Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib

Abstract

Background: Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance.

Methods: We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis.

Results: We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell-receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib.

Conclusions: Resistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell-receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.).

Figure 1. Functional Characterization of Bruton’s Tyrosine Kinase (BTK) with the C481S Mutation

An assay of recombinant nonmutant BTK versus BTK with the C481S mutation (mutant BTK) showed that the mutant form had enhanced kinase activity that could be inhibited by ibrutinib, though at a much higher half-maximal effective concentration than with nonmutant BTK (Panel A). The data shown reflect two independent experiments with triplicate samples for each treatment. After transfection of mutant or nonmutant BTK into HEK 293T cells, administration of ibrutinib did not inhibit B-cell–receptor signaling in cells with mutant BTK to the same degree as in cells with non-mutant BTK. BTK autophosphorylation at tyrosine 223 (Y223) (Panel B) was significantly more inhibited in cells with nonmutant BTK than in cells with mutant BTK, at 0.01 µM, 0.1 µM, and 1 µM of ibrutinib (P<0.001), and the difference in inhibition between nonmutant BTK and mutant BTK was significantly greater with ibrutinib than with dasatinib (P<0.001). The data shown in Panels A and B are normalized. I bars in both panels indicate standard errors. After transfection of nonmutant or mutant BTK into BTK^−/− DT40 cells, B-cell–receptor signaling was inhibited by ibrutinib in cells with nonmutant BTK to a greater degree than in cells with mutant BTK (Panel C). The data shown in Panels B and C reflect at least three independent experiments.

Figure 2. Functional Characterization of PLCγ2 with the R665W and L845F Mutations

After stimulation of DT40 cells with anti-IgM antibody, calcium flux assays showed calcium release in the cells with nonmutant PLCγ2 that can be completely inhibited by ibrutinib. Cells bearing either the R665W mutation or the L845F mutation showed calcium release that is not inhibited by 1 µM ibrutinib (P = 0.62 for R665W and P = 0.43 for L845F). Error bars represent standard errors. RFU denotes relative fluorescence units.

Figure 3. Characterization of R665W PLCγ2 Mutation in a Patient

One patient who was found to have an R665W mutation in PLCγ2 had samples available for immunoblot analysis at baseline, during the period of response to ibrutinib, and at the time of relapse, before ibrutinib was discontinued. At baseline, BTK was phosphorylated, and this phosphorylation was inhibited by ibrutinib both during the period of response to the drug and at the time of relapse (Panel A). However, PLCγ2 showed evidence of sustained activation at the time of relapse through phosphorylation at tyrosine 759 (Y759) (Panel B), a finding that suggests that the R665W mutation is a gain-of-function mutation. At the time of relapse, after the drug had been discontinued, fresh cells were treated with vehicle, plate-immobilized anti- IgM antibody, 1 µM ibrutinib, or 1 µM ibrutinib plus anti-IgM antibody. BTK phosphorylation was inhibited by ibrutinib (Panel C), but PLCγ2 phosphorylation was not (Panel D); these findings show that the R665W mutation is not sensitive to ibrutinib in vitro. Densitometry values, shown below the lanes, reflect the ratio of phosphorylated protein to total protein in Panels B, C, and D; in Panel A, the values reflect the ratio of phosphorylated protein to loading control because total BTK protein expression can change over time during ibrutinib therapy.