Abstract
PurposePediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives.MethodsWe performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional ‘druggable’ GABAAR in group 3 cells.ResultsAnalysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, β3 and γ2 and 3. There are ~ 1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 109 ions/s. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine (‘KRM-II-08’) binds to the α5-GABAAR (0.8 µM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization.ConclusionGABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.
Highlights
Medulloblastoma is a significant cause of cancer-related morbidity and mortality in children [1]
We report on analysis of G ABAAR and MYC expression in 763 primary medulloblastoma patient tumors, characterization of GABAAR in a patient-derived cell line, identification of chemical features critical to α5-GABAAR preferring benzodiazepine potency, and examination of how such benzodiazepines may impair group 3 cell viability
We analyzed GABR and MYC expression across all subgroups in 763 resected primary medulloblastoma tumors [11] (Fig. 1b, c; Online Resource 1, 2; Online Tables 2, 3). This analysis reveals that: (1) all subgroups have shared high expression of select GABR genes; (2) there is subgroupspecific high expression of some GABR genes and some subgroups have GABR expression that is specific to only a subset of patients within the subgroup; (3) there is a positive correlation in expression of GABRA5 and MYC in a subset of group 3 and more surprisingly WNT tumors
Summary
Medulloblastoma is a significant cause of cancer-related morbidity and mortality in children [1]. Medulloblastoma molecular profiling delineated four subgroups, by consensus termed wingless (WNT), sonic hedgehog (SHH), group 3, and group 4 [5,6,7]. WNT and SHH exhibit anomalous expression of genes associated with the Wnt and Shh pathways, consistent with genomic alterations [8,9,10]. Groups 3 and 4, which account for ~ 60% of medulloblastomas and include those with poorest prognosis, do not have shared subgroup-specific genomic alterations. Group 3 tumors are typically TP53 wild-type and its high expression is associated with poor prognosis [12, 13]. Group 3 tumors share high expression of GABRA5, which codes for the α5-subunit of the ligand-gated ionotropic γ-aminobutyric acid type A receptor (GABAAR) [6]
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