Follicular Lymphoma-associated BTK Mutations are Inactivating Resulting in Augmented AKT Activation

Purpose: On the basis of the recent discovery of mutations in Bruton tyrosine kinase (BTK) in follicular lymphoma, we studied their functional properties. Experimental Design: We identified novel somatic BTK mutations in 7% of a combined total of 139 follicular lymphoma and 11 transformed follicular lymphoma cases, none of which had received prior treatment with B-cell receptor (BCR) targeted drugs. We reconstituted wild-type (WT) and mutant BTK into various engineered lymphoma cell lines. We measured BCR-induced signal transduction events in engineered cell lines and primary human follicular lymphoma B cells. Results: We uncovered that all BTK mutants destabilized the BTK protein and some created BTK kinase-dead mutants. The phospholipase C gamma 2 (PLC gamma 2) is a substrate of BTK but the BTK mutants did not alter PLC gamma 2 phosphorylation. Instead, we discovered that BTK mutants induced an exaggerated AKT phosphorylation phenotype in anti-Ig-treated recombinant lymphoma cell lines. The short hairpin RNA-mediated knockdown of BTK expression in primary human nonmalignant lymph node-derived B cells resulted in strong anti-Ig-induced AKT activation, as did the degradation of BTK protein in cell lines using ibrutinib-based proteolysis targeting chimera. Finally, through analyses of primary human follicular lymphoma B cells carrying WT or mutant BTK, we detected elevated AKT phosphorylation following surface Ig crosslinking in all follicular lymphoma B cells, including all BTK-mutant follicular lymphoma. The augmented AKT phosphorylation following BCR cross-linking could be abrogated by pretreatment with a PI3K delta inhibitor. Conclusions: Altogether, our data uncover novel unexpected properties of follicular lymphoma-associated BTK mutations with direct implications for targeted therapy development in follicular lymphoma.