Homeobox A9 (HOXA9) is a transcription factor that is overexpressed in acute myeloid leukemia (AML). It is associated with the pathogenesis and progression of AML, and is a factor responsible for a poor prognosis. Therefore, the development of HOXA9-targeting molecules may contribute to not only better understanding of the mechanism of HOXA9 regulation, but also the development of therapeutic applications. We constructed a reporter assay system using the promoter region of the KBTBD10 gene, to which HOXA9 directly binds and regulates transcription, in the human acute monocytic leukemia cell line THP-1. Using this luciferase gene assay, we screened 1120 plant extracts and a methanol extract of the unripe fruits of Cerbera manghas was found to suppress the reporter gene expression mediated by the KBTBD10 promoter. From the extract, five steroid-type compounds were identified as the active constituents: 7α-neriifolin (1), 17β-neriifolin (2), 17α-digitoxigenin β-d-glucosyl-(1 → 4)-α-l-thevetoside (3), 17β-digitoxigenin β-d-glucosyl-(1 → 4)-α-l-thevetoside (4), and acetylthevetin B (5). Among the five compounds, 17β-neriifolin most potently inhibited HOXA9-dependent gene expression without affecting the HOXA9 mRNA levels, and suppressed cell proliferation by inducing apoptosis. The findings on the structure–activity relationships of the compounds from C. manghas may contribute to the development of small molecule inhibitors of HOXA9.

同源框 A9 (HOXA9) 是一种在急性髓性白血病 (AML) 中过度表达的转录因子。它与AML的发病和进展有关，是导致预后不良的一个因素。因此，开发 HOXA9 靶向分子不仅有助于更好地理解 HOXA9 调控机制，而且有助于开发治疗应用。我们在人急性单核细胞白血病细胞系 THP-1 中使用 KBTBD10 基因的启动子区域构建了一个报告分析系统，HOXA9 直接结合该基因并调节转录。使用这种荧光素酶基因测定法，我们筛选了 1120 种植物提取物和Cerbera manghas未成熟果实的甲醇提取物被发现抑制由 KBTBD10 启动子介导的报告基因表达。从提取物中，确定了五种类固醇类化合物作为活性成分：7 α -neriifolin ( 1 )、17 β -neriifolin ( 2 )、17 α -digitoxigenin β - d -glucosyl- (1 → 4)- α - l -thevetoside ( 3 )、17 β -digitoxigenin β - d -glucosyl- (1 → 4)- α - l -thevetoside ( 4 ) 和 acetylthevetin B ( 5 )。五种化合物中，17 β-neriifolin 最有效地抑制 HOXA9 依赖性基因表达而不影响 HOXA9 mRNA 水平，并通过诱导细胞凋亡抑制细胞增殖。来自C. manghas的化合物的构效关系的发现可能有助于开发 HOXA9 的小分子抑制剂。