单核细胞中Ptpn11（Shp2）磷酸酶激活突变对正常造血干细胞在白血病发生中异常活化的研究

Germline and somatic activating mutations in tyrosine phosphatase Ptpn11 (Shp2) are associated with an increased risk of developing juvenile myelomonocytic leukemia (JMML), a childhood myeloproliferative neoplasm (MPN). Previous studies have demonstrated that CCL3 secreted from Ptpn11 activating mutations of mouse bone marrow (BM) microenvironmental mesenchymal stem/ progenitor cells (MSPCs) greatly promote the development and progression of hematological malignancy through profound detrimental effects on hematopoietic stem cells (HSCs). To further explore the impact of leukemic cell on wild type HSC, we generate the gain-of-function Ptpn11 mutation in the myeloid lineage, by expressing the lysozyme M promoter-driven and Cre-mediated excision of a transcriptional STOP cassette from Ptpn11E76K conditional knock-in mice. Our hypothesis is that this cause an aberrantly accelerated HSC cycling, decreased stem cell pool, leading to the development and progression of MPN. The deleterious impact of Ptpn11 mutation in the myeloid cells on HSCs are attributable to enhancing cytokine/growth factor signaling. The myeloid cells from Ptpn11E76K/+lysM-Cre+ mice are overwhelming the bone marrow compartment and causing excessive production of IL-1β and other proinflammatory cytokines which hyperactivate HSCs, leading to the exacerbated MPN. MPN is also reproduced in the recipient mice transplanted with mixture of Ptpn11E76K/+lysM-Cre+ and wild type whole bone marrow cells. More importantly, the competitive transplanted mice could identify the contribution of mutant myeloid cell to the activation of HSC by testing Mac-1+Gr-1+ population, HSC pool size, and HSC cell cycle from WT BM portion. To test HSC hyperactivation by IL-1β could be rescued by incubation of IL-1 receptor antagonist (IL-1ra), indicating that blocking IL-1β could ameliorate HSC activation. Collectively, our study aims to test that Ptpn11E76K mutation in myeloid cells hyperactivates HSC and disrupts its niche microenvironment homeostasis. Proteomics analysis in myeloid cell Shp2-activated mutant cells changes in secreted proteins, and RNA-Seq and other large data analysis in both myeloid cell and HSC could map the gene regulation and the development of JMML. Thus, gene associated with the establishment of JMML can be used to prevent the recurrence of new target for clinical treatment.

幼年型粒-单细胞白血病（JMML）是一种由于早期造血细胞分化异常导致的儿童恶性血液系统肿瘤。我们的前期工作表明由单核细胞产生的促炎细胞因子导致正常HSC过度活化，并加剧骨髓移植治疗后由供体细胞发生的骨髓增生性肿瘤。目前骨髓移植后复发率高，其机理仍是难点，此课题我们通过表达Ptpn11E76K的溶菌酶M启动子驱动的和Cre介导的转录STOP盒的切除，在髓系谱系中产生激活Ptpn11突变有条件的敲入小鼠。这同样导致异常加速的HSC活化，导致MPN的发展和进展。我们首先分析在突变小鼠中HSC周期、凋亡、信号通路，并确定HSC中并不表达突变Shp2蛋白而是由于单核细胞中Shp2的突变导致的。通过蛋白组学分析单核细胞Shp2激活突变细胞中有改变的分泌蛋白变化，以及RNA-Seq等大数据并绘制调控的基因网络与JMML发生发展的相关性，建立可用于防止JMML的复发的新靶点，为临床治疗提供理论依据。

幼年型骨髓单核细胞白血病(JMML)是儿童骨髓增生性肿瘤(MPN)，本项目重点研究JMML复发的外因，培育转基因小鼠和小鼠骨髓移植等方法从体外体内研究单核系Shp2突变的异常表达对JMML干/祖细胞(LSCs)自我更新、分化功能和骨髓重建能力的影响，为JMML的靶向诊疗提供实验依据和理论基础。本项目在JMML(Ptpn11E76K)中诱导最常见的突变，特别是在造血干细胞(HSC)幸免的骨髓谱系中，结果显示HSC失去了静止状态并变得精疲力尽。当与Ptpn11E76K+BM细胞共同移植到WT小鼠中时，在野生型(WT)供体HSC中观察到类似的结果。共培养测试表明JMML/MPN细胞可有力地加速小鼠和人类正常造血干细胞/祖细胞的分化。细胞因子分析显示Ptpn11E76K+MPN细胞产生过多的IL-1β，但不产生IL-6、TNF-α、IFN-γ、IL-1α或其他炎性细胞因子。在Ptpn11E76K/+/IL-1R-/-双突变小鼠中，IL-1β受体的消耗有效地恢复HSC静止。本项目表明IL-1β信号转导是维持JMML正常造血发育的潜在治疗靶点。研究结果阐释JMIMI发生发展中的微环境因素对正常干细胞的调控机制，建立稳定转染的细胞系，培育转基因小鼠和小鼠骨髓移植等方法，在体外以及体内研究外部环境的异常对JMIMI的干细胞自我更新、分化功能和骨髓重建能力的影响，阐明突变单核细胞对干细胞生物学功能相关的细胞因子表达的分子调控机制。.团队使用机器学习和神经网络等方法，通过对恶性肿瘤的基因组、转录组和临床信息联合分析，建立新的肿瘤分型机制，挖掘恶性肿瘤关键的肿瘤新靶点，用于对恶性肿瘤的诊断、预后预测和指导治疗，对恶性肿瘤的精准治疗有实质的推动。为解决现有恶性肿瘤的诊断和分类的不足，对不同亚型进行更细致有效的治疗，利用反向传播神经网络构建高精度分类器，在临床可精准诊断、分层和指导治疗。.在项目执行期间，发表高水平学术论文9篇，其中SCI收录9篇；申请国家发明专利2项，已授权1项；培养了博士后2名，博士研究生3名，硕士研究生4名，项目特邀报告《激活突变Shp2促进类似JMML样病变》1项，项目期间项目负责人董磊被评为北京理工大学第一届特立学者。