The Role And Mechanism Of Integrin αvβ3/β-catenin/ALDH1A1 Pathway In FLT3-ITD Positive Acute Myeloid Leukemia

Leukemia is a type of hematological malignancy which has a high incidence and mortality.According to the statistics from U.S.,the annual incidence and mortality of leukemia is top ten among all malignant tumors.Acute myeloid leukemia(AML)is the most common hematological malignancy in adult,which has great heterogeneity.FLT3(Fms-like tyrosine 3 kinase)is the most common recurrent gene mutation in AML,with features the higher number of peripheral white blood cells,conventional chemotherapy resistance,higher relapse rate,shorter disease free survival and overall survival.So AML with FLT3 mutation is a unique subtype correlates with poor prognosis.Therefore,it is of great significance to study the onset and maintenance mechanism of AML harboring FLT3 mutation.FLT3 is a member of type III receptor tyrosine kinase family,expressed in hematopoietic precursor cells,and plays an important role in the development of normal hematopoietic and immune system.FLT3 internal tandem duplication(ITD)mutation is the most common mutation in AML,accounted for about 25-35%.The FLT3-ITD mutation causes FLT3 dimer formation,resulting in ligand independent phosphorylation,constitutive activation of downstream Ras/Raf/MEK/ERK,JAK/STAT and PI3K/Akt/m TOR signal transduction pathways,promote cell proliferation,survival and apoptosis inhibition.As a multikinase inhibitor,Sorafenib could target FLT3 by blocking its phosphorylation,inhibiting downstream signaling pathway such as Raf,causing apoptosis of leukemia cells.Sorafenib could improve the remission rate of AML,but it could not clear residual leukemic cells in bone marrow,leading to subsequent relapse and poor overall survival,suggesting that bone marrow microenvironment mediated drug resistance.The mechanism of microenvironment-induced drug resistance is possible mediated through FLT3 mutation independent pathway.Therefore,it is important to explore the mechanism of microenvironment-induced drug resistance and its intervention might improve the therapeutic efficacy.Our previous work confirmed that osteopontin(OPN)is high expressed in the serum of AML patients harboring FLT3-ITD mutation.OPN concentration is high in co-culture system of MV4-11 cells and human bone marrow osteoblast cells,and the integrin αvβ3 mediates the adhesion,migration and chemotherapy resistance,but the mechanism is not clear.In recent years,the theory of leukemia stem cells(LSC)points out that bone marrow microenvironment could shield the leukemia stem cells from chemotherapy,and plays an important role in disease recurrence,suggesting that OPN and its receptor may maintain leukemia stem cell survival and resistance to chemotherapy in bone marrow microenvironment.ALDH1 could be used as a tumor stem cell marker in colorectal cancer,lung cancer,liver cancer,breast cancer and so on,but its role in acute myeloid leukemia especially in FLT3-ITD~+ AML is still unknown.In this study,we try to clarify the role of ALDH1 in acute myeloid leukemia and the relationship between ALDH1 and OPN/αvβ3 pathway.Also we will clarify the role of OPN/αvβ3 pathway in the microenvironment mediated chemoresistance and discusses its mechanism.This study will provide a new insight in the mechanism of microenvironmentinduced drug resistance and give us a new idea in microenvironment related targets intervention.Objectives:1.To clarify the expression and prognostic role of each ALDH1 family members in AML;2.To clarify the relationship between integrin αvβ3 and ALDH1A1;3.To explore the expression and function of ALDH1A1 in AML cell lines;4.To investigate the effect of OPN/αvβ3 on the sensitivity of FLT3-ITD~+ AML to chemotherapy;5.To explore the role and mechanism of OPN/αvβ3 pathway on β-catenin activation and maintenance;6.To investigate the effect of β-catenin/ALDH1A1 on the sensitivity of FLT3-ITD~+ AML to chemotherapy.Methods:1.By analysis TCGA database,investigating the expression and prognostic role of each ALDH1 family member,ALDH1A1,ALDH1A2,ALDH1A3,ALDH1B1,ALDH1L1,and ALDH1L2 in AML.2.By analysis TCGA database,identify the correlation of expression of ITGAV,ITGB3 and ALDH1A1.3.To explore the function of ALDH1A1 in AML cells by constructing and packaging ALDH1A1 sh RNA lentivirus.4.Alphavbeta3 blocking antibody was used to investigate the role of OPN/αvβ3 in sorafenib sensitivity of FLT3-ITD~+ AML in co-culture system of human bone marrow stromal cells and MV4-11 cells(FLT3-ITD mutation).5.Western blot was used to detect the OPN/αvβ3/PI3K/AKT pathway and the activation of β-catenin by using αvβ3 blocking antibody and PI3 K inhibitor.6.To explore the function of β-catenin and sorafenib sensitivity in AML cells by constructing and packaging CTNNB1 sh RNA lentivirus.Results:1.The expression and prognostic role of ALDH1 in AML:(1)The expression level of ALDH1A1 was closely related to NCCN risk stratification;(2)The expression level of ALDH1A1 was negatively correlated with the overall survival of AML patients.2.The relationship between integrin αvβ3 and ALDH1A1:(1)There was no correlation between ITGAV and ALDH1A1 in gene expression;(2)ITGB3 and ALDH1A1 gene expression was positively correlated.3.Expression and function of ALDH1A1 in AML cell lines:(1)ALDH1A1 is highly expressed in FLT3-ITD positive cell line MV4-11;(2)We had successfully constructed ALDH1A1 sh RNA vectors and packaged lentivirus;(3)ALDH1A1 sh RNA lentivirus could effectively knock down the target gene expression;(4)ALDH1A1 sh RNA could inhibit MV4-11 cell proliferation and affect colony formation ability.4.The effect of OPN/αvβ3 on the sensitivity of FLT3-ITD~+ AML to chemotherapy:(1)The sensitivity of MV4-11 cells to sorafenib was decreased in co-culture system,which could be partly reversed by αvβ3 blocking antibody;(2)Alphavbeta3 blocking antibody could cooperate with sorafenib to reduce Bcl-2 expression and increase Bax expression in MV4-11 cells,and promote cell apoptosis;(3)Osteopontin crosslinking reduced the sensitivity of MV4-11 cells to chemotherapy,this effect could be partially reversed by using αvβ3 blocking antibody.5.The role and mechanism of OPN/αvβ3 pathway on β-catenin activation and maintenance:(1)Osteopontin enhanced β-catenin expression and nuclear translocation;(2)Osteopontin enhanced the expression of CCND1 and c-Myc,which are the downstream proteins of β-catenin;(3)Osteopontin enhanced β-catenin expression by αvβ3/PI3K/Akt/GSK3β pathway,αvβ3 blocking antibody and PI3 K inhibitor LY294002 could partly inhibit β-catenin expression;(4)Human bone marrow stromal cells could enhance the expression of β-catenin through activation PI3K/Akt/GSK3β pathway,this effect could be partly reversed by αvβ3 blocking antibody.6.The effect of β-catenin/ALDH1A1 on the sensitivity of FLT3-ITD~+ AML to chemotherapy:(1)We had successfully constructed CTNNB1 sh RNA vectors and packaged lentivirus;(2)CTNNB1 sh RNA lentivirus could effectively knock down the target gene expression;(3)Knocking down CTNNB1 could reduce the IC-50 value of sorafenib;(4)Knocking down CTNNB1 increased sorafenib-induced apoptosis;(5)The expression of ALDH1A1 was decreased after CTNNB1 knocking down.Conclusions:1.ALDH1A1 expression correlated with clinicopathological features,higher ALDH1A1 level indicated poor prognosis,FLT3-ITD~+ AML patients could be further stratified by ALDH1A1 expression;2.ALDH1A1 knocked down could inhibit the proliferation of MV4-11 cells and affect colony forming ability;3.ITGB3 and ALDH1A1 gene expression was positively correlated;4.Bone marrow stromal cells could activate PI3K/Akt/GSK3β/β-catenin pathway through OPN/ αvβ3,and reduce MV4-11 sensitivity to sorafenib;5.β-catenin knocked down could inhibit the proliferation of FLT3-ITD~+ AML cells,enhance sorafenib sensitivity,and could inhibit the expression of ALDH1A1;6.Integrin αvβ3/β-catenin/ALDH1A1 pathway may become a new target for FLT3-ITD~+ AML.