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.

Preclinical Evaluation of a Novel SHIP1 Phosphatase Activator for Inhibition of PI3K Signaling in Malignant B Cells.

PURPOSE: PI3K signaling is a common feature of B-cell neoplasms, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL), and PI3K inhibitors have been introduced into the clinic. However, there remains a clear need to develop new strategies to target PI3K signaling. PI3K activity is countered by Src homology domain 2-containing inositol-5'-phosphatase 1 (SHIP1) and, here, we have characterized the activity of a novel SHIP1 activator, AQX-435, in preclinical models of B-cell malignancies. EXPERIMENTAL DESIGN: In vitro activity of AQX-435 was evaluated using primary CLL cells and DLBCL-derived cell lines. In vivo activity of AQX-435, alone or in combination with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, was assessed using DLBCL cell line and patient-derived xenograft models. RESULTS: Pharmacologic activation of SHIP1 using AQX-435 was sufficient to inhibit anti-IgM-induced PI3K-mediated signaling, including induction of AKT phosphorylation and MYC expression, without effects on upstream SYK phosphorylation. AQX-435 also cooperated with the BTK inhibitor ibrutinib to enhance inhibition of anti-IgM-induced AKT phosphorylation. AQX-435 induced caspase-dependent apoptosis of CLL cells preferentially as compared with normal B cells, and overcame in vitro survival-promoting effects of microenvironmental stimuli. Finally, AQX-435 reduced AKT phosphorylation and growth of DLBCL in vivo and cooperated with ibrutinib for tumor growth inhibition. CONCLUSIONS: Our results using AQX-435 demonstrate that SHIP1 activation may be an effective novel therapeutic strategy for treatment of B-cell neoplasms, alone or in combination with ibrutinib.