One-carbon-mediated purine synthesis underlies temozolomide resistance in glioblastoma

Abstract

Glioblastoma accounts for nearly half of all primary malignant brain tumors in adults, and despite an aggressive standard of care, including excisional surgery and adjuvant chemoradiation, recurrence remains universal, with an overall median survival of 14.6 months. Recent work has revealed the importance of passenger mutations as critical mediators of metabolic adaptation in cancer progression. In our previous work, we identified a role for the epigenetic modifier ID-1 in temozolomide resistance in glioblastoma. Here, we show that ID-1-mediated glioblastoma tumourigenesis is accompanied by upregulation of one-carbon (1-C) mediated de novo purine synthesis. ID-1 knockout results in a significant reduction in the expression of 1-C metabolism and purine synthesis enzymes. Analysis of glioblastoma surgical specimens at initial presentation and recurrence reveals that 1-C purine synthesis metabolic enzymes are enriched in recurrent glioblastoma and that their expression correlates with a shorter time to tumor recurrence. Further, we show that the 1-C metabolic phenotype underlies proliferative capacity and temozolomide resistance in glioblastoma cells. Supplementation with exogenous purines restores proliferation in ID-1-deficient cells, while inhibition of purine synthesis with AICAR sensitizes temozolomide-resistant glioblastoma cells to temozolomide chemotherapy. Our data suggest that the metabolic phenotype observed in treatment-resistant glioma cells is a potential therapeutic target in glioblastoma.

Fig. 1. TMZ enriches for ID-1 high glioblastoma cells with increased expression of 1-C mediated purine synthesis enzymes.

A Western blot analysis ratio of ID-1, DHFR, MTHFD2, PAICS, and ATIC expression in U251 glioblastoma cells after treatment for 3 days with 100 µM TMZ. B Quantitative densitometry ratios of ID-1, DHFR, MTHFD2, PAICS, and ATIC expression from western blot analysis. The data are normalized to untreated control and presented as mean ± SD. (*p ≤ 0.5, **p ≤ 0.05, ***p ≤ 0.01 by two-tailed t-test, N = 3).

Fig. 2. Reduction in ID-1 expression results in changes in 1-C mediated de novo purine synthesis.

A Western blot analysis and quantitative densitometry ratios of 1-C metabolism enzymes DHFR and MTHFD2 in U251, ID-1^−/−.U251.1, and ID-1^−/−.U251.2 glioblastoma cells. B Western blot analysis and quantitative densitometry ratios of de novo purine synthesis enzymes PAICS and ATIC in U251, ID-1^−/−.U251.1, and ID-1^−/−.U251.2 glioblastoma cells. C LC-MS results showing a relative concentration of IMP, AMP, GMP, and AICAR in U251 and ID-1^−/−.U251.1 cells. D Western blot analysis and quantitative densitometry ratios of ADSL/AMPS expression in U25 and ID-1^−/−.U251.1 cells. All data are normalized to untreated control and presented as mean ± SD. (*p ≤ 0.5, **p ≤ 0.05, ***p ≤ 0.01 by two-tailed t-test, N = 3).

Fig. 3. Chemical inhibition of ID-1 results in a reduction in expression 1-C metabolism and de novo purine synthesis enzymes.

A Western blot analysis and quantitative densitometry ratios of ID-1, DHFR, MTHFD2, PAICS, and ATIC expression in U251 glioblastoma cells after 3-day treatment with 5 µM pimozide. B Western blot analysis and quantitative densitometry ratios of ID-1, DHFR, MTHFD2, PAICS, and ATIC expression in GliNS1 glioblastoma cells after 3-day treatment with 5 µM pimozide. C Western blot analysis and quantitative densitometry ratios of ID-1, DHFR, MTHFD2, PAICS, and ATIC expression in G811 glioblastoma cells after 3-day treatment with 5 µM pimozide. All data are normalized to untreated control and presented as mean ± SD. (*p ≤ 0.5, **p ≤ 0.05, ***p ≤ 0.01 by two-tailed t-test, N = 3).

Fig. 4. Exogenous purine supplementation restores proliferative capacity in U251-ID1-null glioblastoma cells.

A Cell proliferation and viability analysis of U251, ID-1^−/−.U251.1, and ID-1^−/−.U251.2 cells after supplementation with 0 µM and 100 µM hypoxanthine for 3 days. The data are normalized to untreated control and presented as mean ± SD. B Cell proliferation and viability analysis of U251, ID-1^−/−.U251.1, and ID-1^−/−.U251.2 cells after supplementation with 100 µM hypoxanthine over the course of 7 days. Viability measurements were made at 1, 3, 5, and 7 days. The data are normalized to untreated control and presented as mean ± SD. C Relative change in viability and proliferation in hypoxanthine-supplemented and unsupplemented U251, ID-1^−/−.U251.1, and ID-1^−/−.U251.2 cells. The data are presented as mean ± SD change in fluorescence intensity. (*p ≤ 0.5, **p ≤ 0.05, ***p ≤ 0.01 by two-tailed t-test, N = 3).

Fig. 5. AICAR accumulation is associated with increased AMPK activation in U251-ID-1-null cells and sensitizes glioblastoma cells to TMZ chemotherapy.

A Western blot analysis and quantitative densitometry ratios of AMPK and p-AMPK (Thr172) expression in ID-1^−/−.U251.1, ID-1^−/−.U251.2, and U251 cells. B Viability analysis of U251 and U251-TR cells treated with 5 µM AICAR, 50 µM TMZ, or a combination (5 µM AICAR + 50 µM TMZ) for 3 days. C Western blot analysis and quantitative densitometry ratios of AMPK and p-AMPK (Thr172) expression in U251 and U251-TR cells after treatment with 5 µM AICAR, 100 µM TMZ, and combined treatment. All data are normalized to untreated control and presented as mean ± SD. (*p ≤ 0.5, **p ≤ 0.05, ***p ≤ 0.01 by two-tailed t-test, N = 3).

Fig. 6. TMZ chemotherapy enriches for ID-1 and 1-C mediated purine synthesis enzymes in glioblastoma patients.

ID-1 and metabolic enzyme expression studies were performed on samples with pathologically confirmed glioblastoma recurrence following temozolomide chemotherapy. ID-1-high: cases with more than a 2-fold increase in ID-1-expression after recurrence, ID-1-low: cases with less than 2-fold increase in ID-1-expression after recurrence. A Representative images from IHC analysis of primary and recurrent glioblastoma cases from ID-1-low (case A) and ID-1-high (case B) tumors stained for PAICS, MTHFD2, and DHFR. B Percentage of cells staining positive for DHFR, MTHFD2, and PAICS from ID-1-low (case A, D, and F) and ID-1-high (case B, C, and E) cases. Blue trendline represents primary cases, orange trendline represents recurrent cases. All data are presented as absolute mean percentage values of 6 fields of view per case ± SD. (ns = non-significant, p > 0.5, *p ≤ 0.5, **p ≤ 0.05, ***p ≤ 0.01 by two-tailed t-test).