Ptpn11 Activating E76K Mutation Leads To Metabolic Energetic Reprogramming Of MSPCs

[Background] Leukemia is one of the most common tumors in the world,and its mortality rate is among the highest in cancer.The occurrence of leukemia is related to many factors,such as physical factors,environmental factors,genetic factors,etc.,but the most common clinical factor is the abnormal cell function caused by mutation of one or more kinds of genes,which eventually leads to leukemia.At present,common oncogenic mutant genes of leukemia include BCR-ABL fusion mutation,FLT3-ITD fusion mutation,Notch1 mutation,JAK2?V617F?mutation,IDH1/2 mutation,DNMTA mutation,Ptpn11 mutation and the like.Our group focused on the occurrence of leukemia caused by Ptpn11 functionally activated mutations.Myeloproliferative neoplasma?MPN?is a type of hematopoietic stem cell clonal disease.It is a group of diseases characterized by persistent abnormal proliferation of one or more lines of cells.Usual MPN includes chronic myeloid leukemia and true red blood cells,Polycythel1 lia vera?PV?,essential thrombocytopenia?ET?,primary myelofibrosis,and some chronic neutrophilic leukemia,chronic eosinophilic leukemia,systemic mastocytosis and other unclassified diseases?some bone marrow malignant tumors of myeloproliferative properties,such as chronic myeloid leukemia and atypical chronic myeloid leukemia?.The clinical manifestations of patients with extramedullary hematopoiesis?which can lead to splenomegaly?,anemia,abnormal white blood cells,bone marrow fibrosis and other symptoms,and some can be converted to acute leukemia.At present,there is no specific and effective treatment for MPN,mainly supportive therapy,in order to reduce excessive white blood cells or spleen,but it does not prolong the life span of patients,and it can't effectively improve their systemic symptoms.The hematopoietic bone marrow microenvironment is a complex cell microenvironment that supports hematopoietic growth and regulates the proliferation and differentiation of hematopoietic stem cells.It is also called hematopoietic stem cell niche?HSC niche?,which mainly contains supporting cells and supporting molecules.Mesenchymal stem cells,endothelial cells,megakaryocytes,osteoblasts,vascular sinus,etc.,and the supporting molecules secreted by these supporting cells,such as CXCL12 and c-kit,can also flexibly regulate the proliferation and differentiation of HSCs.Interestingly,in addition to genetic mutations in HSCs that cause leukemia,recent studies have found that mutations in bone marrow microenvironment cells,especially mesenchymal stem cells,can also cause leukemia.Mutations of various genes in mouse bone marrow mesenchymal stem cells,such as Notch1,ROR?,etc.,also produce a leukemia-like phenotype.Clinical studies have also found that there is a high probability of detecting a gene mutation in mesenchymal stem cells in AML and PMN patients,which is positively correlated with the patient's poor prognosis.Ptpn11 is the first non-receptor tyrosine kinase to be discovered,and its encoded SHP2 protein consists of three domains,N-SH2,C-SH2 and PTP.The downstream phosphorylation signal can be activated by decoupling the N-SH2 domain and the PTP domain.In pathology,more than 50% of Noonan Syndrome?NS?patients have SHP2 activating mutations,which are likely to be leukemia,especially juvenile myelomonocytic leukemia?juvenile myelomonocytic leukemia?.JMML),childhood myeloproliferative neoplasms?juvenile myeloproliferative neoplasm,MPN?.At the same time,it is considered to be a proto-oncogene of leukemia due to its abnormal activation and mutation of SHP2 in different types of leukemia [5-8].It is also considered to be related to neuroblasts in recent years.Tumors are associated with a variety of solid tumors [9-12].Our group found early that in addition to the occurrence of MPN in Ptpn11 mutations in hematopoietic stem cells,Ptpn11 activating mutations in bone marrow mesenchymal stem cells also cause MPN.It was found that it produced the same leukemia phenotype as the gene mutation in HSC,and it was found that this process was due to the recruitment of monocytes by mutated MSCs,which secreted pro-inflammatory factors to cause the initiate of MPN.However,this article focused on a composite effect on the bone marrow microenvironment.The function changes of Ptpn11 mutation in MSCs are not discussed in depth.Therefore,our group will target Ptpn11 mutations in MSCs changes,especially in metabolism.We will use molecular biology,histomorphology,and cell biology to study changes in the MSCs metabolic pathway of Ptpn11 mutations.I hope that through this thorough research,we will increase our understanding of leukemia and find a suitable treatment method to treat leukemia more effectively against MSCs and HSC.[Research methods] 1.PNPN11^E76K/+;Mx1-cre Ptpn11 E76K activating mutant mice and Ptpn11^+/+;Mx1-cre control mice are generated,and induced crease expression by PIPC,induced Ptpn11 E76K mutation in Mx1 positive bone marrow hematopoietic stem cells and mesenchymal stem cells.2.After the CO2 asphyxiation in mice,the femur and tibia were separated,the bone marrow cells were washed out by bone marrow puncture,and the bone tissue was digested with a concentration of 0.25% type I collagenase,and the mesenchymal stem cells in the bone marrow were obtained by adherent culture.3.Proteomics and metabolomics validation of washed bone marrow cells 4.Prepared Frozen and paraffin sections of bones,lipid difference and collagen fiber difference detected by oil red O staining and Masson staining 5.Performed cell experiments after 2-5 passages of adherent cells.MTT was used to detect the proliferative capacity of cells.RT-qPCR was used to detect the expression of hematopoietic-supported chemokines CXCL12 and SCF,and the glucose uptake capacity and ROS level of cells were detected by flow cytometry.6.We used Seahorse analysis of changes in cellular oxygen consumption rate?OCR?and extracellular acidification rate?ECAR?in the Ptpn11 mutant group relative to the control group.7.We detected changes in ATP and lactate levels in cells,then measured changes in cell mitochondria by RT-qPCR and flow cytometry.8.We detected of changes in metabolic-related signaling pathways by Western Blotting 9.Detecting cell mitochondria number and membrane potential changes? 10.Detection of mitochondrial complex differences in cells? [Experimental results] 1.After two months of PIPC injection,the mice in the experimental group showed an obvious leukemia phenotype.The splenomegaly was enlarged,the granulocytes of the bone marrow were significantly increased,and the red blood cells were reduced.At this time,the mutation rate of Ptpn11 E76K in mouse bone marrow reached to 97%.2.Mesenchymal stem cells isolated from bone marrow and bone showed good shape and active.And metabolomics and proteomics of bone marrow have shown differences in multiple metabolic pathways including energy,lipid,and protein metabolism.3.Oil red O staining of bone marrow showed a marked decrease in bone marrow lipids in Ptpn11 activating mutant mice,and Masson staining showed a decrease in bone collagen fibers in Ptpn11 activating mutant mice.4.Ptpn11 E76K mutant MSCs proliferative capacity is enhanced,CXCL12 and SCF m RNA expression levels are decreased,glucose uptake capacity is increased,and oxidative phosphorylation by-product ROS is increased.5.In aerobic oxidation,Ptpn11 activates mutation of murine mesenchymal stem cells increased both basal and maximum oxygen consumption rate.The maximum extracellular acidification rate in anaerobic glycolysis was enhanced,while the basal extracellular acidification rate showed no significant difference.6.Ptpn11 activating mutant mouse mesenchymal stem cells increased intracellular ATP levels,but there was no significant difference in lactate levels,consistent with the results of Seahorse 7.Ptpn11 activating mutation had no effect on the number of mitochondria in MSCs.The expression of phosphorylated AMPK was decreased,and the expression of total m TOR and phosphorylated m TOR was increased.8.The mitochondrial number of Ptpn11^E76K/+ MSCss did not change significantly,and the membrane potential level of the cells increased.9.The activity of mitochondrial electron transport chain complex of Ptpn11^E76K/+ MSCs changed? [Conclusion] Ptpn11 activating mutations increase the level of aerobic oxidation of mesenchymal stem cells,which is not caused by increased mitochondrial numbers,possibly by increased mitochondrial function.