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Abstract

Medulloblastoma (MB), arising in the cerebellum, is one of the most common malignant brain tumors in children. MB comprises four distinct molecular subgroups. One of them, Group 3, is enriched in oncogene MYC amplifications resulting in dismal prognosis and reduced survival rates. The treatment of MB includes surgical tumor-resection, cranio-spinal irradiation, and chemotherapy. However, even if the treatment is effective, a large population of surviving patients suffer from various sequelae. Therefore, there is a necessity for the development of therapeutics and their combinations targeted against the drivers of MB. In this thesis, a combination of two compounds indirectly targeting MYC is discussed.

We and others have previously demonstrated that MYC-amplified MB cells are highly susceptible towards treatment with class I histone deacetylase (HDAC) inhibitors, potentially due to MYC and HDAC2 co-localization in a protein complex in MB cells. We used a class I HDACi entinostat as the first compound in the combination. In order to determine the second candidate, we applied two target discovery techniques, elucidating the protein complex members by mass spectrometry and examining the potential targets in the transcriptional landscape changes induced by entinostat treatment. The elucidated candidate was tested for the selectivity for MYC-amplified MB cells assessing cell metabolic activity, cell cycle, cell death and viability. The interaction between entinostat and a second target inhibitor was examined and validated. Finally, the mechanism of interaction was investigated.

We determined that cell cycle regulator polo-like kinase 1 (PLK1) is a target for the combination therapy in MYC-amplified MB. PLK1 was found overexpressed in MYC-amplification-associated molecular subgroups and subtypes of MB underscoring the translational potential of PLK1 targeting. Moreover, MYC-amplified cells were more susceptible to the PLK1 inhibition as confirmed by cell metabolic activity, viability, and apoptosis induction. Entinostat and PLK1 inhibitors volasertib and GSK461364 were interacting synergistically in clinically relevant concentrations only in MYC-amplified MB cells. In addition, entinostat and volasertib synergistically induced apoptosis and reduced the cell viability in MYC-amplified cells only. Finally, we show that volasertib and its’ combination with entinostat exert their activity via MYC axis. However, the details of interaction mechanism remain to be examined. The validation experiments involving on-target confirmation by PLK1 knockdown as well as in vivo assessment of the combination therapy are planned.

In summary, in this thesis, the synergistic interaction of entinostat and PLK1 inhibitors was demonstrated. This combination offers a potential for clinical development with MYC amplification serving as a predictive biomarker in MB and possibly other entities with MYC overexpression.