Signaling via the aberrantly activated B-cell receptor (BCR) has a critical role in the pathogenesis of B-cell tumors by promoting survival and clonal expansion of malignant B cells. 1, 2 Multiple preclinical studies indicate that blocking various components of the BCR signaling pathway holds a great therapeutic potential in the treatment of B-cell leukemias and lymphomas. 3, 4 This is further supported by clinical data from recent and ongoing clinical trials, which demonstrate considerable activity of SYK inhibitors (fostamatinib and GS-9973), 5 BTK inhibitors (ibrutinib, AVL-292, CC-292 and ONO-4059) 6, 7 and PI3Kδ inhibitor (CAL-101) 8 as single agents or in combination with other therapies in patients with B-cell tumors, for whom rituximab-based regimens have become a standard of care. However, tumor relapses occur in a significant percentage of patients treated even with the most effective modalities. Therefore, combinations of anti-CD20 monoclonal antibodies (mAbs) with targeted therapeutics inhibiting BCR signaling pathways have recently become an active area of investigation. For example, ibrutinib and CAL-101 are currently being investigated in combination with chemotherapy and/or anti-CD20 mAbs (rituximab and ofatumumab) in several phase I, II and III clinical trials (summarized in Kharfan-Dabaja et al. 9). Conspicuously, the antitumor effects of these combinations have not been studied in the preclinical setting. Therefore, we decided to investigate the molecular interactions between anti-CD20 mAbs and R406 (active metabolite of fostamatinib), ibrutinib and CAL-101.

Startlingly, we observed a significant impairment of rituximab-(R-CDC, Figure 1a) and ofatumumab-induced complement-dependent cytotoxicity (O-CDC)(Supplementary Figure 1a) in Raji cells pre-incubated for 48h with increasing concentrations of R406, ibrutinib or CAL-101, when compared with controls. The highest tested concentration (1μM) of R406 and ibrutinib almost completely abrogated both R-CDC and O-CDC, whereas CAL-101 affected anti-CD20 mAb-mediated CDC to a lesser extent. Flow cytometry studies further revealed that pre-incubation of Raji cells with BCR pathway inhibitors severely impaired the binding of various anti-CD20 mAbs (fluorescein isothiocyanate-conjugated L27 clone (Figure 1b), rituximab and ofatumumab (Figure 1c)). All three compounds induced a downregulation of surface CD20 levels in a panel of 26 primary chronic lymphocytic leukemia (CLL) samples pre-incubated for 48h with 1μM of each BCR signaling inhibitor (Figure 1d). Since ibrutinib has been recently approved as a secondline monotherapy for patients with mantle cell lymphoma (MCL), we