Disruption of RUNX1 contributes to malignant transformation and consequently, RUNX1 variants (RUNX1^var) are found in various myeloid hematological malignancies and associated with a poor prognosis. RUNX1 can either activate or repress transcription, depending on its interaction with co-activators or co-repressors and the promoter context. Genetic variants in RUNX1 can be found in the entire gene. N-terminal missense and nonsense variants mostly affect the RUNT domain, while truncating variants often lead to deletion of the transactivation domain, or result in decreased protein expression due to nonsense mediated RNA-decay. In addition, more than a dozen different chromosomal translocations have been described in hematological malignancies that involve either RUNX1 or CBFB. One of the most common translocations in AML is t(8;21)(q22;q22), leading to the fusion protein RUNX1::RUNX1T1, which accounts for approximately 10% of adult AML [1]. The RUNX1::RUNX1T1 is recognized as a AML-defining genetic abnormality in the latest WHO and ICC classifications [2] and defined as favorable risk AML in the European Leukemia Net (ELN) recommendations. In contrast, AML with a RUNX1^var has been categorized as AML with myelodysplasia-related gene mutations in the WHO 2022 classification [3] and as an adverse risk genetic abnormality by the ELN 2022 [4]. The different prognostic value of RUNX1 variants versus RUNX1 translocations is intriguing, but the underlying mechanisms have not been fully elucidated. We have previously identified Transciption factor 4 (TCF4, E2-2) expression as an independent prognostic factor in AML [5] and found that TCF4 expression is a dominant prognostic factor in multivariate analysis over the presence of RUNX1^var or t(8;21) in AML, suggesting that TCF4 mediates the prognostic effect of RUNX1 aberrations in AML. The exact mechanism how the expression of TCF4 is linked to RUNX1 aberrations is still unclear.

To identify the region of the TCF4 promoter which is essential for RUNX1 binding, we divided the RUNX1 binding region into three different parts of similar size (Fig. 1C). Assessing the transcriptional activity of the different parts revealed that the isolated part 3 did not show transcriptional activity. In contrast, both part 1 and 2 showed transcriptional activity, where part 2 displayed higher activity (Supplementary Fig. 1D). Addition of RUNX1^wt reduced TCF4 promoter activity via both regions (Supplementary Fig. 1E). Interestingly, the sum of luciferase activity of the separate parts was limited when compared to the activity of the full promoter, indicating a synergistic effect in the presence of the combined parts. We further cloned the precise region covering the RUNX1 binding region most proximal to the transcriptional start site, containing a TGTGGT RUNX1 consensus binding site (Fig. 1C, purple, chr18:53255032-53255887) in front of luciferase and tested the effect of our different RUNX1^var. Also here, we found that RUNX1^wt repressed transactivation, which was lost by RUNX1^var, and retained by RUNX1-RUNX1T1 (Fig. 2A). In two recent publications, transactivation assays were used to assess the pathogenicity of different (types) of RUNX1^var [9, 10]. These assays were based on several sequences derived from RUNX1 target genes. As increased TCF4 expression has a strong prognostic effect, the transactivation of TCF4 promoter sequences in this context would be valuable. In addition, in these experiments only sequences were tested from genes that were activated by RUNX1, no targets that are repressed by RUNX1 were taken along. Therefore, we further tested several RUNX1^var described previously, and tested their transcriptional effects on the TCF4 promotor in the myeloid erythroleukemia cell line HEL (Fig. 2B). We confirm that the RUNX1 L29S, a benign RUNX1 variant, acts similar to RUNX1^wt, while all other known pathogenic RUNX1^var, lost the repressive effect on the TCF4 promotor [9, 10].

RUNX1 的破坏导致恶性转化，因此，RUNX1 变体 （RUNX1^var） 存在于各种髓系血液系统恶性肿瘤中，并与不良预后相关。RUNX1 可以激活或抑制转录，具体取决于它与共激活因子或共阻遏因子的相互作用以及启动子环境。RUNX1 中的遗传变异可以在整个基因中找到。N 末端错义和无义变体主要影响 RUNT 结构域，而截短变体通常会导致反式激活结构域的缺失，或由于无义介导的 RNA 衰变而导致蛋白质表达降低。此外，在涉及 RUNX1 或 CBFB 的血液系统恶性肿瘤中已描述了十几种不同的染色体易位。AML 中最常见的易位之一是 t（8;21）（q22;q22），导致融合蛋白 RUNX1：：RUNX1T1，约占成人 AML 的 10% [1]。RUNX1：：RUNX1T1 在最新的 WHO 和 ICC 分类中被认定为 AML 定义的遗传异常 [2]，在欧洲白血病网 （ELN） 建议中被定义为有利风险的 AML。相比之下，在 WHO 2022 分类中，带有 RUNX1^{var 的} AML 被归类为具有骨髓增生异常相关基因突变的 AML [3]，在 ELN 2022 中被归类为不良风险遗传异常 [4]。RUNX1 变体与 RUNX1 易位的不同预后价值很有趣，但其潜在机制尚未完全阐明。 我们之前已经确定转录因子 4 （TCF4， E2-2） 表达是 AML 中的独立预后因素 [5]，并发现 TCF4 表达是多变量分析中 RUNX1^var 或 t（8;21） 在 AML 中，表明 TCF4 介导 AML 中 RUNX1 畸变的预后影响。TCF4 表达与 RUNX1 畸变相关的确切机制尚不清楚。

为了确定 TCF4 启动子对 RUNX1 结合至关重要的区域，我们将 RUNX1 结合区域分为三个大小相似的不同部分（图 1C）。评估不同部分的转录活性表明，分离的部分 3 未显示转录活性。相比之下，第 1 部分和第 2 部分都显示出转录活性，而第 2 部分显示出更高的活性（补充图 1D）。添加 RUNX1^wt 降低了两个区域的 TCF4 启动子活性（补充图 1E）。有趣的是，与完全启动子的活性相比，单独部分的荧光素酶活性总和是有限的，这表明在存在组合部分的情况下具有协同效应。我们进一步克隆了覆盖最靠近转录起始位点的 RUNX1 结合区的精确区域，在荧光素酶前面包含一个 TGTGGT RUNX1 共有结合位点 （图 1C，紫色，chr18：53255032-53255887），并测试了我们不同的 RUNX1^变体的效果。同样在这里，我们发现 RUNX1^wt 抑制了反式激活，反式激活被 RUNX1^var 丢失，并被 RUNX1-RUNX1T1 保留（图 2A）。在最近的两篇出版物中，反式激活试验用于评估不同（类型）RUNX1^{var 的}致病性 [9， 10]。这些检测基于源自 RUNX1 靶基因的几个序列。由于 TCF4 表达增加具有很强的预后效应，因此在这种情况下 TCF4 启动子序列的反式激活将是有价值的。此外，在这些实验中，仅测试了来自 RUNX1 激活的基因的序列，没有带走被 RUNX1 抑制的靶标。 因此，我们进一步测试了前面描述的几种 RUNX1^变体，并测试了它们对髓系红白血病细胞系 HEL 中 TCF4 启动子的转录作用（图 2B）。我们证实 RUNX1 L29S 是一种良性 RUNX1 变体，其作用类似于 RUNX1^wt，而所有其他已知的致病性 RUNX1^变体都失去了对 TCF4 启动子的抑制作用 [9， 10]。