Effect Of Bruton's Tyrosine Kinase Inhibitors Combined With Bortezomib On Human Multiple Myeloma Cell Lines And Its Mechanism

Background and Objective: Multiple myeloma(MM)is a malignant tumor in the blood system characterized by clonal accumulation of malignant plasma cells in the bone marrow.In recent years,great advances have been made in the treatment of MM.For example,the introduction of proteasome inhibitor bortezomib has significantly improved overall response rates and prolonged survival.However,few patients are cured and the majority of patients eventually relapse or progress.Except for the patients who are innately resistant to bortezomib,nearly half of the patients who were sensitive to bortezomib in the initial treatment failed to respond to bortezomib after relapse.Thus,continuing efforts to explore new agents and combination treatment regimens in order to improve clinical outcome are still needed.Bruton's tyrosine kinase(BTK)is a cytoplasmic tyrosine kinase and is an important signal transducer of the B-cell antigen receptor(BCR)signaling pathway.BTK is expressed in all stages of B lineage and plays critical roles in the development,differentiation and function of B lymphocytes.Recent studies have shown that protein expression and activity level of BTK is abnormally increased in many subtypes of B-cell malignancies,such as chronic lymphocytic leukemia(CLL),mantle cell lymphoma(MCL)and diffuse large B-cell lymphoma(DLBCL),which facilitates the proliferation and survival of malignant B cells.Ibrutinib is a selective BTK inhibitor that can irreversibly inhibit its enzymatic activity through covalently binding to cysteine-481 residue in the activation loop of BTK protein and blocking its phosphorylation activation.The median inhibitory concentration(IC50)for BTK is 0.5 nmol/L.So far,ibrutinib has exhibited effective anti-tumor activity across a variety of B-cell malignancies in preclinical and clinical studies,including CLL,MCL and DLBCL.AVL-292 is another novel selective inhibitor of BTK with similar mechanism of action to ibrutinib,but the IC50 of AVL-292 for BTK is lower than ibrutinib(<0.5 nmol/L).Plasma cells are terminally differentiated B cells.Normal plasma cells have low or no BTK expression.However,several recent studies found that myeloma cells from many MM patients and some cell lines expressed high levels of BTK.However,studies on the efficacy and mechanism of BTK inhibitors alone and in combination with the commonly used bortezomib in the treatment of MM are rare.Therefore,this study was designed to investigate the effects of BTK inhibitors,ibrutinib and AVL-292 alone or in combination with proteasome inhibitor bortezomib on human multiple myeloma cell lines NCI-H929 and RPMI8226 and to explore its possible mechanism,and hence to provide new therapeutic target and combination treatment regimen for the treatment of MM.Methods: 1.BTK protein expression in MM cell lines NCI-H929 and RPMI8226 was first detected by Western blot.NCI-H929 and RPMI8226 cells were treated with ibrutinib,AVL-292 alone or in combination with bortezomib in vitro.The cell viability after treatment was detected by CCK-8 assay,the apoptosis by flow cytometry,the expression and phosphorylation level of BTK,NF-?B p65,AKT,ERK proteins and apoptosis-related proteins by Western blot.2.Bone marrow mononuclear cells were isolated from MM patients bone marrow using ficoll density gradient centrifugation method and cultured in vitro to generate bone marrow mesenchymal stem cells(BMSCs).We identified the immunophenotype of BMSCs(passage 3)by flow cytometry.We also cultured BMSCs(passage 3)with osteogenic,chondrogenic and adipogenic differentiation medium and then evaluated the in vitro differentiation potential of BMSCs by Alizarin Red staining,Alcian Blue staining and Oil Red O staining,respectively.3.BMSCs were treated with ibrutinib or AVL-292 at indicated doses for 48 h and cell viability of BMSCs was detected by CCK-8 assay and IL-6 mRNA level was detected by quantitative RT-PCR.Then,the co-culture system of MM cells and BMSCs was established.We tested the effects of BMSCs on proliferation of MM cells as well as the effects of ibrutinib and AVL-292 on proliferation of MM cells co-cultured with BMSCs by CCK-8 assay,respectively.Results: 1.Western blot analysis showed BTK protein expression in NCI-H929 and RPMI8226 cells.2.Ibrutinib and AVL-292 inhibited the proliferation of NCI-H929 and RPMI8226 cells in a dose-dependent and time-dependent manner.The 48 h IC50 of ibrutinib to NCI-H929 and RPMI8226 cells was(10.41±3.29)?mol/L and(51.65±13.58)?mol/L respectively,and of AVL-292 was(7.77±2.99)?mol/L and(6.44±1.06)?mol/L respectively.3.The inhibition rates of proliferation of these two cell lines after treatment with ibrutinib(5 ?mol/L,10 ?mol/L)and AVL-292(5 ?mol/L,10 ?mol/L)combined with different concentration bortezomib(5 nmol/L?10 nmol/L?20 nmol/L?50 nmol/L)were significantly higher than corresponding single agent groups(P<0.05,P<0.01);the synergy coefficients of different combinations were all greater than 1.4.After treated with 10 ?mol/L ibrutinib,10 ?mol/L AVL-292 and 20 nmol/L bortezomib alone for 48 h,the apoptosis rates of NCI-H929 cells were(16.47±2.70)%,(20.23±3.95)% and(12.40±1.06)% respectively,which were all significantly higher than that of control(P < 0.05,P < 0.01);the apoptosis rates of RPMI8226 cells were(11.27±1.64)%,(17.50±3.70)% and(6.30±0.92)% respectively,which of ibrutinib and AVL-292 were both significantly higher than that of control(P<0.05,P<0.01);in the 20 nmol/L bortezomib+10 ?mol/L ibrutinib group and 20 nmol/L bortezomib+10 ?mol/L AVL-292 group,the apoptosis rates of NCI-H929 cells were(32.20±2.74)% and(38.70±5.03)% respectively,and RPMI8226 cells(26.60±1.51)% and(35.93±3.11)% respectively;there were significantly synergistic effects between these two BTK inhibitors and bortezomib.5.Western blot analysis showed that treatment with 10?mol/L ibrutinib or 10 ?mol/L AVL-292 for 24 h resulted in inhibition of BTK phosphorylation,inactivation of the NF-?B,AKT and ERK signaling pathways,down-regulation of anti-apoptotic Bclx L protein as well as up-regulation of cleaved caspase-3 in NCI-H929 cells.The changes of the above indexes were more evident after combination treatment with 20 nmol/L bortezomib.6.We successfully obtained bone marrow mesenchymal stem cells of MM patients(MM-BMSCs).The immunophenotype analysis of MM-BMSCs showed that expressions of CD73,CD90 and CD105 were positive,but expressions of hematopoietic cells markers CD34 and CD45 were negative.Additionally,MM-BMSCs could differentiate into osteoblasts,chondroblasts and adipose cells after induction,so BMSCs we got showed the characteristics of mesenchymal stem cells(MSCs).7.Treatment with 10 ?mol/L ibrutinib or 10 ?mol/L AVL-292 for 48 h had no effects on cell viability of BMSCs from both newly diagnosed/progressive and stable MM patients,but reduced IL-6 mRNA level in BMSCs from newly diagnosed/progressive MM patients.8.Adherence of NCI-H929 and RPMI8226 cells to newly diagnosed/progressive BMSCs triggered the increase of MM cell proliferation viability while adherence to stable BMSCs did not significantly affect MM cell proliferation viability.9.Ibrutinib and AVL-292 could inhibit proliferation of NCI-H929 cells adherent to BMSCs.Furthermore,10 ?mol/L ibrutinib and 10 ?mol/L AVL-292 completely blocked the supportive effect of BMSCs.Conclusion: 1.Both ibrutinib and AVL-292 can inhibit proliferation,induce apoptosis and synergistically augment cytotoxicity of bortezomib in human MM cell lines H929 and RPMI8226.The mechanism may be related to inhibition of BTK kinase activity and downstream survival pathways including NF-?B,AKT and ERK,down-regulation of anti-apoptosis protein Bcl-xL and activation of caspase-3 pathway.2.Ibrutinib and AVL-292 inhibite proliferation of NCI-H929 cells in the presence of MM patients-derived bone marrow mesenchymal stem cells and are able to overcome the supportive effects of the bone marrow mesenchymal stem cells for MM cell proliferation.Furthermore,both ibrutinib and AVL-292 potently inhibite the expression of IL-6 gene mRNA in BMSCs from newly diagnosed/progressive MM patients with minimal inhibitory effect on proliferation of BMSCs.