Bruton tyrosine kinase in immunodeficiency and in B-cell malignancy

Abstract

BTK inhibitors have induced high response rates in the treatment of leukemias and lymphomas. Ibrutinib is the first-in-class US Food and Drug Administration (FDA)-approved BTK covalent inhibitor to treat chronic lymphocytic leukemia (CLL). However, a sub-group of patients develops resistance to ibrutinib therapy. The most common resistance mechanism is substitution of cysteine 481 to serine of BTK, the residue to which ibrutinib binds irreversibly. Few other amino acid replacements at this site had been characterized. In paper I, we therefore performed functional analysis of all the possible amino acid replacements at C481 site due to a single nucleotide change. We also included threonine substitution because of its structural similarity to serine. BTK with cysteine-to-threonine substitution retains catalytic activity and cause ibrutinib resistance. Thus, we identified a new potential resistant variant, BTK C481T for BTK irreversible inhibitors. For the replacement with arginine, phenylalanine, tryptophan or tyrosine, BTK enzymatic activity was completely ablated, while glycine substitution still showed some kinase activity, but to a much lower extent.

CLL patients receiving ibrutinib treatment show rapid mobilization of tumor cells from lymph nodes (LN) to peripheral blood (PB). However, the detailed mechanism of ibrutinib-induced tumor cell redistribution has not been clarified. In paper II, we tried to explore this observation by analyzing changes in plasma inflammation-related biomarkers, transcriptional levels in CLL cells, B-cell activation and migration markers, and PB mononuclear cell populations as early as 9h after ibrutinib treatment. We compared the changes between before and at 6 time points after treatment initiation in LN and PB. We observed a significant downregulation of 10 plasma inflammation-related markers, mainly chemokines but not CLL-derived within 9 hours. RNA-sequencing data showed significant and continuous expression changes of genes related to B-cell receptor (BCR) signaling and CLL biology. We conclude that ibrutinib rapidly blocks an ongoing inflammatory response and in particular influences LN CLL cells.

Loss-of-function (LOF) mutations of BTK lead to a severe block in B lineage development, as seen in X-linked agammaglobulinemia (XLA). In paper III, we analyzed a large number of XLA patients, including 108 previously unreported cases. The tolerance to single amino acid replacements was predicted for full-length BTK. Moreover, we compared these germline XLA-causing mutations with those acquired in leukemia and lymphoma. Based on published cases and those reported to a mutation database, BTK mutation spectrum in more than 10,000 BTK-independent tumors was compared to the BTK-dependent malignancies, CLL and mantle cell lymphoma (MCL), and also to BTK-potentially-dependent malignancies, like diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and germinal center (GC)-derived B-cell lymphoma. This analysis for the first time identifies BTK to be a potential tumor suppressor in a subset of DLBCL and FL. Therefore, whether BTK inhibitors, which show highly efficient therapeutic effects in CLL and MCL, might promote tumorigenesis in a subset of other B cell malignancies remains an open question.