1508 Background: Resistance to chemotherapy is major impediment to the successful treatment of human glioblastomas. METHODS: We used an integrated resistance model and genomics tools to globally explore molecular factors and cellular pathways mediating resistance to O(6)-alkylating agents in glioblastoma cells. RESULTS: We identified a transcriptomic signature of 286 genes that predicts a common in vitro and in vivo resistance phenotype to these agents. This signature was significantly enriched for genes with functions in organismal survival (27.5%) and cell death (49.0%), both with P < 0.00001. Modularity was a predominant organizational principle of the signature, with functions being carried out by groups of interacting molecules in overlapping networks. A highly significant network was built around nuclear factor-κB (NF-κB), which included the persistent alterations of various NF-κB pathway elements. Tumor necrosis factor-α-induced protein 3 (TNFAIP3) was identified as a new endogenous regulatory component of a putative cytoplasmic signaling cascade that mediates NF-κB activation in response to DNA damage caused by O(6)-alkylating agents. Expression of the corresponding zinc finger protein A20 closely mirrored the expression of the TNFAIP3 transcript, and was inversely related to NF-κB activation status in the resistant cells. A prediction model based on the resistance signature enabled the subclassification of an independent, validation cohort of 31 glioblastomas into two outcome groups (P = .037). TNFAIP3 expression was a favorable factor in patient prognosis (P = .028), and was part of an optimized four-gene predictor (TNFAIP3, CD44 antigen, syndecan 1, and F-box protein 32) significantly associated with patient survival (P = .022). CONCLUSIONS: Our results offer strong evidence for TNFAIP3 as a key regulator of the cytoplasmic signaling to activate NF-κB en route to O(6)-alkylating agent resistance in glioblastoma cells. This gene may be an attractive target for therapeutic modulation of glioblastomas. No significant financial relationships to disclose.