Altered enhancer-promoter interaction leads to MNX1 expression in pediatric acute myeloid leukemia with t(7;12)(q36;p13) [ACT-Seq]

Acute myeloid leukemia (AML) with translocation t(7;12)(q36;p13) is a subgroup that occurs only in infants or very young children and is characterised by a poor outcome. Deep molecular characterization has been hampered by the rarity of this AML subtype and the lack of model systems. Approximately 50% of AML with t(7;12)(q36;p13) express an MNX1::ETV6 oncofusion transcript, but there is no evidence for the presence of an oncofusion protein. However, a universal feature is the strong RNA and protein expression of MNX1, a homeobox transcription factor that is normally not transcribed in haematopoietic cells. Here we use whole genome sequencing data to precisely map the translocation breakpoints on chromosomes 7 and 12 in these pediatric AML patients to a region downstream of MNX1 on chromosome 7 and either introns 1 or 2 of ETV6 on chromosome 12. We confirm the tight correlation of MNX1 overexpression in t(7;12)(q36;p13) AML in our own samples and three additional cohorts of pediatric AML. Using a CRISPR-engineered iPSC cell line, ChiPSC22t(7;12), harbouring the t(7;12)(q36;p13) translocation, we unravel an enhancer-hijacking event leading to MNX1 overexpression. Identification of hematopoietic enhancer regions in hematopoietic stem and progenitor cells derived from differentiated ChiPSC22t(7;12) cells allowed us to demonstrate their importance for MNX1 expression in knock-out experiments and by measuring the promoter-enhancer distance in confocal microscopy. In contrast to the prevailing dogma in AML, suggesting that translocations lead to the creation of oncofusion genes, t(7;12)(q36;p13) AML is characterized by an enhancer-hijacking event driving MNX1, a novel leukemia oncogene and the haploinsufficiency of ETV6. Overall design: We used the iPSC line CHIPSC22 (Cellartis/Takara Bio Europe AB, Gothenburg, Sweden), both WT and with a t(7;12)(q36;p13) translocation. We included both the iPSC CHIPSC22 and CHIPSC22 differentiated into hematopoietic stem and progenitor cells (HSPC). We used antibodies against H3K4me1, H3K27ac and IgG. For each condition, 3 replicates were used.

携带t(7;12)(q36;p13)易位的急性髓系白血病（Acute myeloid leukemia，AML）是一种仅见于婴儿或极低龄儿童的亚型，其临床预后不良。由于该AML亚型极为罕见且缺乏模型系统，其深度分子表征一直受到限制。约50%的t(7;12)(q36;p13) AML患者可表达MNX1::ETV6癌融合转录本，但尚未检测到癌融合蛋白的存在。不过，该亚型的共同特征为MNX1的高RNA与蛋白表达——MNX1是一种同源盒转录因子（homeobox transcription factor），正常情况下在造血细胞中不发生转录。本研究利用全基因组测序数据，将这些儿童AML患者7号和12号染色体上的易位断裂点精确定位至7号染色体MNX1下游区域，以及12号染色体ETV6的第1或第2内含子区域。我们在自身样本以及另外3个儿童AML队列中，证实了t(7;12)(q36;p13) AML中MNX1过表达的紧密相关性。通过构建携带t(7;12)(q36;p13)易位的CRISPR编辑诱导多能干细胞（induced pluripotent stem cell，iPSC）细胞系ChiPSC22t(7;12)，我们阐明了驱动MNX1过表达的增强子劫持事件。对分化自ChiPSC22t(7;12)细胞的造血干祖细胞（hematopoietic stem and progenitor cell，HSPC）中的造血增强子区域进行鉴定后，我们通过敲除实验以及共聚焦显微镜测量启动子-增强子距离，证实了这些增强子区域对MNX1表达的关键调控作用。与AML领域认为易位会导致癌融合基因产生的主流认知不同，t(7;12)(q36;p13) AML的特征为增强子劫持事件驱动新型白血病致癌基因MNX1的表达，以及ETV6的单倍体剂量不足。实验整体设计：本研究使用了来自瑞典哥德堡Cellartis/ Takara Bio欧洲公司的iPSC系CHIPSC22，包含野生型（wild type，WT）与携带t(7;12)(q36;p13)易位的两种细胞株。我们同时纳入未分化的CHIPSC22 iPSC，以及诱导分化为造血干祖细胞的CHIPSC22细胞。实验使用针对H3K4me1、H3K27ac及IgG的抗体，每个实验条件均设置3个生物学重复。