Panel A shows the relative messenger RNA levels of Hck and Ncf1 genes. The x-axis lists the constructs (PLZF-Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, Retinoic Acid Receptor Alpha, Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, UBC9/Retinoic Acid Receptor Alpha), and the y-axis shows the relative messenger RNA level. Panel B displays the relative messenger RNA levels of the Cyp26a1 gene in progenitors treated with TAK-981 or all-trans retinoic acid for 24 hours. The x-axis lists the constructs (Retinoic Acid Receptor Alpha, Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, UBC9/Retinoic Acid Receptor Alpha), and the y-axis shows the relative messenger RNA level. Panel C shows the relative messenger RNA levels of the Ifng gene in progenitors treated with TAK-981 for 24 hours. The x-axis lists the constructs (Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, PLZF-Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha), and the y-axis shows the relative messenger RNA level. Panel D depicts the percentage of CD11 expression in progenitors treated with all-trans retinoic acid for 2 days. The x-axis lists the constructs (Retinoic Acid Receptor Alpha, Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, UBC9/Retinoic Acid Receptor Alpha), and the y-axis shows the percentage of CD11 expression. Panel E shows the relative colony numbers of progenitors transformed with different constructs. The x-axis lists the constructs (Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, PLZF-Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, Promyelocytic Leukemia Protein-Retinoic Acid Receptor Gamma, UBC9/Retinoic Acid Receptor Alpha), and the y-axis shows the relative colony numbers. Panel F depicts the percentage of annexin V staining in progenitors treated with TAK-981 for 16 hours or 5 days. The x-axis lists the constructs (Retinoic Acid Receptor Alpha, Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, UBC9/Retinoic Acid Receptor Alpha), and the y-axis shows the percentage of annexin V staining. Each graph includes error bars representing standard deviation and annotations indicating statistical significance.
TAK-981 restores RARA target gene expression, promotes differentiation and apoptosis, and impairs colony formation. (A) Real-time PCR of RARA target genes Hck and Ncf1 in primary progenitors transformed with indicated constructs after 16 h of TAK-981 treatment (100 nM) in methylcellulose. N = 3. (B and C) Real-time PCR analysis of Cyp26a1 (B) and Ifng (C) expression in PML::RARA- or PLZF::RARA-transformed progenitors treated with TAK-981 (100 nM, 24 h) in methylcellulose. N = 2. In B, progenitors were treated with ATRA (1 μM) for 24 h in methylcellulose. (D) FACS analysis of differentiation marker CD11 expression in Lin− progenitors transformed with the indicated fusions and treated with ATRA (1 μM) for 2 days. N = 2. (E) Colony counts of primary mouse Lin− progenitors transformed with PML::RARA, PLZF::RARA, PML::RARG, or UBC9/RARA. N = 3. (F) FACS analysis of annexin V staining of RARA-, PML::RARA-, and UBC9/RARA-transformed progenitors treated with TAK-981 (100 nM) for 16 h or 5 days. Data represent the mean ± SD of three technical repeats from one representative experiment out of at least two biologically independent experiments. Statistical significance was assessed by an unpaired t test: *P < 0.05, **P < 0.005, and ***P < 0.001. n.s = not significant.