Inhibitors Of B-Cell Receptor Molecules Affect Surface CD20 and Impair Antitumor Activity Of Anti-CD20 Monoclonal Antibodies
Authors: Bojarczuk Kamil , Winiarska Magdalena , Bobrowicz Malgorzata , Dwojak Michal , Miazek Nina , Zapala Piotr , Efremov Dimitar G , Golab Jakub 
Published: October 2019

Abstract

Background
Anti-CD20 monoclonal antibodies (mAbs) are widely used in the treatment of non-Hodgkin's lymphomas (NHL) and chronic lymphocytic leukemia (CLL). Combining new agents with already used anti-CD20 mAbs seems to be a reasonable approach to further improve current therapeutic options. It seems that signaling via the aberrantly activated B-cell receptor (BCR) plays a key role in the pathogenesis of certain types of B-cell tumors. Blocking BCR pro-survival pathway holds a great therapeutic potential in both NHL and CLL. Several trials are currently being conducted to investigate the effects of combination of BCR-targeting agents with anti-CD20 mAbs–based therapies. To improve these therapeutic approaches in a planned manner it will be utterly important to decipher actual mechanisms of interactions between BCR-targeted therapies and anti-CD20 mAbs in established in vitro models.

Aims
The aim of this study is to elucidate the role of BCR signaling pathways in the regulation of CD20 levels in B-cell-derived tumor cells and antitumor activity of anti-CD20 mAbs.

Methods
The project is undertaken fully in in vitro settings in the models of human lymphoma cells as well as primary cells from patients with B-cell tumors. Cells are pre-incubated for 48h with inhibitors of BCR signaling (SYK, BTK, PI3K, AKT, PLC-γ, PKC, mTOR, ERK 1/2) and subsequently tested using flow cytometry for their susceptibility to antitumor activity of anti-CD20 mAbs. Membrane level of CD20 antigen is assessed with FITC-conjugated anti-CD20 antibody staining, total level of CD20 protein is assessed in Western blotting. Transcription processes are analyzed with qPCR, ChIP and EMSA. Moreover, stably transduced lymphoma cells with silenced or constitutively active proteins of interest are employed.

Results
The results of our preliminary experiments show that blocking BCR network at many stages of the signaling cascade with specific chemical inhibitors or selective shRNA-mediated silencing of SYK or BTK results in considerable down-regulation of CD20 level as determined with flow cytometry. Moreover, a 48-hour incubation with BCR inhibitors leads to a substantial impairment of antitumor activity of anti-CD20 mAbs. Selected inhibitors of BCR signaling considerably decrease CD20 protein level in total cellular lysates as analyzed using Western blotting. In Raji cells incubated with selected BCR inhibitors quantitative real-time PCR shows a significant decrease in CD20 mRNA level. Noteworthy, washout experiments showed that surface CD20 reaches level of control after 96 h from the time that inhibitors were eliminated from the culture media. Studies performed on cell line expressing constitutively active AKT showed up-regulation of CD20 levels at both levels of protein and mRNA. Moreover, constitutively active AKT protects cells from BCR inhibitors-induced decrease of surface CD20.

Summary/conclusions
Blocking BCR complex network on nearly every step of signal initiation and propagation considerably down-regulates CD20 levels what might have extremely important consequences for the anti-cancer therapy that is based on the use of anti-CD20 mAbs. These studies should provide us with extensive knowledge on the biology of CD20 protein and pathways involved in CD20 regulation.
In light of our recent experiments therapeutic combinations of BCR inhibitors and anti-CD20 mAbs-based modalities should be rationally and consciously introduced into clinic in optimized therapeutic schemes. We hypothesize that results of our experiments may lead to identification of the most beneficial therapeutic modalities and schedules that would improve the quality of life of patients suffering from B-cells originating tumors.

Disclosures:
No relevant conflicts of interest to declare.