B-cell receptor signalling, receptor cross-talk and the SHIP1 phosphatase in malignant B cells

Abstract

B-cell receptor (BCR) signalling plays a major role in the pathogenesis of chronic lymphocytic leukaemia (CLL) and other B-cell malignancies. In addition to its ability to promote cell accumulation, BCR signalling also influences the function of other cell surface receptors (receptor cross-talk), including G-protein couple receptors (GPCR). This project addresses two inter-connected areas related to BCR signalling in malignant B cells. First, it explores pathways of cross-talk between the BCR and the GPCRs CXCR4 and EBI2, including the role of kinases and the inositol lipid phosphatase SHIP1. Second, it identifies novel pathways regulated by a chemical SHIP1 activator, AQX-435.
Established B-cell lines were selected to investigate BCRCXCR4 to extend previous studies performed using primary CLL cells. This revealed that, similar to CLL cells, anti-IgM-induced CXCR4 down-modulation was dependent primarily on SYK and PI3K, but not BTK. However, in contrast to CLL cells, activation of SHIP1 using AQX-435 was not sufficient to down-modulate CXCR4 in B-cell lines. One aim of these cell line studies was to perform RNAi transfection studies to investigate the role of individual signalling intermediates in BCRCXCR4 cross-talk. However, it was not possible to achieve sufficient target knock-down with RNAi for reliable characterisation. Investigation of cross-talk was extended to EBI2 which, like CXCR4, is involved in positioning B cells during immune responses. Overall, EBI2 was expressed at low levels on CLL cells compared to normal B cells and this low level did not seem to be sufficient to support signalling or survival responses following ligand stimulation. In contrast to CXCR4, there was no strong evidence that EBI2 was subject to regulation either by anti-IgM or AQX-435.
Analysis of RNA-seq data demonstrated that AQX-435 and the PI3K inhibitor idelalisib shared the ability to reduce anti-IgM-induced changes in gene expression in CLL cells. However, AQX-435 also triggered a unique transcriptional response driven by activation of liver X receptor (LXR), potentially linked to SHIP1 activation. LXR activation drives cholesterol efflux and functional experiments demonstrated that supplementation of cholesterol using mevalonic acid significantly reduced AQX-435-induced CLL cell apoptosis.
Overall, these experiments shed important new light on BCR signalling in malignant B cells, and support the idea that SHIP1 activators, such as AQX-435, are attractive potential therapeutic agents.

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Identifiers

ORCID for Graham Packham: orcid.org/0000-0002-9232-5691

ORCID for Mark Cragg: orcid.org/0000-0003-2077-089X

ORCID for Andrew Steele: orcid.org/0000-0003-0667-1596

ORCID for Francesco Forconi: orcid.org/0000-0002-2211-1831

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