Panel A shows two bar graphs. The x-axis labels include PLZF-Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, Retinoic Acid Receptor Alpha, Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, and UBC9/Retinoic Acid Receptor Alpha, while the y-axis represents the relative messenger RNA level. Panel B presents a bar graph showing the percentage of CD11 positive Gr-1 positive cells after 4 days of TAK-981 treatment, with the x-axis labeling different constructs and the y-axis indicating the percentage. Panel C displays a bar graph of c-Kit positive cell percentages under the same conditions. Panel D includes images of stained cells after 36 hours of TAK-981 treatment, showing morphological changes. Panel E shows images of stained Acute Promyelocytic Leukemia blasts after in vivo treatment with TAK-981 for 3 days. Panel F presents a bar graph of relative messenger RNA levels of Cyp26a1, Pram1, Hck, and Ncf genes in Acute Promyelocytic Leukemia blasts after in vivo treatment. Panel G shows a bar graph of the abundance of lineage-negative progenitors expressing different fusion proteins after 5 days of TAK-981 treatment. Panel H depicts a bar graph of colony counts of primary progenitors transformed with different fusion proteins after treatment and replating. Panel I illustrates a schematic model of Promyelocytic Leukemia Protein-driven sumoylation of Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha-bound interactors enforcing the differentiation block in Acute Promyelocytic Leukemia. The graphs and images collectively demonstrate the reactivation of Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha target genes and induction of differentiation upon TAK-981 treatment.
Inhibition of SUMO conjugation reactivates PML::RARA target genes and induces differentiation. (A) Real-time PCR for RARA target, Cyp26a1 and Pram1, gene expression in primary progenitors transformed with indicated constructs after TAK-981 (100 nM) treatment for 18 h in methylcellulose. N = 3. (B and C) FACS analysis of differentiation marker Gr-1 and CD11 (B) or stemness marker c-Kit (C) expression in progenitors transformed with indicated fusions after 4 days of TAK-981 (50 nM) treatment in methylcellulose. N = 3. (D) MGG staining of progenitors after 36 h of TAK-981 treatment (50 nM) in methylcellulose. (E) MGG staining of primary mouse APL blasts from bone marrow after in vivo intravenous treatment with TAK-981 (15 mg/kg) for 3 days. N = 4. Scale bar, 20 mm. (F) Real-time PCR for RARA target Cyp26a1, Pram1, Ncf, and Hck gene expression in primary mouse APL blasts isolated from bone marrow after in vivo treatment with TAK-981 as in E. (G) Abundance of Lin− progenitors expressing the indicated fusions after 5 days of TAK-981 treatment (50 nM) in methylcellulose. N = 3. (H) Colony counts of primary progenitors transformed with indicated fusions. Cells were first treated with TAK-981 (50 nM) for 5 days in methylcellulose, followed by the subsequent replating without SUMO inhibitor. N = 2. Cells were replated every 5–7 days; MC I, II, III, and IV denote the first, second, third, and fourth methylcellulose replatings. (I) Schematic model illustrating PML-driven sumoylation of PML::RARA-bound interactors enforcing the differentiation block in APL. (A–C and F–H) 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.