) signaling pathway (PI3K/AKT/mTOR) is frequently dysregulated in disorders of cell growth and survival, including a number of pediatric hematologic malignancies. The pathway can be abnormally activated in childhood acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic myelogenous leukemia (CML), as well as in some pediatric lymphomas and lymphoproliferative disorders. Most commonly, this abnormal activation occurs as a consequence of constitutive activation of AKT, providing a compelling rationale to target this pathway in many of these conditions. A variety of agents, beginning with the rapamycin analogue (rapalog) sirolimus, have been used successfully to target this pathway in a number of pediatric hematologic malignancies. Rapalogs demonstrate significant preclinical activity against ALL, which has led to a number of clinical trials. Moreover, rapalogs can synergize with a number of conventional cytotoxic agents and overcome pathways of chemotherapeutic resistance for drugs commonly used in ALL treatment, including methotrexate and corticosteroids. Based on preclinical data, rapalogs are also being studied in AML, CML, and non-Hodgkin's lymphoma. Recently, significant progress has been made using rapalogs to treat pre-malignant lymphoproliferative disorders, including the autoimmune lymphoproliferative syndrome (ALPS); complete remissions in children with otherwise therapy-resistant disease have been seen. Rapalogs only block one component of the pathway (mTORC1), and newer agents are under preclinical and clinical development that can target different and often multiple protein kinases in the PI3K/AKT/mTOR pathway. Most of these agents have been tolerated in early-phase clinical trials. A number of PI3K inhibitors are under investigation. Of note, most of these also target other protein kinases. Newer agents are under development that target both mTORC1 and mTORC2, mTORC1 and PI3K, and the triad of PI3K, mTORC1, and mTORC2. Preclinical data suggest these dual- and multi-kinase inhibitors are more potent than rapalogs against many of the aforementioned hematologic malignancies. Two classes of AKT inhibitors are under development, the alkyl-lysophospholipids (APLs) and small molecule AKT inhibitors. Both classes have agents currently in clinical trials. A number of drugs are in development that target other components of the pathway, including eukaryotic translation initiation factor (eIF) 4E (eIF4E) and phosphoinositide-dependent protein kinase 1 (PDK1). Finally, a number of other key signaling pathways interact with PI3K/AKT/mTOR, including Notch, MNK, Syk, MAPK, and aurora kinase. These alternative pathways are being targeted alone and in combination with PI3K/AKT/mTOR inhibitors with promising preclinical results in pediatric hematologic malignancies. This review provides a comprehensive overview of the abnormalities in the PI3K/AKT/mTOR signaling pathway in pediatric hematologic malignancies, the agents that are used to target this pathway, and the results of preclinical and clinical trials, using those agents in childhood hematologic cancers.

Dataset Information

Novel pharmacodynamic biomarkers for MYCN protein and PI3K/AKT/mTOR pathway signaling in children with neuroblastoma.

Publications

Novel pharmacodynamic biomarkers for MYCN protein and PI3K/AKT/mTOR pathway signaling in children with neuroblastoma.

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