Actinomycin D downregulates Sox2 and improves survival in preclinical models of recurrent glioblastoma

Abstract

Background Glioblastoma (GBM) has been extensively researched over the last few decades, yet despite aggressive multimodal treatment, recurrence is inevitable and second-line treatment options are limited. Here, we demonstrate how high-throughput screening (HTS) in multicellular spheroids can generate physiologically relevant patient chemosensitivity data using patient-derived cells in a rapid and cost-effective manner. Our HTS system identified actinomycin D (ACTD) to be highly cytotoxic over a panel of 12 patient-derived glioma stemlike cell (GSC) lines. ACTD is an antineoplastic antibiotic used in the treatment of childhood cancers. Here, we validate ACTD as a potential repurposed therapeutic for GBM in 3-dimensional GSC cultures and patient-derived xenograft models of recurrent glioblastoma. Methods Twelve patient-derived GSC lines were screened at 10 µM, as multicellular spheroids, in a 384-well serum-free assay with 133 FDA-approved compounds. GSCs were then treated in vitro with ACTD at established half-maximal inhibitory concentrations (IC50). Downregulation of sex determining region Y–box 2 (Sox2), a stem cell transcription factor, was investigated via western blot and through immunohistological assessment of murine brain tissue. Results Treatment with ACTD was shown to significantly reduce tumor growth in 2 recurrent GBM patient-derived models and significantly increased survival. ACTD is also shown to specifically downregulate the expression of Sox2 both in vitro and in vivo. Conclusion These findings indicate that, as predicted by our HTS, ACTD could deplete the cancer stem cell population within the tumor mass, ultimately leading to a delay in tumor progression. Key Points 1. High-throughput chemosensitivity data demonstrated the broad efficacy of actinomycin D, which was validated in 3 preclinical models of glioblastoma. 2. Actinomycin D downregulated Sox2 in vitro and in vivo, indicating that this agent could target the stem cell population of GBM tumors.

Investigating the Central Nervous System Disposition of Actinomycin D: Implementation and Evaluation of Cerebral Microdialysis and Brain Tissue Measurements Supported by UPLC-MS/MS Quantification

“…Nevertheless, comprehensive information on its central nervous system (CNS) disposition is lacking. However, previous studies suggested a potential role of actinomycin D in the treatment of pediatric CNS neoplasms: Preclinical studies revealed efficacy in in vitro models of ependymoma [ 15 ] and, to a certain degree, in orthotopic mouse models of glioblastoma, medulloblastoma, and embryonal tumors with multilayered rosettes (ETMR) [ 16 , 17 , 18 ]. Therefore, our aim was to establish a suitable experimental setup to accurately determine the still unclear CNS disposition of actinomycin D. This setup combined quantification of actinomycin D by ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) in cerebral microdialysis microsamples from freely moving mice with measurements in brain tissue and plasma.…”

Store-Operated Calcium Channels Control Proliferation and Self-Renewal of Cancer Stem Cells from Glioblastoma

“…To further confirm the effects of SOC inhibitors on the stem cell population, the expression of SOX2 was assessed. SOX2, which is a transcription factor necessary for the maintenance of glioblastoma stem cell tumorigenic activity, is commonly used to detect the stem cell population in glioblastoma [ 32 , 33 , 34 ]. Figure 5 C illustrates that SOX2 expression was significantly decreased upon treatment with YM-58483 in all GBM cells tested and with GSK-7975A in 2 of 3 GBM cells tested, confirming a role of SOC in the maintenance or expansion of GSC.…”

An Alternative Pipeline for Glioblastoma Therapeutics: A Systematic Review of Drug Repurposing in Glioblastoma

“…The topic of antineoplastic repurposing will only be briefly discussed given that the mechanisms of action of these drugs against GBM are the same as their original hypothesized mechanism. Many of these agents have been investigated preclinically including hydroxyurea, actinomycin D, carmofur, eribulin and paclitaxel [ 186 , 187 , 188 , 189 , 190 , 191 , 192 , 193 , 194 , 195 ]. With regard to clinical trials, vorinostat, an antineoplastic agent used for cutaneous T-cell lymphoma, was studied in a Phase I study in combination with TMZ [ 117 ].…”