Abstract B008: NFY-mediated regulation of CD20 is targeted by novel salinomycin derivatives to potentiate anti-CD20 immunotherapy

Abstract Background: Our team previously demonstrated that salinomycin (SAL) robustly upregulates CD20 antigen levels on the surface of malignant B cells (Torun et al., 2025, Haematologica). Both monoclonal antibodies (mAbs) and cellular therapies target CD20 to eliminate malignant B cells. Therefore, the upregulation of CD20 would further enhance the therapeutic efficacy of monoclonal antibodies (mAbs) and CAR-T cells. In the current study, we selected several SAL derivatives based on their CD20-upregulating activity and tested their ability to enhance immunotherapy in vitro and in preclinical animal models. Material: B-cell-derived malignant cells, SCID and NSG mice, and a library of SAL derivatives. Methods: Flow cytometry, anti-CD20 CAR-T generation, complement- and NK cell-dependent cytotoxicity assays, RNA-seq, RT-PCR, Western blotting, and CRISPR/Cas9 gene editing. Results: Using flow cytometry screening, we selected several SAL derivatives with modifications in chemical structure (positions C1 and C20) that induced CD20 upregulation more efficiently than the original SAL. The in vitro experiments confirmed that these SAL derivatives were more efficient than SAL in inducing the killing of malignant B cells via the action of anti-CD20 therapeutic mAbs (complement- and NK cell-dependent cytotoxicity assays, CDC and ADCC, respectively) and CAR-T cells. Of note, the application of selected SAL derivatives in combination with the anti-CD20 therapeutic mAb, Rituximab, significantly extended the life of SCID mice with Burkitt’s lymphoma. To characterize the critical molecular events mediating the enhanced CD20 upregulation, we analyzed the RNA-seq data. In addition to the suppression of c-Myc and FOXO (previously reported by us in Torun et al., 2025, Haematologica), we found that the regulation of NFY-A/B/C transcription factors also plays a key role. CRISPR/Cas9-mediated knockout of NFY factors led to robust upregulation of CD20, indicating that NFY factors are significant negative regulators of this antigen. Conclusions: Our findings from in vitro assays and preclinical animal models provide a rationale for the clinical evaluation of SAL derivatives to improve treatment outcomes in patients with B-cell-derived malignancies. Funding: National Science Centre (NCN, Poland; 2019/35/B/NZ5/01445 to BP, 2020/39/B/NZ6/03513 to AZ), Medical Research Agency (ABM, Poland; 2024/ABM/03/KPO/KPOD.07.07-IW.07-0218/24) under the National Recovery and Resilience Plan (KPO), and Polish National Agency For Academic Exchange (NAWA; BPI/PST/2024/1/00110 to BP and AZ). Citation Format: Aleksandra Zdanowicz, Bhaskar Pradhan, Marta Jedrzejczyk, Adam Huczynski, Abdessamad Zerrouqi, Beata Pyrzynska. NFY-mediated regulation of CD20 is targeted by novel salinomycin derivatives to potentiate anti-CD20 immunotherapy [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B008.