不同RUNX1突变协同MLL-PTD 在MDS发生及AML转化作用机制研究

Myelodysplastic syndrome (MDS) is a hematopoietic stem-cell disorder characterized by trilineage dysplasia and susceptibility to acute myelogenous leukemia (AML). Recently, mutations of the transcription factor RUNX1 and MLL-PTD have been identified in 10% to 20% and 2.7% to 4.4% of MDS and MDS/AML. Herein, we hypothesize that RUNX1 mutations and MLL-PTD cooperation contribute to MDS genesis and AML transformation. Therefore, we perform mouse bone marrow transplantation (BMT) using Mll-PTD knock-in mouse bone marrow cells transduced with different type of RUNX1 mutants (RUNX1 D171N, N-terminal mutant; RUNX1 s291fs, C-termina mutant). Underwent BMT, transplanted mice will be monitored for long-term reconstitution of the mutant RUNX1 transduced HSC cells, abnormal stem cell biology and their potential MDS onset. Briefly, engraftment of donor-derived cell and phenotype of hematopoietic stem cell in transplanted mice are assessed monthly. To address the self-renewal in vivo, serial BMT are performed 4 months after primary BMT. Survival is estimated by using Kaplan-Meier survival curve. Gene express profile will be perform on LSK cells from Mll-PTD - RUNX1D171, Mll-PTD - RUNX1s291fs, WT-RUNX1 D171, WT- RUNX1 s291fs and Mll-PTD knock-in mice. The key genes involved in those mouse models will be confirmed. Our proposed study will generate new mouse models for potentially modeling human MDS. RUNX1 mutations cooperate with MLL-PTD might accelerate the disease onset. This will help for the future cooperate events studies and drug tests. The new models will provide us new tools to cross with other available mutant mice models, which are implicated in human MDS at second or third hits.

利用正常干细胞→MDS（白血病前期）干细胞→白血病干细胞这样一个肿瘤干细胞发生、演变自然病史模式，以MDS及AML常见分子生物学异常MLL-PTD及RUNX1突变为着眼点。在MLL-PTD knock-in小鼠骨髓细胞中表达不同类型RUNX1突变，构建移植鼠动物模型。通过供者细胞植入分析、移植鼠造血干细胞自我更新及增殖能力分析、生存曲线分析，比较不同类型RUNX1突变与MLL-PTD 协同作用，探讨MLL-PTD联合RUNX1突变在MDS发生及AML转化中的作用机制。并且通过microarray分析，明确关键靶基因，为后续靶向治疗提供重要基础依据。

随着测序技术的不断进步，越来越多的证据表明MDS是一个多基因异常的异质性造血干细胞疾病，我们利用MDS患者常见的基因突变（RUNX1 和MLL-PTD)构建小鼠MDS动物模型，并且通过基因表达谱、Mll-CHIP seq和RNA array分析，寻找可能的作用机制。我们将包含RUNX1不同突变的逆转录病毒质粒包装后感染Mll-PTD小鼠骨髓细胞，建立移植鼠模型。小鼠逐渐出现大细胞性贫血、血小板减少，骨髓及外周血具有三系发育异常的形态学改变，并且RUNX1S291fs移植鼠骨髓病理检查显示除伴有明显铁颗粒增多外，小鼠同时合并不同程度的骨髓纤维化，提示RUNX1突变与MLL-PTD协同，具有致病作用。通过Mll CHIP-Seq、基因表达array和RNA-Seq，我们寻找其共同变化的通路。分析结果显示在MDS小鼠干细胞中，与细胞增殖、凋亡和糖代谢相关基因通路都存在着激活，通过信息网络研究，我们预测HIF1α可能是在其中起关键作用的基因。为了进一步证实我们的假设，随后我们在MDS患者骨髓病理组织中应用免疫组化的方法发现患者HIF1α蛋白表达增强。同样的，我们在Mll-PTD/RUNX Δ/Δ MDS小鼠中发现其Hif1α蛋白水平明显增高，而在Mll-PTD/RUNX1 Δ/Δ MDS小鼠体内敲除Hif1α，则MDS疾病表型被纠正，进一步证实HIF1α在MDS发生中具有重要作用。此外，我们在MDS移植鼠模型中应用HIF1α抑制剂Echinomycin，体外连续集落培养实验证实Echinomycin能够抑制MDS祖细胞克隆形成，并且体内初步实验结果表明Echinomycin能够显著延长MDS移植鼠生存。同时，我们构建了可诱导HIF1α Triple Point Mutation（TPM）小鼠，经Vav1-Cre在造血组织持续诱导HIF1α和HIF1β后，小鼠出现大细胞性贫血，血小板减少，骨髓形态学证实存在多系发育异常，符合MDS表现。通过流式细胞术研究发现，高表达Hif1α的TPM小鼠CD8+T细胞以及单核巨噬细胞明显激活，导致红系成熟阶段细胞减少，无效造血从而引起贫血发生。我们的研究显示HIF1α是MDS发生的关键因子，通过抑制HIF1α表达有可能成为治疗MDS的重要靶点

内容获取失败，请点击重试