U-2932

In Vitro Efficacy of Tirabrutinib and Downstream Signaling Analysis in U-2932 Cells

Tirabrutinib is a highly selective Bruton's tyrosine kinase (BTK) inhibitor used to treat hematological malignancies. In vitro analyses of the anti-tumor mechanisms were conducted in ABC-DLBCL cells followed by phosphoproteomic and transcriptomic analyses.

Tirabrutinib was shown to inhibit the growth of U-2932 cells (Fig. 1A). For U-2932 cells, the IC₅₀ was 27.6 nM, and the maximum growth inhibition rate was 30.8% at 100 nM. Tirabrutinib inhibited BTK autophosphorylation at Tyr-223 in both cells in a concentration-dependent manner (Fig. 1B), with the IC₅₀ values (12.0 nM) being comparable to the IC₅₀ for growth inhibition. Thus, proliferation appears to be inhibited by tirabrutinib via inhibition of BTK phosphorylation.

Next, a phosphoproteomic analysis was conducted to investigate phosphoregulation induced by 1-h treatment with tirabrutinib in U-2932 cells, which would result in anti-tumor efficacy. In U-2932 cells, more than 5,563 distinct phosphoproteins and 11,493 distinct phosphorylation sites (class I phosphosites) were identified with high confidence (P ≥ 0.75). The overall abundances of Ser(P), Thr(P), and Tyr(P) were 84.6%, 14.8%, and 0.7%, respectively. Only 30 phosphorylation sites from 28 distinct phosphoproteins were suppressed (0.3% of all quantified sites) and four phosphorylation sites from four phosphoproteins were induced (0.04% of all quantified sites). Regarding the downregulated phosphorylation sites, five site-specific phosphorylation events that underwent significant regulation in U-2932 cell lines were identified (Table 1).

Fig. 1 Antiproliferative activity (A) and BTK autophosphorylation inhibitory effects (B) of tirabrutinib. (Kozaki R, et al., 2023)

Table 1. Significantly downregulated phosphosites in U-2932 cells. (Kozaki R, et al., 2023)

Activity of miR-1244, miR-193b-5p, and miR-1231 on U-2932 Cells In Vitro

Around 30-40% of patients with DLBCL suffer an early relapse after standard chemotherapy, but today no prediction of whether a patient belongs to this group is possible. MicroRNA is small nucleotide sequences that regulate cellular functions via post-transcriptional modification of gene expression and can serve as prognostic biomarkers.

Reverse transfection of U-2932 cells with reagent achieved a high percentage of 89.6 ± 0.7% of transfected cells after 48 h. The miRNA negative control (100 nM) (miR control (−)) did not change the viability of U-2932 cells for non-transfected (NT) cells after 48 h of transfection, discarding an adverse effect of the transfection method itself on cell viability (Fig. 2). Transfection of miR-1244, miR-193b-5p or miR-1231 (100 nM) mimics did not significantly change the viability of U-2932 (Fig. 2A) for miR control (−)-transfected cells. However, the inhibition of these three miRNAs of endogenous origin with their respective inhibitors significantly decreased U-2932 cell viability after 48 h by 24.1 ± 5.7%, 28.9 ± 6.5%, and 30.9 ± 3.3%, respectively (Fig. 2B), supporting the idea that these miRNAs are involved in the survival of these tumor cells.

The effect of miR-1244, miR-193b-5p, or miR-1231 on the response of DLBCL cells to CHOP treatment was then evaluated. Transfection of U-2932 with miR control (−) did not change cell sensitivity to CHOP and reduced viability dose-dependently with an IC₅₀ of 1.45 μg/mL in non-transfected and miR control (−)-transfected cells. Transfection of U-2932 cells with miR-1244 or miR-193b-5p mimics increased cell viability in the presence of vehicle (1% DMSO) by 27.4 ± 6.4% and 27.9 ± 8.4% (Fig. 3), respectively. Even more importantly, these miRNAs completely blocked the reduction of cell viability by CHOP (0.3 μg/mL) after 48 h of treatment. On the contrary, the miR-1231 mimic did not significantly change the inhibitory effect of CHOP on U-2932 cell viability at any of the concentrations studied (0.3, 1, and 3 μg/mL).

Fig. 2 Inhibition, but not expression, of miR-1244, miR-193b-5p, or miR-1231 reduced cell viability in U-2932 cells. (Bento L, et al., 2022)

Fig. 3 Expression of miR-1244 and miR-193b-5p, but not miR-1231, blocked the antitumoral effect of CHOP in U-2932 cells. (Bento L, et al., 2022)