Unraveling the Essential Role of Aberrant TET3 Expression in Acute Erythroid Leukemia, a Promising Epigenetic Target for Treatment

Acute erythroid leukemia (AEL) is characterized by uncontrolled proliferation and impaired differentiation of erythroid progenitor cells, with a dismal prognosis and a median survival of only 3 months for pure erythroid leukemia (PEL). Despite the association of AEL with mutations in AML-associated oncogenes and tumor suppressor genes, the underlying biology of erythroid proliferation remains unclear. The Ten-eleven translocation (TET) methylcytosine dioxygenases, including TET3, play a crucial role in regulating DNA methylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby influencing gene expression. TET3 is expressed in pro-, early, late, poly- and orthochromatic erythroblasts with an increase in expression within later stages of differentiation (Yan et al Blood. 2017;129(14):2002-2012). However, its role in AEL has not yet been investigated. Given the expression profile of TET3 during healthy erythroid cell development we searched AEL RNA-Sequencing data from Iacobucci et al. (Nat Genet. 2019 Apr;51(4):694-704) and found a high expression of TET3 in all AEL patient samples with no difference between the cytogenetic and molecular subgroups and a significantly higher expression compared to both other TET genes. Own analyses demonstrated that TET3 is significantly aberrantly overexpressed in primary AEL patient samples (n=11) compared to normal bone marrow (n=3), CD34+ cord blood (CB) (n=3) cells, and AML patient samples from different cytogenetic and molecular subgroups (n=15). Stable knockdown (KD) of TET3 expression in the HEL cell line with two independent shRNAs (shA and shB) resulted in a 60-80% reduction in cell proliferation (n=3, p<0.01) and a 21-72% reduction in the clonogenic potential (n=4, p<0.001). Similar results were obtained in the TF-1 cell line with a 30-50% reduction in cell proliferation (n=3, p< 0.01) and a 40-70% reduction in clonogenicity (n=3, p< 0.01). Quantitative analysis of global 5hmC levels via flow cytometry confirmed that TET3 KD in HEL (TF-1) cells results in 30-75% (30-65%) decreased global 5hmC levels, compared to scrambled control transduced cells (n=5, p<0.05), independent of changes in TET1 and TET2 expression. We assessed whether the effect of TET3 depends on its DNA binding domain by comparing wild-type TET3 with a mutant lacking the CXXC10 binding domain. Both constructs significantly enhanced proliferation in HEL (20-30%, n=3, p<0.01) and TF-1 (40-50%, n=3, p<0.01) AEL cell lines compared to the empty vector control, with no significant difference between them, indicating that the catalytic domain alone is sufficient for the proliferative activity of TET3. To elucidate the role of aberrantly increased expression of TET3 in AEL cells, we performed RNA-Sequencing on TET3 KD AEL cell lines and identified 437 differentially expressed genes (DEGs). These DEGs were enriched in cancer-related pathways, with notable upregulation in apoptosis, IL2-STAT5, IL6-STAT3, and TNFα/NF-κB signaling pathways. Analysing the DEGs using the CellRadar plot, we compared the signatures of the HEL cells after TET3 KD with CB cells overexpressing TET3 (from Pullikottil et al Leukemia. 2022;36(2):416-425). Knockdown of TET3 in HEL cells caused a downregulation of Megakaryocyte-Erythroid progenitor and an upregulation of monocyte and granulocyte-macrophage progenitor genes, indicating that TET3 is involved in lineage choice and differentiation. According to the GSEA analysis TET3 KD in HEL cells resulted in an upregulation of P53 pathway-associated genes. We performed Western blot analyses and detected an increase of the P53 protein upon TET3 KD which demonstrates a possible impact of TET3 on the TP53 pathway. The HEL cell line bears a JAK2 mutation (2V617F) as well as a TP53 missense mutation in the DNA binding domain (c.398T>A, M133K). APR-246 is a therapeutic agent that targets P53 mutated proteins in myelodysplastic syndromes and in AML. APR-246 reactivates the transcriptional activity of P53 mutants by facilitating their binding to DNA target sites. We could detect a 32-41% increase in the drug sensitivity towards APR-246 upon KD of TET3 in the HEL cell line (n=3, p<0.01) indicating a potential benefit for combining drug treatment with TET3 targeting. Our results demonstrate an essential role of aberrant TET3 expression in AEL and identify TET3 as a promising epigenetic target for treatment in this challenging disease.