Combinations of Epigenetic Modifying Agents Accentuate Retinoid-Mediated Gene Expression Over Single Agent Therapies In Acute Myelogenous Leukemia Cells

High cure rates seen in acute promyelocytic leukemia (APL) result from adding the retinoid all trans retinoic acid (ATRA) to chemotherapy. The addition of ATRA to chemotherapy has not consistently improved survival for other acute myelogenous leukemia (AML) subtypes. This likely reflects differences in the degree ATRA overcomes transcriptional repression imposed by the epigenetic mechanisms of histone acetylation and DNA methylation between AML and APL. Purpose To determine if differences in induction of retinoic acid receptor (RAR) β2 accounts for variations in ATRA-responses between APL and AML cells and to determine the effects epigenetic modifying agents have on the expression of RARβ2 and related gene sets in AML cells. Methods Primary AML samples, NB-4 (APL), and OCI/AML-2 (AML) cell lines were cultured in standard conditions. Treatments included ATRA 1μM, valproic acid (VPA) 0.6 mM, and 5-aza-2`-deoxycytidine (5-aza-2CDR) 400 ng/mL. Gene expression was measured by real-time PCR. Histone-acetylation was measured by chromatin immunoprecipitation (ChIP) and DNA methylation by EpiTYPER. Gene expression profiles were analyzed with Affymetrix U133A arrays followed by Gene Set Enrichment Analysis (GSEA) to identify RARβ2 related gene sets. Results Treatment of APL (NB-4 and primary samples) with ATRA induced expression of RARβ2 and expression remained attenuated in similarly treated AML (OCI/AML-2 and primary samples) cells. Histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) can potentially reverse epigenetic-mediated transcriptional repression. Treatment of OCI/AML-2 cells with the HDACi VPA and/or the DNMTi 5-aza-2CDR and treatment of primary AML samples with VPA restored ATRA-inducible RARβ2 expression. In accordance, DNA methylation and predominantly deacetylated histones were seen at the OCI/AML-2 RARβ promoter. Treatment of OCI/AML-2 cells with ATRA and VPA (HDACi) in the absence of 5-aza-2CDR (DNMTi) had little effect on DNA methylation at the RARβ promoter, however, 5-aza-2CDR treatments markedly decreased DNA methylation. An unexpected finding was VPA further reduced DNA methylation when added to 5-aza-2CDR treatment. Histone acetylation increased minimally at the RARβ promoter in ATRA-treated OCI/AML-2 cells. However, combining ATRA with VPA (HDACi) and/or 5-aza-2CDR (DNMTi) markedly increased histone acetylation that correlated with gene-induction. The greatest change in histone acetylation and DNA methylation was seen in OCI/AML-2 cells treated with the combination of ATRA + VPA + 5-aza-2CDR that correlated with the largest induction of RARβ2. Heterogeneous levels of DNA methylation were seen at the RARβ promoter in 26 primary AML samples compared to 4 normal bone marrow (NBM) samples. Levels of DNA methylation were similar to NBM in 13 AML samples (50%), decreased in 5 (19%), both increased and decreased in the same sample in 2 (8%), and increased in 6 (23%) primary AML samples. Next, we used Affymetrix gene arrays and GSEA to delineate differences in ATRA-mediated gene regulation between NB-4 (APL) and OCI/AML-2 (AML) cells and to determine whether treatment with VPA (HDACi) and 5-aza-2CDR (DNMTi) restored expression of gene sets that included RARβ2 signaling. ATRA-treatment induced twelve such gene sets in NB-4 cells and six gene sets in OCI/AML-2 cells. Treatment of OCI/AML-2 cells with VPA alone did not induce expression of RARβ2 associated gene sets, whereas treatment of OCI/AML-2 cells with 5-AZA-2CDR modulated expression of a further five gene sets that were modulated in ATRA-treated NB-4 (APL) cells. Treatment of OCI/AML-2 cells with all three agents ATRA + VPA + 5-aza-2CDR was the only treatment combination that modulated the ligand_dependent_nuclear_receptor_activity gene set. This gene set includes many genes regulating retinoid signaling and are engaged as early as 3 hours after ATRA treatment in NB-4 cells. Conclusions RARβ2 is a frequent target for transcriptional repression by epigenetic mechanisms in AML cells. Combinations of HDACi and DNMTi increase ATRA-mediated induction of RARβ2 and epigenetic modifications that favor transcription over individual treatments. This includes a possible demethylating effect of the HDACi VPA. These treatment combinations also restore modulation of a number of RARβ2 related gene sets in AML cells that are similarly regulated in ATRA-treated APL cells.

No relevant conflicts of interest to declare.