In vitro and in vivo studies of Bruton tyrosine kinase (BTK) mutations & inhibition
Sammanfattning: Bruton tyrosine kinase (BTK) is a non-receptor protein kinase that belongs to the TEC family kinases. It plays an important role in the B-cell receptor signaling pathway (BCR) and its pharmacological inhibition has been demonstrated as an effective strategy for the treatment of B-cell malignancies. Ibrutinib, acalabrutinib and zanubrutinib are small molecules and irreversible BTK binders that have been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of several B-cell malignancies. Irreversible inhibitors block BTK catalytic activity by covalently binding to the cysteine (C) 481 located in the kinase domain. Mutations at this residue abrogate the possibility of forming a covalent bond, thereby decreasing the efficacy of the inhibitor. The most common mutation found in treated patients is the cysteine-481 to serine substitution (C481S). However, other less frequent substitutions have also been identified, such as, T474I and T474S substitutions in the BTK gatekeeper residue or PLCg2 gain-of-function substitutions e.g. S707Y and R665W. In paper I we studied a novel C481S knock-in mouse model. Our analysis of these mice reveled no phenotype alterations, as compared to wild-type mice, and demonstrated that C481S substitution has no detectable effect on BTK´s function or on the development of hematopoietic cells. We demonstrated that isolated B-lymphocytes carrying C481S were resistant to irreversible but sensitive to reversible BTK inhibitors (BTKis). This was achieved by analyzing BTK catalytic activity, cell-viability and expression of cell activation markers. Additionally, we confirmed that irreversible BTKis impaired T-lymphocyte activation in a BTK independent manner. This demonstrates the potential of this mouse model to be used in the study of BTKindependent, both therapeutic and adverse, effects caused by irreversible BTKis. Resistance to BTKis has become one of the most critical concerns in long term ibrutinib treated patients. The cause of the resistance to irreversible BTKis is less frequently associated to the gatekeeper residue, in contrast what is observed for other kinase inhibitors such as the fusionprotein BCR-ABL inhibitor imatininb or the EGFR inhibitor gefitinib. In paper II we aimed to understand the role of gatekeeper and combined gatekeeper/C481 BTK variants in the resistance to reversible and irreversible BTKis. We evaluated protein expression, catalytic activity and susceptibility to BTKis of 16 BTK single and double variants. We found that double T474I/C481S, T474M/C481S and T474M/C481T variants were insensitive to ³16 fold irreversible inhibitor pharmacological serum concentration. On the other hand, reversible BTKis showed a variable inhibition pattern. RN486 seemed to have highest therapeutic potential for patients that develop resistance to combined gatekeeper/C481 BTK variants.
Denna avhandling är EVENTUELLT nedladdningsbar som PDF. Kolla denna länk för att se om den går att ladda ner.