Targeting Metabolic Reprogramming to Radiosensitize Glioblastoma Stem Cells

Abstract

Glioblastoma is the most prevalent and lethal primary intrinsic tumor in the central nervous system. Metabolic reprogramming, a hallmark of cancer, promotes tumor cell proliferation and survival. Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Understanding the metabolic regulation of GSCs may inform novel therapeutic approaches to improved clinical outcome for glioblastoma patients. In current study, we aim to interrogate the aberrant metabolic pattern in GSCs and develop novel targeted approach to sensitize GSC to radiation. Patient-derived GSCs were validated using functional assays. Metabolomic analysis of GSCs and matched differentiated glioblastoma cells (DGCs) was performed using carbon tracing followed by non-targeted mass spectrometry to identify aberrant metabolic pathways upregulated in GSCs. Whole-genome promoter/enhancer analysis with chromatin immunoprecipitation against histone 3 lysine 27 acetylation followed by deep sequencing (H3K27ac ChIPseq) was used to validate these identified metabolic pathways in GSCs. Quantitative RT-PCR and Western blotting were performed assess the relative expression between GSCs and DGCs. Radiation sensitivity was measured using Cs-137 irradiator. Validation of the stem-like function of target metabolic gene was performed using RNA interference followed by in vitro neurosphere formation assay and in vivo tumorigenesis with xenograft model. We previously showed that increased glucose influx is a key survival mechanism in GSCs. Metabolomic profiling identified the upregulation of metabolites of pyrimidine synthesis pathway in GSCs to channel carbon flow from glucose. The activation of pyrimidine synthetic genes in GSCs was validated with unbiased enrichment analysis of whole-genome promoter/enhancer pattern. Targeting the pyrimidine synthetic rate-limiting step enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamyolase, dihydroorotase) or the critical downstream enzyme, DHODH (dihydroorotate dehydrogenase) inhibited the survival, self-renewal, and in vivo tumor initiation of GSCs through the depletion of the pyrimidine nucleotide supply. Core glioblastoma driver mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of GSC tumorigenic capacity and sensitization to radiation. Higher expression of pyrimidine synthesis genes portended a poor prognosis of glioblastoma patients. Our results demonstrate a novel therapeutic approach of sensitizing GSC to radiation through targeting the nexus between driver mutations and metabolic reprogramming in stem-like glioma cells.