Targeting DNA Double-Strand Break Repair to Potentiate Radio- and Chemo Therapy of Glioblastoma

Date

2015-08-03

Authors

Gil del Alcazar, Carlos Rodrigo

Journal Title

Journal ISSN

Volume Title

Publisher

Content Notes

Abstract

Surgical resection followed by radiation and adjuvant temozolomide (TMZ) is the standard of care for glioblastoma (GBM). Not all GBMs respond to therapy, and most of them quickly acquire resistance to TMZ and recur. In order to develop more effective and rational treatments for GBM, it is crucial to understand the mechanisms underlying radio- and chemo-resistance. We find that protracted TMZ treatment of mice bearing orthotopic tumors (generated by xenografting GBM9 neurospheres) leads to acquired-TMZ resistance resulting in tumor recurrence. In order to understand the basis for therapy-driven TMZ resistance, we generated and functionally characterized ex-vivo cultures from the primary and recurrent tumors. We found that cell lines derived from recurrent (TMZ-treated) tumors were more resistant to TMZ in vitro compared to cell lines derived from primary (untreated) tumors. We also found that the increased resistance to TMZ was due to the augmented repair of TMZ-induced DNA double strand breaks (DSBs). TMZ induces DNA replication-associated DSBs that are repaired primarily by the homologous recombination (HR) pathway. We found that cell lines from recurrent cultures exhibited faster resolution of Rad51 foci and higher levels of sister chromatid exchanges (SCEs), which indicated that a higher level of HR was contributing to TMZ resistance in these lines. We have recently shown that CDKs 1 and 2 promote HR in S and G2 phases of the cell cycle, in part, by phosphorylating the exonuclease EXO1. We hypothesized, therefore, that blocking CDKs 1 and 2 might be a viable strategy for re-sensitizing recurrent tumors to TMZ. Indeed, we found that CDK inhibitors, AZD5438 and Roscovitine, could attenuate HR in the recurrent TMZ-resistant cell lines resulting in significant chemo-sensitization to TMZ. While HR is primarily involved in the repair of TMZ-induced DSBs, ionizing radiation induced DSBs would be mainly repaired by the non-homologous end joining (NHEJ) pathway. We, therefore, developed another approach to sensitize these tumors to both radiation and TMZ by using a dual PI3K/mTOR inhibitor, NVP-BEZ235, to block both DNA-PKcs and ATM, key enzymes in the NHEJ and HR pathways, respectively. We found that NVP-BEZ235 inhibited both DNA-PKcs and ATM in tumors generated in mice from GBM9 neurospheres, thus blocking the repair of TMZ- and IR-induced DSBs, and resulting in significant chemo- and radio-sensitization. Next, we established that NVP-BEZ235 can cross the blood-brain barrier and recapitulated its radiosensitizing effects in an intracranial GBM model. We found that NVP-BEZ235, administered with IR, can attenuate DSB repair in these intracranial tumors, attenuate tumor growth, and extend survival of tumor-bearing mice. Importantly, attenuation of DSB repair was more pronounced in tumor cells compared to normal brain cells, thereby providing a larger therapeutic window. In sum, this study indicates that augmented DNA repair may underlie therapy resistance in GBM and provides support for the potential use of DNA repair inhibition as an effective strategy for improving the efficacy of GBM therapy.

General Notes

Table of Contents

Citation

Related URI