Faculty of Medicine, Division of Laboratory Medicine, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi, Japan.
Department of Clinical Laboratory, Tohoku Medical and Pharmaceutical University Hospital, Sendai, Miyagi, Japan.
PLoS One. 2024 May 14;19(5):e0303428. doi: 10.1371/journal.pone.0303428. eCollection 2024.
Differentiation therapy using all-trans retinoic acid (ATRA) for acute promyelocytic leukemia (APL) is well established. However, because the narrow application and tolerance development of ATRA need to be improved, we searched for another efficient myeloid differentiation inducer. Kinase activation is involved in leukemia biology and differentiation block. To identify novel myeloid differentiation inducers, we used a Kinase Inhibitor Screening Library. Using a nitroblue tetrazolium dye reduction assay and real-time quantitative PCR using NB4 APL cells, we revealed that, PD169316, SB203580, SB202190 (p38 MAPK inhibitor), and triciribine (TCN) (Akt inhibitor) potently increased the expression of CD11b. We focused on TCN because it was reported to be well tolerated by patients with advanced hematological malignancies. Nuclear/cytoplasmic (N/C) ratio was significantly decreased, and myelomonocytic markers (CD11b and CD11c) were potently induced by TCN in both NB4 and acute myeloid leukemia (AML) M2 derived HL-60 cells. Western blot analysis using NB4 cells demonstrated that TCN promoted ERK1/2 phosphorylation, whereas p38 MAPK phosphorylation was not affected, suggesting that activation of the ERK pathway is involved in TCN-induced differentiation. We further examined that whether ATRA may affect phosphorylation of ERK and p38, and found that there was no obvious effect, suggesting that ATRA induced differentiation is different from TCN effect. To reveal the molecular mechanisms involved in TCN-induced differentiation, we performed microarray analysis. Pathway analysis using DAVID software indicated that "hematopoietic cell lineage" and "cytokine-cytokine receptor interaction" pathways were enriched with high significance. Real-time PCR analysis demonstrated that components of these pathways including IL1β, CD3D, IL5RA, ITGA6, CD44, ITGA2B, CD37, CD9, CSF2RA, and IL3RA, were upregulated by TCN-induced differentiation. Collectively, we identified TCN as a novel myeloid cell differentiation inducer, and trials of TCN for APL and non-APL leukemia are worthy of exploration in the future.
全反式维甲酸（ATRA）在急性早幼粒细胞白血病（APL）中的分化治疗已得到广泛认可。然而，由于 ATRA 的应用范围较窄且需要改善其耐受性，我们正在寻找另一种有效的髓样分化诱导剂。激酶激活参与白血病生物学和分化阻断。为了鉴定新型髓样分化诱导剂，我们使用了激酶抑制剂筛选文库。通过硝基蓝四唑染料还原测定和 NB4 APL 细胞的实时定量 PCR，我们发现 PD169316、SB203580、SB202190（p38 MAPK 抑制剂）和曲昔匹特（TCN）（Akt 抑制剂）能显著增加 CD11b 的表达。我们专注于 TCN，因为它被报道对晚期血液恶性肿瘤患者具有良好的耐受性。TCN 显著降低核/质（N/C）比值，并在 NB4 和急性髓系白血病（AML）M2 衍生的 HL-60 细胞中强力诱导髓系标志物（CD11b 和 CD11c）的表达。使用 NB4 细胞的 Western blot 分析表明，TCN 促进 ERK1/2 磷酸化，而 p38 MAPK 磷酸化不受影响，表明 ERK 通路的激活参与了 TCN 诱导的分化。我们进一步研究了 ATRA 是否会影响 ERK 和 p38 的磷酸化，发现没有明显的影响，这表明 ATRA 诱导的分化与 TCN 效应不同。为了揭示 TCN 诱导分化涉及的分子机制，我们进行了微阵列分析。使用 DAVID 软件进行的途径分析表明，“造血细胞谱系”和“细胞因子-细胞因子受体相互作用”途径具有高度显著性。实时 PCR 分析表明，这些途径的组成部分包括 IL1β、CD3D、IL5RA、ITGA6、CD44、ITGA2B、CD37、CD9、CSF2RA 和 IL3RA，均被 TCN 诱导分化上调。总之，我们鉴定出 TCN 是一种新型的髓样细胞分化诱导剂，未来值得探索 TCN 在 APL 和非 APL 白血病中的应用。
