Background. Lenalidomide has clinical activity in chronic lymphocytic leukemia (CLL) patients exerting pleiotropic activity on the immune system. The combination of lenalidomide with the anti-CD20 monoclonal antibody ofatumumab induces durable responses in patients with relapsed/refractory CLL. In addition to cell- and complement-mediated B-cell depletion induced by anti-CD20 antibodies and to immunomodulatory activity of lenalidomide, both drugs have been shown to directly inhibit survival and proliferation of malignant B cells. Signal transduction mediated by the B-cell receptor (BCR) promotes CLL survival and proliferation and is considered a key determinant of CLL clinical behavior and target for therapeutic interventions. However, the hypothesis that ofatumumab and lenalidomide could target BCR signal transduction has never been examined in CLL. Aims. In this study, we used phospho-specific flow cytometry to investigate the direct effects of lenalidomide and ofatumumab on BCR signaling in CLL cells from different prognostic groups of treatment-naive patients. Methods. The phosphorylation levels of four proteins downstream of the BCR signaling, namely SYK, ERK1/2, PLC-γ, and NF-κB p65, were analyzed at the single-cell level in 9 CLL cell samples treated in vitro with single-agent ofatumumab, single-agent lenalidomide or their combination, using phospho-specific flow cytometry. Phosphorylation was measured in the basal condition and following BCR stimulation with anti-IgM, resulting in overall 576 signal readouts. The repeated measures Anova with Dunnett's multiple comparison test was used to compare drug effects on protein phosphorylation. Results. Ofatumumab and lenalidomide induced different effects in ex-vivo CLL cells with different prognostic features as defined by the IGHV mutational status. In the IGHV-mutated (M) subset, treatment with ofatumumab, alone or in combination with lenalidomide, induced a significant reduction of SYK and ERK1/2 basal phosphorylation. Remarkably, in the same CLL group, lenalidomide significantly inhibited ERK1/2 basal phosphorylation. On the contrary, in the patient group defined by unmutated IGHV status (UM), treatment with ofatumumab and lenalidomide induced no significant changes in basal phosphorylation of BCR proteins. As expected, BCR stimulation with anti-IgM antibodies induced a signaling response in CLL cells that was statistically significant for ERK1/2, PLC-γ, and NF-κB p65. In the M subset, treatment with ofatumumab, alone or in combination with lenalidomide, significantly inhibited the anti-IgM signaling response of SYK and ERK1/2. Interestingly, lenalidomide significantly reduced the anti-IgM-induced signaling response of SYK. In contrast, in the UM CLL subset, ofatumumab and lenalidomide induced no significant changes in anti-IgM responses of BCR proteins. Conclusions. This study suggests that ofatumumab and lenalidomide can influence the BCR signaling in the basal state as well as under stimulation, with differential effects in distinct prognostic subsets of CLL. Although a study on a larger patient set is needed to extend and confirm these results, overall they indicate that ofatumumab and lenalidomide can inhibit BCR signaling in the M subset of CLL whilst have no significant effect in UM CLL, potentially contributing to the capacity of these drugs to differentially inhibit disease-progression in patients with CLL.
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