Glutamine Transporter ASCT2 Mediates Activating Mutation Ptpn11 (Shp2) Promoting The Development Of Myeloproliferative Neoplasma And Leukemia

BackgroundMyeloproliferative tumor（MPN）is a kind of clonal disease of hematopoietic stem cells.It is a kind of hematological system disease characterized by the continuous abnormal proliferation of primary or multiple cell lines.The common MPN includes chronic myelogenous leukemia,polycythemia vera（PV）,essential thrombocytopenia Thmmbocvthemia,（ET）,primary myelofibrosis,and rare MPN include chronic neutrophilic leukemia,chronic eosinophilic leukemia,systemic mastocytosis and other unclassified diseases（some bone marrow malignant tumors with myeloproliferative characteristics,such as chronic myeloid leukemia and atypical chronic myeloid leukemia）.The clinical manifestations of the patient are extramedullary hematopoiesis（which can lead to splenomegaly）,anemia,abnormal increase of leukocytes,bone marrow fibrosis and other symptoms,which can be transformed into acute leukemia in the late stage of pathology.At present,there is no effective treatment for MPN in clinic.The main treatment for MPN is supportive therapy.The purpose is to reduce the number of excessive leukocytes or splenomegaly,but it cannot prolong the life of patients,and it is difficult to effectively improve their systemic symptoms.Therefore,to explore the pathogenesis of myeloproliferative tumors,including leukemia and other refractory hematological diseases,and to find effective therapeutic targets are still urgent problems.In the cases of MPN,researchers found that a considerable number of patients had abnormal activation and overexpression of protein tyrosine Phosphatase-2.Protein tyrosine phosphatase 2（SHP2）is a non receptor protein tyrosine phosphatase encoded by Ptpn11 gene,which is involved in the activation of various signaling pathways,It has been proved that SHP2 plays a key role in the occurrence and development of a variety of cancers,and SHP2 protein is mostly found in various hematopoietic cells,which plays a very important role in the growth,development,proliferation and differentiation of hematopoietic cells.Mutations in SHP2 are thought to be leukemias,especially juvenile myelomonocytic leukemia,（JMML）,a high risk factor of myeloproliferative tumor（MPN）in children,is also considered to be the proto oncogene of leukemia because of its abnormal mutation and activation in various types of leukemia.Amino acid transporter 2（ASCT2）,is a The neutral amino acid carrier encoded by the gene mainly transports glutamine,serine,alanine and cysteine,among which the glutamine transported into the cell not only participates in the tricarboxylic acid cycle in the cell as the reaction substrate,provides energy for cell growth,but also provides the necessary nitrogen source for cell life synthesis,which is in the state of rapid proliferation Cancer cells play a crucial role.The high expression of ASCT2 has been proved to play a key role in the occurrence and development of a variety of cancers,such as triple negative breast cancer,endometrial cancer and so on.Although there are a lot of researches on the physiological function of glutamine vector ASCT2 in the scientific community,however,there are few researches on the regulatory role of glutamine vector ASCT2,especially in hematological system tumors.However,the role of ASCT2 in the pathogenesis of SHP2 activated mutation has not been reported so far.Therefore,this study intends to use the mouse model of SHP2 activated mutation to explore the effect of ASCT2 protein on the myeloproliferative tumor and leukemia caused by SHP2 activated mutation,and to observe whether ASCT2 inhibitor（GPNA）can inhibit the pathogenesis of myeloproliferative tumor and leukemia.Methods1.Using mice（C57BL/6J）with genotype Mx1-Cre⁺and Ptpn11^E76K/+for breeding,to construct mice with genotype Ptpn11^E76K/+/Mx1-Cre⁺.2.HSCs and MSCs were isolated from normal mice and mutant mice by flow cytometry and cultured in vitro.3.Western blot was used to compare the expression of protein related to cell proliferation and energy metabolism between ASCT2 and p-m TOR,p-ERK,p-AKT,p-S6.4.The contents of glutamine and branched chain amino acids in the cells and serum of the two groups of mice were detected.5.Western blot analysis was used to compare the changes of ASCT2 expression,cell proliferation and energy metabolism in mutant mice and drug treated groups.Result1.Ptpn11^E76K/+/Mx1-Cre⁺activated mutant mouse MPN model was successfully constructed.2.Compared with normal mice,the expression of ASCT2 was increased in the mutant mice,and the proliferation of MSCs was enhanced in the mutant mice.The expression of signal molecules such as p-m TOR,p-ERK,p-AKT and p-S6was enhanced.3.The content of glutamate in bone marrow mesenchymal stem cells and serum of mutant mice was significantly higher.4.The spleen of Ptpn11^E76K/+/Mx1-Cre⁺mutant mice treated with 4asct2 inhibitor was smaller than that of untreated mice,the glutamate content of bone marrow cells was significantly lower,and the AKT/m TOR signal pathway activity was inhibited,indicating that the progression of myeloproliferative tumor and leukemia was blocked after the inhibition of ASCT2.ConclusionThe expression of ASCT2 is closely related to the pathogenesis of myeloproliferative tumor and leukemia caused by SHP2 activation mutation.Inhibition of ASCT2 activation can reduce the uptake of glutamine by MPN and leukemia cells,and effectively inhibit the pathogenesis of SHP2 mutant myeloproliferative tumor and leukemia.ASCT2 may be an effective target for the treatment of this type of disease.