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.