The Synergistic Inhibition of IRF4/NF-κB/STAT3 Pathway by Lenalidomide Combined with BTK Inhibitor Enhances the Effect of Rituximab on Apoptosis in ABC Type Diffuse Large B-cell Lymphoma

Abstract

AIM: Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) has poor response to standard rituximab combined chemotherapy regimen and poor clinical prognosis. The aim of this study is to investigate the in vitro experimental basis for the clinical application of the three-drug combination (Zanubrutinib, Lenalidomide and Rituximab), named as ZR2 regimen by enhancing the apoptotic effect and molecular mechanism of rituximab against ABC type diffuse large B-cell lymphoma. Method: The ABC-DLBCL cell line NU-DUL-1 was used as a cell model in vitro. (1)A culture system consisting of Zanubrutinib, Lenalidomide, and tumor cells was established. Cell proliferation was detected and subsequent experimental concentrations were determined using the CCK-8 method to determine the concentrations and coordinated effects of the two drugs; (2) The culture system of drugs including Zanubrutinib, Lenalidomide and Rituximab, peripheral blood mononuclear cells, serum, and tumor cells .This experiment was set up with blank control group, Zanubrutinib ,Lenalidomide or rituximab monotherapy group, Zanubrutinib combined with lenalidomide two drug group, and triple drug combination group. Flow cytometry was used to detect cell apoptosis, and Western blot was used to detect changes in the IRF4/NF-κB/STAT3 pathway and apoptosis related protein expression. Results: The combination of Zanubrutinib and Lenalidomide can synergistically enhance the anti-tumor effect of rituximab on ABC-DLBCL cells. The combination of three drugs can increase the level of apoptosis, and the overall effect showed a trend of three drug combination being better than two drug combination of Zanubrutinib and Lenalidomide (p<0.05) and rituximab monotherapy (p<0.01), respectively. Simultaneous combination therapy can downregulated the expression of IRF4 and inhibited the phosphorylation activation of NF-κB and STAT3, upregulated the expression of Cleaved caspase-3, and reduced the anti-apoptotic ability of tumor cells. Conclusions: Zanubrutinib combined with Lenalidomide can enhance the in vitro anti-tumor effect of rituximab (ZR2 regimen) on ABC-DLBCL cells, and its mechanism may be related to synergistic inhibition of IRF4/NF-κ B/STAT3 resistance signaling axis activation.