Regulatory function of ten‑eleven translocation‑2 in transcriptional mechanisms of demethylated myeloma cells

Multiple myeloma (MM) is a malignant plasma cell disorder characterized by clonal expansion of plasma cells in bone marrow.Genetic aberrations in MM are well-established, while epigenetic alterations, particularly dna methylation, are increasingly recognized as key regulators of gene expression.The modified bases 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) occur in gene promoters and regulatory regions.While the presence of 5-mC in the promoter is associated with transcriptional silencing, 5-hmC, generated by oxidation of 5-mC through ten-eleven translocation (TET), is associated with gene activation.The present study investigated promoter-specific 5-hmC and 5-mC levels in TET1, TET2 and TET3 genes in patients with MM and myeloma cell lines.Quantification of methylation was performed using a glucosylation and enzyme digestion-based method, bisulfite sequencing and nanopore sequencing.TET mrna expression levels were assessed using reverse transcription-quantitative PCR and interactions with specificity protein (Sp)-1 Sp1 and Sp3 transcription factors were analyzed using chromatin immunoprecipitation.TET promoter-wide 5-mC and 5-hmC profiles were compared in CD138 + sorted cells from newly diagnosed and relapsed patients with MM and in five myeloma cell lines treated with 5-azacytidine and/or 5-aza-2'-deoxycytidine.In newly diagnosed patients, TET1 mRNA expression levels were increased in comparison with TET2 and TET3, which corresponded to a reduced ccGG site methylation of the TET1 promoter (5-10%) and increased TET3 methylation (21-50%).Of the MM cell lines, the 5-azacytidine-treated KMS12-PE cell line exhibited significantly higher TET2 gene expression levels when compared with TET1 and TET3.Subsequently, immunoprecipitation with Sp3 demonstrated increased TET2 recruitment, which suggested a potential interaction between Sp3 and TeT2.The present findings indicated dynamic regulation of TET genes through ccGG site methylation and hydroxymethylation of their promoter in MM and demonstrated that demethylating agents selectively modulate TET gene expression and promoter occupancy, highlighting their potential impact on epigenetic gene activation.Notably, the divergent expression patterns observed suggest that TET1 acts as an oncogenic driver, while the inducible response of TET2 highlights its role as a potential tumor suppressor in myeloma biology.Consequently, the present results provide a rationale for the pharmacological restoration of TET2 expression as a strategic approach to suppress tumor progression through epigenetic reprogramming.