Targeted removal of PML-RARα protein is required prior to inhibition of histone deacetylase for overcoming all-trans retinoic acid differentiation resistance in acute promyelocytic leukemia

All-trans retinoic acid (tRA)-induced differentiation in NB4 cells, a cell line derived from an acute promyelocytic leukemia patient with t(15;17) translocation, is markedly facilitated by sodium butyrate (NaB), a histone deacetylase inhibitor (HDACI), or by hexamethylene bisacetamide (HMBA), a non-HDACI tRA-differentiation inducer, as determined by nitroblue tetrazolium reduction. The tRA-induced expression of RIG-G, Bfl-1/A1, and p21^waf1 and, to a lesser extent, of CCAAT/enhancer binding protein-ε (C/EBPε) are also enhanced by such combined treatments. Both responses are associated with a facilitated diminution of the leukemogenic PML-RARα protein and retained ΔPML-RARα, a cleavage product. Treatment with tRA in tRA differentiation-resistant NB4 subclones R4 and MR-2 does not result in PML-RARα diminution and the tested gene expressions. Moreover, the addition of HMBA or NaB with tRA results in only minimal increase of differentiation in the tRA differentiation-resistant subclones. The increases in acetylated histone H3 (AcH3) and AcH4 in NaB-treated NB4, R4, and MR-2 cells are similar and do not correlate with the extent of differentiation induction when NaB and HMBA are given in combination with tRA. Arsenic trioxide (As₂O₃) treatment results in the total degradation of PMLRARα without increasing AcH3 or AcH4 or inducing differentiation in R4 cells. AS₂O₃ in combination with tRA induces gene (Bfl-1/A1 and C/EBPε) expression and partial differentiation. Both NaB and HMBA addition to As₂O₃-plus-tRA-treated R4 cells further enhances differentiation. These results suggest that elimination of the dominant negative PML-RARα protein is required prior to inhibition of histone deacetylase to fully overcome tRA- differentiation resistance in APL cells.