Limited antitumor activity of combined BET and MEK inhibition in neuroblastoma.

BACKGROUND: The treatment of high-risk neuroblastoma continues to present a formidable challenge to pediatric oncology. Previous studies have shown that Bromodomain and extraterminal (BET) inhibitors can inhibit MYCN expression and suppress MYCN-amplified neuroblastoma in vivo. Furthermore, alterations within RAS-MAPK (mitogen-activated protein kinase) signaling play significant roles in neuroblastoma initiation, maintenance, and relapse, and mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors demonstrate efficacy in subsets of neuroblastoma preclinical models. Finally, hyperactivation of RAS-MAPK signaling has been shown to promote resistance to BET inhibitors. Therefore, we examined the antitumor efficacy of combined BET/MEK inhibition utilizing I-BET726 or I-BET762 and trametinib in high-risk neuroblastoma. PROCEDURE: Utilizing a panel of genomically annotated neuroblastoma cell line models, we investigated the in vitro effects of combined BET/MEK inhibition on cell proliferation and apoptosis. Furthermore, we evaluated the effects of combined inhibition in neuroblastoma xenograft models. RESULTS: Combined BET and MEK inhibition demonstrated synergistic effects on the growth and survival of a large panel of neuroblastoma cell lines through augmentation of apoptosis. A combination therapy slowed tumor growth in a non-MYCN-amplified, NRAS-mutated neuroblastoma xenograft model, but had no efficacy in an MYCN-amplified model harboring a loss-of-function mutation in NF1. CONCLUSIONS: Combinatorial BET and MEK inhibition was synergistic in the vast majority of neuroblastoma cell lines in the in vitro setting but showed limited antitumor activity in vivo. Collectively, these data do not support clinical development of this combination in high-risk neuroblastoma.

Parvalbumin interneuron vulnerability and brain disorders.

Parvalbumin-expressing interneurons (PV-INs) are highly vulnerable to stressors and have been implicated in many neuro-psychiatric diseases such as schizophrenia, Alzheimer's disease, autism spectrum disorder, and bipolar disorder. We examined the literature about the current knowledge of the physiological properties of PV-INs and gathered results from diverse research areas to provide insight into their vulnerability to stressors. Among the factors that confer heightened vulnerability are the substantial energy requirements, a strong excitatory drive, and a unique developmental trajectory. Understanding these stressors and elaborating on their impact on PV-IN health is a step toward developing therapies to protect these neurons in various disease states and to retain critical brain functions.

Robust Expression of Functional NMDA Receptors in Human Induced Pluripotent Stem Cell-Derived Neuronal Cultures Using an Accelerated Protocol.

N-methyl-D-aspartate (NMDA) receptors are critical for higher-order nervous system function, but in previously published protocols to convert human induced pluripotent stem cells (iPSCs) to mature neurons, functional NMDA receptors (NMDARs) are often either not reported or take an extended time to develop. Here, we describe a protocol to convert human iPSC-derived neural progenitor cells (NPCs) to mature neurons in only 37 days. We demonstrate that the mature neurons express functional NMDARs exhibiting ligand-activated calcium flux, and we document the presence of NMDAR-mediated electrically evoked postsynaptic current. In addition to being more rapid than previous procedures, our protocol is straightforward, does not produce organoids which are difficult to image, and does not involve co-culture with rodent astrocytes. This could enhance our ability to study primate/human-specific aspects of NMDAR function and signaling in health and disease.

Reduction of Leukocyte Microvascular Adherence and Preservation of Blood-Brain Barrier Function by Superoxide-Lowering Therapies in a Piglet Model of Neonatal Asphyxia.

Background: Asphyxia is the most common cause of brain damage in newborns. Substantial evidence indicates that leukocyte recruitment in the cerebral vasculature during asphyxia contributes to this damage. We tested the hypothesis that superoxide radical ( O 2 ⋅ _ ) promotes an acute post-asphyxial inflammatory response and blood-brain barrier (BBB) breakdown. We investigated the effects of removing O 2 ⋅ _ by superoxide dismutase (SOD) or C3, the cell-permeable SOD mimetic, in protecting against asphyxia-related leukocyte recruitment. We also tested the hypothesis that xanthine oxidase activity is one source of this radical. Methods: Anesthetized piglets were tracheostomized, ventilated, and equipped with closed cranial windows for the assessment of post-asphyxial rhodamine 6G-labeled leukocyte-endothelial adherence and microvascular permeability to sodium fluorescein in cortical venules. Asphyxia was induced by discontinuing ventilation. SOD and C3 were administered by cortical superfusion. The xanthine oxidase inhibitor oxypurinol was administered intravenously. Results: Leukocyte-venular adherence significantly increased during the initial 2 h of post-asphyxial reperfusion. BBB permeability was also elevated relative to non-asphyxial controls. Inhibition of O 2 ⋅ _ production by oxypurinol, or elimination of O 2 ⋅ _ by SOD or C3, significantly reduced rhodamine 6G-labeled leukocyte-endothelial adherence and improved BBB integrity, as measured by sodium fluorescein leak from cerebral microvessels. Conclusion: Using three different strategies to either prevent formation or enhance elimination of O 2 ⋅ _ during the post-asphyxial period, we saw both reduced leukocyte adherence and preserved BBB function with treatment. These findings suggest that agents which lower O 2 ⋅ _ in brain may be attractive new therapeutic interventions for the protection of the neonatal brain following asphyxia.

Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells.

Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 10(6) white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.