FOXO1 promotes resistance of non-Hodgkin lymphomas to anti-CD20-based therapy

Abstract

Diminished overall survival rate of non-Hodgkin lymphoma (NHL) patients treated with a combination regimen of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) has been recently linked to recurrent somatic mutations activating FOXO1. Despite of the clinical relevance of this finding, the molecular mechanism driving resistance to R-CHOP therapy remains largely unknown. Herein, we investigated the potential role of FOXO1 in the therapeutic efficacy of rituximab, the only targeted therapy included in the R-CHOP regimen. We found CD20 transcription is negatively regulated by FOXO1 in NHL cell lines and in human lymphoma specimens carrying activating mutations of FOXO1. Furthermore, both the expression of exogenous mutants of FOXO1 and the inhibition of AKT led to FOXO1 activation in lymphoma cells, increased binding to MS4A1 promoter and diminished CD20 expression levels. In contrast, a disruption of FOXO1 with CRISPR/Cas9 genome-editing (sgFOXO1) resulted in CD20 upregulation, improved the cytotoxicity induced by rituximab and the survival of mice with sgFOXO1 tumors. Accordingly, pharmacological inhibition of FOXO1 activity in primary samples upregulated surface CD20 levels. Importantly, FOXO1 was required for the downregulation of CD20 levels by the clinically tested inhibitors of BTK, SYK, PI3K and AKT. Taken together, these results indicate for the first time that the AKT-unresponsive mutants of FOXO1 are important determinant of cell response to rituximab-induced cytotoxicity, and suggest that the genetic status of FOXO1 together with its transcriptional activity need further attention while designing anti-CD20 antibodies based regimens for the therapy of pre-selected lymphomas.

Keywords: CD20; FOXO1; R-CHOP; lymphoma; rituximab.

Figure 1.

Ablation of FOXO genes and its effects on CD20 levels and rituximab efficacy in vitro and in vivo. (A) Western blotting showing FOXO1, FOXO3 and CD20 proteins levels in Raji cell clones that were previously transduced with lentiviruses encoding sgRNA (CRISPR/Cas9 technology) targeting either FOXO1 or FOXO3 loci. Clones with empty vector or sgEGFP were used as controls. β-actin level was used as loading control. (B) FACS analysis of cell surface levels of CD20 (left panel, example of graph from FlowJo software; right panel, quantification of MFI values) in clones of Raji cells characterized in panel A. (C) CDC (complement-dependent cytotoxicity) assay showing improved response of sgFOXO1 cell clones to low concentrations of rituximab in the presence of human serum. (D) Kaplan-Meier survival plot of mice inoculated intravenously with Raji cells (either mix of 3 control clones or mix of 3 clones with sgFOXO1) expressing Red Firefly luciferase. Mice (n = 7) were then injected intraperitoneally with rituximab (10 mg/kg) three times a week.

Figure 2.

Pharmacological inhibition of FOXO1 activity elevates CD20 levels in Raji cell line and tumor cells from CLL patients ex vivo. (A) Schema illustrating signaling of PTEN/AKT/FOXO pathway, combined with PTEN inhibitor (SF1760), FOXO1 inhibitor (AS1842856) and our hypothesis concerning negative regulation of CD20 expression by FOXO1. (B-C) FACS analysis showing increased levels of cell surface CD20 antigen 48 h upon incubation of Raji cells with different concentrations of either SF1760 (panel B) or AS1842856 (panel C). (D) FACS analysis of cell surface CD20 antigen in tumor cells (CD19-positive) isolated from blood of CLL patients and treated for 48 h ex vivo with different concentrations of AS1842856. Statistical significance was determined with 1-way Anova, *** p < 0.001 vs controls.

Figure 3.

AKT inhibitors activate FOXO1, affect CD20 levels and impair rituximab efficacy in vitro. (A) The levels of FOXO1, FOXO3 and AKT (either total or phosphorylated) in Raji cells incubated for 6–24 hours with MK-2206 or GDC-0068 (left panel) followed by nuclear/cytoplasmic fractionation. Oct-2 and EEA1 were used as nuclear and cytoplasmic markers, respectively. Quantification of nuclear FOXO1 upon incubation with either MK-2206 (1 μM) or GDC-0068 (2.5 μM) was performed, normalized to Oct-2 using the ImageStudio software and depicted on graphs (right panel). (B) FACS analysis showing decreased levels of cell surface CD20 antigen 48 h upon incubation of Raji cells with inhibitors of PI3K (idelalisib) and AKT (MK-2206 and GDC-0068) (left panel) and CDC assay showing impaired response of Raji cells to different concentrations of rituximab upon pre-incubation with 1 μM idelalisib, 1 μM MK-2206 or 2.5 μM GDC-0068 for 48 hours (right panel). (C) FACS analysis of cell surface levels of CD20 in clones of Raji cells (top panel) and SU-DHL4 (lower panel) with sgFOXO1, sgFOXO3 and control clones (with either empty vector or sgEGFP) incubated with AKT inhibitors for 48 h (same clones as in Fig. 1). (D) Impaired response of control clone (with sgEGFP), but not the clone with sgFOXO1 to low concentrations of rituximab in the presence of human serum, as estimated by CDC assay. Cells were incubated with either MK-2206 (left panel) or GDC-0068 (right panel) for 48 hours before rituximab application and estimation of cell survival.

Figure 4.

Binding of FOXO1 to the promoter and transcriptional regulation of MS4A1 expression are necessary for the downregulation of CD20 levels by AKT inhibitors. (A) The mRNA levels of MS4A1 (CD20) estimated by RT-PCR in Raji and SU-DHL4 cells 18 hours upon incubation with AKT inhibitors (MK-2206 and GDC-0068). (B) Luciferase reporter assays showing reduced activity of MS4A1 promoter (−394/+89 bp) cloned into pGL4 reporter and introduced by nucleofection into Raji and SU-DHL4 cells. Assays performed 24 hours upon incubation with AKT inhibitors. (C) Increased activity of FOXO transcription factors 24 h upon expression of exogenous wild-type FOXO1, but not the FOXO1-H215R mutant, measured in luciferase reporter assays in Raji cells transfected with 3xIRS-Luc construct (left panel). Luciferase reporter assays showing increased activity of MS4A1 promoter (−394/+89 bp) upon expression of FOXO1-H215R mutant relative to FOXO1-WT (right panel). (D) ChIP-Seq data from independent experiments conducted by Dominguez-Sola et al., 2015, using anti-FOXO1 antibody, showing approximate FOXO1 binding site at MS4A1 locus near the transcription start site (TSS). ChIP-seq reads are mapped mostly to upstream region located immediately before the TSS. (E-G) ChIP experiments showing increased binding of acetylated histone H3 (acetyl-K27) (E) and FOXO1 (F) to fragment −182/−88 bp of MS4A1 promoter as well as to the promoters of known FOXO transcriptional targets (GADD45a and IL7R) (G) upon incubation of SU-DHL4 and Raji cells with GDC-0068 for 24 hours.

Figure 5.

Expression of mutated FOXO1 results in decreased levels of CD20 antigen. (A) Averaged normalized expression of MS4A1 mRNA in malignant lymphoma samples collected by International Cancer Genome Consortium (ICGC, MALY-DE project), including samples with wild-type FOXO1 (n = 10) and with N-terminal mutations of FOXO1 (n = 6). Results were visualized using GraphPad Prism software. Statistical significance was determined with Mann Whitney test, *p < 0.05 (B) FOXO transcriptional activity was measured using luciferase assays with 3 × IRS-Luc reporter (IRS, insulin-responsive signal), introduced to cells together with constructs encoding either wild-type FOXO1 (FOXO1-WT) or FOXO1-AAA mutant, mixed with constructs encoding either myr-AKT1 and GFP or GFP alone (as control). (C-D) FACS analysis showing levels of surface CD20 in Raji cells (panel C) and SU-DHL4 cells (panel D) expressing exogenous FOXO1 (WT or mutant). Cells were cotransfected with construct encoding EGFP followed by the FACS analysis of CD20 surface level (mean fluorescence intensity; MFI) in EGFP-positive cells (48 h post transfection) and visualization using FlowJo software (left panel in D). (E) Luciferase reporter assays showing reduced activity of MS4A1 promoter (−394/+89 bp) introduced by nucleofection into Raji cells (left panel) and SU-DHL4 cells (right panel), together with constructs encoding either wild-type or mutant FOXO1.