NEW ALKBH2 AND ALKBH5 INHIBITORS FOR TREATING GLIOBLASTOMA

Glioblastoma (GBM) is the most aggressive and deadly adult brain tumor and the development of new therapeutic strategy for its treatment represents one of the millennium challenges. Despite considerable efforts to develop new therapeutic approaches, after decades of research, today the standard treatment for newly diagnosed GBM contemplates surgical resection followed by postoperative focal conformal radiotherapy (RT) and chemotherapy based on the alkylating agent temozolomide (TMZ) and 6 cycles of adjuvant TMZ (Stupp regimen) [1]. However, GBM recurrence is inevitable, and patients have a median survival of about 15 months [2]. The reasons for this very high mortality are various, including inter- and intra-tumor heterogeneity, drug resistance, very high invasiveness, and very intricate interaction between GBM (principally glioblastoma stem cells, GSCs) and tumor microenvironment (including immune cells, blood vessels, astrocytes and extracellular matrix). As part of a multi-disciplinary study in which we tested possible anti-tumor molecules, we demonstrated that the methyl-3-propyl-5H-imidazo[1,2c][1,3]benzoxazin-5-thione MV1035 can inhibit the migration and invasiveness of glioblastoma U87 cells [3]. Moreover, through in silico analysis (SPILLO-PBSS software) and cell free studies we identified the RNA demethylase ALKBH5 as the direct target inhibited by MV1035. Interestingly, MV1035 has also a synergic effect in combination with TMZ both on U87 cells and on patient-derived (PD) GSCs [4]. We demonstrated that its effect is also due to inhibition of alkylated DNA repair protein 2 (ALKBH2). Moreover, in the PD GCS7 cell line, MV1035 has proven to be effective inducing methylation of O6-methylguanine-DNA methyltransferase (MGMT) promoter and consequently reducing MGMT protein expression. Considering MV1035 ability to inhibit both ALKBH5 (crucial for maintaining GSCs tumorigenicity) and ALKBH2 (involved in TMZ resistance), this compound was considered a good candidate to be further developed. Starting from MV1035 we modified the original structure with the aim to identify the key motifs that are responsible for its activity against the DNA repair proteins ALKBH2 and ALKBH5 to gather information eventually leading to more potent inhibitors.Such modifications were made both on the tricyclic imidazobenzoxazine original core and the alkyl chains connected to the imidazole ring.We synthesized new molecules, and they have been tested through a MTT assay, with the aim to assess cell viability, through a cell-free ALKBH2 activity assay, to evaluate the inhibition of active recombinant ALKBH2 protein, and through a dot blot analysis of methylated RNA, to evaluate the inhibition of active recombinant ALKBH5. We found that compound MV3009 is able to significantly reduce the activity of ALKBH2 and to inhibit ALKBH5 in a dose dependent manner as well as the most effective in reducing the viability of U87-MG cells in a time and dose dependent way, giving us important information to identify the key motifs that are responsible for the activity of these inhibitors. This work was supported by Fondazione Giovanni Celeghin (www.fondazioneceleghin.it).