Co-Targeting of BTK and TrxR as a Therapeutic Approach to the Treatment of Lymphoma

Abstract

Diffuse large B-cell lymphoma (DLBCL) is a haematological malignancy representing the most diagnosed non-Hodgkin's lymphoma (NHL) subtype. Despite the approved chemotherapies available in clinics, some patients still suffer from side effects and relapsed disease. Recently, studies have reported the role of the Trx system and the BCR signalling pathway in cancer development and drug resistance. In this regard, we assessed a potential link between the two systems and evaluated the effects of [Au(d2pype)₂]Cl (TrxR inhibitor) and ibrutinib (BTK inhibitor) alone and in combination on the cell growth of two DLBCL lymphoma cell lines, SUDHL2 and SUDHL4. In this study, we show higher expression levels of the Trx system and BCR signalling pathway in the DLBCL patient samples compared to the healthy samples. The knockdown of TrxR using siRNA reduced BTK mRNA and protein expression. A combination treatment with [Au(d2pype)₂]Cl and ibrutinib had a synergistic effect on the inhibition of lymphoma cell proliferation, the activation of apoptosis, and, depending on lymphoma cell subtype, ferroptosis. Decreased BTK expression and the cytoplasmic accumulation of p65 were observed after the combination treatment in the DLBCL cells, indicating the inhibition of the NF-κB pathway. Thus, the co-targeting of BTK and TrxR may be an effective therapeutic strategy to consider for DLBCL treatment.

Keywords: B-cell receptor signaling pathway; DLBCL; bruton’s tyrosine kinase; thioredoxin reductase.

Figure 1

Trx system and BCR signalling pathway-related gene expression in DLBCL and healthy samples. (A,B), mRNA expression in diffuse large B-cell lymphoma (DLBCL) patients (n = 47) and healthy samples (n = 444) was analysed. The significance levels are marked as *** p < 0.001.

Figure 2

Correlation analysis of expression of genes of the Trx system and BCR signalling pathway. Expression of TXNRD1, TXN, and PRDX1 in comparison with BTK, RelA, and XIAP expression in DLBCL (n = 48). (A–C), correlation analysis between BTK and TXNRD1 (A), TXN (B), and PRDX1 (C); (D–F), correlation analysis between RelA and TXNRD1 (D), TXN (E), and PRDX1 (F); (G–I), correlation analysis between XIAP and TXNRD1 (G), TXN (H), and PRDX1 (I). The correlation coefficient^® was calculated using Spearman’s r correlation analysis, and p values are displayed.

Figure 3

TrxR1 knockdown decreases BCR/NF-κB pathway-related mRNA and protein expression. SUDHL2 and SUDHL4 cells were transfected with TrxR1 specific siRNA (siTrxR) and a scrambled siRNA (control). (A,B), RT-qPCR was performed 48 h after transfection to determine the mRNA expression of TrxR1, BTK, p65, and surviving in SUDHL2 and SUDHL4 cells. (C,D), Western blotting was performed 48 h after transfection to determine the protein levels of BTK and TrxR in SUDHL2 and SUDHL4 cells. (E,F), The BTK expression was detected after treating cells with the indicated [Au(d2pype)₂]Cl after 24 h in SUDHL2 and SUDHL4 cells. B-actin was used as the loading control for BTK protein blots, and Vinculin was used as the loading control for the TrxR protein blots. Western blots are representative of 3 independent experiments. Significance levels are marked as * p < 0.05 and ** p < 0.005.

Figure 4

The cell proliferation and the isobologram analysis in DLBCL cell lines treated with ibrutinib with or without [Au(d2pype)₂]Cl. SUDHL2 (A) and SUDHL4 (B) were treated with indicated concentrations of ibrutinib for 24 or 48 h. (C) SUDHL2 and (D) SUDHL4 cells were treated with ibrutinib alone or in combination with 0.25 µM of [Au(d2pype)₂]Cl for 24 h. In the figures, the IC₅₀ of ibrutinib alone is presented as IC_50wo, and the IC₅₀ of ibrutinib with [Au(d2pype)₂]Cl is presented as IC_50w. (E,F), isobolograms between [Au(d2pype)₂]Cl and ibrutinib in the lymphoma cell lines. The plots showing the FIC values indicate the drug interaction and the dashed lines mark the FIC = 1. Two independent experiments with three technical replicates were performed in each lymphoma cell line.

Figure 5

Caspase-3 activity and cell inhibition in DLBCL cell lines after treatment. (A,B), the two DLBCL cell lines were treated with the indicated concentrations of ibrutinib with or without [Au(d2pype)₂]Cl for 24 h. (A) SUDHL2 and (B) SUDHL4 cells were cotreated with 0.25 µM of [Au(d2pype)₂]Cl. (C,D), lymphoma cells were treated with the indicated concentrations of ibrutinib with or without [Au(d2pype)₂]Cl plus 25 µM zVAD-fmk, or 10 µM Ferrostatin-1 for 24 h. Significance levels are marked as * p < 0.05 and ** p < 0.005.

Figure 6

Expression of BTK and NF-κB signalling pathway after treatment. Cells were treated with 5 µM of ibrutinib (SUDHL4) or 10 µM of ibrutinib (SUDHL2) combined with 0.25 µM of [Au(d2pype)₂]Cl. (A), protein levels of BTK were detected using Western blotting after 24 h treatment in SUDHL2 and SUDHL4 cells. (B), RT-qPCR was performed to detect the mRNA expression levels of TrxR, BTK, p65, and Survivin after 24 h in SUDHL2 and SUDHL4 cells. (C), Western blotting was carried out to determine the protein levels of p-IκB after 24 h treatment. (D), immunofluorescence staining was performed after 12 h with indicated treatment in SUDHL2 cells. Significance levels are marked as * p < 0.05 and ** p < 0.005.