Novel mouse model resistant to irreversible BTK inhibitors: a tool identifying new therapeutic targets and side effects

Abstract

Pharmacological inhibitors of Bruton tyrosine kinase (BTK) have revolutionized treatment of B-lymphocyte malignancies and show great promise for dampening autoimmunity. The predominant BTK inhibitors tether irreversibly by covalently binding to cysteine 481 in the BTK catalytic domain. Substitution of cysteine 481 for serine (C481S) is the most common mechanism for acquired drug resistance. We generated a novel C481S knock-in mouse model and, using a battery of tests, no overt B-lymphocyte phenotype was found. B lymphocytes from C481S animals were resistant to irreversible, but sensitive to reversible, BTK inhibitors. In contrast, irreversible inhibitors equally impaired T-lymphocyte activation in mice, mimicking the effect of treatment in patients. This demonstrates that T-lymphocyte blockage is independent of BTK. We suggest that the C481S knock-in mouse can serve as a useful tool for the study of BTK-independent effects of irreversible inhibitors, allowing for the identification of novel therapeutic targets and pinpointing potential side effects.

Comparative Analysis of BTK Inhibitors and Mechanisms Underlying Adverse Effects

“…In contrast, when acalabrutinib or spebrutinib are in vitro evaluated against EGFR ( Barf et al, 2017 ) or EGFR/JAK3 kinases, respectively ( Evans et al, 2013 ), results from biochemical and cellular data correlate, demonstrating that neither kinase inhibits EGFR and that only spebrutinib inhibits JAK3 kinase activity. Apart from biochemical and cellular assays, we have recently generated a knock-in mouse, which carries a cysteine 481 to serine mutation in BTK, enabling adequate in vivo off-target analysis for all irreversible inhibitors ( Estupiñán et al, 2020 ).…”

“…Ibrutinib potently inhibits BTK causing reduced BCR signaling ( Honigberg et al, 2010 ), but it also targets many other cellular processes through the roles of BTK outside of the BCR ( Nore et al, 2000 ). The direct inhibition of other kinases impacts upon normal processes in T lymphocytes (no contribution of BTK) as well as macrophages and platelets (both cell types express BTK and also other kinases interacting with BTKis) ( Feng et al, 2015 ; Byrd et al, 2016 ; Barf et al, 2017 ; Nicolson et al, 2018 ; Estupiñán et al, 2020 ).…”

B cell signalling pathways—New targets for precision medicine in chronic lymphocytic leukaemia

“…Interestingly, a BTK C481S knock-in mouse model was recently generated. 124 This cysteine to serine substitution is the most common mechanism for acquired resistance to BTK inhibitors, and these mice are resistant to irreversible BTK inhibitors. This model may prove useful to identify novel therapeutic targets.…”

“…This model may prove useful to identify novel therapeutic targets. 124 Together, the available PDX and transgenic mouse models are valuable hypothesis-testing tools and provide novel insights on tumour biology and drug mechanisms. Nevertheless, their use in precision medicine, where treatment decisions may be required with some urgency, is currently limited.…”

“…Data generated from these models should therefore be interpreted with care. Interestingly, a BTK C481S knock‐in mouse model was recently generated 124 . This cysteine to serine substitution is the most common mechanism for acquired resistance to BTK inhibitors, and these mice are resistant to irreversible BTK inhibitors.…”