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Letters to the Editor
Vecabrutinib inhibits B-cell receptor signal transduction in chronic lymphocytic leukemia cell types with wild-type or mutant Bruton tyrosine kinase
Ibrutinib as monotherapy or in combination has trans- formed the treatment landscape of chronic lymphocytic leukemia (CLL).1,2 This drug covalently tethers to the cys- teine-481 residue in Bruton tyrosine kinase (BTK), which is a pivotal enzyme in the B-cell receptor (BCR) pathway.3 Ibrutinib treatment results in long-term overall survival in patients with CLL; however, disease can relapse, particu- larly in previously treated patients. At a median of 3.4 years of follow-up, the cumulative incidence of progres- sion was 19%, and 85% of these patients had acquired mutations of BTK or PLCG2.4,5 The predominant BTK mutation is cysteine to serine (BTKC481S), with the second most frequent alteration being cysteine to arginine (BTKC481R), both of which preclude covalent bond forma- tion by ibrutinib, resulting in resistance to this drug.4 Reversible BTK inhibitors, such as vecabrutinib, have been developed that bind to BTK and maintain inhibitory activity against WT and mutant BTK. In this study, we characterized the activity of vecabrutinib on the BCR pathway using a CLL cell line model system engineered to overexpress BTK C481 WT or two mutant variants, BTKC481S and BTKC481R. 6 We also used primary CLL cells resistant to ibrutinib to further extend our investigations of vecabrutinib. A phase 1b clinical trial was recently completed in B-cell malignancies in which vecabrutinib was well-tolerated with some evidence of activity includ- ing in CLL patients with the C481S mutation.7
Vecabrutinib is a highly selective reversible BTK inhibitor (half maximal inhibitory concentration [IC50] = 3 nM). In a panel of 234 kinases and kinase variants, vecabrutinib demonstrated a biochemical IC50 of <100 nM for seven kinases (Figure 1A; Online Supplementary Figure S1A, B). The IC50 of vecabrutinib against WT BTK was similar to that of ibrutinib3 while it was more potent than ibrutinib on ITK and TEC kinases. In a direct kinase assay, vecabrutinib inhibited WT and mutant C481S vari- ants with similar potency. Data from healthy donors’ whole blood (n=145) further established the potency of vecabrutinib in inhibiting BTK, although there was a high degree of variability (mean ± standard deviation values, 50 nM ± 39 nM, range 2.8 nM – 216 nM) (Figure 1B). Vecabrutinib inhibited phosphorylation of the BTK downstream target PLCg2 in Ramos Burkitt lymphoma cells with IC50 values of 13 ± 6 nM (Figure 1C). Collectively, these data suggest that vecabrutinib inhibits phosphorylation of BTK and of PLCg2 at nanomolar con- centrations.
In MEC-1, a CLL cell line, treatment with both vecabrutinib and ibrutinib did not alter cell cycle profiles and resulted in 15-20% cell death at 1 mM (Online Supplementary Figure S1C-E). Vecabrutinib decreased BTK phosphorylation at a dose of 0.1 mM. Consistent with the observed decline in BTK phosphorylation, decreases in phosphorylation of PLCg2 and ERK were also observed. Phospho-S6 levels did not change in the MEC-1 cell line after treatment with vecabrutinib (Online Supplementary Figure S1F).
To mimic ibrutinib resistance, we transduced MEC-1 to generate cell lines that stably expressed green fluorescent protein and overexpressed either WT BTK (BTKWT) or the mutated variant C481S (BTKC481S), or C481R (BTKC481R) BTK.6 Cell death induced by ibrutinib or acalabrutinib has been limited when the drugs have been tested in vitro
in B-cell lines or primary CLL cells,8,9 and similarly, vecabrutinib did not affect cell viability or cell cycle pro- file (Online Supplementary Figure S2A-D). Several known proteins within the BCR signal transduction pathway, including BTK, can be used as biomarkers to monitor ibrutinib response or biological activity, including ERK and S6.8,10 We evaluated these proteins by immunoblot for phosphorylated proteins to show response to vecabrutinib and ibrutinib in cell lines that harbor WT overexpression or mutant BTK overexpression. Vecabrutinib at a dose of 1 mM decreased phospho-ERK more effectively than ibrutinib did in ibrutinib-resistant MEC-1 cells that overexpress mutant BTK (Figure 1D-F). This was observed in both BTKC481S and BTKC481R vari- ants. Changes in phospho-ERK were consistent with those in our previous study, in which we showed that phospho-ERK is a superior biomarker to determine ibru- tinib response upon overexpression of mutant BTK in MEC-16 (Figure 1E, F).
It is important to note that ibrutinib also decreased phospho-proteins in cells with mutated BTK. There are two explanations for this finding. First, the cell lines express endogenous WT BTK and second, ibrutinib can bind reversibly to BTK, albeit with reduced potency. In the clinic, ibrutinib’s poor pharmacokinetic properties (peak plasma level along with initial and terminal elimi- nation half-lives) preclude activity as a noncovalent inhibitor. Vecabrutinib treatment at a dose of 1 mM decreased Bcl-2 levels in cells harboring BTKC481S and Mcl-1 in cells overexpressing BTKC481R (Online Supplementary Figure S2E-G).
To evaluate protein changes more extensively as well as to compare the effects of vecabrutinib with those of ibrutinib, we compared protein profiles using the reverse- phase protein array (RPPA). Since we observed the largest effect of vecabrutinib after treatment at 1 mM, in all cell lines we compared this concentration with samples treat- ed with dimethylsulfoxide (DMSO) vehicle. The top ten canonical pathways were identified by Ingenuity Pathway Analysis (Online Supplementary Figure S3A-C). Several pathways were commonly affected in all three cell types. The extent of change and significance were different (Figure 2A). The top three canonical pathways with maximal change after vecabrutinib in cells with BTKWT overexpression were FLT3 signaling in hematopoietic progenitor cells, EGF and HGF signaling (Online Supplementary Figure S3A), whereas in cells with BTKC481S overexpression they were HGF signaling, regu- lation of epithelial-mesenchymal transition by growth factors pathway and B-cell receptor signaling (Online Supplementary Figure S3B) and in cells with BTKC481R over- expression they were epithelial-mesenchymal transition, ERK/MAPK signaling, and a senescence pathway (Online Supplementary Figure S3C). We also classified the types of target proteins using pie charts (Online Supplementary Figure S3D-F). Kinase and transcription regulator protein groups constituted >60% of proteins affected by vecabru- tinib in all BTK subtypes.
BCR pathway inhibition generally affects signal trans- duction, measured as phospho-proteins, proteins involved in transcription factors, cell proliferation, B-cell proteins, and apoptosis. Among the 258 proteins evaluat- ed by RPPA, eight phospho-proteins and five other pro- teins were affected by vecabrutinib and ibrutinib (Figure 2B). SHP-2 (PTPN11), a phosphatase that plays a critical role at several junctures in the BCR pathway, has been shown to interact with many proteins.11 The tyrosyl phosphorylation of this protein has been shown to be
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haematologica | 2022; 107(1)