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Inhibition of B-cell receptor signaling disrupts cell adhesion in mantle cell lymphoma via RAC2.

ABSTRACT: Inhibition of the B-cell receptor (BCR) signaling pathway is highly effective in B-cell neoplasia through Bruton tyrosine kinase inhibition by ibrutinib. Ibrutinib also disrupts cell adhesion between a tumor and its microenvironment. However, it is largely unknown how BCR signaling is linked to cell adhesion. We observed that intrinsic sensitivities of mantle cell lymphoma (MCL) cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion signatures were simultaneously downregulated by ibrutinib in the ibrutinib-sensitive, but not ibrutinib-resistant, cells. Among the differentially expressed genes, RAC2, part of the BCR signature and a known regulator of cell adhesion, was downregulated at both the RNA and protein levels by ibrutinib only in sensitive cells. RAC2 physically associated with B-cell linker protein (BLNK), a BCR adaptor molecule, uniquely in sensitive cells. RAC2 reduction using RNA interference and CRISPR impaired cell adhesion, whereas RAC2 overexpression reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib exhibited slower tumor growth, with reduced RAC2 expression in tissue. Finally, RAC2 was expressed in ?65% of human primary MCL tumors, and RAC2 suppression by ibrutinib resulted in cell adhesion impairment. These findings, made with cell lines, a xenograft model, and human primary lymphoma tumors, uncover a novel link between BCR signaling and cell adhesion. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development.

SUBMITTER: Wu W

PROVIDER: S-EPMC7805322 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Inhibition of B-cell receptor signaling disrupts cell adhesion in mantle cell lymphoma via RAC2.

Wu Wenjun W Wang Weige W Franzen Carrie A CA Guo Hui H Lee Jimmy J Li Yan Y Sukhanova Madina M Sheng Dong D Venkataraman Girish G Ming Mei M Lu Pin P Gao Anhui A Xia Chunmei C Li Jia J Zhang Liang Leo LL Jiang Vivian Changying VC Wang Michael L ML Andrade Jorge J Zhou Xiaoyan X Wang Y Lynn YL

Blood advances 20210101 1

Inhibition of the B-cell receptor (BCR) signaling pathway is highly effective in B-cell neoplasia through Bruton tyrosine kinase inhibition by ibrutinib. Ibrutinib also disrupts cell adhesion between a tumor and its microenvironment. However, it is largely unknown how BCR signaling is linked to cell adhesion. We observed that intrinsic sensitivities of mantle cell lymphoma (MCL) cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell ...[more]

PMID: 33570628

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Project description:Aberrant B-cell receptor (BCR) signaling is a key driver in lymphoid malignancies. Bruton tyrosine kinase (BTK) inhibitors that disrupt BCR signaling have received regulatory approvals in therapy of mantle cell lymphoma (MCL). However, responses are incomplete and patients who experience BTK inhibitor therapy failure have dire outcomes. CG-806 (luxeptinib) is a dual BTK/SYK inhibitor in clinical development in hematologic malignancies. Here we investigated the pre-clinical activity of CG-806 in MCL. In vitro treatment with CG-806 thwarted survival of MCL cell lines and patient-derived MCL cells in a dose-dependent manner. CG-806 blocked BTK and SYK activation and abrogated BCR signaling. Contrary to ibrutinib, CG-806 downmodulated the anti-apoptotic proteins Mcl-1 and Bcl-xL, abrogated survival of ibrutinib-resistant MCL cell lines, and partially reversed the pro-survival effects of stromal microenvironment-mimicking conditions in primary MCL cells. Dual BTK/SYK inhibition led to mitochondrial membrane depolarization accompanied by mitophagy and metabolic reprogramming toward glycolysis. In vivo studies of CG-806 demonstrated improved survival in one of the two tested aggressive MCL PDX models. While suppression of the anti-apoptotic Bcl-2 family proteins and NFκB signaling correlated with in vivo drug sensitivity, OxPhos and MYC transcriptional programs were upregulated in the resistant model following treatment with CG-806. BAX and NFKBIA were implicated in susceptibility to CG-806 in a whole-genome CRISPR-Cas9 library screen (in a diffuse large B-cell lymphoma cell line). A high-throughput in vitro functional drug screen demonstrated synergy between CG-806 and Bcl-2 inhibitors. In sum, dual BTK/SYK inhibitor CG-806 disrupts BCR signaling and induces metabolic reprogramming and apoptosis in MCL. The Bcl-2 network is a key mediator of sensitivity to CG-806 and combined targeting of Bcl-2 demonstrates synergy with CG-806 warranting continued exploration in lymphoid malignancies.

Project description:Lymphoma cells interact with specific microenvironment cell subsets, such as stromal cells, from which they receive survival and proliferation stimuli. These interactions negatively influence therapeutic response and increase the risk for drug resistance and relapse. It is still not clear why clinically similar tumors in different patients respond differently to established treatment regimens. Objective: The main aim of this study was to identify and compare key signaling pathways related to stromal cell adhesion in different mantle cell lymphoma (MCL) cell lines.Methods: Human MCL cells (JeKo1 and REC1) were cultured alone or in co-culture with mouse MS-5 stromal cells. Differentially expressed genes in adherent cells were identified by RNA sequencing and species-specific read sorting. The cell surface levels of proteins encoded by selected differentially expressed genes were quantified using flow cytometry in adherent and suspension cells. Selective inhibitors or siRNA were used to assess their significance for adherence to stromal cells. Results: Jeko1 and REC1 cells exhibited different time-dependent patterns of adhesion to stromal cells. 590 genes that were differently regulated upon adhesion in each cell line individually, showed significant differences in the extent or direction of regulation between the two cell lines. From this set, there was a significant increase in transcript levels for B-cell Receptor (BCR) pathway signature genes in adherent JeKo1 cell but not in adherent REC1 cells. Inhibition of BCR signaling using siRNA or the clinically approved inhibitor, Ibrutinib, decreased the number of adherent JeKo1 cells but had no such effect on REC1 cells. The cell surface levels of CXCR4 were higher in JeKo1 cells compared to REC1 cells and CXCR4 inhibition disrupted the adhesion of JeKo1 but not REC1 cells to the stromal cells. ICAM1 cell surface levels were induced in JeKo1 cells upon adhesion while REC1 showed a high level of ICAM1 in non-adherent cells. Depletion of ICAM1 levels by siRNA reduced the stromal cell adhesion of both JeKo1 and REC1 cells. Conclusions: The results suggest that different MCL cell lines exhibit different signaling profiles and pathway dependencies during microenvironment interaction. In this study BCR signaling pathways were shown to be important for stromal cell adhesion of JeKo1 cells but not REC1 cells. This provides a possible mechanism to account for the differential responsiveness of MCL patients to treatment with the Bruton’s Tyrosine Kinase inhibitor, Ibrutinib, an inhibitor of BCR signaling that is clinically approved for MCL.

Dataset Information

Inhibition of B-cell receptor signaling disrupts cell adhesion in mantle cell lymphoma via RAC2.

Publications

Inhibition of B-cell receptor signaling disrupts cell adhesion in mantle cell lymphoma via RAC2.

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