BTK gatekeeper residue variation combined with cysteine 481 substitution causes super-resistance to irreversible inhibitors acalabrutinib, ibrutinib and zanubrutinib

Abstract

Irreversible inhibitors of Bruton tyrosine kinase (BTK), pioneered by ibrutinib, have become breakthrough drugs in the treatment of leukemias and lymphomas. Resistance variants (mutations) occur, but in contrast to those identified for many other tyrosine kinase inhibitors, they affect less frequently the "gatekeeper" residue in the catalytic domain. In this study we carried out variation scanning by creating 11 substitutions at the gatekeeper amino acid, threonine 474 (T474). These variants were subsequently combined with replacement of the cysteine 481 residue to which irreversible inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib, bind. We found that certain double mutants, such as threonine 474 to isoleucine (T474I) or methionine (T474M) combined with catalytically active cysteine 481 to serine (C481S), are insensitive to ≥16-fold the pharmacological serum concentration, and therefore defined as super-resistant to irreversible inhibitors. Conversely, reversible inhibitors showed a variable pattern, from resistance to no resistance, collectively demonstrating the structural constraints for different classes of inhibitors, which may affect their clinical application.

Fig. 1. Expression and catalytic activity of BTK gatekeeper variants in COS-7 cells.

Thirty-six hours post transfection, the cells were serum starved for 4 h and subsequently activated with serum and pervanadate for 5 min at room temperature. The cell lysates were immunoblotted for total and tyrosine (Y) 223 phosphorylated BTK protein. For densiometric quantification, background signal was subtracted and β-actin utilized as an internal loading control as well as for normalization of total BTK. Values displayed for BTK phosphorylation were normalized both to total protein and wild-type BTK. Four categories were used to quantify the relative BTK activation of the gatekeeper variants compared to wild-type, low (<0.5-fold), intermediate low (0.51–1.0-fold), intermediate high (1.01–1.5-fold) and high (>1.51-fold compared to wild-type). Two-way ANOVA, 95% confidence interval, p value ⁽*⁾ < 0.05 and **** < 0.0001.

Fig. 2. Catalytic activity of BTK variants.

COS-7 cells were transfected with wild-type BTK and the enzymatically active BTK gatekeeper variants. Thirty-six hours post transfection, the cells were serum starved for 4 h, BTK inhibitor treated for 1 h and activated for 5 min. BTK protein expression and phosphorylation of Y223 and Y551 were evaluated by immunoblotting. Numbers below bands indicate ratio of phosphorylated protein to total protein as obtained by densiometric quantification with background signal subtracted. β-actin was utilized as an internal loading control and values displayed for BTK phosphorylation were normalized to wild-type. A Gatekeeper variants inhibited at 0.5 μM of ibrutinib. B Ibrutinib-resistant variants requiring higher ibrutinib concentration. C Ratio of Y223 phosphorylated protein over total protein (as quantified by densiometric analysis) from C481S, T474A, T474S/C481S, and T474A/C481S variants. Washout (wo) was performed three times in serum-free medium prior to activation.

Fig. 3. BTK and PLCG2 inhibition by RN486, fenebrutinib and CGI-1746.

COS-7 cells were transfected with wild-type BTK and ibrutinib-resistant gatekeeper variants. Thirty-six hours post transfection, the cells were serum starved for 4 h and treated with the BTK inhibitor for 1 h. Activation was performed for 5 min at room temperature using serum and pervanadate. Total (BTK and PLCG2) and phosphorylated protein sites (Y223 and Y551 for BTK; Y753 for PLCG2) were measured using immunoblotting. A Ibrutinib, RN486, fenebrutinib and CGI-1746 were tested in transfected cells with wild-type BTK and resistant variants C481S and C481T. B Non-covalent BTK inhibitors in COS-7 cells transfected with the resistant variants T474M/C481T, T474I/C481S, and T474M/C481S. Ratio of phosphorylated protein over total protein (as quantified by densiometric analysis). C Representative western-blot for T474I/C481S. Western-blots for T474M/C481S and T474M/C481T are included in Supplementary Fig. 6.

Fig. 4. Comparison of the BTK phosphorylation activity blocked by covalent and non-covalent BTK inhibitors in the double variant T474I/C481S.

COS-7 cells were transfected with T474I/C481S variant, serum starved and treated with the covalent and non-covalent BTK inhibitors. Activation was performed using serum and pervanadate. Total BTK and the amount of BTK phosphorylated at Y223 were measured using immunoblotting. Ratio of phosphorylated protein over total protein (as quantified by densiometric analysis) are displayed.

Fig. 5. Binding of covalent and non-covalent inhibitors to BTK and effects of amino acid substitutions.

The entire kinase domain-ibrutinib complex structure is in the left-center with ibrutinib in the catalytic site. The N-terminal lobe is in light gray and the C-terminal lobe in dark gray. Side chains are shown for threonine (T) 474, top, and cysteine (C) 481, below, in blue. Top row shows the chemical structures and indicates binding of covalent inhibitors and the bottom row of non-covalent inhibitors. Original residues at positions 474 and 481, T and C, respectively, are in blue. Substitutions at 474 methionine (yellow) and isoleucine (green), at 481 serine (yellow) and threonine (green). For the panels 474 and 481 are indicated in the middle. Ovals in light and dark orange indicate the sites of formation of hydrogen bonds and the covalent bond to cysteine, respectively [36, 41, 47, 50, 51, 60].

Fig. 6. Structural explanations for substitutions.

Ibrutinib, top, and RN-484, bottom, are used as examples for covalent and non-covalent inhibitors, respectively. Substitutions of C481 prevent covalent binding of inhibitors, and the side chains can affect the positioning of the inhibitor. Methionine and isoleucine replacements at gatekeeper 474 collide with the covalent inhibitors and affect binding affinity. Non-covalent inhibitors have different binding mode and side chains at 481 are further away from the inhibitor. The gatekeeper residue is important for binding of these inhibitors, as well.

Fig. 7. Effects of ibrutinib and fenebrutinib on BTK activity in single and double variants.

(top panel) Schematic representation of the BCR-BTK signaling pathway and the (middle panel) identification of ibrutinib super-resistant double mutants. Gatekeeper T474P mutant is not phosphorylated on Y551 and hence evaluation of pY223 cannot be made. Variants in bold represent mutations found in treated CLL patients. (bottom panel) Selected findings regarding fenebrutinib sensitivity or resistance. Other covalent BTK inhibitors acalabrutinib and zanubrutinib were also tested at very high concentration and super-resistance was confirmed (Supplementary Figs. 4 and 5). Fenebrutinib was also tested for C481T, T474M/C481S, T474I/C481S and T474M/C481T, see Fig. 3. While other covalent inhibitors are not mentioned in the figure, they behave similar to ibrutinib.