Studies of Bruton’s tyrosine kinase inhibitors in B-cell malignancies

Despite significant advances, the prognosis in relapsed/refractory (R/R) B-cell malignancies remains poor. In the Phase I study of the selective Bruton’s Tyrosine Kinase inhibitor (BTKi) tirabrutinib in R/R B-cell malignancies, I showed that targeting BTK demonstrated remarkable clinical responses and tolerability in Chronic Lymphocytic Leukaemia and Mantle Cell Lymphoma. Targeted DNA sequencing demonstrated that no mutations were associated with a lack of response in CLL. However, in activated B-cell like diffuse large B-cell lymphoma (ABC DLBCL), only 35% of patients responded to treatment and median duration of response was 12 weeks. This prompted my laboratory studies to explore mechanisms of resistance to BTKi in ABC DLBCL. Using BTKi resistant cell lines TMD8 RO and TMD8 RI, I undertook biological and genetic studies. BTK expression and subcellular localisation was not altered in the resistant cell lines. Apoptosis induced by the BTKi ibrutinib and tirabrutinib occurred 24 hours following drug exposure. A decrease in the expression of the anti-apoptotic proteins MCL1, BCLxL and BCL2A1 was observed in TMD8 but not TMD8 RO following treatment with tirabrutinib, consistent with modulation of the BCR pathway. No significant change was identified in apoptotic gene expression. Study of the BCR signalling pathway showed an increase in cell surface expression of sIgM and CD20 in the resistant cell lines. No change in IgM RNA levels nor CD20 were observed. However, gene expression of IGJ was downregulated in the resistant cell lines. Both TMD8 RO and TMD8 RI showed increased basal levels of phosphor-tyrosine phosphorylation and amplified BCR signalling following BCR ligation. Collectively, these studies indicate hyperactivation of the BCR signalling pathway in the development of resistance to BTKi, with changes occurring upstream of BTK. Further studies to characterise changes at the cell surface are required to identify novel therapeutic approaches to the development of BTKi resistance.