Tet2 and relevant potential intervention in cancer

Thesis

5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) are products of TET enzyme activity, which initiates at 5-methylcytosine (5mC) in DNA. 5hmC has been found to be globally depleted in cancer, while TET2 is highly mutated. The first objective of this work was the investigation of the fate of the associated nucleosides in the context of nucleotide metabolism. Moreover, since many nucleoside analogs are used in cancer therapy to induce replication arrest and DNA demethylation, the effects on proliferation induced by the administration of nucleosides with bases, which are implicated in DNA demethylation, were evaluated. Finally, the TET2 protein interaction network was investigated to elucidate its lost function in cancer and shed light on possible means of intervention. It is demonstrated that DNA polymerase is capable of accepting modified cytidine triphosphates for polymerization. However triphosphate generation is impaired by substrate selectivity of CMPK1. 5hmdC and 5fdC given as media supplements to a number of cancer cell lines caused selective inhibition of proliferation without obvious defects in DNA methylation. By analyzing gene expression datasets, cytidine deaminase (CDA) overexpression was identified in sensitive cell lines. Overexpression and knockdown experiments demonstrated that CDA is necessary and sufficient to confer sensitivity to 5hmdC. Furthermore, CDA was able to deaminate in vitro 5hmdC and 5fdC, but not 5cadC, and a deaminated version (5hmU) was detected in the DNA of treated cells. 5hmU and 5fU in the DNA correlated with S/G2 phase arrest and increased levels of γH2AX at 72 h, suggesting that defects in proliferation relate to DNA damage. 5hmdC and 5fdC administration to mice (IP route) resulted in no overt toxicity and some degree of antitumor activity. Furthermore, TET2 was found to interact with WDR61, a member of the PAF complex (involved in transcriptional elongation), possibly explaining its role in 5hmC deposition over gene bodies and in oncogenesis, given the documented role of PAF in leukaemia. In conclusion, this study identifies the salvage route of new biological cytidine variants, a new avenue with which to target CDA overexpressing cancers, and a possible mechanism to explain the role of TET2 in leukemogenesis.

Authors: Zauri, M

• Publication date: 2014
• DOI: 10.5287/ora-1zyp2mkzz
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: Oxford University, UK
• Language: English
• Keywords: Tet2; epigenetic; metabolism; Paf; cancer; drug; nucelotides
• Subjects: Genetics (life sciences); Biology; Cell Biology (see also Plant sciences); Medical Sciences; DNA damage signalling; Biology (medical sciences); Biochemistry; Metabolism