Trancriptomic analysis of leukemic bone marrow cells with GFI1 wildtype, GFI1 SNP variant (GFI1-36N) and low level of GFI1 expression

GFI1 is a transcriptional repressor protein that plays an essential role in HSCs development, lymphoid and myeloid differentiation and Acute Myeloid Leukaemic (AML) pathogenesis. Low expression levels of GFI1 is associated with a poor prognosis in AML development. In addition, a single nucleotide polymorphism (SNP) variant of GFI1 results in the generation of GFI1 protein with asparagine (N) instead of serine (S) at the 36th amino acid position, known as GFI136N. Expression of the GFI1-36N allele leads as well to poor prognosis and promotes AML development. In this study, we demonstrated with the help of RNAseq transcriptomic analysis that the presence of GFI1-36N is associated with increased frequency of chromosomal aberrations and mutational burden in murine and human AML cells. In particular, GFI1-36N modulates DNA repair pathways, O6-methylguanine-DNA-methyltransferase (MGMT)-mediated repair and homologous recombination repair (HR). Mechanistically, GFI1-36N exhibits impaired binding to Ndrg1 promoter element compared to GFI1-36S (wild type), causing decreased NDRG1 levels, consequently leading to suppression of MGMT expression, imprinted at the transcriptome and proteome, thus leaving the AML cells vulnerable to DNA damaging agents. Furthermore, we showed that a low expression level of GFI1 in leukemic cells is associated with high OXPHOS and enhanced glutamine metabolism. However, we hypothesise that the observed metabolic phenotype is mediated through FOXO1 protein. RNAseq transcriptomic analysis revealed higher Foxo1 mRNA expression levels with lower GFI1 expression, providing the first hint of Foxo1 as a potential target gene of GFI1 protein. The mRNA and protein levels of high Foxo1 with reduced GFI1 expression was confirmed by RT-PCR and western blot, respectively. In addition, CHIPseq and ATACseq analysis further proved that Foxo1 is a potential target gene of GFI1. In summary, we show that GFI1 plays a role during DNA repair and metabolism and thus provides critical insights into a novel therapeutic option for AML patients carrying the GFI1-36N variant or having a low expression level of GFI1. Overall design: The current study includes three mouse models: GFI1-KI mice, which express human GFI1 at the murine Gfi1 locus; GFI1-KD mice, expressing 10-20 % of the human GFI1 gene; and GFI1-36N mice, which express 36N variant of human GFI1 gene. The lineage depleted cells from GFI1-KI, GFI1-KD, and GFI1-36N mice were isolated and retrovirally transduced with MLL-AF9 oncogene, coexpressing GFP reporter protein. The GFP positive cells were FACS sorted and transplanted into lethally irradiated (7+3 Gy) C57BL/6 mice. The total bone marrow cells from the leukemic mice were serially transplanted into sublethally irradiated mice (3 Gy), and the total bone marrow cells from the quaternary transplanted leukemic mice were used for transcriptomic analysis. For the transcriptome analysis of temozolomide treated cells, leukemic bone marrow cells were treated with 50 µg/ml temozolomide 20 h before RNA isolation.

GFI1是一种转录抑制蛋白，在造血干细胞（Hematopoietic Stem Cells，HSCs）发育、淋巴系与髓系分化以及急性髓系白血病（Acute Myeloid Leukemia，AML）的发病过程中发挥核心作用。GFI1低表达与AML发生中的不良预后密切相关。此外，GFI1的单核苷酸多态性（Single Nucleotide Polymorphism，SNP）变异会导致其编码的蛋白在第36位氨基酸位点发生丝氨酸（S）替换为天冬酰胺（N）的突变，该变异体被称为GFI136N。GFI1-36N等位基因的表达同样会导致不良预后，并促进AML的发生发展。

本研究通过RNA测序（RNA sequencing，RNAseq）转录组分析证实，GFI1-36N的存在与小鼠和人AML细胞中染色体畸变频率升高及突变负荷增加显著相关。具体而言，GFI1-36N可调控DNA修复通路，包括O6-甲基鸟嘌呤-DNA-甲基转移酶（O6-methylguanine-DNA-methyltransferase，MGMT）介导的修复以及同源重组修复（Homologous Recombination Repair，HR）。

机制层面，与野生型GFI1-36S相比，GFI1-36N与Ndrg1启动子元件的结合能力受损，导致NDRG1水平下调，进而抑制MGMT的表达——该调控效应在转录组和蛋白质组层面均有体现，最终使AML细胞对DNA损伤剂更为敏感。

此外，本研究发现白血病细胞中GFI1低表达与高氧化磷酸化（Oxidative Phosphorylation，OXPHOS）及增强的谷氨酰胺代谢相关，但本研究推测上述代谢表型由叉头框蛋白O1（Forkhead Box O1，FOXO1）介导。RNAseq转录组分析显示，GFI1低表达时Foxo1的mRNA水平更高，这首次提示Foxo1可能是GFI1蛋白的潜在靶基因。研究人员分别通过逆转录聚合酶链式反应（Reverse Transcription-Polymerase Chain Reaction，RT-PCR）和蛋白质印迹（Western Blot）验证了GFI1低表达时Foxo1的mRNA和蛋白水平升高。此外，染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing，ChIP-seq）与转座酶可及性测序（Assay for Transposase-Accessible Chromatin using sequencing，ATAC-seq）分析进一步证实Foxo1是GFI1的潜在靶基因。

综上，本研究证实GFI1在DNA修复及代谢调控中发挥重要作用，为携带GFI1-36N变异或GFI1低表达的AML患者提供了全新的治疗思路。

研究整体设计：本研究包含三种小鼠模型：①GFI1敲入（GFI1-KI）小鼠，可在小鼠Gfi1基因座表达人源GFI1；②GFI1敲低（GFI1-KD）小鼠，其表达的人源GFI1基因仅为正常水平的10%~20%；③GFI1-36N小鼠，可表达人源GFI1的36N变异体。研究人员分离上述三种小鼠的谱系耗竭细胞，通过逆转录病毒转导携带共表达绿色荧光蛋白（Green Fluorescent Protein，GFP）报告蛋白的MLL-AF9癌基因；随后通过流式细胞术（Fluorescence Activated Cell Sorting，FACS）分选GFP阳性细胞，并将其移植至接受致死剂量辐射（7+3 Gy）的C57BL/6小鼠体内。之后，将白血病小鼠的全骨髓细胞连续移植至接受亚致死剂量辐射（3 Gy）的受体小鼠体内，最终取第四代移植白血病小鼠的全骨髓细胞用于转录组分析。针对替莫唑胺（Temozolomide）处理细胞的转录组分析，研究人员在RNA提取前20小时，用50 μg/ml的替莫唑胺处理白血病骨髓细胞。