Exploring hTERT promoter methylation in cutaneous T‐cell lymphomas

Abstract

Cutaneous T-cell lymphomas (CTCL) are telomerase-positive tumors expressing hTERT, although neither gene rearrangement/amplification nor promoter hotspot mutations could explain the hTERT re-expression. As the hTERT promoter is rich in CpG, we investigated the contribution of epigenetic mechanisms in its reexpression. We analyzed hTERT promoter methylation status in CTCL cells compared to healthy cells. Gene-specific methylation analyses revealed a common methylation pattern exclusively in tumor cells. This methylation pattern encompassed a hypermethylated distal region from-650bp to-150bp and a hypomethylated proximal region from-150bp to +150bp. Interestingly, the hypermethylated region matches with the recently named TERT Hypermethylated Oncogenic Region (THOR). THOR has been associated with telomerase reactivation in many cancers, but it has so far not been reported in cutaneous lymphomas. Additionally, we assessed the effect on THOR of two histone deacetylase inhibitors (HDACi), romidepsin and vorinostat, both approved for CTCL treatment as well as a DNA methyltransferase inhibitor (DNMTi) 5-azacytidine, unapproved for CTCL. Contrary to our expectations, the findings reported herein revealed that THOR methylation is relatively stable under these epigenetic drugs' pressure, whereas these drugs reduced the hTERT gene expression.

Targeting Epigenetic Modifiers Can Reduce the Clonogenic Capacities of Sézary Cells

“…In all treated cells (TC), a decrease in hTERT mRNA levels occurred with no change in the methylation status of hTERT promoter. This result is in compliance with our initial observations ( 14 ) and in line with previous findings in the literature where DNMTi (5-azacytidine or decitabine) have been reported to alter the expression of some genes (other than hTERT ) without changing their promoter methylation status ( 16 ). This effect may result from demethylation of upstream genes (like transcription factors) or regulatory elements (like enhancers) or from secondary responses to DNA damage or repair mechanism caused by DNMTi toxicity ( 16 ).…”

“…With this aim in mind, we treated SS cells with romidepsin or vorinostat using IC50 values (Romidepsin: 1.56nM, 1.85nM and 21.40nM; Vorinostat: 0.254µM, 0.830µM and 2.44µM; in HuT78, L2 and L4, respectively) and we evaluated first the effect of HDACi on hTERT expression. Consistent with our previous finding, hTERT promoter methylation status remained unaffected by HDACi pressure ( 14 ) even though we observed a drop in hTERT expression. Indeed, compared to NTC, the hTERT expression levels in HuT78 decreased by 37% with romidepsin and 36% with vorinistat.…”

“…Three SS cell lines were investigated: one commercially-available cell line HuT78 (ATCC, France) and two cell lines (SS patient-derived-cells): L2 and L4 ( 15 ) recently developed and well-characterized at our laboratory. Cells were cultured as previously reported ( 14 , 15 ). The half maximal inhibitory concentrations (IC50) for 5-azacytidine or HDACi were determined at 72h or 48h, respectively, using the luminescent cell viability assay CellTiter-Glo ® (Promega, USA).…”

“…Our team, on the other hand, examined the effect of these DNA packaging modifiers at the level of a specific gene promoter sequence, the human telomerase reverse transcriptase gene ( hTERT ). We had previously demonstrated that in SS, the telomerase ( hTERT ) expression holds a critical role in telomeres maintenance and tumorigenesis ( 13 , 14 ). We then reported that each of romidepsin, vorinostat and 5-azacytidine can reduce hTERT expression while maintaining hTERT’ s promotor methylation status ( 14 ).…”

“…We had previously demonstrated that in SS, the telomerase ( hTERT ) expression holds a critical role in telomeres maintenance and tumorigenesis ( 13 , 14 ). We then reported that each of romidepsin, vorinostat and 5-azacytidine can reduce hTERT expression while maintaining hTERT’ s promotor methylation status ( 14 ). We herein, report on the effect of the aforementioned DNA packaging modifiers on the tumorigenic capacities of Sézary cells in vitro , along with the changes in hTERT gene expression.…”