In vitro testing of histone deacetylase inhibitors (HDACi) in combination with temozolomide radio-chemotherapy as a treatment strategy for pediatric high-grade gliomas (pedHGG)
by Sophie Therese George
Date of Examination:2025-12-15
Date of issue:2025-11-03
Advisor:Prof. Dr. Christof Kramm
Referee:Prof. Dr. Christof Kramm
Referee:Prof. Dr. Ralf Dressel
Persistent Address:
http://dx.doi.org/10.53846/goediss-11592
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Abstract
English
Pediatric high-grade gliomas (pedHGG) are highly malignant pediatric brain tumors associated with a fatal prognosis and include, among others, diffuse intrinsic pontine gliomas (DIPG), which in 65 % carry an H3.3K27M mutation, leading to an epigenetic imbalance resulting in global hypotrimethylation and hyperacetylation at H3K27. Due to the lack of effective conventional temozolomide radio-chemotherapy, new combined treatment options using epigenetic drugs might be promising. Therefore, the present in vitro drug study aimed to analyze the use of five HDAC inhibitors (HDACi) – vorinostat, domatinostat, mocetinostat, resminostat, and panobinostat – as therapeutic adjuvants together with temozolomide (TMZ) and irradiation (IRR) in pedHGG/DIPG. For this purpose, tumor cell lines derived from primary pediatric glioblastoma (H3WTpedHGG) and DIPG cells (H3.3K27M-DIPG) differing in their H3 mutation status were employed. Western blot analyses were conducted to investigate the epigenetic effect of HDACi on histone modifications. As a focus of this study cell viability assays and synergy analyses have been performed to investigate if HDACi combined with TMZ and IRR led to an increased therapeutic efficacy in terms of sensitivity and synergy. The study confirmed that all five HDAC inhibitors led to histone hyperacetylation in H3WTpedHGG and H3.3K27M-DIPG cells, regardless of H3 mutation status. Notably, domatinostat (no longer available on the market) and mocetinostat, both benzamide class inhibitors, demonstrated the highest growth-inhibitory capacities and exhibited additive and sensitizing effects mainly in combination with TMZ across all H3WTpedHGG/H3.3K27M-DIPG cell lines, while radio-sensitization effects with these two inhibitiors were mainly observed with H3WT-pedHGG cells. These findings suggest that benzamides may be promising drug candidates for combination therapies in these tumors. Vorinostat, resminostat, and panobinostat, all hydroxamic acid inhibitors, revealed therapeutic efficacy and synergistic cytotoxicity together with TMZ and IRR in H3WTpedHGG cells, while H3.3K27M-DIPG cells became resistant to resminostat and panobinostat. In general, H3.3K27M-DIPG cells were less susceptible to any treatment than H3WT-pedHGG cells. This findings may hint to an overall more resistant cellular phenotype, impeding the success of any or most single and combinatory treatment regimens. The crucial limiting factors of this in vitro study are the presence of various cell line specific effects, potential off-target toxicities, and the development of resistance upon HDACi treatment making a definite prediction on H3 mutation dependence difficult. Overcoming these limiting properties, by using isogenic DIPG cell lines and performing experiments on functional characteristics with and without temozolomide radio-chemotherapy, would be an essential approach to further investigate HDAC enzymes as probable epigenetic targets in pedHGG/DIPG. Future preclinical trials on pedHGG should aim to combine standard radio-chemotherapy with different novel treatment approaches, including other tumor-specific epigenetic targeted therapies and immunotherapeutic strategies, to achieve synergistic drug combinations thatideally can cross the blood brain barrier. To this end, additional in vivo drug experiments should be considered in the future. Most importantly, oncological research on pedHGG should be conducted independently of adult glioblastoma research, as the mission statement of every pediatric clinician already states: “Children are not little adults.”
Keywords: pediatric high-grade glioma; DIPG; HDACi; H3K27M
