The Mechanism Of EphB4/ephrinB2 Mediated IM Resistance In Chronic Myelogenous Leukemia Through Microenvironment In Bone Marrow

Background and purposeChronic myelogenous leukemia(CML)is a kind of tumors derived from the clonal myeloproliferative tumors which are characterized by 9 and 22 chromosome translocation of Ph chromosome.This type of chromosomes forms Bcr-Abl fusion genes encoding proteins that have with abnormal tyrosine kinase activity and lead to the onset of CML.Imatinib(IM)is a type of Bcr-Abl tyrosine kinase inhibitor,which has become primary drug used in the clinical treatment of CML.Most of patients with chronic phase CML significantly improve their 5-year-survival by applying Imatinib,however,some of them are still suffering recurrence of the disease.So,now IM-drug resistance has become one of major challenges in the clinical treatment.The traditional research methods on IM resistant mechanism mainly focus on the leukemia stem cells itself since the cause of the point mutations in Abl kinase domain structure inhibited the effect combination of the kinase and IM.Other possible mechanism mainly includes the amplification of Bcr-Abl gene sequence,excessive expression of Bcr-Abl protein and the decline concentration of intracellular drug.The recent research has seen that defects in HSC or abnormal signals in bone marrow change the crosstalk between HSC and niche.The above change further breaks the balance of normal bone marrow microenvironment and turns it into one to protect residual leukemia stem cells(LSC).The above process is called the microenvironment-mediated resistance,which is one of the main causes of leukemia residue,recurrence,and therefore,has become a research hotspot in recent years.Bone marrow hematopoietic microenvironment,also called niche,refers to the specific microenvironment that is suitable for HSC,including bone marrow stromal cells,extracellular matrix(ECM)and a variety of hematopoietic growth factors.The above three factors constitute a highly complex but effective regulatory networks,which play an important role to support and regulate the proliferation and differentiation of hematopoietic stem cells.Osteoblast is an important stromal cells in bone marrow microenvironment.It not only maintains the resting state of HSC but also support and sustain hematopoietic stem cell differentiation and function.It has been found that transplanting HSCs and osteoblasts together to allogeneic mouse in bone marrow transplantation can increase HSCs' survival rate,but lose the hematopoietic function in mice when osteoblast is missing.Osteoblasts in bone marrow are derived from mesenchymal stem cells(MSC),a type of stromal cells having multiple potential differentiation.MSC mainly resides within the bone marrow around the blood vessels and is the main source of osteogenesis progenitor cells[65].It plays the key role in maintaining bone homeostasis.MSCs undergo the osteoblast differentiation participated with parathyroid hormone other related factors and secrete functional protein such as osteopontin,osteocalcin,etc.So far,the mechanism of MSC migration from the area of vascular niche to the bone surface and eventually form bone specific is still unclear.Eph,the largest family of protein tyrosine kinase receptor,plays an important role in regulation of the cell movement,positioning,sorting,migration and the formation of boundary.Through the contact of cells,receptor and the ligand on the surface of cell bind and activate receptor's forward pathway and(or)ligands-reverse pathways to biological functions.Literature shows that in recent years,Eph/ephrin in a variety of proteins are involved in the regulation of hematopoietic cells in bone marrow microenvironment migration and formation of bone steady state.Currently,a large amount of efforts in literature mainly study the influence of Eph/ephrin molecules on normal MSC cell biology behavior.The purpose of our research is to study the impact of Eph/ephrin molecules on chronic myeloid leukemia MSC biological behaviors,so as to further explore its role in CML resistance.Our preliminary experiments demonstrated that in chronic myeloid leukemia refractory/patients relapse,snap CML cell line K562 and all have EphB4 high expression,therefore,we hypothesized that impact of leukemia cells through EphB4 membrane receptor on the biological characteristics of MSCS in the bone marrow niche lays the foundation for the change of bone marrow microenvironment.Starting from the impact of EphB/ephrinB pathways on the MSC osteogenetic differentiation,cell stretch and migration as the breakthrough point,this work systematically explores the role of EphB in this phenomenon and its mechanism.Methods:Results:1?CCP-derived MSCs cell morphology,proliferation capability,osteogenic and adipogenic differentiation capability showed significant difference from CBC-derived MSCs and normal MSCs.2?Compared with the normal and CBC-derived MSCs,CCP-derived MSCs cell ALP,RUNX2mRNA and expression level of OCN protein increased significantly.3?CCP marrow-derived MSCs cell EphB4 ephrinB2 EphB2 ephrinB1mRNA was highly expressed.Its protein expression was higher than the normal and CBC-derivedMSCs.4?EphB4-Fc promoted CCP-derived MSCs cell osteogenic differentiation through ephrinBl or ephrinB2 reverse pathway activating STAT3 protein.5.EphB4-Fc mediated MSCs cell sprend throughephrinB2 reverse pathway activating PI3K and JNKprotein rather than Src protein,6?EphrinB2-Fc mediated MSCs cell migration via EphB4 forward pathway activating Src and Abl protein rather than PI3K protein.7?K562-R with EphB4 highexpression promoted the expression of CCP-MSCs ALP\RUNX2 mRNA via direct contact way under the condition of osteogenic differentiation.8?CCP-MSC abnormal osteogenesis differentiation induced by EphB4-Fc promoted K562 cell chemoresistance to IM,changed K562 cell cycle,reduced K562 apoptosis rate.9?Acute leukemia model was established respectively by two ways:intra-bone marrow injection and hypodermic injection into the NOG(NOD/SCID/?-/-)mice.Subcutaneous transplantation tumor technology proved successful and stable.10?Proteins of marrow cavity and subcutaneous transplantation tumor were expressed differentially.11?RUNX2 and VEGF genetic expression of marrow cavity mice were all higher than subcutaneous transplantation tumor.Runx2 expression between different groups showed significant difference,and was intimate relativity with MSC and EphB4 expression level.Runx2 high expression was significant chemoresistance to IM.12?In subcutaneous transplantation tumor,VEGFexpression intimate relativity with MSC and EphB4 expression level.And high VEGF expression was significant chemoresistance to IM.Conclusion:1?CML-CP-derived MSCs showed osteogenic differentiation function abnormalities.2?The abnormal osteogenic differentiation was induced by leukemic cells and MSC activated through EphB4/ephrinB abnormal signals.3.The abnormal osteogenic differentiation can further promote leukemic cells chem ores i stance.