Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, United Kingdom.
Department of Molecular Physiology and Cell Signalling, University of Liverpool, Institute of Systems, Molecular and Integrative Biology, 5(th) Floor Nuffield Building, Crown Street, Liverpool L69 3BX, United Kingdom.
Cell Signal. 2022 Aug;96:110358. doi: 10.1016/j.cellsig.2022.110358. Epub 2022 May 18.
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 Y and Y 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.
BTK 抑制剂（BTKi）显著改善了慢性淋巴细胞白血病（CLL）和某些 B 细胞淋巴瘤患者的预后。然而，需要新的策略来增强反应。在这里，我们使用来自 CLL 患者的恶性原代细胞和 B 细胞淋巴瘤细胞系，对 BTKi 对 B 细胞受体（BCR）诱导的信号转导的影响进行了详细分析。尽管 BTK 被认为是 PLCγ2 的关键激活剂，但 BTKi（依鲁替尼和阿卡替尼）未能完全抑制具有强 BCR 信号转导能力的 CLL 样本中的钙反应。这种 BTKi 抗性钙信号足以参与下游钙依赖性转录，并抑制 CLL 细胞凋亡，并且完全独立于 BTK，而不仅仅是其激酶活性，因为使用 BTK 降解 PROTAC 也获得了类似的结果。在两种 B 细胞淋巴瘤细胞系中也观察到 BTK 独立的钙信号，BTKi 对钙反应的初始阶段几乎没有影响，但确实加速了随后的细胞内钙下降。与 BTKi 相反，SYK 的抑制完全阻断了 CLL 和淋巴瘤细胞中的钙反应。BTK 独立的钙反应的参与与 CLL 和淋巴瘤细胞中 PLCγ2 的 Y 和 Y 上 BTK 独立的磷酸化有关。此外，在 CLL 样本中，抑制 RAC（可介导 BTK 独立激活 PLCγ2）与依鲁替尼合作抑制钙反应。BTK 独立的钙信号可能限制 BTKi 抑制 BCR 信号转导反应的有效性，我们的结果表明抑制 SYK 或 BTK 和 RAC 的双重抑制可能是增强途径阻断的替代策略。
