The therapeutic potential of repurposed mebendazole, alone and in synergistic combination with ONC201, in the treatment of diffuse midline glioma.

H3K27-altered diffuse midline glioma (DMG) is a universally fatal disease with no available therapeutic strategies apart from palliative radiotherapy. Repurposing marketed non-cancer drugs in oncology is emerging as a fast-tracking approach to speed up the development of new treatment options, urgently needed for DMG. Repurposed anthelmintic mebendazole (MBZ) is in the spotlight against brain tumors, because it joins promising anticancer properties with high neuropenetrance, favorable pharmacokinetic and safety profile. Although MBZ is undergoing Phase I/II trials against brain tumors, including DMG, MBZ anticancer properties and the underlying mechanisms of actions have poorly been characterized in DMG preclinical models. We found that MBZ robustly reduced cell viability in six out of seven DMG cell lines with either K27M-mutated or wild-type H3. All IC(50) values (range 102 to 958 nM) fell in a clinically attainable range. The antiproliferative MBZ properties were mediated by an arrest of DMG cells in the G(2)/M phase with a concomitant upregulation of the key cell cycle regulators p21 and p27, whereas p53 upregulation and activation were cell context-dependent. At the same growth-inhibitory concentrations, MBZ triggered apoptotic cell death, as evidenced by higher levels of the apoptotic markers caspase-3 and PARP cleavage. Consistently, Annexin V-Propidium iodide (PI) double staining showed MBZ dose-dependent increase in both stages of apoptosis. Of interest, the combination of MBZ with the first-in-class imipridone ONC201 sinergistically increased the antiproliferative effects in two DMG cell lines as assessed by combination scores with different algorithms, showing additive effects in two others cell lines. Mechanistically, the combination potentiated the proapoptotic activity of either MBZ or ONC201, while not changing the cytokinetic perturbations induced by the single drugs. Finally, one pair of ONC201-sensitive and ONC201-resistant DMG cell lines with acquired resistance showed same responsiveness to MBZ with similar values of IC(50) and E(max). In conclusion, MBZ demonstrates high growth-inhibitory/proapoptotic activity, chemosensitization property to ONC201 and the ability to overcome ONC201 resistance in DMG cell cultures, proposing as a new low-toxicity therapeutic for DMG, with a potential to be used in second-line treatment and/or in combination protocols.