CD20 is a cell surface glycoprotein, encoded by the MS4A1 gene, which is highly expressed on most B-cells. Although CD20 is suggested to be involved in calcium signaling, its exact function is unknown. The monoclonal antibody rituximab, which targets CD20, has revolutionized the treatment of B-cell lymphoma. Despite the widespread use of CD20 antibodies across lymphoma subtypes, little is known about the endogenous regulation of CD20. The tight correlation between CD20 expression and responsiveness to CD20 antibody suggests that increasing CD20 expression levels could improve rituximab sensitivity. This could be particularly useful for B-cell lymphoma with low CD20 surface expression (e.g. chronic lymphocytic leukemia).
The main goal of the project is to dissect and understand the CD20 regulation using an unbiased RNAi approach and to exploit this knowledge to improve the efficacy of anti-CD20 therapy.
In order to discover novel regulators of CD20 expression, we performed a genome-wide RNAi screen. The shRNA library targets over 5000 mRNAs, with 5 to 6 shRNAs per target. The CD20 regulatome was studied in a Burkitt lymphoma cell line (Raji) characterized by medium expression of CD20.
MS4A1 was the most significant screen hit that decreased CD20 surface levels with three shRNAs and served as positive control. Based on stringent selection criteria, we identified hits involved in diverse molecular mechanisms such as signaling, trafficking, chromatin remodeling and cell membrane composition. In total, 21 genes targeted with two non-overlapping shRNAs were selected for validation: 12 decreasing and 9 increasing CD20 levels. For every gene, two shRNAs were synthesized and cloned into shRNA expression vectors. Every shRNA was investigated in separate experiments for its effect on CD20 expression levels on Raji cells. We were able to validate and confirm the influence of those genes on CD20 phenotype on Raji cells, as well as on other B-cell lymphoma cell lines (SuDHL5, BL2 and BL60). The group of genes decreasing CD20 expression can provide an insight into physiological regulation and the machinery involved in CD20 trafficking and processing. More relevant from a therapeutic viewpoint are genes where silencing increases the expression of CD20. In order to investigate a potential therapeutic synergy of our hits with Rituximab, we investigated complement-mediated lysis triggered by Rituximab in vitro after silencing of several hits. In this assay we observed direct correlation between CD20 surface levels with the sensitivity towards Rituximab for our hits: increased CD20 levels led to more Rituximab induced lysis (up to two fold increase), and vice versa. One of the main aims of the screen was the discovery of druggable targets that can be potentially used in combination therapy with Rituximab. Indeed, a chemical inhibitor targeting one of our screen hits was able to increase CD20 expression levels on Raji cells.
With an unbiased shRNA screen we identified potent regulators of CD20 expression levels. Our approach led to the discovery of novel druggable targets, which modulate CD20 levels. This may provide the rationale for novel combinatory therapies, which might improve the efficiency of anti-CD20 therapy in B-cell lymphoma.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.