Genome-wide CRISPR screening identifies CRL1 as a therapeutic target for restoring CD19 expression and improving immunotherapy efficacy in  B-cell malignancies

Abstract Background: Antigen escape through CD19 downregulation is one of the key resistance mechanisms to CD19-targeted immunotherapies such as chimeric antigen receptor (CAR) T cells. While genetic alterations in CD19 are rare, relapsed patients frequently exhibit a loss of CD19 surface expression despite stable mRNA levels, pointing to post-transcriptional or post-translational regulation. However, the mechanisms that govern CD19 protein stability, intracellular trafficking, and degradation remain poorly defined. Aims: To identify molecular regulators of CD19 protein stability and to test therapeutic strategies to restore CD19 surface expression and immunotherapy based target cell killing in B-cell malignancies. Methods:We performed a genome-wide CRISPR/Cas9 knockout screen in CD19⁺ Ramos cells cocultured with primary CD19 CAR-T cells at a low E:T ratio that preserves most B cells, while resulting in their CD19 loss. We then sorted out B cells retaining CD19 expression under this immune pressure and detected their genes knocked out using NGS. Among the top hits, we identified Cullin 1 (CUL1), a scaffold component of the CRL1 (Cullin RING Ligase 1) E3 ubiquitin ligase complex, which is responsible for targeting numerous proteins for ubiquitin mediated degradation. CUL1 was validated with individual sgRNAs across independent biological replicates. Pharmacologic inhibition of the CRL1 complex was achieved using pevonedistat, a clinical-grade inhibitor of the NEDD8 activating enzyme (NAE), which inactivates Cullin RING ligases by blocking neddylation. CD19 expression was assessed using both surface and intracellular flow cytometry. Immune cytotoxicity was quantified using flow based assays in cocultures with CAR-T cells, CAR-NK cells, and CD16⁺ NK effectors combined with the therapeutic CD19 monoclonal antibody Tafasitamab for antibody dependent cellular cytotoxicity (ADCC). Experiments were extended to multiple B cell malignancy models (Ramos, HG3, MEC1, Raji) and primary chronic lymphocytic leukemia (CLL) patient samples. Results: CRISPR screening revealed CUL1 as one of the key regulators of CD19 surface expression upon CAR-T cell mediated pressure. CUL1 knockout resulted in a significant upregulation of CD19 at both the surface and total protein levels, without any significant changes in mRNA expression. Treatment with pevonedistat mimicked the CUL1 knockout, significantly enhancing CD19 expression across various B cell lines. Functional assays with pevonedistat treated B cells exhibited increased susceptibility to immune killing by CAR-T, CAR-NK, and Tafasitamab mediated ADCC compared to CAR-T or CAR-NK alone. Notably, pevonedistat enhanced immune mediated cytotoxicity also in primary CLL samples using both primary T cells engineered with CD19 CAR and CD19CAR-NK cells, underscoring the therapeutic relevance of this approach. Across models, CD19 mRNA levels remained unchanged, providing strong support for post-translational regulation via the CRL1 axis. Conclusions: Our study identifies CRL1 as a novel post-translational regulator of CD19 protein stability, with implications for immune escape in B cell malignancies. Pharmacologic inhibition of CRL1 through pevonedistat stabilises CD19 and significantly enhances immune cytotoxicity mediated by CAR-T cells, CAR-NK cells, and antibody dependent effector mechanisms. These findings pave the way to overcome antigen escape and enhance CD19 targeted immunotherapies in resistant or relapsed B-cell leukemias and lymphomas. This research was supported by the grantfrom Czech Science Foundation (project no. 22-35273S), project MUNI/A/1685/2024 and project NICR (EU program EXCELES, No. LX22NPO5102).