Implementing Patient-Derived Xenografts to Assess the Effectiveness of Cyclin-Dependent Kinase Inhibitors in Glioblastoma.

Glioblastoma (GBM) is the most common primary brain tumor with no available cure. As previously described, seliciclib, a first-generation cyclin-dependent kinase (CDK) inhibitor, down-regulates the anti-apoptotic protein, Mcl-1, in GBM, thereby sensitizing GBM cells to the apoptosis-inducing effects of the death receptor ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we have assessed the efficacy of seliciclib when delivered in combination with the antibody against human death receptor 5, drozitumab, in clinically relevant patient-derived xenograft (PDX) models of GBM. A reduction in viability and significant levels of apoptosis were observed in vitro in human GBM neurospheres following treatment with seliciclib plus drozitumab. While the co-treatment strategy induced a similar effect in PDX models, the dosing regimen required to observe seliciclib-targeted responses in the brain, resulted in lethal toxicity in 45% of animals. Additional studies showed that the second-generation CDK inhibitor, CYC065, with improved potency in comparison to seliciclib, induced a significant decrease in the size of human GBM neurospheres in vitro and was well tolerated in vivo, upon administration at clinically relevant doses. This study highlights the continued need for robust pre-clinical assessment of promising treatment approaches using clinically relevant models.

Sensitization of glioblastoma cells to TRAIL-induced apoptosis by IAP- and Bcl-2 antagonism.

Due to the lack of effective treatments for glioblastoma (GBM), we here studied the responsiveness of GBM cell lines to the combination of death ligand, TRAIL and the IAP antagonist, TL32711 (Birinapant). Responses were highly heterogeneous, with synergistic apoptosis as well as treatment resistance observed. Caspase-8 and Bid, together with caspase-3, form a nonlinear signalling hub that efficiently induced apoptosis in responder cell lines. Cells resistant to TRAIL/TL32711 expressed low amounts of procaspase-8 and Bid and poorly activated caspase-3. We therefore hypothesised that improving caspase-8 activation or sensitising mitochondria to truncated Bid (tBid) could convert non-responder GBM cell lines to responders. Mathematical simulations of both strategies predicted mitochondrial sensitization to tBid would outperform enhancing caspase-8 activation. Indeed, antagonising Bcl-2 by ABT-199 allowed TRAIL/TL32711 response synergies to manifest in otherwise TRAIL resistant cell lines. These findings were further corroborated in experiments with a translationally relevant hexavalent TRAIL variant. Our study therefore demonstrates that a high caspase-8/Bid signature is associated with synergistic TRAIL/TL32711-induced apoptosis in GBM cells and outlines Bcl-2 antagonism as a highly potent intervention to sensitize highly TRAIL-resistant GBM cells to TRAIL/TL32711 combination treatment.

Analysis of the Impact of CD200 on Phagocytosis.

One factor that impacts on microglial activation is the interaction between the ubiquitously expressed CD200 and CD200R, which is expressed only on microglia in the brain. Decreased signalling through CD200R, when CD200 expression is reduced, results in microglial activation and may, at least in part, explain the increased cell activity that is observed with age, in models of Alzheimer's and Parkinson's disease as well as in the human diseases. There is evidence of increased microglial activation in CD200-deficient mice, and isolated microglia prepared from these mice are more reactive to inflammatory stimuli like Toll-like receptor 2 and 4 agonists, and interferon-γ. Here, we examined the impact of CD200 deficiency on amyloid-ß (Aß)-induced changes in microglia and report, perhaps unexpectedly, that the effect of Aß was attenuated in microglia prepared from CD200-deficient mice. The evidence indicates that this is a consequence of increased phagocytosis, associated with increased lysosomal activity in CD200-deficient microglia. The data suggest that mTOR-related signalling is decreased in these cells and that inhibiting mTOR by rapamycin increases phagocytosis. Thus, while the findings to date have emphasized the anti-inflammatory effects of CD200-CD200R interaction, the present evidence indicates a previously unreported impact on lysosomal function.

Targeting Autophagy in Glioblastoma.

The role of autophagy in cancer cell survival and cell death has received much attention in recent years; however, scientists are still trying to unravel the complex relationship that exists between autophagy as a tumor suppressor mechanism and as a promoter of tumor progression. In glioblastoma (GBM), the most fatal tumor of the central nervous system, mounting evidence suggests that autophagy processes are tightly intertwined with GBM tumorigenesis and the development of different molecular subtypes. This has led to exciting prospects that autophagy-targeted therapies may improve the efficacy of conventional therapies as well as therapies targeted at specific genetic alterations in individual GBM patients.

Predicting the cell death responsiveness and sensitization of glioma cells to TRAIL and temozolomide.

Genotoxic chemotherapy with temozolomide (TMZ) is a mainstay of treatment for glioblastoma (GBM); however, at best, TMZ provides only modest survival benefit to a subset of patients. Recent insight into the heterogeneous nature of GBM suggests a more personalized approach to treatment may be necessary to overcome cancer drug resistance and improve patient care. These include novel therapies that can be used both alone and with TMZ to selectively reactivate apoptosis within malignant cells. For this approach to work, reliable molecular signatures that can accurately predict treatment responsiveness need to be identified first. Here, we describe the first proof-of-principle study that merges quantitative protein-based analysis of apoptosis signaling networks with data- and knowledge-driven mathematical systems modeling to predict treatment responsiveness of GBM cell lines to various apoptosis-inducing stimuli. These include monotherapies with TMZ and TRAIL, which activate the intrinsic and extrinsic apoptosis pathways, respectively, as well as combination therapies of TMZ+TRAIL. We also successfully employed this approach to predict whether individual GBM cell lines could be sensitized to TMZ or TRAIL via the selective targeting of Bcl-2/Bcl-xL proteins with ABT-737. Our findings suggest that systems biology-based approaches could assist in personalizing treatment decisions in GBM to optimize cell death induction.

The endocannabinoid, anandamide, augments Notch-1 signaling in cultured cortical neurons exposed to amyloid-ß and in the cortex of aged rats.

Aberrant Notch signaling has recently emerged as a possible mechanism for the altered neurogenesis, cognitive impairment, and learning and memory deficits associated with Alzheimer disease (AD). Recently, targeting the endocannabinoid system in models of AD has emerged as a potential approach to slow the progression of the disease process. Although studies have identified neuroprotective roles for endocannabinoids, there is a paucity of information on modulation of the pro-survival Notch pathway by endocannabinoids. In this study the influence of the endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol, on the Notch-1 pathway and on its endogenous regulators were investigated in an in vitro model of AD. We report that AEA up-regulates Notch-1 signaling in cultured neurons. We also provide evidence that although Aß(1-42) increases expression of the endogenous inhibitor of Notch-1, numb (Nb), this can be prevented by AEA and 2-arachidonoylglycerol. Interestingly, AEA up-regulated Nct expression, a component of γ-secretase, and this was found to play a crucial role in the enhanced Notch-1 signaling mediated by AEA. The stimulatory effects of AEA on Notch-1 signaling persisted in the presence of Aß(1-42). AEA was found to induce a preferential processing of Notch-1 over amyloid precursor protein to generate Aß(1-40). Aging, a natural process of neurodegeneration, was associated with a reduction in Notch-1 signaling in rat cortex and hippocampus, and this was restored with chronic treatment with URB 597. In summary, AEA has the proclivity to enhance Notch-1 signaling in an in vitro model of AD, which may have relevance for restoring neurogenesis and cognition in AD.

The fatty acid amide hydrolase inhibitor URB597 exerts anti-inflammatory effects in hippocampus of aged rats and restores an age-related deficit in long-term potentiation.

BACKGROUND: Several factors contribute to the deterioration in synaptic plasticity which accompanies age and one of these is neuroinflammation. This is characterized by increased microglial activation associated with increased production of proinflammatory cytokines like interleukin-1ß (IL-1ß). In aged rats these neuroinflammatory changes are associated with a decreased ability of animals to sustain long-term potentiation (LTP) in the dentate gyrus. Importantly, treatment of aged rats with agents which possess anti-inflammatory properties to decrease microglial activation, improves LTP. It is known that endocannabinoids, such as anandamide (AEA), have anti-inflammatory properties and therefore have the potential to decrease the age-related microglial activation. However, endocannabinoids are extremely labile and are hydrolyzed quickly after production. Here we investigated the possibility that inhibiting the degradation of endocannabinoids with the fatty acid amide hydrolase (FAAH) inhibitor, URB597, could ameliorate age-related increases in microglial activation and the associated decrease in LTP. METHODS: Young and aged rats received subcutaneous injections of the FAAH inhibitor URB597 every second day and controls which received subcutaneous injections of 30% DMSO-saline every second day for 28 days. Long-term potentiation was recorded on day 28 and the animals were sacrificed. Brain tissue was analyzed for markers of microglial activation by PCR and for levels of endocannabinoids by liquid chromatography coupled to tandem mass spectrometry. RESULTS: The data indicate that expression of markers of microglial activation, MHCII, and CD68 mRNA, were increased in the hippocampus of aged, compared with young, rats and that these changes were associated with increased expression of the proinflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor-α (TNFα) which were attenuated by treatment with URB597. Coupled with these changes, we observed an age-related decrease in LTP in the dentate gyrus which was partially restored in URB597-treated aged rats. The data suggest that enhancement of levels of endocannabinoids in the brain by URB597 has beneficial effects on synaptic function, perhaps by modulating microglial activation.

The multiplicity of action of cannabinoids: implications for treating neurodegeneration.

The cannabinoid (CB) system is widespread in the central nervous system and is crucial for controlling a range of neurophysiological processes such as pain, appetite, and cognition. The endogenous CB molecules, anandamide, and 2-arachidonoyl glycerol, interact with the G-protein coupled CB receptors, CB(1) and CB(2). These receptors are also targets for the phytocannabinoids isolated from the cannabis plant and synthetic CB receptor ligands. The CB system is emerging as a key regulator of neuronal cell fate and is capable of conferring neuroprotection by the direct engagement of prosurvival pathways and the control of neurogenesis. Many neurological conditions feature a neurodegenerative component that is associated with excitotoxicity, oxidative stress, and neuroinflammation, and certain CB molecules have been demonstrated to inhibit these events to halt the progression of neurodegeneration. Such properties are attractive in the development of new strategies to treat neurodegenerative conditions of diverse etiology, such as Alzheimer's disease, multiple sclerosis, and cerebral ischemia. This article will discuss the experimental and clinical evidence supporting a potential role for CB-based therapies in the treatment of certain neurological diseases that feature a neurodegenerative component.

Endocannabinoids prevent ß-amyloid-mediated lysosomal destabilization in cultured neurons.

Neuronal cell loss underlies the pathological decline in cognition and memory associated with Alzheimer disease (AD). Recently, targeting the endocannabinoid system in AD has emerged as a promising new approach to treatment. Studies have identified neuroprotective roles for endocannabinoids against key pathological events in the AD brain, including cell death by apoptosis. Elucidation of the apoptotic pathway evoked by ß-amyloid (Aß) is thus important for the development of therapeutic strategies that can thwart Aß toxicity and preserve cell viability. We have previously reported that lysosomal membrane permeabilization plays a distinct role in the apoptotic pathway initiated by Aß. In the present study, we provide evidence that the endocannabinoid system can stabilize lysosomes against Aß-induced permeabilization and in turn sustain cell survival. We report that endocannabinoids stabilize lysosomes by preventing the Aß-induced up-regulation of the tumor suppressor protein, p53, and its interaction with the lysosomal membrane. We also provide evidence that intracellular cannabinoid type 1 receptors play a role in stabilizing lysosomes against Aß toxicity and thus highlight the functionality of these receptors. Given the deleterious effect of lysosomal membrane permeabilization on cell viability, stabilization of lysosomes with endocannabinoids may represent a novel mechanism by which these lipid modulators confer neuroprotection.

A role for p53 in the beta-amyloid-mediated regulation of the lysosomal system.

Beta-amyloid accumulates around neurons in Alzheimer's disease and is thought to contribute to the neurodegenerative process. This study examined the role of the tumour suppressor protein, p53, in the neurodegenerative pathway, with focus on the interaction of p53 with the lysosomal system. beta-Amyloid increased expression of p53 and its transcription target, Bax, in cultured cortical neurons. In addition, A beta increased the association of phospho-p53(ser15) with the lysosomal compartment and this correlated with destabilization of the lysosomal membrane and a concomitant increase in cytosolic cathepsin-L activity. These effects of beta-amyloid were abolished by the p53 inhibitor, pifithrin-alpha, and siRNA-mediated knockdown of p53, demonstrating that p53 is a critical regulator of lysosomal integrity and the induction of cathepsin-L protease activity. In addition, activation of the apoptotic cascade was abolished by pifithrin-alpha. We conclude that p53 associates with the lysosome to regulate a lysosomal branch of the apoptotic cascade which contributes to beta-amyloid-mediated neurodegeneration.