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BTK-independent regulation of calcium signalling downstream of the B-cell receptor in malignant B-cells

BTK-independent regulation of calcium signalling downstream of the B-cell receptor in malignant B-cells
BTK-independent regulation of calcium signalling downstream of the B-cell receptor in malignant B-cells
BTK inhibitors (BTKi) have dramatically improved outcomes for patients with chronic lymphocytic leukaemia (CLL) and some forms of B-cell lymphoma. However, new strategies are needed to enhance responses. Here we have performed a detailed analysis of the effects of BTKi on B-cell receptor (BCR)-induced signalling using primary malignant cells from CLL patients and B-lymphoma cell lines. Although BTK is considered as a key activator of PLCγ2, BTKi (ibrutinib and acalabrutinib) failed to fully inhibit calcium responses in CLL samples with strong BCR signalling capacity. This BTKi-resistant calcium signalling was sufficient to engage downstream calcium-dependent transcription and suppress CLL cell apoptosis and was entirely independent of BTK and not just its kinase activity as similar results were obtained using a BTK-degrading PROTAC. BTK-independent calcium signalling was also observed in two B-lymphoma cell lines where BTKi had little effect on the initial phase of the calcium response but did accelerate the subsequent decline in intracellular calcium. In contrast to BTKi, calcium responses were completely blocked by inhibition of SYK in CLL and lymphoma cells. Engagement of BTK-independent calcium responses was associated with BTK-independent phosphorylation of PLCγ2 on Y753 and Y759 in both CLL and lymphoma cells. Moreover, in CLL samples, inhibition of RAC, which can mediate BTK-independent activation of PLCγ2, cooperated with ibrutinib to suppress calcium responses. BTK-independent calcium signalling may limit the effectiveness of BTKi to suppress BCR signalling responses and our results suggest inhibition of SYK or dual inhibition of BTK and RAC as alternative strategies to strengthen pathway blockade.
B-cell receptor, BTK, Calcium, Chronic lymphocytic leukaemia, Lymphoma
0898-6568
Arthur, Rachael Louise
b595bf5a-7c08-426e-9992-8b12c5e95817
Wathen, Alexander
5de40d6d-7462-4b67-be63-8c2114bc2420
Lemm, Elizabeth
f363493b-47ce-4f8e-9e94-272486397cb3
Stevenson, Freda
ba803747-c0ac-409f-a9c2-b61fde009f8c
Forconi, Francesco
ce9ed873-58cf-4876-bf3a-9ba1d163edc8
Linley, Adam J
52b02551-8e9a-4d09-9ebb-99853b970d92
Steele, Andrew
4349f6aa-2e3a-49a8-be73-7716056ae089
Packham, Graham
fdabe56f-2c58-469c-aadf-38878f233394
Valle-argos, Beatriz
4fddaa71-c0aa-4b95-b464-8bdb592428a2
Arthur, Rachael Louise
b595bf5a-7c08-426e-9992-8b12c5e95817
Wathen, Alexander
5de40d6d-7462-4b67-be63-8c2114bc2420
Lemm, Elizabeth
f363493b-47ce-4f8e-9e94-272486397cb3
Stevenson, Freda
ba803747-c0ac-409f-a9c2-b61fde009f8c
Forconi, Francesco
ce9ed873-58cf-4876-bf3a-9ba1d163edc8
Linley, Adam J
52b02551-8e9a-4d09-9ebb-99853b970d92
Steele, Andrew
4349f6aa-2e3a-49a8-be73-7716056ae089
Packham, Graham
fdabe56f-2c58-469c-aadf-38878f233394
Valle-argos, Beatriz
4fddaa71-c0aa-4b95-b464-8bdb592428a2

Arthur, Rachael Louise, Wathen, Alexander, Lemm, Elizabeth, Stevenson, Freda, Forconi, Francesco, Linley, Adam J, Steele, Andrew, Packham, Graham and Valle-argos, Beatriz (2022) BTK-independent regulation of calcium signalling downstream of the B-cell receptor in malignant B-cells. Cellular Signalling, 96, [110358]. (doi:10.1016/j.cellsig.2022.110358).

Record type: Article

Abstract

BTK inhibitors (BTKi) have dramatically improved outcomes for patients with chronic lymphocytic leukaemia (CLL) and some forms of B-cell lymphoma. However, new strategies are needed to enhance responses. Here we have performed a detailed analysis of the effects of BTKi on B-cell receptor (BCR)-induced signalling using primary malignant cells from CLL patients and B-lymphoma cell lines. Although BTK is considered as a key activator of PLCγ2, BTKi (ibrutinib and acalabrutinib) failed to fully inhibit calcium responses in CLL samples with strong BCR signalling capacity. This BTKi-resistant calcium signalling was sufficient to engage downstream calcium-dependent transcription and suppress CLL cell apoptosis and was entirely independent of BTK and not just its kinase activity as similar results were obtained using a BTK-degrading PROTAC. BTK-independent calcium signalling was also observed in two B-lymphoma cell lines where BTKi had little effect on the initial phase of the calcium response but did accelerate the subsequent decline in intracellular calcium. In contrast to BTKi, calcium responses were completely blocked by inhibition of SYK in CLL and lymphoma cells. Engagement of BTK-independent calcium responses was associated with BTK-independent phosphorylation of PLCγ2 on Y753 and Y759 in both CLL and lymphoma cells. Moreover, in CLL samples, inhibition of RAC, which can mediate BTK-independent activation of PLCγ2, cooperated with ibrutinib to suppress calcium responses. BTK-independent calcium signalling may limit the effectiveness of BTKi to suppress BCR signalling responses and our results suggest inhibition of SYK or dual inhibition of BTK and RAC as alternative strategies to strengthen pathway blockade.

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ARTHUR ET AL main text v2 CLEAR - Accepted Manuscript
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Accepted/In Press date: 14 May 2022
e-pub ahead of print date: 18 May 2022
Published date: August 2022
Additional Information: Funding Information: This work was supported by the John Goldman Fellowship for Future Science from Leukaemia UK (Grant ID: 153896 ) and grants from the University of Southampton , Cancer Research UK ( C2750/A23669 , C42023/A29370 ) and the Southampton Experimental Cancer Medicine and Cancer Research Centres ( C24563/A15581 , C34999/A18087 ). Publisher Copyright: © 2022
Keywords: B-cell receptor, BTK, Calcium, Chronic lymphocytic leukaemia, Lymphoma
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Identifiers

Local EPrints ID: 468989
URI: http://eprints.soton.ac.uk/id/eprint/468989
DOI: doi:10.1016/j.cellsig.2022.110358
ISSN: 0898-6568
PURE UUID: 238a1539-c331-426d-afb3-fa7a1a5753c7
ORCID for Freda Stevenson: ORCID iD orcid.org/0000-0002-0933-5021
ORCID for Francesco Forconi: ORCID iD orcid.org/0000-0002-2211-1831
ORCID for Andrew Steele: ORCID iD orcid.org/0000-0003-0667-1596
ORCID for Graham Packham: ORCID iD orcid.org/0000-0002-9232-5691

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Date deposited: 02 Sep 2022 19:10
Last modified: 17 Mar 2024 07:26

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Contributors

Author: Rachael Louise Arthur
Author: Alexander Wathen
Author: Elizabeth Lemm
Author: Freda Stevenson ORCID iD
Author: Francesco Forconi ORCID iD
Author: Adam J Linley
Author: Andrew Steele ORCID iD
Author: Graham Packham ORCID iD
Author: Beatriz Valle-argos

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