Responses to the Selective Bruton's Tyrosine Kinase (BTK) Inhibitor Tirabrutinib (ONO/GS-4059) in Diffuse Large B-cell Lymphoma Cell Lines

Abstract

Bruton's tyrosine kinase (BTK) is a key regulator of the B-cell receptor signaling pathway, and aberrant B-cell receptor (BCR) signaling has been implicated in the survival of malignant B-cells. However, responses of the diffuse large B-cell lymphoma (DLBCL) to inhibitors of BTK (BTKi) are infrequent, highlighting the need to identify mechanisms of resistance to BTKi as well as predictive biomarkers. We investigated the response to the selective BTKi, tirabrutinib, in a panel of 64 hematopoietic cell lines. Notably, only six cell lines were found to be sensitive. Although activated B-cell type DLBCL cells were most sensitive amongst all cell types studied, sensitivity to BTKi did not correlate with the presence of activating mutations in the BCR pathway. To improve efficacy of tirabrutinib, we investigated combination strategies with 43 drugs inhibiting 34 targets in six DLBCL cell lines. Based on the results, an activated B-cell-like (ABC)-DLBCL cell line, TMD8, was the most sensitive cell line to those combinations, as well as tirabrutinib monotherapy. Furthermore, tirabrutinib in combination with idelalisib, palbociclib, or trametinib was more effective in TMD8 with acquired resistance to tirabrutinib than in the parental cells. These targeted agents might be usefully combined with tirabrutinib in the treatment of ABC-DLBCL.

Keywords: BCR signaling; BTK; DLBCL; combination therapy.

Figure 1

Effect of tirabrutinib on B-cell lines. (a) Six cell lines were exposed to different concentrations of tirabrutinib before an analysis of viability at 72 h using a CellTiterGlo^® Assay. Data shown here are the mean + SD of the six sensitive cell lines (n = 4–6). (b) TMD8 and REC1 cells were exposed to different concentrations of tirabrutinib for 72 h before analysis of apoptosis by AnnexinV-FITC staining by flow cytometry. Data shown are mean + SD (n = 6). (c) Electron microscopy of TMD8 cells left untreated (control) or exposed to 300 nM of tirabrutinib for 48 h. Cells display typical signs of apoptosis, including chromatin condensation, membrane blebbing, and mitochondrial changes. No other obvious morphologies were observed. (d) TMD8 cells were treated with 300 nM of tirabrutinib and/or the caspase inhibitor zVAD.fmk (25,000 nM, grey bars) or Q-VD-Oph (10,000 nM, black bars) for 72 h before analysis of apoptosis by AnnexinV-FITC staining by flow cytometry. Data shown are mean + SD (n = 3). (e) TMD8 cells were treated for 72 h before undergoing Western blot analysis.

Figure 2

BTK inhibitor mechanism of resistance in ABC-DLBCL cell lines. Two sensitive (TMD8, OCI-LY10) and two resistant (OCI-LY3, TMD8R) cell lines were treated with 300 nM of tirabrutinib for 4 h. Cells were lysed and analyzed through Western blotting using anti-P-BTK, anti-P-ERK, anti-P-STAT3, and their non-phosphorylated forms. Data are representative of three individual experiments.

Figure 3

Anti-tumor activity of tirabrutinib in TMD8 and TMD8R xenograft models. SCID mice were injected with 1 × 10⁷ of (a) TMD8 or (b) TMD8R cells in Matrigel, subcutaneously. After randomization, mice were administered with a vehicle or tirabrutinib via mixed diet for three weeks (TMD8) or two weeks (TMD8R), respectively. The tumor volumes measured for nine mice for TMD8 and ten mice for TMD8R at each measurement time point are presented as the mean ± standard error. The Dunnett-test was used to compare data on tumor volume in the 0.0037%, 0.012%, and 0.037% group, versus the vehicle. A p value of less than 5% was considered statistically significant. * p < 0.05. ** p < 0.01. *** p < 0.001.

Figure 4

A combination of tirabrutinib and idelalisib or lenalidomide can overcome BTK-inhibitor resistance. TMD8R cells were treated with indicated doses of tirabrutinib combined with 100 nM of idelalisib or 1000 nM of lenalidomide for 72 h. Apoptosis was determined by AnnexinV-FITC staining by flow cytometry. The combination index was calculated using Calcusyn (Biosoft, Ferguson, MO, USA).