Study On The Activation Of BTK And The Mechanical Regulation Mechanism Of BTK-mediated Tumor Cell Adhesion And Proliferation

More and more attention has been paid to the role of the fluid shear stress in the development of tumors.Multiple myeloma（MM）is a plasma cell neoplasm,the MM cells in the peripheral blood can circulate and migrate to new bone marrow areas and proliferate,which cause of recurrence of the disease and difficulty of treatment.Therefore,understanding the mechanical regulation mechanism and molecular mechanism in the processes of metastasis and the post-metastasis of MM cells will be crucial to solve the current problems.Metastatic and post-metastatic development of MM cells involve two processes:adhesion during metastasis and proliferation after metastasis.However,the mechanical regulation mechanism of these two processes is not clear.BTK plays an important role in the proliferation,development,transport,homing and other functions of mature B cells.However,the influence mechanism of BTK on MM cell adhesion and proliferation is not clear.In addition,inhibitors of BTK are used in the clinical treatment of many B-cell tumors.However,drug resistance caused by mutations is currently a problem.Therefore,understanding the activation mechanism of BTK can provide more effective target sites and ideas for solving this problem.We explored the above processes by three means.First,we used parallel plate flow chamber to explore the adhesion behavior mediated by the CXCL12-BTK-α₄β₁ signaling pathway under different fluid shear stresses.Secondly,we used a circulatory system to simulate the stress situation in the process of cell metastasis to explore the influence mechanism of force and BTK on the proliferation behavior of MM cells.Finally,we studied the activation mechanism of BTK through free molecular dynamics（FMD）simulation using three mutations.Our results show that with the increase of fluid shear stress,the trend of the adhesion probability was increasing first and then decreasing,which is mainly reflected in the probability of rolling and firm adhesion events.In addition,the maximum value appears at 0.3 dyn/cm²,which is the same in the mean stop frequency and mean stop time of rolling.Those results represent the cell adhesion is regulated by the fluid shear stress.More specifically,the fluid-enhanced adhesion under the condition of less than 0.3 dyn/cm²,and the fluid-weakened adhesion under the condition of greater than 0.3 dyn/cm².Inhibition of BTK can significantly inhibit the adhesion level of cells,increase the rolling velocity of cells,and reduce stop frequency and stop time.From the fluid point of view,the trend that the firm adhesion probability increases first and then decreases is abolished and the rolling state is also changed because of the inhibition of BTK.These results indicated that inhibition of BTK disrupted the activation of integrinα₄β₁ by the chemokine CXCL12,thus affecting the interaction betweenα₄β₁ and VCAM-1,and also showed that the effect of BTK on cell adhesion behavior was also regulated by fluid shear stress.At the cell proliferation level,we found that the fluid shear stress had a certain killing effect on cells,the higher the force and the higher the rate of cell death.But we also found that the force had a certain promoting effect on the proliferation of the remaining cells,and with the increase of the force,the proliferation amplitude of the cells increased first and then decreased,which means the lower force promote cell proliferation is more apparent.However,we also found that over time,the proliferation rate of cells under high force increased significantly,especially after 48h,indicating that the effect of stress on cell proliferation was a continuous process.Inhibition of BTK can observably inhibit the promotion effect of force on cell proliferation,and the inhibition effect is more significant than that under static condition,indicating that BTK plays a more prominent role in cell proliferation after fluid action.Through computer simulation,we found that T474M mutation changed the structure of A-loop and stabilized the ATP binding site.However,there was no similar structural transformation in the two structures of loss-of-function mutation G414R and R525Q,marking that the local structural change induced by T474M mutation was the structural basis for promoting kinase activity.In conclusion,this study aims at the process of MM cell metastasis to explore the regulatory mechanisms of fluid shear stress and BTK on cell adhesion and proliferation,and at the same time to explore the structural basis of BTK activation,so as to provide a more effective treatment idea for multiple myeloma and drug development of BTK.