Oncogenic Nexus of TET2 with Thpok in Myeloid Neoplasia
Authors: Ujoatu Ejike , Mukhopadhyay Sohini , Gurnari Carmelo , Visconte Valeria , Maciejewski Jaroslaw , Basu Jayati , Jha Babal K. , Grimes H. Leighton , Sykes Stephen , Kappes Dietmar 
Published: December 2024

ThPOK (encoded by Zbtb7b gene) is a well-known transcription factor in lymphopoiesis and acts as a proto-oncogene in T cells to promote thymic lymphoma. We recently discovered an alternative role of ThPOK in the regulation of myeloid lineage commitment that serves as a brake for neutrophil commitment and differentiation during normal hematopoiesis. We previously showed that lineage-specific ThPOK transcription is primarily regulated by an evolutionarily conserved CpG-enriched silencer element residing upstream of the ThPOK promoter (Basu J et al. Nat Immunol. 2023) Methylation at CpG dinucleotides plays a critical role in gene regulation. Hyper methylation of ThPOK silencer elements potentially can abrogate the break on ThPOK expression.
The methylation of CpG islands in our genome is controlled by TET family of enzymes that progressively catalyze the oxidation of 5-methylcytosine (mC) to demethylate promoter/enhancer/silencer associated mCpG. Loss-of-function TET2 mutations (TET2MT) and TET deficiency are frequently observed in a large proportion of myeloid neoplasms (Guan Y et al. Blood Cancer Discov. 2020). TET2MT causes early myeloid clonal ontogenesis often acting as founder hits as supported by their frequent occurrence in clonal hematopoiesis. TET2 alone accounts for &gt;1/2 of TET activity in HSPCs (Guan Y et al. Blood Cancer Discov. 2020). TET2-mediated demethylation of mCpG is critical for HSPC proliferation, and differentiation, therefore functional inactivation via TET2 MT leads to abnormal accumulation of 5mC in enhancer and silencer causing differentiation block and transcriptional repression of tumor suppressors and overexpression of proto-oncogenes resulting in a volatile pre-neoplastic state, a hallmark of myeloid neoplasm.
We analyzed data from a large cohort of Cleveland Clinic patients with myeloid neoplasia (MN; n= 1246) that included high (n=239) and low-risk (541) MDS and primary and secondary AML (n= 541). We assessed the correlation between disease types and ThPOK expression levels and methylation status of the ThPOK silencer. We generated n HSC-specific ThPOK overexpressing transgenic (Vav transgenic ThPOK) mouse model to investigate the effect of overexpression of ThPOK on the development of MN. We further assessed the leukemogenic potential of ThPOK deficient Lin- bone marrow cells using in vitro assays with Hoxa9/Meis1, AML1 -ETO, and MLL-AF9. In addition, we generated isogenic TET2ko AML cell lines from K562 and THP1 using CRISPR/Cas9 gene editing and the expression of ZBTB7B were determined using qRT-PCR and western blot analysis for THPOK protein, non-targeting Sg RNA were used as control.
Analysis of Patients samples revealed that hypermethylation of the ThPOK silencer and increase in ThPOK mRNA expression are strongly associated with MDS patients bearing TET2 MT as well as several genetically distinct forms of AML (NPM1 mutant, MLL-AF9, and AML1-ETO). Higher expression of ThPOK mRNA correlated with poor prognosis in AML. Interestingly, ThPOK over-expressing mice developed MPN within weeks of age with high penetrance. Conversely, ThPOK-deficient Lin-bone marrow cells are protected from in vitro leukemogenesis by Hoxa9/Meis1, AML1-ETO, and MLL-AF9. Knock out of TET2 from AML cell line resulted in significant increase in the expression of ZBTB7B expression as observed in the qRT-PCR experiment
Our findings present a novel role of ThPOK's activity as an oncogene that promotes myeloid oncogenesis and hence can be a therapeutic target for treating MN.