PML-RARα and AML1-ETO translocations are rarely associated with methylation of the RARβ2 promoter

The acute promyelocytic leukemia-specific PML-RARα fusion protein is a dominant-negative transcriptional repressor of retinoic acid receptor (RAR) target genes, which recruits HDAC and corepressor proteins and inhibits coactivators. Another oncogenic transcription factor, AML1-ETO, was proposed to cause an HDAC-dependent repression of RAR target genes. The RAR target RARβ2 gene has been reported to be frequently silenced by hypermethylation in many types of cancer cells. We examined the methylation status of the RARβ2 and asked if demethylation could reverse ATRA resistance in ATRA-resistant PML-RARαand AML1-ETO-positive cells. PML-RARα positive NB4 and its ATRA-resistant subvariant MR2 and AML1-ETO expressing Kasumi-1 cells had heterozygous methylation of RARβ2. Although DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine partially reversed RARβ2 CpG methylation in these cells, it did not significantly enhance ATRA-induced RARβ2 mRNA expression and induction of maturation. However, the histone acetylase inhibitor SAHA combined with ATRA significantly reactivated RARβ2 mRNA both in NB4 and MR2 cells with degradation of PML-RARα which was associated with maturation. In contrast, SAHA did not affect AML1-ETO levels and failed to induce RARβ2 expression and maturation in Kasumi-1 cells. In primary AML samples, RARβ2 expression was uniformly low; however, no specific correlation was observed between the methylation of the RARβ2 gene and expression of the fusion proteins, PML-RARα and AML1-ETO. These results demonstrate that oncogenic PML-RARα and AML1-ETO translocations are rarely associated with RARβ2 promoter methylation in primary AML samples.