Novel BTK Mutations Conferring Resistance to Non-Covalent BTK Inhibitors and Alternative Treatment Strategy

Abstract

Inhibitors of Bruton tyrosine kinase (BTK) denote an important class of molecules for treatment of chronic lymphocytic leukemia (CLL) and other B-cell malignancies. While resistance to covalent BTK inhibitors (BTKi) is predominantly mediated by BTK mutations at cysteine 481 and activating PLCG2 mutations, mechanisms of resistance to non-covalent BTKi are not fully characterized. Recently, Wang E et al. (NEJM 2022) identified novel BTK mutations in patients resistant to the non-covalent BTKi pirtobrutinib. Here，we generated and comprehensively characterized BTK and PLCG2 mutations conferring resistance to ibrutinib and five different non-covalent BTKi namely pirtobrutinib (LOXO-305), vecabrutinib (SNS-062), nemtabrutinib (ARQ-531), fenebrutinib (GDC-0853), and RN-486. Long-term in vitro dose escalation method was performed to generate resistance using multiple parallel lines of mantle cell lymphoma REC-1 cells. REC-1 were selected due to their high sensitivity to BTKi. Targeted next generation sequencing was used to identify acquired mutations in BTK and PLCG2. While all the six lines of REC-1 cells treated long term with ibrutinib gained the C481F BTK mutation, in cells resistant to the non-covalent BTKi we identified six different mutations in BTK (variant allele frequency (VAF) 22% to 99%) and three mutations in PLCG2 (VAF 10%, 30% and 53%). Four out of six lines of REC-1 cells resistant to vecabrutinib acquired BTK G409R mutation while one line acquired a L845F PLCG2 mutation. Three different BTK mutations, L528S, G480R and D539H were found in three independent lines of REC-1 cells resistant to fenebrutinib. RN-486-resistant REC-1 cells acquired G480R and V416L BTK mutations in two out of three different lines. One of the six pirtobrutinib-resistant lines acquired a BTK A428D mutation, while two other resistant lines gained R727L and S1079R PLCG2 mutations. Interestingly, mutations in three BTK residues A428, L528, and V416 reported to mediate pirtobrutinib resistance in CLL patients were also identified in the REC-1 cell lines that acquired resistance to pirtobrutinib, fenebrutinib, and RN-486, respectively. Of note, nemtabrutinib-resistant REC-1 cells, despite the increased IC50, did not acquire a BTK or PLCG2 mutation, indicating the role of other mechanisms in the generation of resistance. To functionally analyze BCR signaling capabilities of the different BTK mutant REC-1 cells, we performed measurement of intracellular [Ca2+] ([Ca2+]i) upon stimulation with anti-IgM. Except for A428D, all BTK mutant REC-1 cell lines showed a similar or enhanced [Ca2+]i compared to wild type (WT) cells. Western blot analyses showed decreased anti-IgM-mediated activation of BTK in all the different BTK mutant cell lines compared to WT. However, the downstream activation of phospho-AKT was enhanced in all the mutant lines, similar to earlier reports, while phospho-ERK levels changed marginally. Moreover, by studying sensitivity of the BTK mutated cells to a panel of nine different covalent and non-covalent BTKi, we identified G409R and D539H mutants to be sensitive to covalent BTKi while the other BTK mutants were resistant to both covalent and non-covalent BTKi. The BCL2 inhibitor venetoclax is an important treatment option for CLL, however, the response to venetoclax in the context of non-covalent BTKi resistance is unknown. Flow cytometry based BH3 profiling assay was performed to study apoptotic priming in the BTK mutants. Except for G409R, all BTK mutants showed increased apoptotic priming to BAD peptide exposure, indicating enhanced BCL2 dependency compared to WT. In line with this, the non-covalent BTKi resistant REC-1 cells showed enhanced sensitivity to venetoclax in vitro while G409R BTK mutant had a similar IC50 as WT. In summary, we identified novel BTK protein kinase domain mutations that confer resistance to non-covalent BTKi. Our findings highlight the importance of sequencing either all exons or at least the kinase domain (Exons 13 to 19) of BTK in the context of progression under non-covalent BTKi treatment. We also found that cells harboring these novel BTK mutations showed differential sensitivity to the covalent vs. non-covalent BTKi. We further demonstrate the potential of venetoclax as follow up treatment upon resistance to non-covalent BTKi. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal