Targeted DNA repair gene expression study in glioblastoma patient biopsies: clinical impact and characterisation of the NEIL3 DNA glycosylase, a novel candidate target for therapy. (Doctoral thesis)

Despite surgical resection and the combination of ionizing radiation and chemotherapy with temozolomide (TMZ), a DNA methylating agent introduced in 2005, glioblastoma (GBM) remains a lethal disease associated with poor prognosis, treatment resistance and inevitable relapse. Resistance to chemoradiation is mediated in great part by complex and redundant DNA repair mechanisms.
The main aim of this project was to propose novel molecular targets and strategies in the fight against GBM, based on the identification and characterization of the DNA repair machineries that i) are altered during glioblastomagenesis and ii) mediate chemoresistance and tumor relapse. To this end, we have embarked on the expression analysis of a selection of genes encompassing the major DNA repair pathways and cell cycle-related factors, in a clinicallyrelevant cohort of paired primary and recurrent biopsies from GBM patients.
In the first part of this thesis, we report the generation and validation of a DNA repair and cell cycle gene signature that clustered GBM specimens in two major groups displaying an inverse expression profile of the signature and a third, less defined group. Specific analysis of the tumor pairs revealed that GBM recurrences frequently displayed a gene expression profile different from that of the matched primary biopsy, indicating that tumor progression is associated with significant deregulation of DNA repair and cell cycle pathways. Furthermore, the gene signature expression pattern observed at relapse was linked to progression-free survival. Finally, our signature exposed therapeutic group-specific vulnerabilities to inhibitors of the DNA damage response and/or genotoxicants, as well as specific alterations in key core GBM pathways. Thus, our gene signature bears clinical relevance, with the prospect of better patient stratification and personalized therapeutic strategies.
In the second part of this thesis, we have exploited our gene expression dataset to uncover DNA repair genes specifically deregulated in GBM. We report the upregulation of NEIL3 encoding a member of the NEIL DNA glycosylase family, and the downregulation of NEIL1 and NEIL2, the other members of this family. In view of the documented role of NEIL3 in promoting repair of oxidative DNA damage at telomeres, we investigated the impact of depleting NEIL3 in GBM cell lines. We found that NEIL3 knockdown resulted in telomere shortening, downregulation of the shelterin factor TRF1 and deregulation of chromosomespecific telomeric repeat-containing RNAs (TERRAs). In parallel, we observed an increase in telomere dysfunction-induced foci (TIFs) after NEIL3 loss, suggesting the activation of the DNA damage response at telomeres. Finally, NEIL3 depletion was associated with increased sensitivity to oxidative DNA damage as well as TMZ. Hence, we propose that NEIL3 could represent an attractive therapeutic target for improved treatment of GBM.