Identification and overcoming rituximab resistance in diffuse large B-cell lymphoma using next-generation sequencing.

Abstract

Although rituximab, an antiCD20 monoclonal antibody, has dramatically improved the clinical outcomes of diffuse large B-cell lymphoma, rituximab resistance remains a challenge. We developed a rituximab-resistant cell line (RRCL) by sequential exposure to gradually increasing concentrations of rituximab in a rituximab-sensitive cell line (RSCL). When the same dose of rituximab was administered, RRCL showed a smaller decrease in cell viability and apoptosis than RSCL. To determine the differences in gene expression between RSCL and RRCL, we performed next-generation sequencing. In total, 1,879 differentially expressed genes were identified, and in the over-representation analysis of Consensus- PathDB, mitogen-activated protein kinase (MAPK) signaling pathway showed statistical significance. MAPK13, which encodes the p38δ protein, was expressed more than four-fold in RRCL. Western blot analysis revealed that phosphop38 expression mainwas increased in RRCL, and when p38 inhibitor was administered, phosphop38 expression was significantly decreased. Therefore, we hypothesized that p38 MAPK activation was associated with rituximab resistance. Previous studies have suggested that p38 is associated with NF-κB activation. Deferasirox has been reported to inhibit NF-κB activity and suppress phosphorylation of the MAPK pathway. Furthermore, it also has cytotoxic effects on various cancers and synergistic effects in overcoming drug resistance. In this study, we confirmed that deferasirox induced dose-dependent cytotoxicity in both RSCL and RRCL, and the combination of deferasirox and rituximab showed a synergistic effect in RRCL at all combination concentrations. We suggest that p38 MAPK, especially p38δ, activation is associated with rituximab resistance, and deferasirox may be a candidate to overcome rituximab resistance.