激活突变Ptpn11 (Shp2)调控小鼠造血干细胞骨髓微环境促进骨髓增殖性肿瘤发生及其机制

The hematological malignancy has become a serious health problem in human because its incidence and mortality rates continue to rise among all malignancies, however, the underlying molecular mechanisms remain unclear. It had been reported that SHP2 activating mutations were associated with leukemia. In our previous study, we generated the conditional SHP2E76K/+ knock-in mice tissue specific in bone marrow mesenchymal stem/progenitor cell bearing SHP2 activating mutation. We found this mutated mice developed a profound Myeloproliferative neoplasma (MPN) and subsequently progress to acute leukemias. Further study showed that BM primitive hematopoietic progenitors and stem cells which express wild-type SHP2 were markedly decreased in the cell number and displayed aberrant activation and accelerated differentiation. In the meantime, stem cell microenvironment showed abnormal changes: SHP2E76K/+ activating mutations in mesenchymal stem/progenitor cells (MSPCs) cause excessive production of inflammation/ chemotactic factor in the culture supernatants of bone marrow and mesenchymal stem/progenitor cells ; lots of monocytes were recruited to the vicinity where HSCs reside. All the results suggested that SHP2E76K/+ activation mutations drive MPN by regulating the bone marrow microenvironment. In this application, we are going to further define that whether the SHP2E76K/+ mutation affect the bone marrow microenvironment and the mutated microenvironment played a major role in driving/enhancing the development of MPN by using the obtained SHP2E76K/+ activating mutation mice model and activating mutations in other microenvironmental cell type mice model. We will set up animal transplantation experiments and cytokine array analysis combined with clinical samples analysis to illustrate above issues and further explore the molecular mechanism. We expect that the results from this study will provide new clues and target for the clinical prevention and treatment of MPN and leukemia.

血液系统肿瘤因发生率和死亡率持续上升而严重危害人类健康，且机制不清。已有研究表明SHP2的异常活化与白血病的发生密切相关。我们前期获得条件性基因敲入小鼠模型，特异性在骨髓间充质干细胞中发生SHP2E76K/+激活突变。发现该小鼠出现骨髓增殖性肿瘤（MPN）并可演变成急性白血病。进一步研究发现表达野生型SHP2的骨髓造血干细胞（HSCs）数量减少且功能异常（呈高度激活耗竭状态）；同时HSCs微环境出现异常变化：骨髓及间充质干细胞培养上清中均发现某些炎症/趋化因子异常表达；HSCs周围聚集了大量的单核细胞。提示激活突变SHP2通过调控骨髓微环境导致骨髓增殖性肿瘤。本课题拟进一步利用已建立的突变小鼠模型及在其他微环境细胞中突变的小鼠模型，通过动物移植试验及细胞因子阵列分析等, 结合临床样本，明确激活突变SHP2对骨髓微环境及MPN发生发展的影响，并阐明其机制，为临床MPN及白血病防治提供新思路。

PTPN11激活突变可导致血液系统肿瘤，但是，其机制尚不清楚。我们研究发现PTPN11E76K/+激活突变的小鼠出现骨髓增殖性肿瘤（MPN）并可演变成急性白血病，进一步深入研究并通过单细胞测序分析不同分化细胞群体的转录组和染色质可及性，然后进行发育轨迹模拟。我们发现PTPN11E76K激活突变导致造血干细胞耗尽，而增殖的髓系祖细胞（GMPs）和随之分化成熟的髓系细胞增多。scRNA-seq和ATAC-seq分析显示PTPN11E76K/+突变的GMP的谱系转化和基因表达模式发生异常变化。PTPN11E76K/+激活突变鼠的GMP和单核细胞在转录调控方面有很大差异。同时，PTPN11E76K/+中性粒细胞倾向于在翻译和蛋白质修饰方面异常变化。RNA-seq和ATAC-seq的结合分析显示，嗜中性白细胞和单核细胞的生物学功能发生了不同变化。因此，我们描述PTPN11E76K/+突变鼠的髓样谱系分化过程，并鉴定其许多基因表达形式和基序变化。.同时我们还研究Ptpn11E76K/+;Mx1-cre激活突变骨髓来源的外泌体在MPN进程中发挥的作用。将Ptpn11E76K/+激活突变骨髓来源的外泌体与正常造血干细胞进行体外培养，再通过尾静脉注射到正常小鼠体内观察外泌体在体内外所发挥的作用，并检测突变骨髓来源的外泌体中细胞因子的变化。结果发现突变骨髓来源的外泌体中CCL2、CCL3以及CCL4等炎性因子增加，正常小鼠髓系分化增加，脾脏增大。CCR1和CCR2受体拮抗剂使用后小鼠MPN症状有所缓解，外泌体中细胞因子的表达也随之降低。因此Ptpn11E76K/+激活突变骨髓来源的外泌体中细胞因子含量改变导致MPN发生，外泌体及其携带的炎性因子都可以作为MPN治疗靶点。以上工作明确了激活突变SHP2对骨髓微环境及MPN发生发展的影响并阐明其机制，为临床MPN及白血病防治提供了新的思路和靶点。