Mechanisms of O6-methylguanine induced senescence and transcriptional repression in glioblastoma cells

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An important group of genotoxic agents acts via methylation at the O6-position of guanine, giving rise to the formation of O6-methylguanine (O6MeG) in the DNA. Among these agents is the anticancer drug temozolomide (TMZ), which is used in the 1st line therapy of high-grade gliomas, notably glioblastomas. In our previous work we showed that TMZ induces in glioblastoma cells not only apoptosis and autophagy, but also senescence, which was triggered by O6MeG. In extended (unpublished) background studies we also show that senescence occurs in G2 phase cells, which is dependent on p21 activity. Since the success of glioblastoma therapy is related to the DNA repair capacity of the tumor, we addressed the question whether TMZ-induced senescence leads to alterations in DNA repair capacity. These studies showed that TMZ-induced senescence is strongly associated with transcriptional repression of the DNA repair proteins EXO1, MSH2, MSH6 und RAD51, with p21 playing a role also in this repression process. In this grant proposal we wish to analyze in two intertwined subprojects (TP1, Kaina; TP2, Christmann) the molecular mechanisms underlying TMZ-induced senescence, focusing on the role of persistent DNA damage and sustained DDR activation. Specifically we want to address the questions of a) which signaling mechanisms activated by O6MeG trigger senescence, b) what makes activation of DDR following primary DNA damage in a fraction of cells permanent, c) can sustained DDR be alleviated, d) are senescent cells prone to DNA damaging agents, e) can senescent cells regain propliferation capacity, f) is DNA repair capacity and drug sensitivity of recovered senescent cells comparable or different from parental cells that have not undergone senescence? Further we wish to study g) the molecular mechanism underlying senescence associated p21 mediated transcriptional repression of repair genes using ChiP-Seq and mass-spectrometry to find p21 interaction partners, h) whether transcriptional repression of DNA repair genes EXO1, MSH2, MSH6 und RAD51 is affected by epigenetic mechanisms, i.e. alterations in the chromatin structure or promoter hypermethylation. The experimental data will be assessed as to whether the DNA repair capacity in senescent cells can be harnessed in order to prevent their escape from the senescent state, eliminating them selectively by anticancer drug treatment.