TERT promoter mutations and cancer aggressiveness

Telomere maintenance represents a hallmark of tumour cells inevitable for unlimited cell division and proliferation. Accordingly, the majority of malignancies including glioblastoma multiforme (GBM), and malignant pleura mesothelioma (MPM) exhibit telomerase re-activation. In that regard, the identification of non-coding mutations in the telomerase reverse transcriptase (TERT) promoter across multiple cancer types strengthened the importance of telomerase hyperactivation during malignant transformation and progression. Indeed, several studies including our own demonstrated not only an association of TERT promoter mutations with enhanced gene expression but also with dismal prognosis in GBM and melanoma. The majority of tumours lacking TERT promoter mutations exhibit telomerase activation. This suggests an oncogenic function of promoter mutations exceeding mere telomere stabilisation. However, the underlying biological mechanisms are still speculative. Accordingly, this project follows the hypothesis that TERT promoter mutations facilitates a more aggressive cancer cell type irrespective of the affected tissue. To address this, the impact of TERT promoter mutations and their interaction with a nearby single nucleotide polymorphism (SNP; rs2853669) on aggressiveness will be investigated in GBM and compared to MPM cells. Moreover, we hypothesize that according to the TERT promoter sequence secondary DNA structures can be altered subsequently influencing sensitivity towards promoter targeting agents (G- quadruplex stabilizers). To validate these hypotheses, first TERT promoter activity will be analysed using genetically engineered reporter constructs with variable promoter sequences in GBM and MPM cells harbouring differing endogenous TERT promoter variants. In addition, these experiments will be performed in isogenic cell lines of both tumour entities generated by the CRISPR/Cas9 technology. To discover mechanisms responsible for TERT promoter hyperactivation, the recruitment of regulatory transcription factors (TF) to wild-type and mutated promoter sequences will be compared in GBM cell lines and isogenic cell models. Promising TF will be knocked-down and the impact on telomerase activity and cell aggressiveness investigated. In addition, this project addresses the question whether telomere- unrelated functions of the TERT gene foster the enhanced malignant phenotype in both wild-type and mutated GBM cells. Subsequently, alterations of TF landscapes and non-canonical signalling cascades in promoter wild-type and mutated backgrounds will be verified performing in silico analyses of GBM tumours. Finally, the impact of TERT promoter status on sensitivity towards experimental telomerase inhibitors will be elucidated in vitro and in vivo. This project gives an insight into factors driving the high aggressiveness of TERT promoter-mutated GBM and MPM cases. Our results will clarify whether this enhanced malignancy is a consequence of more robust telomere stabilisation, augmented non-canonical TERT functions or reflects different malignant transformation processes guided by the TERT promoter mutation status. The obtained knowledge shall contribute to the discovery of new therapy targets and subsequently support the development of feasible treatment strategies necessary to combat immortalized cancer cells.

Immortalization mechanisms are major drivers of malignant transformation in diverse tumor types, including high-risk brain tumors and malignant pleura mesothelioma. Thus up to 90% of these malignancies are characterized by unlimited replication and immortalization mainly based on telomerase re-activation. Accordingly, this project aimed to investigate molecular mechanisms driving aggressiveness of telomerase-driven high-risk brain tumors and of malignant pleura mesothelioma. In general, the project wanted to clarify the impact of immortalization mechanisms, in particular telomerase re-activation via TERT promoter mutations or epigenetic mechanisms, on malignant cell transformation as well as on stem cell features and how this knowledge can be translated into novel therapeutic interventions. The project uncovered that progressive malignant meningioma as well as human malignant pleura mesothelioma frequently harbor TERT promoter mutations negatively impacting patient prognosis in this tumor types. In case of meningioma, we successfully targeted this driver mutation by an ETS-factor inhibitor in preclinical cell models. Comparable results have been reached for malignant BRAFV600E-mutated high-grade gliomas. In detail, we uncovered that ETS1 transcription factor binding drives telomerase reactivation, which can be blocked by an ETS inhibitor. Accordingly, we suggest considering compounds targeting ETS1 as an innovative personalized therapeutic treatment strategy for TERT promoter-driven meningioma and BRAF-altered high-grade gliomas. As our data already demonstrated, that replicative immortality by reactivation of telomerase is one major driver of stem cell-like characteristics in ependymoma, we dissected genetic and epigenetic mechanisms maintaining this stemness features in high-risk ependymoma. Based on single cell RNA-sequencing analyses of ependymoma tumors, we uncovered fibroblast growth factor receptors (FGFR) being enriched in undifferentiated stem cell-like subpopulations. Genetically or pharmacological inhibition of FGFR signaling impaired both, ependymoma cell survival and induced cellular differentiation. Based on our preclinical results, a pediatric patient suffering from progressive high-risk ependymoma was treated with an FGFR inhibitor showing stable disease for several months. According to this data, we assume that therapeutic regimens inducing differentiation represent novel treatment options for high-risk pediatric brain tumors. In case of glioblastoma, a synergistic effect between FGFR and integrin/focal adhesion kinase inhibition was observed, suggesting this combination therapy as novel therapeutic concept in "FGFR4-high" GBM patients. Summarizing this project comprehensively dissected the impact of telomerase reactivation on patient prognosis and gave first evidence of TERT representing a novel suitable drug target in aggressive brain tumors. In parallel, we uncovered the biological complexity of stem cell-like characteristics driven by aberrant telomerase and FGFR signaling activation and translated this knowledge into innovative pre-clinical treatment approaches for high-risk brain tumors.