DNA Methylation Independent Silencing of the RARα Gene Expression in Acute Myeloid Leukemia.

All- trans -retinoic acid (ATRA) and the gene encoding retinoic acid receptor-α (RARα) have been implicated in the pathogenesis and treatment of acute promyelocytic leukemia (APL). Nevertheless, the role of these molecules in the pathogenesis and therapy of non-APL acute myeloid leukemias (AMLs) remains unclear. Previously we have shown that expression of the ATRA-inducible RARα2 isoform is downregulated in a variety of AML cell lines and increases with hematopoietic differentiation along the myelomonocytic lineage. Using quantitative real-time PCR we have now investigated expression of the RAR α gene in primary AML cells (n=23) and report that as in AML cell lines the levels of RARα2 mRNAs are markedly reduced (by 48 fold in APL, p≤0.05, and by 52 fold in non-APL AML, p≤0.01) relative to cord blood (CB) derived CD33 (or CD34) positive cell population (n=4). However, in contrast to the AML cell lines and normal hematopoietic progenitors, the expression of the RARα1 isoform was also significantly reduced in primary AML samples (up to 26 fold, p≤0.05). Examination of potential mechanisms underlying the silencing of the RAR α gene expression in AML revealed that the RAR α 2 promoter possesses two small CpG islands that are fully methylated in all AML cell lines examined. Consistent with the expression pattern of RARα1 a single CpG island in the RARα1 promoter region was unmethylated in all these samples. As expected from such results expression of RARα2, but not RARα1, could be stimulated with a DNA demethylating agent 5-aza-2′deoxycytidine and synergistic effects between 5-aza-2′deoxycytidine and ATRA were observed on both RARα2 expression and cellular differentiation of APL and non-APL AML cell lines. Extending this analysis to clinical material we have surprisingly discovered that RARα1 and RARα2 CpG islands are unmethylated in all AML patient samples, including 3 APL cases, suggesting that DNA methylation may not play a significant role in silencing of the RAR α gene expression in primary AML cells. Chromatin immunoprecipitation of the RARα2 regulatory region with antibodies to specific histone modifications revealed presence of other negatively acting chromatin states in primary AML samples (relative to normal CD33 positive CB cells), including decreased histone H3 acetylation as well as decreased di- and tri-methylation of histone H3 lysine 4. A possibility that microRNAs targeting sequences in the common 3′-UTR regions of the RARα1 and α2 isoforms may also contribute to silencing of both RARα1 and α2 expression in AML cannot be excluded. These results underscore the complexities of mechanisms that are responsible for silencing of gene expression in AML and support the notion that diminished RAR α expression contributes to leukemogenesis.