Salinomycin expands the cytotoxicity of both anti-CD20 monoclonal antibodies and natural killer cells towards non-Hodgkin lymphomas

The primary hurdle in non-Hodgkin lymphoma (NHL) treatment is the resistance to the recommended R-CHOP regimen which is composed of anti-CD20-targeted immunotherapy, Rituximab or RTX, and multiple chemotherapeutic drugs: cyclophosphamide, doxorubicin, vincristine and prednisone. In many patients, the resistance is correlated to the reduced level of CD20 antigen on the surface of tumor B cells. While testing drugs that overcome this resistance, we sought of testing whether Salinomycin, a drug that preferentially act on cancer cells over healthy cells, and selectively target cancer stem cells, is able to improve the sensitivity of NHL to Rituximab. Herein, we investigated its effect on NK cell- and complement-dependent cytotoxicity on NHL cells and its mechanism of action. Methods: Antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and degranulation assays were used to assess the sensitivity of NHL cell lines and primary cancer cells upon treatment with salinomycin plus rituximab. Serum and PBMCs of human healthy donors were used as source of complement and Natural Killer cells, respectively. Flow cytometry was used to quantify cell death rate and cell surface protein levels. RNA-seq and qPCR were used to identify and confirm the differentially expressed genes upon salinomycin treatment. GSEA, an online resource of the Broad Institute MA, was used to screen the differentially expressed genes and major signaling pathways. Results and Conclusions: In addition to previously reported ability of Salinomycin to kill cancer stem cells, our data show that the sub-lethal doses of Salinomycin greatly increased surface CD20 (protein target for Rituximab therapy) on both lymphoma cell lines and primary CLL samples. Lymphoma cell death induced by rituximab and mediated by complement and NK cell cytotoxicities was significantly increased upon treatment with salinomycin. Transcriptomic analysis of cells treated with salinomycin identified a sets of differentially expressed genes (including CD20-encoding gene) and markedly activated/affected pathways that are potentially involved in enhancing the sensitivity of Rituximab-treated NHL cells. The anticipated signaling pathways influencing NK cell activity will be further examined. These data indicate that salinomycin is an interesting therapeutic agent when combined with therapeutic anti-CD20 monoclonal antibodies. This particular combination would ensure higher NK cell-mediated cytotoxicity and offset the NHL resistance to R-CHOP regimen. Aknowledgements: National Science Centre (NCN), ID: 2016/23/B/NZ5/02622; Ministry of Science and Higher Education, ID: DI2014007344. Citation Format: Abdessamad Zerrouqi, Nina Miazek-Zapala, Anna Torun, Piotr Zapala, Jakub Golab, Magdalena Winiarska, Beata Pyrzynska. Salinomycin expands the cytotoxicity of both anti-CD20 monoclonal antibodies and natural killer cells towards non-Hodgkin lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-261.