BTKCys481Ser drives ibrutinib resistance via ERK1/2 and protects BTKwild-type MYD88-mutated cells by a paracrine mechanism

Acquired ibrutinib resistance due to BTK^Cys481 mutations occurs in B-cell malignancies, including those with MYD88 mutations. BTK^Cys481 mutations are usually subclonal, and their relevance to clinical progression remains unclear. Moreover, the signaling pathways that promote ibrutinib resistance remain to be clarified. We therefore engineered BTK^Cys481Ser and BTK^WT expressing MYD88-mutated Waldenström macroglobulinemia (WM) and activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL) cells and observed reactivation of BTK-PLCγ2-ERK1/2 signaling in the presence of ibrutinib in only the former. Use of ERK1/2 inhibitors triggered apoptosis in BTK^Cys481Ser-expressing cells and showed synergistic cytotoxicity with ibrutinib. ERK1/2 reactivation in ibrutinib-treated BTK^Cys481Ser cells was accompanied by release of many prosurvival and inflammatory cytokines, including interleukin-6 (IL-6) and IL-10 that were also blocked by ERK1/2 inhibition. To clarify if cytokine release by ibrutinib-treated BTK^Cys481Ser cells could protect BTK^WT MYD88-mutated malignant cells, we used a Transwell coculture system and showed that nontransduced BTK^WT MYD88-mutated WM or ABC DLBCL cells were rescued from ibrutinib-induced killing when cocultured with BTK^Cys481Ser but not their BTK^WT-expressing counterparts. Use of IL-6 and/or IL-10 blocking antibodies abolished the protective effect conferred on nontransduced BTK^WT by coculture with BTK^Cys481Ser expressing WM or ABC DLBCL cell counterparts. Rebound of IL-6 and IL-10 serum levels also accompanied disease progression in WM patients with acquired BTK^Cys481 mutations. Our findings show that the BTK^Cys481Ser mutation drives ibrutinib resistance in MYD88-mutated WM and ABC DLBCL cells through reactivation of ERK1/2 and can confer a protective effect on BTK^WT cells through a paracrine mechanism.