Germline variant GFI1-36N affects DNA repair in AML cells resulting in sensitivity to DNA damage and repair therapy

Transcriptional repressor Growth factor independence 1 (GFI1) is a key regulator of haematopoiesis. We previously established that the germline variant GFI1-36N promotes acute myeloid leukemia (AML) development, however the mechanism is not full elucidated. Here using multi-omics approach, we show GFI1-36N expression impedes DNA repair in leukemic cells. We demonstrate 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) 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. Targeting MGMT via temozolomide and HR via olaparib caused specifically extensive lethality in in vitro and ex vivo human and AML samples expressing GFI1-36N. Whereas the effects were insignificant on non-malignant GFI1-36S or GFI1-36N cells. Further, mice transplanted with GFI1-36N leukemic cells treated with combination of temozolomide and olaparib had a significantly longer AML-free survival than mice transplanted with GFI1-36S leukemic cells. In summary, we show that GFI1-36N disturbs DNA repair activity via NDRG1-MGMT axis and thus provides critical insights into novel therapeutic option for AML presented with GFI1-36N variant. Key Points Presence of GFI1-36N impedes Homologous DNA and MGMT DNA repair selectively in AML cells via the NDRG1-MGMT axis. Use of temozolomide and olaparib allows selectively targeting GFI1-36N leukemic cells. Introduction Gfi1 is a transcription factor which regulates the development of haematopoietic cells as well as neuronal and intestinal epithelial cells 1-5. We reported that a variant of GFI1, denominated GFI1-36N (characterized by an exchange of serine to asparagine at position 36), has a prevalence of 5-7% in a healthy control population but is found at an increased frequency of 10-15% among MDS and AML patients 6,7. The expression of germline variant GFI1-36N predisposes the carriers to develop de novo AML and MDS and correlates with a poor prognosis 6,7. Recently, we and other showed that malignant cells with GFI1-36N variant have increased H3K9-acetylation at target genes resulting in higher expression of genes required for cell survival and proliferation 8. GFI1 exerts its repressive role by recruiting histone-modifying enzymes (deacetylases HDAC1-3, demethylase LSD1, methyl transferase G9a) and regulates the accessibility of DNA to its target genes such as Hoxa9, Pbx1, Meis1, CSF1 and CSFR1 9-15. We also showed that GFI1 regulates apoptosis through its regulation of p53 in lymphoblastic leukemia 16 and we have demonstrated that GFI1 facilitates DNA repair 17. However, it is not known how these activities are affected in the GFI1-36N variant and whether the ability of GFI1 to regulate DNA repair pathways is maintained and how this might affect the development of myeloid malignancies. In this study, we leveraged multi-omics profiling to gain mechanistic insights into the molecular architecture that drives leukemia in the presence of GFI1-36N. We provide evidence that GFI1-36N interferes with DNA in leukemic myeloid cells, which leads to a higher frequency of genetic aberrations in MDS/AML cells. We also show that GFI1-36N myeloid leukemic cells are more sensitive to targeting MGMT and HR repair deficient cells, which opens a new selective therapeutic window to treat AML/MDS.

转录抑制因子生长因子独立因子1（GFI1）是造血作用的关键调控因子。本团队前期研究证实，生殖系变异体GFI1-36N可促进急性髓系白血病（AML）的发生发展，但其具体分子机制尚未完全阐明。本研究采用多组学方法，证实GFI1-36N的表达会阻碍白血病细胞的DNA修复过程。研究发现，GFI1-36N的存在与小鼠及人类AML细胞中染色体畸变频率升高、突变负荷增加显著相关。具体而言，GFI1-36N可调控两类DNA修复通路：O6-甲基鸟嘌呤-DNA-甲基转移酶（MGMT）修复通路与同源重组修复（HR）通路。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蛋白水平下调，进而抑制MGMT的表达——该调控效应在转录组与蛋白质组层面均得到验证，最终使AML细胞对DNA损伤剂的敏感性显著升高。

体外及离体的人类AML样本实验显示，通过替莫唑胺靶向抑制MGMT、奥拉帕利靶向阻断HR通路，可特异性诱导表达GFI1-36N的白血病细胞发生大规模死亡；而对非恶性的GFI1-36S或GFI1-36N细胞，上述治疗的效应并不显著。进一步的体内实验表明，移植GFI1-36N阳性白血病细胞的小鼠，经替莫唑胺联合奥拉帕利治疗后，其无AML生存期显著长于移植GFI1-36S阳性白血病细胞的小鼠。

综上，本研究证实GFI1-36N可通过NDRG1-MGMT轴扰乱DNA修复活性，为携带GFI1-36N变异体的AML患者提供了全新的治疗策略。

**核心要点**：
1. GFI1-36N可通过NDRG1-MGMT轴，选择性阻断急性髓系白血病细胞的同源重组修复与MGMT介导的DNA修复；
2. 替莫唑胺联合奥拉帕利可实现对GFI1-36N阳性白血病细胞的选择性靶向杀伤。

**引言**：
Gfi1是一类转录因子，可调控造血细胞、神经元及肠上皮细胞的发育[1-5]。本团队此前报道，GFI1的一处变异体GFI1-36N（表现为第36位丝氨酸突变为天冬酰胺）在健康对照人群中的患病率为5%~7%，但在骨髓增生异常综合征（MDS）与AML患者中的检出率升高至10%~15%[6,7]。携带该生殖系变异体的个体易罹患原发性AML与MDS，且预后较差[6,7]。近期本团队及其他研究团队证实，携带GFI1-36N变异体的恶性细胞在靶基因位点的H3K9乙酰化水平升高，进而上调细胞存活与增殖相关基因的表达[8]。GFI1通过招募组蛋白修饰酶（包括去乙酰化酶HDAC1-3、去甲基化酶LSD1、甲基转移酶G9a）发挥转录抑制作用，并调控DNA与靶基因（如Hoxa9、Pbx1、Meis1、CSF1及CSFR1）的染色质可及性[9-15]。此外，本团队还发现GFI1可通过调控p53通路影响淋巴白血病的细胞凋亡[16]，且能促进DNA修复[17]。但目前尚不明确GFI1-36N变异体如何影响上述功能，以及该变异体是否保留GFI1调控DNA修复通路的能力，及其对髓系恶性肿瘤发生发展的具体影响。

本研究通过多组学分析，解析GFI1-36N阳性白血病发生的分子机制。研究证实，GFI1-36N可干扰髓系白血病细胞的DNA修复过程，进而升高MDS/AML细胞的遗传畸变频率。同时，GFI1-36N阳性髓系白血病细胞对MGMT及HR修复缺陷靶点的靶向治疗更为敏感，这为AML/MDS的治疗提供了全新的选择性治疗窗口。