Abstract LB206: Screening with a library of salinomycin derivatives identifies strong upregulators of CD20 antigen in malignant B cells

Abstract Background: Our previous data revealed that K+/Na+ cation carriers, including salinomycin, robustly upregulate CD20 antigen levels on the surface of malignant B cells via suppression of c-Myc and FOXO, two negative transcriptional regulators of CD20 expression (Torun et al., 2024 Haematologica). The therapeutic efficacy of anti-CD20 monoclonal antibodies (mAbs) against malignant B cells depends, among other factors, on the levels of CD20 expression. Therefore, treatment with cation carriers, leading to CD20 upregulation, translates to more efficient recognition and elimination of B cell-derived tumor cells upon treatment with immunotherapy, such as anti-CD20 mAbs and CAR-T cells. Due to the broad interest in the anticancer properties of salinomycin (SAL), numerous derivatives of SAL have been synthesized, characterized by minimal systemic toxicity and enhanced specificity toward cancer and cancer stem cells. Using a library of SAL derivatives, we have conducted a screening to evaluate the efficiency of these new compounds in upregulating the CD20 antigen. Material and methods: Burkitt lymphoma cell lines and primary chronic lymphocytic leukemia (CLL) samples. Methods: Flow cytometry, complement- and NK cell-dependent cytotoxicity assays, and animal studies. Results: A library of 40 salinomycin derivatives with modifications in chemical structure (positions C1 and C20) of the original SAL has been generated. Raji cells (Burkitt lymphoma cell line) were treated with these compounds for 48h, followed by an estimation of the surface level of CD20 antigen by flow cytometry. A dozen SAL derivatives were more efficient in CD20 upregulation than SAL, with some of them exhibiting maximum upregulation at concentrations 5-10 times lower than the original SAL. Interestingly, the other dozen SAL derivatives exhibited no ability to upregulate CD20 expression, which was consistent with their failure to downregulate c-Myc. Some SAL derivatives with enhanced CD20 upregulation activity were selected as prospective therapeutic compounds for further studies. These compounds kept the ability of the original SAL to enhance the cytotoxic activity of NK cells. They appeared more efficient than SAL in complement-dependent cytotoxicity (CDC) assays and in raising the CD20 levels on the primary CLL samples. Encouraged by the positive in vitro data, we initiated the animal studies and the characterization of the critical molecular events mediating the enhanced CD20 upregulation. Conclusions: We characterized a library of SAL derivatives in the context of their ability to induce CD20 antigen upregulation and found a dozen more efficient regulators. This discovery provides a rationale for the clinical evaluation of SAL derivatives to obtain better therapeutic responses in patients with B cell-derived malignancies. Funding: National Science Centre (NCN, Poland; grants no 2019/35/B/NZ5/01445 to BP and 2020/39/B/NZ6/03513 to AZ). Citation Format: Beata Pyrzynska, Aleksandra Zdanowicz, Bhaskar Pradhan, Marta Jedrzejczyk, Dimitar G. Efremov, Adam Huczyński, Abdessamad Zerrouqi. Screening with a library of salinomycin derivatives identifies strong upregulators of CD20 antigen in malignant B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB206.