Abstract
We recently demonstrated that obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab compared to rituximab (RTX) mediates significantly enhanced antibody-dependent cell cytotoxicity (ADCC) in vitro and increased overall survival in a Burkitt lymphoma (BL) xenograft non-obese diabetic severe combined immunodeficiency gamma (NSG) model. In this study we compared the phosphoproteomic changes by pathway analysis following obinutuzumab vs RTX against RTX-sensitive (Raji) and -resistant BL (Raji4RH). Phosphoproteomic analyses were performed by mass-spectrometry (MS)-based label-free quantitative phosphoproteomic profiling. We demonstrated that 418 proteins in Raji and 377 proteins in Raji 4RH, were differentially phosphorylated (>1.5-fold) after obinutuzumab vs. RTX. Proteins that were significantly differentially phosphorylated included the B cell antigen receptor (BCR) (PLCG2, BTK and GSK3B), Fc gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF1), and natural killer cell-mediated cytotoxicity (MAPK1, RAF1, PLCG2 and MAPK3) signaling pathways. Differential phosphorylation of BCR or cytotoxicity pathway proteins revealed significant up-regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab compared to RTX. Silencing of PLCG2 in the BCR and MAPK1 in the cytotoxicity pathway significantly increased BL proliferation and decreased BL cytotoxicity after obinutuzumab compared to RTX. These results in combination with our previous results demonstrating a significant improvement in in vitro BL cytotoxicity and in vivo BL survival by obinutuzumab compared to RTX may in part be due to differential effects on selected BL protein signaling pathways.
Highlights
Non-Hodgkin lymphoma (NHL) is the seventh most common cause of cancer in the United States [1]
We recently demonstrated that obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab compared to rituximab (RTX) mediates significantly enhanced antibodydependent cell cytotoxicity (ADCC) in vitro and increased overall survival in a Burkitt lymphoma (BL) xenograft non-obese diabetic severe combined immunodeficiency gamma (NSG) model
We demonstrated that the knockdown of B cell antigen receptor (BCR) signaling pathway protein, PLCG2, significantly enhanced BL proliferation and reduced cell death after obinutuzumab compared to RTX
Summary
Non-Hodgkin lymphoma (NHL) is the seventh most common cause of cancer in the United States [1]. CD20 has been demonstrated to be expressed in ≥ 98% of mature B-cell lymphomas in children and adolescents including BL/ leukemia and approximately 35-45% of pediatric precursor B-cell acute lymphoblastic leukemia (pre-B-ALL) [5, 6]. Based on their mechanism of action, anti-CD20 monoclonal antibodies (mAbs) may be divided into two major sub-types, type-I and type-II. Both type-I and type-II anti-CD20 antibodies can be distinguished by their ability to redistribute CD20 into lipid rafts within the cell membrane [7, 8]
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