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Replication-associated single-ended DNA double-strand breaks (seDSBs) are repaired predominantly through RAD51-mediated homologous recombination (HR). Removal of the non-homologous end-joining (NHEJ) factor Ku from resected seDSB ends is crucial for HR. The coordinated actions of MRE11-CtIP nuclease activities orchestrated by ATM define one pathway for Ku eviction. Here, we identify the pre-mRNA splicing protein XAB2 as a factor required for resistance to seDSBs induced by the chemotherapeutic alkylator temozolomide. Moreover, we show that XAB2 prevents Ku retention and abortive HR at seDSBs induced by temozolomide and camptothecin, via a pathway that operates in parallel to the ATM-CtIP-MRE11 axis. Although XAB2 depletion preserved RAD51 focus formation, the resulting RAD51-ssDNA associations were unproductive, leading to increased NHEJ engagement in S/G2 and genetic instability. Overexpression of RAD51 or RAD52 rescued the XAB2 defects and XAB2 loss was synthetically lethal with RAD52 inhibition, providing potential perspectives in cancer therapy., Graphical Abstract Graphical AbstractXAB2 promotes Ku eviction from single-ended DNA double-strand breaks independently of the ATM kinase.

Unrepaired O6-methylguanine lesions induced by the alkylating chemotherapy agent temozolomide lead to replication-associated single-ended DNA double-strand breaks (seDSBs) that are repaired predominantly through RAD51-mediated homologous recombination (HR). Here, we show that loss of the pre-mRNA splicing and DNA repair protein XAB2 leads to increased temozolomide sensitivity in glioblastoma cells, which reflects abortive HR due to Ku retention on resected seDSBs. XAB2-dependent Ku eviction also occurred at seDSBs generated by the topoisomerase I poison campthotecin and operated in parallel to an ATM-dependent pathway previously described. Although Ku retention elicited by loss of XAB2 did not prevent RAD51 focus formation, the resulting RAD51-ssDNA associations were unproductive, leading to increased engagement of non-homologous-end-joining in S/G2 and genetic instability. Overexpression of RAD51 or the single-stranded DNA annealing factor RAD52 rescued the XAB2 defects. RAD52 depletion led to severe temozolomide sensitivity, whereas a synthetic lethality interaction was observed between RAD52 and XAB2.

Despite surgical resection and genotoxic treatment with ionizing radiation and the DNA alkylating agent temozolomide, glioblastoma remains one of the most lethal cancers, due in great part to the action of DNA repair mechanisms that drive resistance and tumor relapse. Understanding the molecular details of these mechanisms and identifying potential pharmacological targets have emerged as vital tasks to improve treatment. In this review, we introduce the various cellular systems and animal models that are used in studies of DNA repair in glioblastoma. We summarize recent progress in our knowledge of the pathways and factors involved in the removal of DNA lesions induced by ionizing radiation and temozolomide. We introduce the therapeutic strategies relying on DNA repair inhibitors that are currently being tested in vitro or in clinical trials, and present the challenges raised by drug delivery across the blood brain barrier as well as new opportunities in this field. Finally, we review the genetic and epigenetic alterations that help shape the DNA repair makeup of glioblastoma cells, and discuss their potential therapeutic impact and implications for personalized therapy.

Introduction: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.

Glioblastoma multiforme (GBM) is an aggressive brain tumor with poor prognosis (the median survival of GBM patients is 14.6 months). Current GBM treatment, which consists of surgical resection followed by radiation therapy and temozolomide (TMZ) chemotherapy, is faced with the development of resistance. The DNA-alkylating agent TMZ exerts its cytotoxic function by damaging DNA and inducing tumor cell death. However, efficient repair and/or tolerance of TMZ-induced DNA lesions can mediate resistance to chemotherapy. One such important factor is the O-6-methylguanine-DNA methyltransferase (MGMT). In addition to mediating the cellular response to anti-cancer therapeutics, DNA repair pathways also help cancer cells to cope with the extra load of endogenous DNA damage that occurs as a consequence of the tumorigenic process. Which DNA repair molecules are important for GBM survival in the absence of chemotherapeutic treatment and/or in response to anti-cancer genotoxics remains, however, vastly unknown. In this project, we have addressed this question by developing a large-scale differential shRNA screen specifically targeting components of the DNA-damage response (DDR). This includes not only DNA repair genes, but also genes involved in cell cycle checkpoints, DNA damage tolerance pathways and chromatin factors that facilitate DNA repair. Pooled shRNAs will be introduced in selected GBM-derived, cancer stem like cells (GSCs), as well as control non-cancer cells. Genes that appear essential to the survival of GSCs, but dispensable to non-cancer cells, either in the absence of treatment, or following exposure to TMZ, will be selected. Subsequently, promising candidates will be validated individually in vitro, and the best candidates will be further validated in vivo, using patient derived xenograft animal models developed in the laboratory. This will allow us to monitor, in a pre-clinical setting, the impact of depleting selected DDR factors, on tumor formation and response to TMZ. Long-term perspectives include determining the potential of the selected DDR components as druggable, therapeutic targets in the treatment against GBM.