Alkylpurine–DNA–N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients
Sameer Agnihotri, Aaron S. Gajadhar, Christian Ternamian, Thierry Gorlia, Kristin L. Diefes, Paul S. Mischel, Joanna Kelly, Gail McGown, Mary Thorncroft, Brett L. Carlson, Jann N. Sarkaria, Geoffrey P. Margison, Kenneth Aldape, Cynthia Hawkins, Monika Hegi, Abhijit Guha
Sameer Agnihotri, Aaron S. Gajadhar, Christian Ternamian, Thierry Gorlia, Kristin L. Diefes, Paul S. Mischel, Joanna Kelly, Gail McGown, Mary Thorncroft, Brett L. Carlson, Jann N. Sarkaria, Geoffrey P. Margison, Kenneth Aldape, Cynthia Hawkins, Monika Hegi, Abhijit Guha
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Research Article Oncology

Alkylpurine–DNA–N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients

Abstract

Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). O6-methylguanine–DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O6-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine–DNA–N-glycosylase (APNG), which repairs the cytotoxic lesions N3-methyladenine and N7-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.

Authors

Sameer Agnihotri, Aaron S. Gajadhar, Christian Ternamian, Thierry Gorlia, Kristin L. Diefes, Paul S. Mischel, Joanna Kelly, Gail McGown, Mary Thorncroft, Brett L. Carlson, Jann N. Sarkaria, Geoffrey P. Margison, Kenneth Aldape, Cynthia Hawkins, Monika Hegi, Abhijit Guha

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Figure 2

APNG confers resistance to GBM cells in vitro.

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APNG confers resistance to GBM cells in vitro. (A) Cell viability of A17...
(A) Cell viability of A172 cells expressing EV, MGMT, APNG, or both MGMT and APNG in response to varying amounts of TMZ. (B) Representative Western blot of A172 cells stably expressing APNG, MGMT, or both under 100 μM TMZ. Cleaved PARP was used as a measure of apoptosis. Densitometry values (cleaved PARP relative to β-ACTIN) are shown below. (C) Western blot and cell viability in response to TMZ of site-directed APNG mutant R182A compared with WT APNG. (D) Western blot and cell viability in response to TMZ of site-directed MGMT mutant C145G compared with WT MGMT. (E) Activated cleaved caspase 3/7 levels of A172 cells expressing APNG, MGMT, or both. (F) Comet tail assay measuring DNA damage of A172 cells exposed to 100 μM TMZ. Comet tail length, indicative of less DNA damage, was reduced by expression of APNG and/or MGMT, but not their corresponding catalytic dead mutants. *P < 0.05, **P < 0.01.