| 1. |
- Bensberg, Maike, 1993-
(författare)
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DNA methylation in T cell leukaemia
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- T cell acute lymphoblastic leukaemia (T-ALL) is a predominantly paediatric cancer that stems from malignant transformation of developing T cells. While the disease has an overall survival rate of 80%, the intense chemotherapy treatment causes severe toxicity and long-term side effects. Furthermore, the survival rate for patients in relapse is less than 25%. Consequently, there is a need for improved therapy options to reduce treatment-related side effects and improve the survival rate of relapsed patients. Targeting aberrant DNA methylation with hypomethylating agents (HMAs) has been successful in the treatment of myelodysplastic syndromes and acute myeloid leukaemia but has not been routinely used in the treatment of T-ALL, despite DNA hypomethylation being observed in T-ALL patients. In this work, we employed a comprehensive set of molecular and sequencing-based techniques to explore the possibilities of HMAs as a treatment option for T-ALL.We made the discovery that the DNA demethylating enzyme ten-eleven translocation 2, TET2, is downregulated or completely silenced in primary T-ALL. Moreover, the TET2 promoter was highly methylated in a group of patients, suggesting that TET2 itself can be silenced through DNA methylation in T-ALL. By treatment with HMAs, TET2 was demethylated in T-ALL cell lines and was one of few genes that was activated upon loss of DNA methylation, indicating that TET2 expression is regulated by DNA methylation in T-ALL cell lines. The development of a novel HMA, the DNMT1-specific inhibitor GSK-3685032, offers a tool to reveal the mechanism of action of the traditional HMAs, 5- azacytidine and decitabine, and to study the effects of acute loss of DNA methylation on cancer cells. We found that 5-azacytidine and decitabine are cytotoxic to T-ALL cells primarily by creating DNA double strand breaks. In contrast, GSK did not prompt a DNA damage response and instead reduced global DNA methylation to as little as 18% with limited cytotoxicity only occurring after levels of DNA methylation had dropped below 30%, a level of demethylation not achieved with DEC or AZA.T-ALL is more than two times more common in boys than girls and mutations in X-linked tumour suppressor genes that escape X inactivation, have been suggested as an underlying cause for the observed sex-bias. In theory, these aberrations would be more detrimental in XYmale cells than in XX-female cells due to the presence of an extra protective copy of the gene in females. We profiled DNA methylation during T cell development and created a map of sex-specific gene expression and expression from the inactive X chromosome, finding that some, but not all, suggested tumour suppressor genes in fact escape X inactivation. These results highlight the importance of profiling the healthy cells that T-ALL arises from to correctly judge the functional impact of gene dysregulation in cancer.In the last study, we aimed to investigate the role of N6-adenine methylation (6mdA) during T cell differentiation. While 6mdA is common in bacteria it is much rarer in humans. Nevertheless, 6mdA has previously been associated with several cellular processes, including cancer progression. Our study calls the presence of 6mdA in mammals into question by exposing limitations of the techniques used in its analysis. We show that contamination with bacterial DNA or 6mAcontaining RNA, nonspecific antibody binding, and low precision of third-generation sequencing techniques all hinder the detection and investigation of rare DNA modifications, such as 6mdA.Together, this work is an in-depth study of the function and the potential of DNA methylation in the biology of healthy and malignant T cells.
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| 2. |
- Bensberg, Maike, et al.
(författare)
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TET2 as a tumor suppressor and therapeutic target in T-cell acute lymphoblastic leukemia
- 2021
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 118:34
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Tidskriftsartikel (refereegranskat)abstract
- Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is an aggres-sive malignancy resulting from overproduction of immature T-cells in the thymus and is typified by widespread alterations in DNA methyl-ation. As survival rates for relapsed T-ALL remain dismal (10 to 25%), development of targeted therapies to prevent relapse is key to improv-ing prognosis. Whereas mutations in the DNA demethylating enzyme TET2 are frequent in adult T-cell malignancies, TET2 mutations in T-ALL are rare. Here, we analyzed RNA-sequencing data of 321 primary T-ALLs, 20 T-ALL cell lines, and 25 normal human tissues, revealing that TET2 is transcriptionally repressed or silenced in 71% and 17% of T-ALL, respec-tively. Furthermore, we show that TET2 silencing is often associated with hypermethylation of the TET2 promoter in primary T-ALL. Impor-tantly, treatment with the DNA demethylating agent, 5-azacytidine (5-aza), was significantly more toxic to TET2-silenced T-ALL cells and resulted in stable re-expression of the TET2 gene. Additionally, 5-aza led to up-regulation of methylated genes and human endogenous ret-roviruses (HERVs), which was further enhanced by the addition of phys-iological levels of vitamin C, a potent enhancer of TET activity. Together, our results clearly identify 5-aza as a potential targeted therapy for TET2-silenced T-ALL.
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| 3. |
- Douvlataniotis, Karolos, et al.
(författare)
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No evidence for DNA N-6-methyladenine in mammals
- 2020
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Ingår i: Science Advances. - Washington, DC, United States : American Association for the Advancement of Science (A A A S). - 2375-2548. ; 6:12
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Tidskriftsartikel (refereegranskat)abstract
- N-6-methyladenine (6mdA) is a widespread DNA modification in bacteria. More recently, 6mdA has also been characterized in mammalian DNA. However, measurements of 6mdA abundance and profiles are often very dissimilar between studies, even when performed on DNA from identical mammalian cell types. Using comprehensive bioinformatics analyses of published data and novel experimental approaches, we reveal that efforts to assay 6mdA in mammals have been severely compromised by bacterial contamination, RNA contamination, technological limitations, and antibody nonspecificity. These complications render 6mdA an exceptionally problematic DNA modification to study and have resulted in erroneous detection of 6mdA in several mammalian systems. Together, our results strongly imply that the evidence published to date is not sufficient to support the presence of 6mdA in mammals.
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