Distinct BTK inhibitors differentially induce apoptosis but similarly suppress chemotaxis and lipid accumulation in mantle cell lymphoma

Zhuojun Liu,Jian Yu,Mingxia Shi,Shuhua Yue,Yuanshi Xia,Shuo Zhang,Jia Liu,Xiaofang Chen,Tianming Zhang,Lin Li,Jing Zhang,Hao Jia

doi:10.1186/s12885-021-08475-3

Zhuojun Liu, Jian Yu + Show 10 more

Open Access

https://doi.org/10.1186/s12885-021-08475-3

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Abstract

BackgroundThe more selective second-generation BTK inhibitors (BTKi) Acalabrutinib and Zanubrutinib and the first-generation BTKi Ibrutinib are highlighted by their clinical effectiveness in mantle cell lymphoma (MCL), however, similarities and differences of their biological and molecular effects on anti-survival of MCL cells induced by these BTKi with distinct binding selectivity against BTK remain largely unknown.MethodsAlamarBlue assays were performed to define cytotoxicity of BTKi against MCL cells, Jeko-1 and Mino. Cleaved PARP and caspase-3 levels were examined by immunoblot analysis to study BTKi-induced apoptotic effects. Biological effects of BTKi on MCL-cell chemotaxis and lipid droplet (LD) accumulation were examined in Jeko-1, Mino and primary MCL cells via Transwell and Stimulated Raman scattering imaging analysis respectively. Enzyme-linked immunoassays were used to determine CCL3 and CCL4 levels in MCL-cell culture supernatants. RNA-seq analyses identified BTKi targets which were validated by quantitative RT-PCR (qRT-PCR) and immunoblot analysis.ResultsAcalabrutinib and Zanubrutinib induced moderate apoptosis in Ibrutinib high-sensitive JeKo-1 cells and Ibrutinib low-sensitive Mino cells, which was accompanied by cleaved PARP and caspase-3. Such effects might be caused by the stronger ability of Ibrutinib to upregulate the expression of pro-apoptotic genes, such as HRK, GADD45A, and ATM, in JeKo-1 cells than in Mino cells, and the expression of such apoptotic genes was slightly changed by Acalabrutinib and Zanubrutinib in both JeKo-1 and Mino cells. Further, Acalabrutinib, Zanubrutinib and Ibrutinib reduced MCL-cell chemotaxis with similar efficiency, due to their similar abilities to downmodulate chemokines, such as CCL3 and CCL4. Also, these three BTKi similarly suppressed MCL-cell LD accumulation via downregulating lipogenic factors, DGAT2, SCD, ENPP2 and ACACA without significant differences.ConclusionBTKi demonstrated differential capacities to induce MCL-cell apoptosis due to their distinct capabilities to regulate the expression of apoptosis-related genes, and similar biological and molecular inhibitory effects on MCL-cell chemotaxis and LD accumulation.

Highlights

The more selective second-generation Bruton tyrosine kinase (BTK) inhibitors (BTKi) Acalabrutinib and Zanubrutinib and the first-generation BTK inhibitors (BTKi) Ibrutinib are highlighted by their clinical effectiveness in mantle cell lymphoma (MCL), similarities and differences of their biological and molecular effects on anti-survival of MCL cells induced by these BTKi with distinct binding selectivity against BTK remain largely unknown
BTKi induced differential cytotoxicity against MCL cell lines We firstly aimed to compare the cytotoxic effectiveness of Ibrutinib, Acalabrutinib and Zanubrutinib observed in MCL
RNA-seq identified apoptosis-related targets of BTKi To understand the comprehensive mechanisms involved in MCL-cell apoptosis, we analyzed differentially expressed genes (DEGs) in Jeko-1 and Mino cells treated with Ibrutinib, Acalabrutinib or Zanubrutinib, versus untreated cells, via whole-transcriptome RNA sequencing (RNA-seq)

Summary

Introduction

The more selective second-generation BTK inhibitors (BTKi) Acalabrutinib and Zanubrutinib and the first-generation BTKi Ibrutinib are highlighted by their clinical effectiveness in mantle cell lymphoma (MCL), similarities and differences of their biological and molecular effects on anti-survival of MCL cells induced by these BTKi with distinct binding selectivity against BTK remain largely unknown. Ibrutinib could potently induce apoptosis via inhibiting BCR signaling activated canonical NF-κB signaling in MCL [4], or altering the expression of anti-apoptotic gene, MCL-1 [5]. The production of chemokines, CCL3 and CCL4, is increased upon BCR activation in MCL [6], chronic lymphocytic leukemia (CLL) [7] and diffuse large B cell lymphoma (DLBCL) [8], and such effects can be impaired by Ibrutinib treatment, which significantly decreases chemokine receptor CXCR4 expression in MCL [6] and CLL [9]. It has been reported that BCR signaling activation could increase the level of lipoprotein lipase (LPL), a protein essential for fatty acid metabolism providing cells with energy and survival advantage, which can be impaired by Ibrutinib treatment via reducing LPL level in CLL [11]

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