B-cell receptor signalling in malignant B cells: Analysis of BTK “by-pass” pathways and time-resolved, single-cell quantification of Ca2+ mobilisation

Abstract

The B-cell receptor (BCR) is the key functional unit of normal B cells and continues to exert an important influence post-transformation in various B-cell malignancies, including chronic lymphocytic leukaemia (CLL). BCR activation initiates formation of the signalosome, a multi-protein signalling complex which includes BTK, a kinase which is the target for drugs used to treat CLL and other B-cell malignancies, including ibrutinib and acalabrutinib. BTK phosphorylates PLCg2 leading to intracellular calcium (iCa2+) mobilisation and downstream pro-survival/proliferation responses. However, the ability of ibrutinib to interfere with anti-IgM-induced iCa2+ mobilisation in CLL cells varies between samples revealing the presence of alternate, BTK-independent signalling to iCa2+. Identification of this alternate BTK “by-pass” signalling pathway may allow the development of new strategies to more effectively block BCR signalling in malignant B cells. Flow cytometric analysis of iCa2+ release is widely used as a measure of BCR signalling capacity. Strong sIgM signalling capacity is associated with shorter survival and this assay may therefore have value as a biomarker to predict CLL patient outcomes. However, there are several limitations of flow cytometry, including the inability to study single-cells over time. This might mask intraclonal heterogeneity, whereby a potentially rare subset of cells might have ‘atypical’ signalling and/or inhibitor responses contributing to disease progression and/or treatment failure. Thus, new approaches to quantify iCa2+ release may improve the prognostic power of BCR signal analysis. This project had two main aims both focused on the critical BTKàPLCg2àiCa2+ signalling pathway. First, to identify the nature and importance of the BTK “by-pass” signal, and second, to determine whether the method of analysing iCa2+ mobilisation could be improved by using microwell arrays to study single-cell, time-resolved iCa2+ mobilisation. Analysis of primary CLL samples demonstrated that BTK by-pass signalling was a drug class-effect as it was observed with both ibrutinib and acalabrutinib. It was most evident in samples with the strongest anti-IgM signalling capacities suggesting that acquisition of BTK “by-pass” signalling is responsible for stronger iCa2+ responses per se. Analysis using multi-inhibitor beads and target phosphorylation demonstrated that BTK “by-pass” occurred despite full occupancy of the BTK active site by inhibitor, and use of a BTK-targeting PROTAC revealed that “by-pass” signalling was not due to kinase-independent functions of BTK. Mapping of PLCg2 phopshorylation sites demonstrated that retained anti-IgM-induced iCa2+ mobilisation was associated with BTK-independent activatory phosphorylation of PLCg2 at Tyr753 and Tyr759 in both CLL samples and Blymphoma cell lines. Since PLCg2 Tyr753 and Tyr759 can be phosphorylated by SYK and a SYK inhibitor fully blocked anti-IgM-induced iCa2+ responses in all samples, direct SYK-mediated phosphorylation of PLCg2 may be one potential pathway of BTK “by-pass” signalling. Microwell arrays consist of thousands of wells designed to trap single-cells and, coupled with fluorescent microscopy, have the potential to be used to analyse time-resolved iCa2+ mobilisation. Microwell size is crucial for optimal trapping of single cells, and the device was fabricated and optimised to trap a high percentage of single CLL cells. Responses to ionomycin could be measured in primary CLL cells, and there was a variable reponse to anti-IgM in some samples, validated by effective inhibition of these responses following kinase inhibitor pretreatment. Despite substantial technical challenges that remain to be addressed, including increasing the sensitivity of the assay, the arrays hold promise for more in-depth analysis of iCa2+ mobilisation. Overall, the results provide new insight into mechanisms of BTK “by-pass” signalling, and some evidence to tentatively support the hypothesis that microwell arrays may be an attractive approach to assess iCa2+ flux in malignant B cells.

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

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