Rituximab and Obinutuzumab Induce Direct B-Cell Death Via B-Cell Receptor (BCR) Signaling, but Rituximab Elicits Stronger BCR-Derived Pro-Survival Signals Diminishing Apoptosis
Authors: Edelmann Jennifer , Dokal Arran , Holzmann Karlheinz , Britton David James , Vilventhraraja Emma , Smith Ryan Jack , Cragg Mark S , Braun Andrejs , Döhner Hartmut , Cutillas Pedro , Gribben John G. 
Published: November 2019

The anti-CD20 monoclonal antibody (mAb) rituximab in combination with chemotherapy has improved outcomes for patients with CD20+ B-cell lymphoma. Obinutuzumab was developed as an anti-CD20 mAb with enhanced induction of direct B-cell death and antibody-dependent cellular cytotoxicity. In direct comparison to rituximab, improved outcomes with obinutuzumab have been observed for chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL), but not for diffuse large B-cell lymphoma (DLBCL). The molecular basis behind these discrepancies remained unknown.
Our aim was to define intracellular signaling events in the induction of direct B-cell death upon rituximab and obinutuzumab treatment.
We performed LC-MS/MS phosphoproteomics on SU-DHL4 lymphoma cells treated with rituximab or obinutuzumab (0, 1 and 24 h time-points). Kinase activities were inferred by kinase-substrate enrichment analysis linking kinases to their phosphosite substrates. Immunoblotting was done where necessary to understand the nature of specific signaling events.
The activity of 41 and 40 protein kinases was altered upon rituximab or obinutuzumab treatment, respectively, 32 of which were affected by both mAbs. Pathway enrichment analyses revealed up-regulated B-cell receptor (BCR) signaling and down-regulated cell cycle progression with both treatments. To delineate differences between the two mAbs we investigated signaling events in the BCR cascade in more detail.
The proximal BCR kinase SYK was strongly phosphorylated on Tyr352 by both mAbs, whereas SYK Tyr525/526 phosphorylation, essential for normal kinase function, was detectable at a low level only with rituximab. Phospho-SYK Tyr352 in the absence of phospho-SYK Tyr525/526 has been associated with autoimmune checkpoint activation and B-cell apoptosis.
Both mAbs phosphorylated the key BCR kinase BTK on Tyr551 but not on Tyr223. The latter site was essential for full kinase activity and, in line with incomplete BTK activation, downstream PCLγ2 phosphorylation was delayed. No evidence was found for PLCγ2 dependent de-phosphorylation of NFAT2/NFAT3 and PKCβ activation. Instead, we observed NFAT1 de-phosphorylation and PKCδ activation, both of which have been associated with an anergic B-cell reaction.
The MAPK pathway and MYK were strongly activated by both mAbs serving as potent inducers of cell death in the absence of pro-survival signals. To determine if pro-survival signals were generated, we assessed PI3K and NF-κB activation. While we found no evidence for NF-κB activation, differences in the phosphorylation status of PI3K binding sites on CD19 and BCAP suggest stronger PI3K activation by rituximab than obinutuzumab. In keeping with this, the PI3K effector AKT was much more strongly activated by rituximab. AKT activity was likely also increased by Ca2+-flux following rituximab but not obinutuzumab treatment and modified by differential SHIP1 phosphorylation, a phosphatase that hydrolyzes PI3K-generated PIP3.
A central AKT target in apoptosis regulation is BAD. We found increased BAD Ser99 and Ser118 phosphorylation only after rituximab treatment. Rituximab thereby sequestered BAD in the cytosol and impaired its inhibitory effects on anti-apoptotic BCL-2 and BCL-xL. Moreover, signaling events protecting against caspase 2 activation were exclusively found for rituximab.
In summary, here we show that rituximab and obinutuzumab both induce BCR signaling capable of promoting B-cell apoptosis. However, rituximab generates signals that increase anti-apoptotic BCL-2 effects and diminish B-cell death, whereas obinutuzumab more readily overcomes resistance through BCL-2 overexpression characteristic for FL and CLL. The differences found between rituximab and obinutuzumab are likely less pronounced in cases with strong tonic BCR signaling, which is more prevalent in DLBCL. Thus, our findings provide a mechanistic rationale for the observations made in clinical trials comparing rituximab and obinutuzumab head-to-head. We believe that our results will help to identify new patient subgroups benefitting from obinutuzumab and to better assess the role of anti-CD20 treatment in combination therapies.

Disclosures
Vilventhraraja: Janssen Pharmaceutical Companies: Employment. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Gribben:Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.