Buonaiuto, Michela (2025) Targeted inhibition of the lysine methyltransferase SETD8 decreases tumor growth and impairs the autophagic flux in Glioblastoma. [Tesi di dottorato]

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Abstract

Epigenetics is widely improving cancer therapies by exploiting epigenetic factors as potential therapeutic targets. Recently, the lysine methyltransferase SETD8 has been identified as a promising target in various cancers, in which the protein was found overexpressed. SETD8 methylates different proteins. Its main target is the lysine 20 of histone 4 (H4K20). H4K20 mono-methylation (H4K20me1) is key in mediating DNA damage repair. Alteration in the DNA damage-repair process could cause cell-cycle arrest and trigger autophagy. Before our work, the role of SETD8 in adult-type diffuse gliomas (Glioblastoma IDH-wildtype, GB) had never been investigated. GB is one of the worst forms of brain tumor. The standard therapeutic approach involves surgical resection followed by radio- and chemotherapy (with Temozolomide, TMZ). Unfortunately, the prognosis of GB patients usually remains poor. Thus, we aimed to investigate the role of SETD8 in GBs, focusing on its overexpression and the cellular response to its inhibition. Specifically, we examined how SETD8 disruption impacts cell-cycle progression and the activation of autophagy in GB cells. We found that SETD8 was overexpressed in higher-grade adult-type diffuse gliomas, correlating with poor survival and a worse prognosis. We observed that SETD8 inhibition or silencing led to the accumulation of DNA damage, which triggered cell-cycle arrest in both GB cell lines and primary GB cells. To enhance the death of the damaged cells, we combined Adavosertib (Adv) a cell-cycle checkpoint inhibitor, with SETD8 inhibitor (UNC0379). The combination forced the cells to enter mitosis, where they subsequently died by a caspase-mediated death called “mitotic catastrophe”. We found that UNC0379 synergized with Adv. We observed that the combination caused GB growth reduction in Xenograft models. 4 In the meantime, we observed that UNC0379 determined the accumulation of large vesicles in GB-treated cells. Given the DNA damage accumulation due to UNC0379, we hypothesized that the drugs could induce the autophagy process. We thus investigated the nature of the vesicles, evaluating the autophagy markers (particularly LC3II and p62), and confirming their autophagosome nature. We found increasing levels of p62 and the accumulation of LC3-II after UNC0379 treatment. We also treated the cells with Bafilomycin (BAF) and Rapamycin, an inhibitor and an activator of the autophagic flux. All these experiments supported the hypothesis that the autophagic flux was blocked due to the inhibition of SETD8 activity. Furthermore, focusing on p62 increment after SETD8 inhibition, we investigated PHF8, an H4K20 mono-methylation remover, still giving increased levels of p62. This suggests that the de-methylation of H4K20me1 could be the key to enhancing p62 transcription. Finally, we decided to combine UNC0379 with TMZ. The aim was to determine whether TMZ-induced DNA damage and UNC0379-induced autophagic blockage can trigger a significant anti-tumor response. Indeed, autophagy can protect tumor cells from damage, promoting their survival despite treatments. Preliminary results showed that this combination led to a promising increase in GB cell death. The combination of UNC0379 with Adv or TMZ demonstrates promising effects, paving the way for novel therapeutic strategies in GB treatment. Further studies are required to develop more effective approaches for inducing cell death in GB.

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