AbstractIntroduction:Despite surgical resection and genotoxic treatment with ionizing radiation (IR) and the DNA alkylating agent temozolomide (TMZ), glioblastoma remains one of the most lethal cancers, due in great part to the action of complex DNA repair mechanisms that drive resistance and tumour relapse. One such mechanism involves the DNA-repair protein O6-methylguanine-DNA methyltransferase (MGMT) which mediates the direct removal of O6-methylguanine, the most highly cytotoxic lesion induced by TMZ. Importantly, silencing of MGMT through promoter methylation, which is observed in about 40% of GBM patients, confers a small but significant survival benefit in patients treated with TMZ and IR compared to IR only. In addition to MGMT, several DNA repair pathways have been involved in the repair of TMZ-induced lesions. Understanding the molecular details of these mechanisms and identifying potential pharmacological targets have emerged as vital tasks to improve treatment. At the same time, deciphering the genetic and epigenetic alterations that shape the “DNA repair makeup” of GBM cells should help tailor therapy to individual patients.Approach and Results:We have undertaken complementary approaches to understand the molecular basis behind chemoresistance, tumour progression and relapse in GBM patients. Firstly, we have measured the mRNA expression levels of a selection of DNA repair and cell cycle factors in paired, primary and recurrent GBM biopsies from patients treated or not with TMZ. Classification of the deregulated genes led to the identification of a gene signature that segregated 3 groups of biopsies. Inspection of these groups suggests that one route to tumour progression is associated with profound alterations in cell cycle genes as well as genes encoding crucial DNA repair factors. We have further exploited our differential gene expression analysis to learn how cancer cells modulate the expression of specific pathways in response to glioblastomagenesis and genotoxic treatment, and propose novel therapeutic strategies based on the inhibition of DNA repair factors. Lastly, we have carried out a large-scale shRNA screen of DNA repair/chromatin factors to identify those gene silencings that sensitize GBM cells to TMZ, as well as synthetic lethal interactions with MGMT. We are currently validating the most promising candidates in vitro as well as in vivo, using orthotopic xenograft models of GBM.