Mechanisms of Resistance to Noncovalent Bruton's Tyrosine Kinase Inhibitors

Abstract

Background: Covalent (irreversible) Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood.

Methods: We performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors.

Results: Among 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors.

Conclusions: Resistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.).

Figure 1.. BTK Mutations in Patients with Chronic Lymphocytic Leukemia with Acquired Resistance to Noncovalent BTK Inhibitors.

Panel A shows non-C481 Bruton’s tyrosine kinase (BTK) mutations found at the time of relapse during treatment, as revealed by serial targeted gene sequencing of specimens from patients with chronic lymphocytic leukemia treated with the noncovalent BTK inhibitor pirtobrutinib. The timing of specimen collection is shown on the x axis, and the cancer-cell fraction of the non-C481 BTK mutations is shown on the y axis. Panel B shows BTK mutations outside the BTK C481 residue found in patients with resistance to pirtobrutinib. Each individual occurrence of a mutation is depicted as an arrowhead. PH denotes pleckstrin homology domain, and SH Src homology domain. Panels C and D are fish-plot representations of single-cell mutational data from Patient 3 (Panel C) and Patient 4 (Panel D) before pirtobrutinib therapy and at relapse during treatment. Patient 3 had a phospholipase C gamma 2 (PLCγ2) mutation before pirtobrutinib therapy and was found to have acquired BTK L528W mutations at relapse after 9 months of treatment. Patient 4 had the BTK C481S mutation before pirtobrutinib therapy, and it was suppressed during treatment; this patient was found to have acquired BTK L528W at relapse after 11 months of treatment. Mutations in XPO1 (exportin 1), FBXW7 (F-box and WD repeat—containing protein 7), and BIRC3 (baculoviral IAP repeat–containing protein 3) were also found.

Figure 2.. Resistance to BTK Inhibitors Conferred by BTK Mutations Outside the C481 Residue.

Panel A shows the locations of non-C481 BTK mutations (V416L, A428D, M437R, T474I, and L528W) identified in patients with pirtobrutinib resistance mapped onto the crystal structure of the BTK kinase domain (gray ribbon). Mutated amino acids are shown as red spheres. Panel B shows the chemical structures of ibrutinib, pirtobrutinib, and ARQ-531 overlaid onto the crystal structure of the BTK kinase domain to illustrate the interactions between noncovalent BTK inhibitors and the non-C481 BTK mutations identified in specimens from patients with pirtobrutinib resistance. Panel C shows the results of experiments in which TMD8 cells transduced with mutant BTK were treated with pirtobrutinib or ibrutinib for 72 hours and the half-maximal inhibitory concentrations (IC₅₀) determined with the use of cell-viability assays. Panel D shows a heat map of IC₅₀ values at 72 hours after treatment of TMD8 cells transduced with mutant BTK with ibrutinib or a panel of noncovalent BTK inhibitors (pirtobrutinib, fenebrutinib, vecabrutinib, or ARQ-531). These data show that BTK C481S is resistant to ibrutinib but sensitive to noncovalent BTK inhibitors, whereas cells expressing BTK mutants V416L, A428D, M437R, T474I. and L528W are less sensitive to noncovalent BTK inhibitors. The data in Panels C and D are from three independent replicates and have been normalized to values obtained with a vehicle control (dimethylsulfoxide).

Figure 3.. Effect of BTK Resistance Mutations on Binding of Noncovalent and Covalent Inhibitors to BTK and on B-Cell–Receptor Signaling.

Panel A shows the binding affinities (K_D) of each noncovalent BTK inhibitor to purified wild-type or mutant BTK protein, determined with the use of surface plasmon resonance technology. On the right are K_inact/K_| values for covalent BTK inhibitors on the enzymatic activity of each wild-type or mutant BTK protein. The K_inact/K_| value indicates the efficiency of covalent bond formation between each drug and BTK protein. Red values indicate mutants that decreased drug-binding efficiency by a factor of at least 10. The K_D values for the covalent inhibitors to BTK C481S are reported. Panel B shows the inhibitory effects of TMD8 cells transduced with BTK mutants (C481S, A42ED, M437R, V416L, L528W, and T474I) on BTK autophosphorylation at Y223 and PLCγ2 phosphorylation at Y1217, On IgM stimulation of TMD8 cells, BTK mutants activated AKT (phosphorylated S473) and extracellular signal-related kinase (ERK; phosphorylated Y202 and Y204) signaling pathways similarly to wild-type BTK. (The prefix “p” indicates phosphorylation.) However, as shown in Panel C, intracellular calcium was elevated to higher levels in cells expressing BTK mutants than in those expressing wild-type BTK. The Ca²⁺ ratio is based on flow cytometry performed with lndo-1 (Life Technologies); higher values indicate greater concentrations of intracellular calcium.