title: Elucidating BCR-mediated regulation of FOXO1 in chronic lymphocytic leukaemia: a role for deubiquitinase proteins?
Creators: name: Almuhanna, Hassan Nasser B.
Publisher: name: University of Glasgow
Year: 2024

description: The B-cell receptor (BCR) activity in chronic lymphocytic leukaemia (CLL) cells is vital for disease progression, driving cell survival, proliferation, and chemoresistance. Forkhead box protein O1 (FOXO1), a transcription factor widely considered as a tumour suppressor in B-cell malignancies, is inactivated downstream of BCR activation. Previously, we demonstrated that FOXO1 expression is significantly upregulated in lymph node (LN) biopsies of poor prognostic CLL patients. However, FOXO1 cytoplasmic localisation and deregulation of FOXO target genes, including cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin-dependent kinase inhibitor 1B (CDKN1B), and cyclin D1 (CCND1), may suggest functional inactivity of FOXO1 in these CLL patients. This finding indicates that FOXO1 possesses tumour-suppressive function in CLL cells. Aligning with previous studies, our data demonstrated that FOXO1 is an effector of BCR crosslinking in vitro, promoting FOXO1 inactivation and nuclear exclusion through phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT)-dependent phosphorylation of FOXO1. This was further confirmed using the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, which restored FOXO1 nuclear localisation and subsequently increased FOXO1 DNA binding and transcriptional activities, as indicated by the modulation of FOXO target genes, including the upregulation of Bcl-2-binding component 3 (BBC3) and the downregulation of cyclin D2 (CCND2). Furthermore, the levels of phosphorylated and total FOXO1 protein were transiently upregulated upon BCR crosslinking, peaking at 30 minutes and sustaining up to 2 hours. Therefore, we investigated FOXO1 regulation upon BCR crosslinking in relation to post-translational modifications involving the ubiquitination-proteasome system (UPS) pathway, particularly deubiquitinase (DUB) proteins.

Little is known about the role of individual DUB family members in CLL. Our analysis revealed that expression levels of DUB proteins in patient CLL cells were largely upregulated, including the expression of ubiquitin-specific protease 7 (USP7) and ubiquitin-specific protease 9, x-linked (USP9x). FOXO1 co-immunoprecipitation (co-IP) demonstrated USP7 interaction with FOXO1 in primary CLL cells and cell lines. The FOXO1-USP7 interaction was largely unaffected by BCR crosslinking, while inhibition with ibrutinib increased this interaction, suggesting that the PI3K/AKT pathway may play a role beyond modulating FOXO1 phosphorylation, potentially including modulation of FOXO1 interaction with DUB proteins. Treating CLL cells with the pan-DUB inhibitor PR-619 downregulated AKTˢ⁴⁷³ and FOXO1ᵀ²⁴ phosphorylation. However, USP7 inhibitors (P5091 and HBX19818) and the USP9x inhibitor (WP1130) were largely less effective at inhibiting the PI3K/AKT pathway. This suggests that DUB proteins have a regulatory role in the activity of the PI3K/AKT signalling pathway, but the inhibition of an individual DUB protein was not sufficient to exert a significant effect on PI3K/AKT activity. Additionally, the inhibitors induced the accumulation of MEC1 cells in the G0/G1 phase but did not impact cell proliferation. The combination of ibrutinib and DUB inhibitors enhanced CLL response to ibrutinib, leading to a greater reduction in cell viability, proliferation, and cell cycle accumulation at the G0/G1 phase.

DUB inhibitors or knockdown (KD) of USP7 or USP9x demonstrated no effect on total FOXO1 protein expression, while FOXO1 transcriptional activity was increased in MEC1 cells by HBX19818 or USP7/USP9x KDs, as indicated by the upregulation of FOXO target genes, including CDKN1B and BBC3. This effect was further enhanced by the combination of HBX19818 with ibrutinib. The nuclear localisation of FOXO1 while only modestly regulated by the inhibition of DUB proteins, particularly PR-619 and HBX19818 was enhanced when combined with ibrutinib. Additionally, USP7 or USP9x KD alone or in combination with ibrutinib increased FOXO1 DNA binding activity. This suggests that a reduction of USP7 interaction with FOXO1 may facilitate the promotion of FOXO1 to its DNA binding site, resulting in increased FOXO1 transcriptional activity.

Proteome and subsequent co-IP analysis of FOXO1 novel interactors revealed an interaction between FOXO1 and the E3 ligase tripartite motif containing 21 (TRIM21), with TRIM21 being predominantly cytoplasmic. TRIM21 KD resulted in a reduction of total FOXO1 and FOXO1 nuclear localisation, suggesting that FOXO1 is a substrate of TRIM21, which plays a role in regulating FOXO1 stability, localisation, and potentially its activity.

Our findings suggest that unleashing FOXO1 anti-tumour activity by simultaneously inhibiting BCR-mediated phosphorylation and USP7 deubiquitination of FOXO1 may present an alternative therapeutic strategy for CLL patients.