XAB2 promotes Ku eviction from single-ended DNA double-strand breaks independently of the ATM kinase

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.publishedVersio

Identification of DNA damage response genes as targets for personalized treatment of Glioblastoma through RNA interference screens

Despite surgical resection and genotoxic treatment with ionizing radiation and the DNA alkylating agent temozolomide (TMZ), glioblastoma (GBM) remains one of the most lethal cancers, due in part to the action of DNA repair factors that drive resistance and lead to tumor relapse. Important features of these mechanisms include the inherent redundancy and complexity of the many DNA repair pathways activated as part of the DNA damage response (DDR). One important DDR factor is encoded by the O-6-methylguanine-DNA methyltransferase (MGMT) gene whose epigenetic silencing is a strong predictive marker for favorable outcome in GBM patients treated with TMZ. However, even patients displaying MGMT promoter methylation succumb to tumor relapse, indicating that other DNA repair mechanisms promote resistance to the treatment. The main aim of this work was to identify essential DDR factors that support GBM cell survival under TMZ treatment in order to provide novel targets and thus, novel insights into drug combinations that could overcome GBM chemoresistance. The strategy to achieve this goal was to use well-characterized GBM cell lines in focused loss of function shRNA screens performed under different conditions. Indeed, screens were designed to uncover DDR genes that are essential for GBM cells i) under normal conditions, ii) under TMZ treatment, iii) and under TMZ treatment in MGMT negative contexts. Pooled shRNAs targeting more than 500 DDR genes were introduced into GBM cells reflecting two clinically-relevant GBM contexts: a MGMT-positive context using MGMT-expressing cells and a negative one, using cell lines in which MGMT was depleted hrough pharmacological inhibition or RNA interference. Potential DDR targets that could sensitize GBM cells to TMZ have been identified and some of them validated. Surprisingly, some of these targets are not only involved in pathways that are commonly known to drive TMZ resistance (i.e. Double strand break repair, Direct repair), but also in repair pathways such as transcription-coupled nucleotide excision repair (TC-NER) or the early steps of the Fanconi Anemia pathway (FA). While components of the early FA pathway might be attractive targets especially in MGMT-negative contexts, it may be interesting to target factors involved in the early steps of the TC-NER to sensitize MGMTpositive cells to TMZ. So in addition to its predictive and prognostic value, MGMT might be a relevant stratification marker to treat patients in a personalized way. Supporting this hypothesis, the screen analyses revealed that the curative inhibition of some genes in combination with TMZ treatment might turn to be deleterious depending on MGMT expression level. One of the promising target will be further investigated in vitro as well as in vivo to elucidate the molecular mechanisms by which this gene operates to confer cellular resistance to TMZ. This study opens up new horizons for combinatorial therapies and hopefully, will be used to improve therapeutical approaches, particularly for MGMT-positive GBM patients, who currently do not benefit at all from chemotherapy

DNA repair mechanisms and their clinical impact in glioblastoma.

peer reviewedDespite 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

XAB2 promotes Ku eviction from single-ended DNA double-strand breaks independently of the ATM kinase

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

Maximizing the Efficacy of MAPK-Targeted Treatment in PTENLOF/BRAFMUT Melanoma through PI3K and IGF1R Inhibition

The introduction of MAPK pathway inhibitors paved the road for significant advancements in the treatment of BRAF-mutant (BRAF(MUT)) melanoma. However, even BRAF/MEK inhibitor combination therapy has failed to offer a curative treatment option, most likely because these pathways constitute a codependent signaling network. Concomitant PTEN loss of function (PTEN(LOF)) occurs in approximately 40% of BRAF(MUT) melanomas. In this study, we sought to identify the nodes of the PTEN/PI3K pathway that would be amenable to combined therapy with MAPK pathway inhibitors for the treatment of PTEN(LOF)/BRAF(MUT) melanoma. Large-scale compound sensitivity profiling revealed that PTEN(LOF) melanoma cell lines were sensitive to PI3Kβ inhibitors, albeit only partially. An unbiased shRNA screen (7,500 genes and 20 shRNAs/genes) across 11 cell lines in the presence of a PI3Kβ inhibitor identified an adaptive response involving the IGF1R-PI3Kα axis. Combined inhibition of the MAPK pathway, PI3Kβ, and PI3Kα or insulin-like growth factor receptor 1 (IGF1R) synergistically sustained pathway blockade, induced apoptosis, and inhibited tumor growth in PTEN(LOF)/BRAF(MUT) melanoma models. Notably, combined treatment with the IGF1R inhibitor, but not the PI3Kα inhibitor, failed to elevate glucose or insulin signaling. Taken together, our findings provide a strong rationale for testing combinations of panPI3K, PI3Kβ + IGF1R, and MAPK pathway inhibitors in PTEN(LOF)/BRAF(MUT) melanoma patients to achieve maximal response