BTK Inhibitor Downregulates IL-17 Secretion and Enhances CD20mb Sensitivity to ABC Type Diffuse Large B-cell Lymphoma via CYLD/NF-kB Signaling Pathway

AIM: To explore whether two novel Bruton’s tyrosine kinase (BTK) inhibitors, acalabrutinib and zanubrutinib, in combination with Rituximab (RTX), enhance the cytotoxic effects on diffuse large B-cell lymphoma (DLBCL) cell lines and to elucidate the underlying mechanisms. Method: We selected the activated B-cell-like (ABC) DLBCL cell lines NU-DUL-1 and SU-DHL-2 as parental lines, and their corresponding Rituximab (CD20mb)-resistant lines NU-DUL-1-R as resistant strains. Using 20% fresh normal human serum as the source of complement, we employed 7-aminoactinomycin D (7-AAD) flow cytometric staining to assess the cytotoxic effects of BTK inhibitors combined with RTX via complement-dependent cytotoxicity (CDC) on tumor cells. Tumor cells were labeled with carboxyfluorescein diacetate N-succinimidyl ester (CFSE) and co-cultured with peripheral blood mononuclear cells (PBMCs). The cytotoxic effects of BTK inhibitors combined with RTX via antibody-dependent cell-mediated cytotoxicity (ADCC) were evaluated using 7-AAD and PE-Annexin V flow cytometric staining to determine whether sensitivity to RTX was increased. Flow cytometry was used to detect the expression of CD20 on DLBCL cell lines and the proportions of CD4+IL-17+ T cells in PBMNCs, granzyme B and TNF-α in CD8+ T cells in the PBMCs-tumor cell co-culture system. ELISA was employed to measure the concentrations of related cytokines. qPCR was used to assess the effects of BTK inhibitors on the mRNA expression of retinoic acid-related orphan receptor (RORC) and interleukin-17 (IL-17). Western blotting was performed to detect the protein expression levels of p-NF-κB-p65 in CD4+ T cells after treatment with BTK inhibitors. We also established a mouse model of BALB/c B-cell lymphoma using A20 cells overexpressing human CD20. Tumor volume was measured and recorded. Flow cytometry was used to detect the proportion of Th17 cells in peripheral blood and spleen, and ELISA was employed to measure the expression levels of IL-17 in serum. Results: (1) The novel BTK inhibitors had little effect on the expression of CD20 on the surface of NU-DUL-1, NU-DUL-1-R, and SU-DHL-2 cells. (2) When RTX exerted its CDC effect, compared with the monotherapy group, the combination of acatinib and RTX significantly enhanced the cytotoxic effects on NU-DUL-1, NU-DUL-1-R, and SU-DHL-2 cells (P<0.05), with a dose-dependent effect observed in NU-DUL-1 and NU-DUL-1-R cells (P<0.05). The combination of zanubrutinib and RTX also significantly enhanced the cytotoxic effects on NU-DUL-1 and NU-DUL-1-R cells (P<0.05), but the effect on SU-DHL-2 cells was not statistically significant at a zanubrutinib concentration of 10 µmol/L (P>0.05). (3) When RTX exerted its ADCC effect, compared with the monotherapy group, the combination of acalabrutinib and RTX significantly enhanced the cytotoxic effects on NU-DUL-1, NU-DUL-1-R, and SU-DHL-2 cells (P<0.05). The combination of zanubrutinib and RTX significantly enhanced the cytotoxic effects on NU-DUL-1 and SU-DHL-2 cells (P<0.05), but the effect on NU-DUL-1-R cells was not statistically significant at a zanubrutinib concentration of 3 µmol/L (P>0.05). (4) Flow cytometry results showed that both acalabrutinib and zanubrutinib significantly downregulated the proportion of Th17 cells and upregulated the secretion of granzyme B and TNF-α in CD8+ T cells (P<0.05). (5) Compared with the control group, RTX alone significantly upregulated the proportion of Th17 cells and increased IL-17 secretion. In contrast, the combination of acalabrutinib or zanubrutinib with RTX significantly downregulated the proportion of Th17 cells and reduced IL-17 secretion (P<0.05). (6) Western blot results showed that both acalabrutinib and zanubrutinib inhibited the phosphorylation of NF-κB-p65 in CD4+ T cells (P<0.05). (7) Both novel BTK inhibitors downregulated the expression of RORC and IL-17 mRNA in CD4+ T cells and reduced the secretion of IL-17, significantly inhibiting the differentiation of Th17 cells (P<0.05). (8) In vivo experiments demonstrated that zanubrutinib significantly reduced tumor volume and downregulated the proportion of Th17 cells and IL-17 levels in the spleen and peripheral blood of mice (P<0.05). Conclusions: BTK inhibitors reduce IL-17 secretion via the CYLD/NF-κB pathway and enhance the sensitivity of ABC - DLBCL to CD20 mb The combination of novel BTK inhibitors with RTX may be an effective strategy to overcome RTX resistance in ABC -DLBCL.