U2AF1 is a haplo-essential gene required for hematopoietic cancer cell survival in mice

Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained wild-type allele is expressed, suggesting that mutant hematopoietic cells may require the residual wild-type allele to be viable and cause disease. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knock-out mice was similar to control mice, but that deletion of the wild-type allele in U2AF1 (S34F) heterozygous mutant expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of wild-type U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1 (S34F) expressing cells were also more sensitive to reduced, but not absent, expression of wild-type U2AF1 than non-mutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the wild-type U2af1 allele was deleted compared to when it was not deleted. These results suggest that selectively targeting the wild-type U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations. Mouse E14.5 fetal liver cells or adult bone marrow were harvested and RNA was then isolated from sorted hematopoietic progenitor cells (Lin- cKit+ Sca1-) using the NucleoSpin RNA Plus XS kit (Macherey-Nagel), per manufacturer’s instructions. The RNA-sequencing libraries were prepared using KAPA RNA Hyper Prep Kit with RiboErase, per manufacturer’s protocol. After amplification, ~300 bp fragments were sequenced on Illumina NovaSeq 6000, generating 2 x 150bp paired-end reads. The reads were then mapped using HISAT2 (version 2.1.0) against the GRCm38 version of the mouse genome from Ensembl consortium. Differentially expressed genes were identified using DESeq2, and splicing event classification was performed using rMATS (version 4.1.0). For Fetal liver samples due to experimental limitations few samples have potential confounding batch effects that may impact differential gene expression. Therefore, we focused using samples within each batch for splicing analysis only.

剪接体（spliceosome）基因U2AF1的体细胞突变在骨髓增生异常综合征（myelodysplastic syndromes）患者中较为常见。编码最常见氨基酸替换的U2AF1突变始终为杂合型，且保留的野生型等位基因（wild-type allele）会得以表达，这提示突变造血细胞可能需要残留的野生型等位基因以维持存活并致病。本研究证实，U2af1杂合敲除小鼠的造血功能与RNA剪接均与对照组小鼠相似，但在表达U2AF1（S34F）杂合突变的造血细胞中删除野生型等位基因（即形成半合子突变体）会导致小鼠死亡。上述结果确认，U2AF1突变的造血细胞在体内存活依赖于野生型U2AF1的表达，且U2AF1属于单倍必需致癌基因。表达突变型U2AF1（S34F）的细胞对野生型U2AF1表达量降低（而非完全缺失）的敏感性也高于非突变细胞。此外，与未删除野生型U2af1等位基因的组别相比，移植了表达突变型U2AF1的白血病细胞的小鼠在删除野生型U2af1等位基因后，肿瘤负荷显著降低且生存期得以延长。上述结果提示，在杂合突变细胞中选择性靶向野生型U2AF1等位基因可诱导癌细胞死亡，有望成为携带U2AF1突变患者的治疗策略。收集小鼠胚胎14.5天（E14.5）的胎肝细胞或成年骨髓细胞，随后依照制造商说明书，使用NucleoSpin RNA Plus XS试剂盒（Macherey-Nagel）从分选的造血祖细胞（Lin⁻ cKit⁺ Sca1⁻）中提取RNA。采用搭载RiboErase的KAPA RNA Hyper Prep试剂盒，依照制造商方案构建RNA测序文库。扩增完成后，在Illumina NovaSeq 6000平台对约300 bp的片段进行测序，生成2×150 bp双端读段。使用HISAT2（版本2.1.0）将读段比对至Ensembl联盟发布的小鼠基因组GRCm38版本。通过DESeq2鉴定差异表达基因，采用rMATS（版本4.1.0）进行剪接事件分类。由于实验条件限制，胎肝样本中仅有少量样本存在可能干扰差异基因表达分析的混杂批次效应，因此本研究仅使用每一批次内的样本开展剪接分析。