Anti-CD20 Monoclonal Antibodies Hijack the B-Cell Receptor Signaling Cascade Thereby Activating the NOTCH1 Signaling Pathway

NOTCH1 is a cell surface receptor, regulation of which depends on the integrity and subsequent cleavage of its inhibitory domain. Subtle mechanical forces transmitted after ligand-binding [Wang et al., 2013] or removal of Ca2+-ions [Rand et al., 2000] make the site accessible for cleavage, resulting in release of the transcription factor NICD1. Mutations in NOTCH1 that prolong NICD1 activity have been found in chronic lymphocytic leukemia (CLL) with enrichment in up to 30% of Richter transformation (RT). Clinical trials have revealed that NOTCH1 mutant CLL derives no benefit from the addition of type I anti-CD20 monoclonal antibodies (mAb) such as rituximab. This lack of benefit has not been observed with the type II anti-CD20 mAb obinutuzumab.To understand how anti-CD20 mAbs influence NOTCH1, we studied intracellular signaling events induced by rituximab or obinutuzumab and assessed their implications on NOTCH1 signaling.As a model for this, mass spectrometry-based phosphoproteomic analysis was applied to rituximab or obinutuzumab treated SU-DHL4 B-lymphoma cells (1h and 24h time-points) and >8500 phosphorylation sites measured relative to untreated controls. Activation of kinase pathways was inferred by kinase-substrate enrichment analysis (KSEA). NOTCH1 receptor activity was assessed by western blot for NICD1 and qPCR for HES1 expression (NICD1 target gene). We used rituximab F(ab')2 fragments, trastuzumab as an isotype control and SB2H2 to cross-link the IgG B-cell rec...
Source: Blood - Category: Hematology Authors: Tags: 641. CLL: Biology and Pathophysiology, excluding Therapy: Mechanisms of Action and Resistance to Targeted Agents Source Type: research