1001 - Identification of novel targets that upregulate CD20 expression in rituximab-resistant cells with the use of genome-wide CRISPR screening

Background: CD20 antigen has been used as a target of monoclonal antibodies (mAb) such as rituximab (RTX) in the therapy of B-cell malignancies for more than two decades. However, CLL B cells tend to express lower levels of CD20 on their surface or downregulate it following the mAb treatment, resulting in mAb resistance and subsequently therapy failure. Therefore, it is crucial to investigate the regulation of CD20 in order to restore sensitivity to anti-CD20 mAb and enhance its efficacy. The aim of this project was to perform genome-wide CRISPR/Cas9 knockout screening to identify genes whose disruption upregulates CD20 surface expression in the resistant cells with low CD20 levels.

Methods: To create a model mimicking the situation in patients who had developed resistance to mAb therapy, we generated an RTX-resistant CD20-low B-lymphoid cell line by chronic exposure to increasing concentrations of rituximab. These cells were then transduced by the GeCKO (Genome-Scale CRISPR Knock-Out) lentiviral library to obtain a collection of single-gene knockouts. Following a 2.5-week cultivation period allowing the full manifestation of gene knockouts, the top 5% of cells with the highest surface expression of CD20 were sorted out. Using next-generation sequencing and the MAGeCK algorithm, we identified gene knockouts responsible for CD20 upregulation. The most interesting of these genes were subsequently validated using newly designed gRNAs, and the CD20 expression was assessed using flow cytometry and western blot. To determine the response to the anti-CD20 mAb, cells were incubated with rituximab in the presence of human serum for 24 hours and the viability was measured using the CellTiter-Glo assay.

Results: Our CRISPR/Cas9 knockout screening revealed several interesting genes whose disruption led to the increase in CD20 surface expression. As the top hit we identified gene CSK, encoding a negative regulator of Src kinases. CSK is a key kinase in the activation of the B-cell receptor (BCR) pathway - one of the most important pathways in B cells. Another significantly enriched gene involved in BCR signalling was PTEN, a well-known tumour suppressor. Interestingly, among the top ten hits we identified four genes (SSR1, SSR2, SSR3, SSR4) encoding all four subunits of the translocon-associated protein (TRAP) complex, endoplasmic reticular complex involved in protein translocation across ER membrane and other ER processes. The presence of STT3A among the top hits suggests the involvement of another ER complex, oligosaccharyltransferase (OST). OST consists of two subunits encoded by genes STT3A and STT3B and is involved in glycosylation. These results indicate that both BCR signalling and ER play an important role in CD20 regulation. The last gene selected for validation was CD37. It encodes a surface protein, expressed predominantly in B cells, which is involved in immune response, but its function has not been fully described. We indeed observed the upregulation of CD20 upon CD37 disruption. But on the contrary, the loss of CD20 surprisingly led to a decrease in CD37 expression. This suggests the presence of a regulatory loop between these two genes.
We generated knockout cell lines for genes CSK, SSR1 and CD37 in our RTX-resistant cells and successfully validated the results from the screening in these cell lines. In each cell line, we observed an increase in both surface and total levels of CD20 compared to wild-type cells. Moreover, in all three cases, cells restored their sensitivity to rituximab. The validation of other selected hits is currently ongoing.

Conclusion: In summary, we have identified several promising genes involved in CD20 regulation and whose disruption may be used to upregulate CD20 expression. The thorough validation and investigation of underlying mechanisms could provide a way for a potential enhancement of anti-CD20 mAb therapy.