Abstract B51: FOXO1 determines non-Hodgkin lymphomas response to anti-CD20-based therapy
Authors: Zerrouqi Abdessamad , Pyrzynska Beata , Dwoyak Michal , Morlino giulia , Zapala Piotr , Miazek Nina , Golab Jakub , Winiarska Magdalena 
Published: May 2018

Abstract
Lack of remission or early relapse remains a major clinical concern in diffuse large B-cell lymphoma (DLBCL), with a third of patients failing to respond to current regimens or relapsing with resistant disease. Genome and transcriptome sequencing studies have identified FOXO1 as one of the recurrent targets for somatic mutations in DLBCL associated with patient short survival. These mutations enhance FOXO1 nuclear localization and activity on target genes.
Herein, we determine the role FOXO1 (wild-type and mutated) on the expression of the rituximab-target, CD20. The effect of active mutated FOXO1 on CD20 expression was determined upon inhibition of AKT, exogenous expression of active myristolated AKT, and mutated FOXO1 at its N-terminal region. FOXO1 binding activity to CD20 promoter was assessed using various assays combined with an in silico analysis of a publicly available Chip-Seq data. Our results show that the activation of FOXO1 using AKT inhibitors (MK-2206 and GDC-0068) led to a significant decrease of CD20 transcript and protein levels, which resulted in a significant resistance to rituximab-mediated complement-dependent cytotoxicity (CDC). Consistently, the overexpression of either wild-type or mutated FOXO1 repressed CD20 expression. Unlike the activity of exogenous wild-type FOXO1 that can be inhibited by AKT1, the activity of mutated FOXO1-AAA (Thr24, Ser256 and Ser319 mutated to Ala and that cannot be phosphorylated) markedly inhibits CD20 expression. Furthermore, CD20 levels remained high and unchanged by the DNA-binding defective mutant of FOXO1-H215R, suggesting a FOXO1-mediated regulation of CD20 transcription through FOXO1 binding to CD20 promoter, as confirmed by ChIP, EMSA, and the analysis of ChIP-Seq data using anti-FOXO1 antibody. These results show that FOXO1 mutants that are insensitive to AKT1 kinase activity effectively suppress CD20 expression in comparison to wild-type FOXO1, through a binding to CD20 promoter. In addition, the disruption of FOXO1 with CRISPR/Cas9 genome editing resulted in CD20 upregulation and improved the cytotoxicity induced by rituximab and survival of mice with sgFOXO1 tumors. Consistently, in primary samples pharmacologic inhibition of FOXO1 activity upregulated surface CD20 levels.
In summary, these results establish FOXO1 as an important determinant of cell response to complement-dependent rituximab-induced cytotoxicity. In addition, our findings indicate that the genetic status of FOXO1 together with its transcriptional activity needs further attention while designing rituximab-based regimens in the therapy of selected lymphomas.
Supported by National Science Center, Poland (NCN#2013/11/B/NZ5/03240), STREAM project (Horizon 2020,#692180).
Citation Format: Abdessamad Zerrouqi, Beata Pyrzynska, Michal Dwoyak, giulia Morlino, Piotr Zapala, Nina Miazek, Jakub Golab, Magdalena Winiarska. FOXO1 determines non-Hodgkin lymphomas response to anti-CD20-based therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B51.