Functional Analyses of BTK Mutations in Follicular Lymphoma

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. Recently, novel mutations in the Bruton's Tyrosine Kinase (BTK) have been identified in FL (Krysiak et al, Blood 2017). Additional mutations in other components of BCR signaling pathways in FL have been reported as well but functional consequences have not been fully explored.

Methods: Using a combination of WES on N=82 FLand BTK resequencing in an extended FL panel we uncovered novel BTK mutations in FL. These BTK mutations are all distinct from the previously identified mutations suggesting that a full complement of BTK mutations has not yet been identified. Using crispr-Cas9-mediated BTK gene targeting in multiple lymphoma cell lines, we generated novel BTK^-/- null lines. We introduced all BTK mutations by site-directed mutagenesis into a reference BTK cDNA. Subsequently, we re-introduced HA-tagged wild type and mutated BTK constructs using lentiviral transduction into the BTK^-/- cell lines and also into BTK^-/- DT40 cells. We measured signal transduction pathway components following BCR cross linking. The location of BTK mutations was modeled onto a 3D-model assembled from various published BTK substructures.

Results: Mutations in BTK are distributed along the entire length of the gene and are targeting the PH-, SH2- and catalytic domain, with the catalytic domain carrying the majority of BTK mutations. Interestingly, occasional frame shift mutations as reported and a splice acceptor site mutation by genetic criteria suggest gene inactivation. The 3D modeling data show that all BTK mutations are on the surface of the BTK protein within the various functional domains. The quantitation of BTK protein after reconstitution into various engineered BTK^-/- cell lines uncovered lower steady state levels of various mutants especially for mutations targeting the catalytic domain. PLCγ2 remains the best studied BTK substrate and various sites have been reported to be phosphorylated by BTK in vivo (PLCγ2 amino acids 753, 759 and 1217). Following BCR crosslinking in engineered BTK^-/- lymphoma cell lines we found only a modest reduction in the phosphorylation of PLCγ2 residue 759 or 1217 demonstrating that these sites are under the control of various tyrosine kinases and not exclusively targeted by BTK. After BCR crosslinking in the BTK^-/- cells lines we detected a substantial increase in PLCy2 residues 759 or 1217 phosphorylation over baseline and this increase was abolished by ibrutinib pre-treatment. Therefore, PLCγ2 residue 759 or 1217 phosphorylation is due to multiple BCR stimulated and ibrutinib sensitive tyrosine kinases. In engineered BTK^-/- lymphoma cell lines reconstituted with HA-tagged wild type and mutated BTK we did not detect major effects by the BTK mutants on the phosphorylation state of PLCγ2 residue 759 or 1217. In addition, phosphorylation of BTK at residue 223 (a major autophosphorylation site) was reduced or absent in multiple BTK mutants. Summarizing these findings, FL-associated BTK mutations do not activate the BTK kinase; instead, multiple BTK mutants are inactivating.

Focusing on BCR-regulated signaling pathways, we measured ERK and AKT phosphorylation in the engineered BTK^-/- lymphoma cell lines reconstituted with HA-tagged wild type and mutated BTK. Following BCR crosslinking, we detected increased p-AKT-473 phosphorylation in most BTK mutants in a total of 4 separate cell lines. In contrast, p-ERK was largely unaffected. Recent reports implicated AKT activation as a major factor in tonic BCR signaling in germinal center type DLBCL. We did not detect elevated AKT phosphorylation prior to BCR stimulation in our BTK reconstituted cell systems but an exaggerated response following BCR engagement. The effects of BTK mutations on protein-protein interactions within the BTK signalosome as well as effects on sensitivity to ibrutinib will be updated at the meeting.

Conclusion : Our initial efforts at functional characterization of FL-associated BTK mutations uncovered that despite reduced stability and impaired catalytic activity most BTK mutations resulted in enhanced AKT activation following BCR crosslinking. Given the established pro-growth and pro-survival function of AKT, the data provide a clear reason why FL would select for such mutations.