Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor in adults.Current therapy includes surgery,radiation and chemotherapy with temozolomide (TMZ).Major determinants of clinical response to TMZ include methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter and mismatch repair (MMR) status.Though the MGMT promoter is methylated in 45% of cases,for the first nine months of follow-up,TMZ does not change survival outcome.Furthermore,MMR deficiency makes little contribution to clinical resistance,suggesting that there exist unrecognized mechanisms of resistance.We generated paired GBM cell lines whose resistance was attributed to neither MGMT nor MMR.We show that,responding to TMZ,these cells exhibit a decoupling of DNA damage response (DDR) from ongoing DNA damages.They display methylation-resistant synthesis in which ongoing DNA synthesis is not inhibited.They are also defective in the activation of the S and G2 phase checkpoint.DDR proteins ATM,Chk2,MDC1,NBS1 and gammaH2AX also fail to form discrete foci.These results demonstrate that failure of DDR may play an active role in chemoresistance to TMZ.DNA damages by TMZ are repaired by MMR proteins in a futile,reiterative process,which activates DDR signaling network that ultimately leads to the onset of cell death.GBM cells may survive genetic insults in the absence of DDR.We anticipate that our findings will lead to more studies that seek to further define the role of DDR in ultimately determining the fate of a tumor cell in response to TMZ and other DNA methylators. Current determinations of TM6 include methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) are two of the most aggressive primary brain tumors in adults. Current therapy includes surgery, radiation and chemotherapy with temozolomide (TMZ) promoter and mismatch repair (MMR) status.Though the MGMT promoter is methylated in 45% of cases, for the first nine months of follow-up, TMZ does not change survival outcome. Frthermore, MMR deficiency makes little contribution to clinical resistance, suggesting that there exist unrecognized mechanisms of resistance. We generated paired GBM cell lines whose resistance was attributed to neither MGMT nor MMR.We show that, responding to TMZ, these cells exhibit a decoupling of DNA damage response (DDR) from ongoing DNA damages.They display methylation-resistant synthesis in which ongoing DNA synthesis is not inhibited. They are also defective in the activation of the S and G2 phase checkpoint. DDR proteins A TM, Chk2, MDC1, NBS1 and gammaH2AX also fail to form discrete foci. These results demonstrate that failure of DDR may play an active role in chemoresistance to TMZ. DNA damages by TMZ are repaired by MMR proteins in a futile, reiterative process, which activates DDR signaling network that ultimately leads to the onset of cell death. GBM cells may survive genetic insults in the absence of DDR. We anticipate that our findings will lead to more studies that seek to further define the role of DDR in determining the fate of a tumor cell in response to TMZ and other DNA methylators.