Abstract 1012: CD20 supports BCR signaling in an intra-clonal aggressive chronic lymphocytic leukemia subpopulation of cells and rituximab primarily targets these BCR-proficient B cells in vivo
Authors: Pavlasova Gabriela , Borsky Marek , Svobodova Veronika , Oppelt Jan , Cerna Katerina , Novotna Jitka , Musilova Katerina , Seda Vaclav , Vojackova Eva , Brychtova Yvona , Doubek Michael , Pospisilova Sarka , Mayer Jiri , Mraz Marek 
Published: August 2018

Abstract
The hallmark of chronic lymphocytic leukemia (CLL) cells is their re-circulation between peripheral blood and immune niches to obtain pro-proliferative and pro-survival signals. CLL cells that have recently exited the immune niches to the peripheral blood are characterized by low cell-surface levels of chemokine receptor CXCR4 and high levels of activation molecule CD5. These CXCR4dimCD5bright CLL cells have a ~2-fold higher CD20 expression due to the activation of the CXCR4/SDF-1 axis (Pavlasova et al., Blood, 2016). We hypothesized that CD20 up-regulation in the context of a microenvironment is required for some functional regulation. We hypothesized that CD20 expression is of importance for B-cell receptor (BCR) signaling as we observed that CXCR4dimCD5brightCD20bright CLL cells have also ~2-fold higher surface IgM levels (P&lt;0.0001). This was coupled with higher responsiveness to BCR-crosslinking with anti-IgM (P=0.0015). CD20 levels directly affect BCR-induced calcium flux and the phosphorylation of BCR/PI3K-associated molecules (LYN, SYK, ERK, GAB1) after BCR-crosslinking. The CXCR4dimCD5brightCD20bright subpopulation contains more proliferative (Ki67+) cells, higher levels of pAKT/pERK/pCD79a (P&lt;0.001), and their gene expression signature (NGS Illumina) is significantly enriched for genes involved in BCR and MAPK signaling, migration, and actin cytoskeleton organization (P&lt;0.0001). Finally, we have shown that rituximab primarily and potently eliminates the CXCR4dimCD5brightCD20bright CLL cells (P&lt;0.0001). Overall, rituximab was ~9-fold more efficient in eliminating CXCR4dimCD5brightCD20bright CLL cells than CXCR4brightCD5dimCD20dim cells (P=0.03) during FCR therapy in vivo. Altogether, we described that higher CD20 expression supports BCR signaling and contributes to the activated phenotype and aggressiveness of an intra-clonal subpopulation of CXCR4dimCD5brightCD20bright cells. This is a first mechanistic explanation of CD20 function in CLL cells. Additionally, it is tempting to speculate that rituximab's clinical success is at least partially attributed to the preferential elimination of the intra-clonal proliferative subpopulation of BCR-proficient CLL cells. Supported by: the Ministry of Education, Youth and Sports of the Czech Rep. under the project CEITEC 2020 (LQ1601); Czech Science Foundation (project No. 16-13334Y); the Ministry of Health of the Czech Rep., grant No. 16-29622A. All rights reserved. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 692298. This study reflects only the author's view and the Research Executive Agency is not responsible for any use that may be made of the information it contains. MH CZ-DRO (FNBr, 65269705); MUNI/H/0865/2016; MUNI/A/0968/2017.
Citation Format: Gabriela Pavlasova, Marek Borsky, Veronika Svobodova, Jan Oppelt, Katerina Cerna, Jitka Novotna, Katerina Musilova, Vaclav Seda, Eva Vojackova, Yvona Brychtova, Michael Doubek, Sarka Pospisilova, Jiri Mayer, Marek Mraz. CD20 supports BCR signaling in an intra-clonal aggressive chronic lymphocytic leukemia subpopulation of cells and rituximab primarily targets these BCR-proficient B cells in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1012.