Temozolomide-Derived AIC Is Incorporated into Purine Synthesis in Glioblastoma

Abstract

Glioblastoma (GBM) is a lethal brain tumor with limited therapeutic options. Temozolomide (TMZ), a standard-of-care chemotherapeutic agent, exerts its cytotoxicity by alkylating DNA, which triggers a DNA damage response and depletes ATP and NAD⁺. However, TMZ also releases the byproduct 4-amino-5-imidazole carboxamide (AIC), which is believed to be a benign metabolite. We considered the possibility that AIC from TMZ could enter the de novo purine synthesis pathway, contributing to AMP and NAD⁺ synthesis and thus potentially antagonizing the anticancer activity of TMZ. The purpose of this article is to determine if AIC from TMZ can be incorporated into cellular purines. Using mass spectrometry with isotope-labeled TMZ, we demonstrate that the AIC derived from TMZ is incorporated into AMP and NAD⁺ in glioblastoma cell lines. Further, we performed an analysis of publicly available transcriptomic data from the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Our analyses demonstrate that de novo purine synthesis is upregulated in GBM relative to the normal brain. Collectively, our findings demonstrate that a drug metabolite of TMZ, AIC, can be incorporated into de novo purine synthesis, which is upregulated in GBM.

1

Hydrolysis of Temozolomide forms AIC and its active methyldiazonium alkylating agent.

2

TMZ-derived AIC incorporated into purine biosynthesis. Here, we represent the de novo (light blue) and purine salvage synthesis (magenta) pathways as well as the proposed TMZ-derived AIC salvage pathway (dark blue). Abbreviations for the purine metabolites are the following: PRPP: phosphoribosyl pyrophosphate, 5-PRA: 5-phosphoribosylamine, GAR: glycinamide ribonucleotide, AIR: aminoimidazole ribonucleotide, SAICAR: succinylaminoimidazole carboxamide ribonucleotide, AIC: 4-amino-5-imidazole carboxamide, AICAR: aminoimidazole carboxamide ribonucleotide, FAICAR: formamidoimidazole carboxamide ribonucleotide, IMP: inosine monophosphate, AMP: adenosine monophosphate, GMP: guanosine monophosphate, ADP: adenosine diphosphate, ATP: adenosine triphosphate, dATP: deoxyadenosine triphosphate, NAD⁺: nicotinamide adenine dinucleotide.

3

Isotope-labeled AIC + 3 is incorporated into AMP and NAD⁺ in U87 cells grown under adherent or serum-free tumorsphere-forming conditions. (A,B) U87 cells were grown in the presence of serum for adherent cell conditions or serum-free for stem-like conditions that form tumorspheres. See Materials and Methods for details. (C,D) Cells were incubated with increasing concentrations of AIC + 3 for 24 h in either adherent or tumorsphere conditions. Metabolites were extracted, and isotopic enrichment of AMP and NAD⁺ was measured by LC–MS/MS, as described in the Materials and Methods section. Error bars represent four independent replicate injections.

4

Isotope-labeled AIC + 3 is incorporated into AMP and NAD⁺ in patient-derived glioblastoma stem cell lines. (A) Representative brightfield images of three different patient derived xenograft (PDX) cell lines, GBM10, GBM39, and GBM85. Cells were grown under various concentrations of AIC + 3 for 24 h. Metabolites were extracted, and isotopic enrichment of AMP and NAD⁺ was measured by LC–MS/MS, as described in the Materials and Methods section (B,C). The cell lines were obtained from the Mayo clinic and were characterized with the following phenotype and genotypes. All cell lines were IDH1/2 wild-type and had TERT promoter mutations at either C228T or C250T. GBM10: Mesenchymal phenotype, EGFR amplified, MGMT unmethylated. GBM39: Mesenchymal phenotype, EGFR VIII, MGMT methylated. GBM85: Proneural phenotype, EGFR mosaic gain, MGMT methylated. Error bars represent four independent replicate injections.

5

Temozolomide-derived AIC as well as hypoxanthine are incorporated into the purine synthesis pathway. (A) Our synthetic scheme for the synthesis of isotopically labeled TMZ involved making isotope-labeled hypoxanthine and AIC intermediates. See Materials and Methods and Supporting Information for details on synthesis and characterization. (B) Isotope-labeled hypoxanthine, AIC, and TMZ were incubated at various concentrations with U87 cells for 24 h grown under serum-containing adherent conditions. Metabolites were extracted, and isotopic enrichment of AMP and NAD⁺ was measured by LC–MS/MS, as described in the Materials and Methods section. We confirm our prior data using commercially purchased AIC + 3 as it aligns with our synthesized AIC + 1. Similarly, using isotope-labeled TMZ + 1, incorporation of TMZ-derived AIC was nearly identical to that of AIC + 1. When we also tested if Hx could be salvaged via the purine salvage pathway, U87 cells were even more efficient at using Hx than AIC to generate AMP and NAD⁺. Error bars represent four independent replicate injections.

6

De novo and purine salvage biosynthesis are upregulated in GBM patients. (A) Purine biosynthesis pathway scheme with upregulated enzymes and downregulated enzymes shown in red and green, respectively. Abbreviations of each metabolite are described in the legend of Figure . (B) Using the UCSC Xena platform, we analyzed gene expression for these genes in GBM tumors from the TCGA study compared to both adjacent normal tissue as well as normal brain tissue derived from the Genotype-Tissue Expression (GTEx) study. RNA expression values were normalized using DESeq2, log₂-transformed, and expressed as log₂(RSEM expected count (DESeq2) + 1). Normal brain cortex tissue of noncancer patients (n = 105) from the GTEx database was compared to tissue samples from IDHWT TCGA-GBM patients. Gene expression values were also obtained from the TCGA-GBM database from normal adjacent tissue (n = 5), primary GBM tumor (n = 140), and recurrent GBM tumors (n = 13). Statistical significance was determined by ANOVA with Tukey’s Multiple Comparison Test. Asterisks denote significance level: *p < 0.05, ***p < 0.001, ****p < 0.0001.