Rituximab, a therapeutic anti-CD20 monoclonal antibody, is effective against B-cell malignancies, when combined with chemotherapy, such as cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP therapy). However, in case of 30-40% patients the resistance to rituximab-based therapy appears to be a serious concern. One of the major mechanisms of resistance relies on the reduced level of CD20 antigen on the surface of tumor B-cells.
While exploring the molecular mechanisms affecting the level of CD20, we found that transcription factor FOXO1 is a negative regulator of MS4A1 expression, the gene encoding CD20 antigen. Using the CRISPR/Cas9 genome-editing technology, we disrupted the loci of FOXO genes in lymphoma cell lines and discovered that ablation of FOXO1 (but not FOXO3) was sufficient for upregulation of the surface level of CD20 by 3-fold. Consistently, the complement-dependent cytotoxicity (CDC), induced by rituximab, was significantly improved in cell clones with abrogated expression of FOXO1, but not FOXO3. Importantly, the treatment with pharmacological inhibitor of FOXO1 activity, AS1842856, resulted in increased levels of CD20 on the surface of both lymphoma cell lines and patients-derived chronic lymphocytic leukemia (CLL) cells cultured ex vivo.
In order to verify our findings in the animal model, we inoculated SCID mice intravenously with Raji cells, what resulted in the development of lymphoma-like tumors. We demonstrated that mice treated systemically with rituximab, administered at a dose of 10 mg/kg, survived longer when inoculated with sgFOXO1-transduced Raji cells as compared with mice inoculated with control Raji cells (median survival 49 days versus 29 days, respectively). These results confirmed that FOXO1 ablation in lymphoma cells resulted in higher efficacy of rituximab treatment in vivo.
Taken together, these results establish FOXO1 as an important determinant of cell response to complement-dependent rituximab-induced cytotoxicity and indicate that FOXO1 inhibitors could be exploited for the therapeutic purposes, in combination with anti-CD20 monoclonal antibodies. Novel FOXO1 inhibitors with improved potency and selectivity are however urgently needed for further exploration of our discoveries in the near future.
Acknowledgments: NCN, Poland (grant no: 2013/11/B/NZ5/03240) and H2020 (STREAM project, CSA action, grant no: 692180).
Citation Format: Beata Pyrzynska, Abdessamad Zerrouqi, Michal Dwojak, Giulia Morlino, Piotr Zapala, Nina Miazek, Agnieszka Zagozdzon, Kamil Bojarczuk, Malgorzata Bobrowicz, Marta Siernicka, Marcin M. Machnicki, Joanna Barankiewicz, Ewa Lech-Maranda, Przemyslaw Juszczynski, Dinis Calado, Jakub Golab, Magdalena Winiarska. FOXO1 is transcriptional regulator of malignant B-cell surface antigen CD20, the target for therapeutic monoclonal antibodies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B17.