1022 - FOXO4 is required for the promotion of CLL growth and survival

Chronic lymphocytic leukaemia (CLL) pathogenesis is supported in part by ‘tonic’ B-cell receptor (BCR) signalling, through activation of oncogenic signalling pathways such as the phosphoinositide-3-kinase (PI3K)-AKT/mTOR pathway. This pathway is critical in regulating a plethora of cell functions ranging from cell size to oxidative damage, with increased activity promoting cell survival and proliferation via AKT-mediated negative regulation of the Forkhead Box (FOX) class O (FOXO) transcription factor family. This is achieved via AKT-mediated phosphorylation of FOXO at conserved sites, negating DNA binding via cytoplasmic relocalisation. FOXO family members (FOXO1, FOXO3, FOXO4 and FOXO6) are implicated in the regulation of numerous cellular processes, however it is their primary role in cell cycle arrest and apoptosis that has depicted FOXOs as ‘bona fide’ tumour suppressors. This can be achieved via FOXO-mediated upregulation of pro-apoptotic and anti-proliferative target genes such as BCL2L11 (BIM) and CDK1NB (p27kip1) respectively. However, FOXOs also regulate processes such as DNA and oxidative damage responses and thus exhibit ‘bimodal’ characteristics, either promoting or preventing cell survival in a context-dependent manner. This behaviour is mimicked in B-cell malignancies, where FOXO activity can hinder or promote lymphomagenesis in a disease-specific context. Nevertheless, the strong tumour suppressive effects induced by FOXO activity demonstrate clinical relevance in utilising FOXO activity as a novel therapeutic target for CLL treatment.
We previously demonstrated that while FOXO1 expression is upregulated and inactive (cytoplasm sequestered) in primary CLL patient samples, treatment of cells with the dual mTOR inhibitor AZD8055 resulted in FOXO1 nuclear accumulation and activation resulting in cell cycle arrest and cell death. We now demonstrate that pharmacological inhibition of FOXO1 nuclear accumulation using a selective FOXO1 inhibitor (AS1842856) attenuates an AZD8055-mediated proliferative block and cell kill, further demonstrating that FOXO1 behaves in a tumour suppressive manner in CLL. FOXO3 activity has also been reported to be anti-tumourigenic in CLL; microenvironmental chemokines (e.g. CCL19, CCL21) promote CLL cell survival through PI3K-AKT-mediated FOXO3 inactivation, indicating that the CLL tumour microenvironment can promote CLL proliferation and survival in part through AKT-mediated FOXO inactivation.
However, no clear role has been identified for FOXO4 in CLL proliferation and survival. Initial investigations revealed upregulated FOXO4 expression compared to healthy B-cell donor samples, suggesting a potential role for FOXO4 in CLL. We generated a bulk RNA-seq dataset profiling 5 primary CLL patient samples co-cultured on a pro-proliferative co-culture (NTL-CD40L/IL4; 24 hr) in the presence or absence of AZD8055, ibrutinib (BTKi), or a combination. One striking feature of the dataset was that primary CLL cells treated with AZD8055 exhibit significant increases in FOXO4 expression (2.5 fold), likely due to AKT inhibition and subsequent FOXO-mediated upregulation of FOXO4. This finding was supported in AZD8055-treated MEC1 and HG3 cell lines, both exhibiting significant FOXO4 upregulation. As pharmacological mTOR inhibition also increases the activity of FOXO1 and FOXO3, we generated shRNA-mediated FOXO4 depletion in the MEC1 cell line to investigate distinct FOXO4-mediated processes. Proliferation (CellTrace Violet) analysis revealed a significant reduction in proliferative capacity in cells with depleted FOXO4, while apoptosis (Annexin/7-AAD) analysis showed significant increases in AZD8055/Ibrutinib-induced cell death, coinciding with significant increases in the pro-apoptotic FOXO target gene BCL2L11. These data suggest FOXO4 is required for CLL cell survival and chemoresistance.
The importance of FOXO4 in CLL homeostasis is supported by investigation of the DNA damage response. Initial gene profiling showed a significant downregulation of GADD45A expression in FOXO4 depleted cells, suggesting a role for FOXO4 in the DNA damage response. Further investigation via intracellular staining revealed that p-γH2AX - a phosphorylated histone crucial for forming repair foci at sites of dsDNA breaks - is significantly upregulated following AZD8055/Ibrutinib treatment, further supporting FOXO4 as a mediator of DNA damage repair and chemoresistance. There is also significant upregulation of SESN3 in FOXO4-depleted cells, suggesting a role for FOXO4 in maintaining intracellular ROS levels. Further investigation of the role of FOXO4 in the oxidative damage response, as well as investigation into the role of FOXO4 in primary patient samples, are currently ongoing. Collectively, these findings demonstrate the opposing roles of individual FOXO family members and indicate a putative role for FOXO4 in promoting CLL cell survival, proliferation and the cellular response to intracellular damage.