De Novo Purine Synthesis Is a Targetable Vulnerability That Confers Radiation Resistance and Inferior Patient Survival in IDH-Wild Type Diffuse Glioma

Abstract

We sought to understand how metabolic phenotypes mediate RT resistance in isocitrate dehydrogenase wild type diffuse glioma (IDHwtGBM) to define genomic-independent metabolic radiosensitization strategies. Steady state metabolomics and clonogenic survival curves were performed in 23 IDHwtGBM cell lines during exponential growth. Relationships between metabolites and RT resistance were determined using metabolic pathway analysis and Spearman correlations. γH2AX foci were enumerated using immunofluorescence. Patient-derived IDHwtGBM neurospheres were grown under stem cell conditions. Treatment of GBM xenografts was with RT (2 Gy x 5), mycophenolate mofetil (MMF, 120 mg/kg x 6 doses) or the combination. Increased abundance of metabolites involved in purine metabolic pathways (guanine and inosine) was positively correlated with RT resistance, while increased abundance of those involved in energy production (e.g., glycolysis and lipid metabolism) was negatively correlated (p<0.05 for all). RT resistance was also associated with the ability to repair γH2AX foci after RT but not targetable transcriptionally-defined pathways, cell-cycle distribution or proliferation rates. Supplementing purine-poor radiosensitive IDHwtGBM cell lines with cell-permeable purine precursors (nucleosides) decreased γH2AX foci staining 2-3 fold following RT and protected cells from RT (enhancement ratios 0.6-0.8). Mycophenolic acid (MPA), an inhibitor of de novo guanine synthesis, depleted GTP by 90%, increased RT-induced γH2AX staining 2-3 fold and radiosensitized multiple purine-rich IDHwtGBM cell lines and neurospheres (enhancement ratios 2-3). These effects were rescued by exogenous nucleosides. Inhibition of de novo pyrimidine synthesis using teriflunomide had no effect on radiosensitivity or γH2AX resolution. Treatment with MMF (an FDA-approved, orally-bioavailable prodrug of MPA) modestly slowed GBM xenograft growth in vivo (increased median tumor tripling time from 8 to 11 days), significantly sensitized tumors to RT (tumor tripling time not reached) and depleted intratumoral GTP 3-fold when combined with RT. In 232 patients from the Pan-Cancer Atlas with newly diagnosed IDHwtGBM, increased expression (>median) of IMPDH1, the rate limiting enzyme in de novo guanine synthesis and target of MPA, was associated with inferior overall survival (HR 0.59, 95% CI 0.43-0.81, p=0.001). Increased expression of the rate limiting enzymes of de novo pyrimidine or adenine synthesis were not associated with inferior survival (HR 1.3, 95% CI 0.96-1.77 or HR 1.5, 95% CI 1.09-2.04). RT resistance in IDHwtGBM is mediated in part by elevated purine levels, which can be overcome by inhibition of de novo purine synthesis. Because many of the genomic drivers of IDHwtGBM stimulate de novo purine synthesis, its inhibition represents a promising metabolic strategy to radiosensitize this genomically heterogeneous disease.