FOXO1 Activation Is an Effector of SYK and AKT Inhibition in Tonic BCR Signal-Dependent Diffuse Large B-Cell Lymphomas

Introduction: In normal B lymphocytes, B-cell receptor (BCR)-induced activation of PI3K-AKT kinases and subsequent inactivation of FOXO1 is a critical pro-survival component of tonic BCR signaling. In murine models, conditional deletion of FOXO1 protected quiescent peripheral B cells from apoptosis mediated by inducible loss of the BCR, demonstrating that PI3K-AKT-FOXO1 axis plays a central role in B-cell homeostasis. Disruption of the BCR signaling by SYK inhibitor leads also to the apoptosis of BCR-dependent DLBCLs, at least in part via a mechanism involving decreased activity of PI3K/AKT axis. Herein, we investigated the role of FOXO1 in toxicity of BCR pathway/SYK inhibition in human BCR-dependent lymphomas.

Methods: BCR-dependent DLBCL cell lines (DHL4, DHL6, Ly1, Ly7) were incubated with SYK inhibitor R406 (4 µM) and AKT phosphorylation, FOXO1 activation and transcriptional activity were assessed by phospho-specific flow cytometry, Western Blot and qPCR. Toxicity of active FOXO1 was assessed by transducing DLBCL cells with a constitutively nuclear FOXO1 mutant. FOXO1 knock-down in DLBCL cell was achieved with shRNA. DREAM cleavage and HRK expression were assessed by Western Blot and qPCR, respectively, in the absence or presence of caspase inhibitors. DLBCL cell viability and apoptosis after incubation with SYK and/or AKT inhibitors R406 and MK2206, were assessed by MTS assay and Annexin V/PI staining. Drug interactions were quantitated by CompuSyn software. The expression of p-SYK and FOXO1 in DLBCL samples was assessed by immunohistochemistry (IHC) in 60 DLBCL patients.

Results: Since FOXO1 is a major effector of tonic BCR signaling, we assessed the activity of FOXO1 in DLBCL cells after SYK inhibition. In all tested cell lines, AKT and FOXO1 phosphorylations decreased after incubation with SYK inhibitor. Diminished FOXO1 phosphorylation resulted in its nuclear relocalization and induction of FOXO1-dependent expression of p27, BIM, TRAIL and GADD45A. To assess whether the increased activity of FOXO1 is sufficient to induce apoptosis of DLBCL cells, we transduced DHL4 cells with a constitutively nuclear and transcriptionally active FOXO1-3A mutant. The mutant induced G1/S cell cycle arrest and triggered apoptosis, whereas wild-type FOXO1 did not change proliferation or cellular viability, demonstrating that FOXO1 activation is sufficient to induce apoptosis of DLBCL cells. Next, we assessed the toxicity of SYK inhibitor in cells lacking FOXO1. Cells with depleted FOXO1 exhibited 70% lower sensitivity to SYK inhibitor than control cells (p<0.0001). Since in previous studies expression of the proapoptotic member of BCL2 family, HRK, was required for SYK-inhibitor induced cell death in DLBCL cells, we determined the role of FOXO1 activation in HRK expression. HRK expression was dramatically increased in SYK-inhibitor treated control cells, but not in FOXO1-deficient cells. Consistent with this, SYK inhibitor triggered cleavage of HRK transcriptional repressor DREAM only in control cells, but not in FOXO1-depleted cells. HRK induction was blocked by caspase inhibitors. Since AKT is major kinase regulating both FOXO1 activity and HRK induction, we assessed the combined effects of the AKT inhibitor MK-2206 with R406 and found markedly synergistic toxicity (combination index [CI] 0.5-0.8). Combination of inhibitors in FOXO1-depleted cells did not trigger cell death, highlighting the critical effector role of FOXO1. FOXO1 expression was present in 80% of primary DLBCL samples and correlated with SYK activity (p=0.009). High levels of FOXO1 protein expression were associated with longer OS (log rank, p=0.04).

Conclusions: Taken together, our results demonstrate that targeting the BCR signaling at multiple levels is a rational therapeutic strategy. Proapoptotic activity of FOXO1 is an important effector of SYK and AKT inhibition in DLBCLs and its expression is required for SYK-and AKT inhibitor-induced toxicity. The underlying mechanism linking FOXO1 activation and cell death involves caspase-dependent cleavage of transcriptional repressor DREAM and subsequent induction of a proapoptotic BCL2 family member, HRK.