Reversing intrinsic cancer cell resistance to alkylating agents by histone deacetylase inhibition
Final Report Abstract
This study focused on identifying possible weaknesses of glioblastoma (GBM) for exploitation during therapy. GBM, defined by the World Health Organization as the most aggressive diffuse glioma of an astrocytic lineage, has long posed a significant therapeutic problem. The reasons for this are legion. To name a few, the location of this cancer. GBM is a brain tumour and is therefore not easily or safely accessible for surgical removal. It is a tumour with undefined edges and it penetrates the surrounding normal tissue, further complicating its removal. It is separated from the body by the blood-brain barrier that prohibits many chemotherapeutics from reaching it and GBM cells contain significant innate cellular resistance to the treatment moieties. Current therapy for patients suffering from GBM includes surgical resection followed by radiotherapy (RT) concomitant with adjuvant chemotherapy using temozolomide (TMZ). Even with these aggressive therapeutic options, the median survival of GBM patients is only around 10 months. This prompted us to attempt to identify an Achilles heel of GBM cells for exploitation. From our groups' previous work, we had a hint that this weakness may be found in how GBM cells tolerate the DNA damage induced by TMZ. Very simply put, TMZ induces DNA damage that kills replicating cancer cells if not repaired or tolerated. A significant proportion of GBM cells cannot repair the TMZ-induced DNA damage O6-methylguanine (O6MeG), so we focused on targeting how these cells tolerate these DNA lesions. A cellular O6MeG tolerance mechanism is homologous recombination (HR). The processing of unrepaired O6MeG by mismatch repair blocks GBM cells in S-phase because it leads to DNA structures that require HR for its bypass. To further validate this tolerance mechanism, the first part of this project demonstrated that the HR protein XRCC3 is required for TMZ tolerance in GBM cells, which we published in Cancer Letters. To demonstrate XRCC3’s role in TMZ resistance we had to make use of RNA interference (RNAi) methods, which is not currently a viable option for targeting this pathway during therapy. The second part of the project, therefore, focused on targeting this bypass and tolerance pathway mediated by HR indirectly by making use of more viable methods. Histone deacetylases (HDACs), and more specifically class I HDACs, are overexpressed in GBM and these HDACs have been shown to play a possible role in the regulation of HR. For our study, we chose a blood-brain barrier penetrating HDAC inhibitor (HDACi), namely Entinostat (MS-275), and were able to demonstrate that combining this HDACi with TMZ caused a much better cell-killing response in GBM cells while not affecting normal astrocytes. Making use of a broad spectrum of cell biology, biochemistry and molecular biology methods, coupled with database mining we were able to show that HDAC inhibition suppresses the expression of a protein called RAD18. Loss of RAD18 prevents GBM cells from initiating HR-dependent O6MeG bypass in S-phase leading to cell death. In addition to these findings, when exposing glioma initiating cells to the HDACi on its own, the cells started to differentiate away from their glioma initiating “stem cell” state. Combining the HDACi with TMZ, therefore, not only made TMZ more effective in killing GBM cells but the HDACi on its own was also able to target this subfraction of malignant cancer-initiating cells. These findings were published in Cell Death & Disease. To put our findings into the bigger context of GBM therapy. It has been shown that RAD18 plays a role in ionizing radiation-induced cell death. In our study, we showed that RAD18 plays a role in TMZ- induced cell death and in inducing differentiation of glioma initiating cells. However, the most important finding was that these GBM resistance pathways can be targeted by a blood-brain barrier penetrant HDAC inhibitor. Therefore, the findings generated in this study suggest that HDAC inhibition could overcome the RAD18-mediated TMZ- and IR-resistance during glioblastoma therapy, potentially creating a new viable therapy option for glioblastoma patients that could drastically improve survival rates.
Publications
- (2018) XRCC3 contributes to temozolomide resistance of glioblastoma cells by promoting DNA double-strand break repair. Cancer Letters, 424, 119-126
Roos WP, Frohnapfel L, Quiros S, Ringel F and Kaina B
(See online at https://doi.org/10.1016/j.canlet.2018.03.025) - (2022) Class I HDAC overexpression promotes temozolomide resistance in glioma cells by regulating RAD18 expression. Cell Death & Disease, 13, 293
Hanisch D, Krumm A, Diehl T, Stork CM, Dejung M, Butter F, Kim E, Brenner W, Fritz G, Hofmann TG and Roos WP
(See online at https://doi.org/10.1038/s41419-022-04751-7)
