Investigation of the role of mutated TET2 in marginal zone lymphomas
- Zhang, Fangwen
- Bertoni, Francesco (Degree supervisor)
- 2025
PhD: Università della Svizzera italiana
English
The family of ten-eleven translocation dioxygenases (TETs) consists of TET1, TET2, and TET3. TET proteins regulate dioxygenase activity-dependent DNA demethylation and dioxygenase activity-independent histone modification. They control the dynamic differentiation and lineage commitment of hematopoietic stem/progenitor cells (HSPCs)by regulating the access of key transcription factors (TFs) to the enhancers of target genes. TET2 mutations, acquired as first hit or during tumor development, cause abnormal epigenetic landscape changes, resulting in multiple stages of lineage commitment/differentiation defects and genetic instability in HSPCs. At varying frequencies, TET2 somatic mutations are commonly detected in multiple hematopoietic malignancies, including several lymphoma subtypes. Among lymphomas, TET2 mutations occur in almost all marginal zone lymphoma (MZL) types, but their prevalence is highest in lymphomas originating in the thyroid. Targeted mutational knock-in at single alleles is starting to be used to model diseases caused by mutations. We use the ribonucleoprotein (RNP) method to co-transfer the gRNA and Cas9 protein of the CRISPR/Cas9 system and the Donor oligonucleotide carrying the target point mutation into MZL wild-type cells (VL51) by nuclear transfection. After the gRNA and Cas9 complex caused a double strand break (DSB) in the target site DNA, the cell used the Donor template for homology-directed repair (HDR) to recombine the target point mutation into the genomic target site. Conversely, we reversed the MZL TET2 mutated HC1 cell line to a TET2 wild-type status. Using this approach, we generated and mimicked human MZL cells with TET2 mutation and established a disease model to explore the biological function sustained by the presence of the TET2 mutation. The edited cells were cultured for clonal analysis, and the gene-phenotype was verified by Sanger sequencing and immunoblotting experiments. Bulk RNA-seq and single-cell sequencing were then performed to analyze the changes in the transcriptome before and after the cells were edited. Cell lines carrying TET2 mutations had altered growth phenotypes and changed at the transcriptome level. In addition, when we combined RNA-seq with DNA methylation results, we found that the methylation status of some genes was associated with TET2 mutations, and the differential gene expression was also associated with the methylation of the gene promoter region. To some extent, it shows that the TET2 mutation changes the gene promoter's methylation and gene expression. The abnormal expression of some genes is related to the proliferation phenotype of cells, especially in the context of TET2 mutation. Research reports in TET2 mutant tumor cells, TET1 and TET3 partially compensate for TET2 activity and contribute to the pathogenesis of TET2 mutant hematopoietic malignancies. TET2 mutant hematopoietic malignancies might be targeted pharmacologically by restoring or inhibiting dioxygenase activity. We explored the inhibitory effect of a newly designed TET inhibitor, TETi76, on several lymphoma cell lines. The results demonstrated a wide anti-tumor activity, and it was more active in cells with TET2 mutations. We then validated its effect also on our MZL cells before and after TET2 gene editing. In addition, we used a custom drug library to perform pharmacological screening in the parental wild type, and TET2 edited mutant monoclonal population of MZL cells to explore possible approaches for the TET2 mutant cells. Several drugs were identified that were more active in TET2 mutant cells, and their targets largely agreed with what was observed at the transcriptome level. –In conclusion, we built TET2 mutants and artificial wild-type cell lines that rescued TET2 mutants through CRISPR/Cas9 editing. Cell lines carrying TET2 mutations had gained growth phenotypes, altered at the transcriptome level, and acquired more sensitivity to some drugs.
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- Language
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- English
- Classification
- Medicine
- License
- License undefined
- Open access status
- green
- Identifiers
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- NDP-USI 2025BIOMED001
- ARK ark:/12658/srd1330841
- URN urn:nbn:ch:rero-006-122611
- Persistent URL
- https://n2t.net/ark:/12658/srd1330841
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