Surgical implantation of Gliadel, wafers coated with 1,3-Bis(2-Chloroethyl)-1-NitrosoUrea (BCNU), after tumor resection is shown to improve survival in patients with GlioBlastoma Multiforme (GBM). However, because of either rapidly acquired or intrinsic cellular resistance, the overall survival after aggressive treatment for GBM remains poor. Cellular resistance to BCNU is largely attributed to the activity of O6-AlkylGuanine DNA alkylTransferase (O6-AGT), an enzyme that restores the BCNU alkylated guanine to its native state and, thereby, prevents the formation of DNA cross-links. Here we identify the Fanconi Anemia (FA) pathway as another cellular mechanism conferring resistance to BCNU. The FA pathway was initially identified by virtue of its inactivation in patients with Fanconi Anemia, a rare autosomal recessive disease characterized by developmental abnormalities and cancer predisposition. Subsequent studies revealed that the FA pathway is crucial for general cellular response to DNA cross-link inducing agents, including cisplatin and mitomycin C. Genes involved in the FA pathway (FANCA, FANCC, FANCE, FANCF, and FANCG) encode for proteins that form a nuclear complex responsible for monoubiquitination of the downstream FANCD2 in response to DNA damage. The modified FANCD2, in turn, interacts with recombination machinery as well as specialized polymerases to repair the damaged DNA. To test whether the FA pathway mediates resistance to BCNU, isogenic FA deficient and retrovirally corrected pairs were treated with BCNU. BCNU treatment of FA proficient cell lines led to dose-dependent and time-dependent increase in the monoubiquitination of FANCD2 and the assembly of FANCD2 nuclear foci, both measures of the FA pathway activation. Importantly, FA deficient cells were more sensitive to BCNU relative to their corrected counter part. To extend these observations to human GBM cell lines, we demonstrate that a GBM cell line (HT15) defective in FA pathway activation exhibited increased BCNU sensitivity relative to FA proficient GBM cell lines. Moreover, a small molecule inhibitor of the FA pathway, curcumin, inhibited FANCD2 monoubiquitination in a dose dependent manner and caused increased sensitivity to BCNU in an FA proficient GBM cell line (U343). These results indicate that the integrity of the FA pathway is one of the important determinants of BCNU resistance in GBM cell lines and that inhibitors of the FA pathway constitute potential adjuvant therapeutic options in BCNU based combination therapy in the treatment of brain cancer.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]