谷氨酰胺载体ASCT2在激活突变Ptpn11(Shp2)致骨髓增殖性肿瘤和白血病中的作用及机制

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 hepotapoietical stem cells and 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 the levels of glutamine in plasma and intracellular glutamine uptake were higher in SHP2 gain of function mutation mice. The results were confirmed in bone marrow cells of juvenile myelomonocytic leukemia（JMML）patient which bearing SHP2 mutation. In the meantime, the oxygen consumption rate was increased in SHP2 activating mutation mice when they were fed glutamine as a fuel and α-ketoglutarate levels were increased. However, glycolysis was enhanced and the ATP levels were increased. Depletion of ASCT2 from SHP2E76K/+ knock-in mice can rescue the procession of the disease and changes of metabolism. All the results suggested that ASCT2 is involved in SHP2 activation mutations driving MPN and leukemia. In this application, we are going to further define that the roles of metabolic changes in bone marrow stem cell and microenvironment in driving/enhancing the development of leukemia by using the obtained mice model which bear SHP2 activating mutation and ASCT2 depletion combined with the inhibitor experiment (in vivo and in vitro). We will further explore the molecular mechanism involved in leukemia progression..

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异常活化与白血病的发生密切相关。我们前期利用造血干细胞HSCs及间充质干细胞表达SHP2E76K/+激活突变的模型，发现该小鼠出现骨髓增殖性肿瘤并可演变成急性白血病。深入研究发现突变鼠血浆中谷氨酰胺含量增高，HSCs胞内摄取增多，谷氨酰胺载体ASCT2表达增高，并在SHP2突变的JMML病人细胞中得到证实；突变鼠细胞以谷氨酰胺为底物的线粒体氧化磷酸化水平增高，α-酮戊二酸增高，糖酵解增强，ATP增加。将SHP2激活突变鼠的ASCT2基因敲除后可以逆转疾病的发生及代谢改变。提示ASCT2介导激活突变SHP2的发病过程。本研究拟通过激活突变SHP2和ASCT2敲除的双突变鼠模型，结合ASCT2抑制剂的体内、外实验，进一步验证造血干细胞及骨髓微环境的代谢改变与白血病发生的关系，并深入探讨其发病的分子调控机制。

PTPN11激活突变可导致代谢改变及血液系统肿瘤，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，增殖的髓系祖细胞（GMPs）和随之分化的髓系细胞增多。造血干细胞（HSC）体外增殖能力增强且依赖培养环境中的谷氨酰胺，Ptpn11E76K/+/Mx1-Cre+激活突变小鼠的血浆、骨髓细胞中谷氨酰胺含量高于正常小鼠，骨髓细胞中谷氨酰胺转运蛋白ASCT2的表达也高于正常小鼠。Ptpn11E76K激活突变鼠的细胞能量代谢增强，能量应激及增殖相关的常见信号分子表达增强, 而抑制ASCT2对谷氨酰胺的摄取，可遏制Ptpn11E76K激活突变鼠的发病进程。所以，谷氨酰胺转运蛋白ASCT2介导激活突变Ptpn11( SHP2)促进骨髓增殖性肿瘤的发生。同时我们揭示了Ptpn11E76K/+/Mx1-Cre+激活突变小鼠骨髓细胞来源的外泌体在骨髓微环境及骨髓增殖性肿瘤发生中的作用，Ptpn11E76K/+激活突变骨髓来源的外泌体中CCL2、CCL3以及CCL4等炎性因子增加促进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。同时我们利用单细胞测序研究揭示Ptpn11激活突变小鼠中骨髓髓系祖细胞的分化异质性及代谢改变。以上工作明确了激活突变Ptpn11(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。.同时我们在深入地研究中发现，Ptpn11(Shp2)在骨髓间充质干细胞激活突变后可导致间充质干细胞（MSC）的恶性转化，MSC细胞注射小鼠后可发生肉瘤，机制研究证实SHP2 E76K突变的MSC细胞中线粒体高代谢和线粒体复合物蛋白I和III活性增高，抑制线粒体复合物蛋白I和III的活性可以抑制肿瘤的生长，进一步深入探讨发现SHP2 E76K突变的MSC细胞可以发生相分离，从而引起线粒体复合物活性的改变，而SHP2小分子抑制剂抑制相分离后，线粒体复合物蛋白I和III的活性也降低，从而可抑制肿瘤的生长，为肿瘤治疗提供了重要的临床指导意义。