Study On The Effect Of AML1-ETO Fusion Gene On The Self-renewal And Differentiation Ability Of Pre-leukemic Hematopoietic Stem And Progenitor Cells And Its Metabolic Mechanis

Background and objective:AML1-ETO(RUNX1::RUNX1T1)fusion gene,derived from the chromosomal translocation between 8q22 and 21q22,is one of the most frequent alterations in AML.It accounts for approximately 15%of all AMLs.Compared with other subtypes of AML,AML patients with AML1-ETO fusion gene have a relatively better prognosis after chemotherapy.However,around 30%of patients relapse within 1 year,and a 5-year overall survival rate is about 51%.Recent evidence indicated that leukemia-initiating cells played a critical role in the processes of leukemogenesis and chemotherapy resistance.However,there is a deficient understanding of the fumdamental biology and potent therapeutic target of this subtype of leukemia.An inducible conditional AML1-ETO knock-in murine model was established to investigated the biological function and the metabolic characteristics of AML1/ETO HSPCs after AE successful induction in the pre-leukemic stage.The targeting therapeutic strategy was explored by interrupting the excessive fatty acid supply during the process from LT-HSCs to GMPs.Methods:(1)The biological characteristics of AML1-ETO induced expression in AML1/ETO mice were demonstrated by the survival,changes of hemogram,spleen morphology,and cell proportion changes of bone marrow.(2)Colony-forming unit(CFU)assay was performed to evaluate the repopulation ability of HSCs in vitro.And competitive transplantation experiment was used to elucidate the role of AML1-ETO induction on hematopoietic reconstitution.(3)Micro-bulk RNA-seq was performed to gain underlying insight into the functional impact of AML1-ETO derived HSPCs.The results were verified by cell cycle experiments.(4)To confirm the role of AML1-ETO on mitochondrial metabolism,Seahorse extracellular flux assay was performed to evaluate oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)in HSPCs.To understand the basis of the changes in OCR and ECAR,we analyzed metabolite levels in HSPCs freshly isolated from primary AML1/ETO mice and control mice using LS-MS/MS.(5)The changes of metabolic pathway were associated with the changes of HSC function,and the unique metabolic pattern of HSCs induced by AML1-ETO was used to further elucidate abnormalities of stemness in HSCs.(6)Limitation of fatty acid uptake or Fatp3 deletion were performed to verify whether they could partially eliminate the blocking of hematopoietic differentiation of HSPCs in AML1/ETO mice.Results:Our results showed that the induction of AML1-ETO resulted in excessive accumulation of hematopoietic stem cells(HSCs)and differentiation blockage from HSCs to hematopoietic progenitor cells(HPCs).AML1-ETO induction impaired the capacity of normal hematopoietic reconstitution and increased aberrant self-renewal ability.Moreover,the oxidative phosphorylation(OXPHOS)and glycolysis metabolism of AML1-ETO expressed HSPCs decreased obviously accompanied by enhanced demand for fatty acid compared with that of normal HSPCs.Limiting exogenous lipid uptake by a fat-free diet or Fatp3 gene deletion was able to partially eliminate the blocking of hematopoietic differentiation without impairing normal HSCs.Conclusion:Our study uncovered the stage of malignant transformation in hematopoietic stem and progenitor cells by a conditional inducible AML1-ETO murine model.AML1-ETO can result in the HSC accumulation and differentiation blockage.When competed with normal HSCs,AML1/ETO-derived HSCs have reduced BM reconstitution ability,meanwhile,have increased quiescence and reduced cell cycling leading to a dormant status with lower OXPHOS and ROS level.Our results identify the key role of fatty acid metabolism in hematopoiesis differentiation blockage induced by AML1-ETO and suggest Fatp3 as a promising target that can be used in the treatment of this subtype of leukemia.