1027 - B cell receptor-mediated signals modulate rapid FOXO1 upregulation in chronic lymphocytic leukaemia: a role for deubiquitinate proteins?

B cell receptor (BCR) engagement of CLL cells is vital for disease progression, driving cell survival, proliferation, and chemo-resistance. Forkhead box protein, subgroup O-1 (FOXO1), a transcription factor widely regarded as a tumour suppressor in B cell malignancies, is inactivated downstream of BCR activation. Previously, we demonstrated that the expression of FOXO1 is significantly upregulated in lymph node biopsies of poor prognostic patients and that FOXO1 activation induces chemosensitivity of BCR-targeted drug activity, positively regulating apoptosis, indicating that FOXO1 possesses a tumour suppressive function in CLL cells. We now demonstrate that FOXO1 expression is rapidly and significantly upregulated within 30 minutes of BCR crosslinking, with upregulation sustained for up to 2 hr; the BTK inhibitor ibrutinib inhibited BCR-mediated FOXO1 upregulation. Due to the rapid changes of FOXO1 protein expression upon BCR crosslinking and the known role of deubiquitinase (DUB) enzymes in regulating its function, we investigated whether DUB proteins were involved in the regulation of FOXO1 expression in CLL cells, with a view to identifying potential novel drug targets that could enhance current CLL treatment.
Little is known about the role of individual DUB family members in CLL. Our analysis revealed dysregulated expression of DUB family members USP7, USP8, USP9x, USP10, and USP14 in freshly isolated CLL cells compared to healthy B-cells, all showing significant upregulation in CLL cells. Stratification of CLL patient samples by Binet stage also revealed that these DUB family members were upregulated in poor prognostic (Stage C) patients compared with Stage A patients, suggesting that DUB proteins may represent a novel biomarker for CLL progression.
Treatment of the CLL cell line MEC1 and primary CLL patient samples with DUB inhibitors including PR-619 (pan-DUBi), P5091 (USP7i) and HBX19818 (USP7i) alone reduces the phosphorylation of AKTS437 and FOXO1T24, indicating that DUBs play a role in inhibiting FOXO1 activity. These effects of DUBi were further enhanced upon combination with ibrutinib treatment, indicating a potential benefit of combining DUB inhibition with current ibrutinib therapy. Furthermore, we demonstrated that the DUBi/ibrutinib combination was synergistic in terms of inducing a 10% enhancement in apoptosis compared with single treatments, and significantly reduced proliferation in MEC1 cells at 72hr.
USP7 has previously been shown to regulate a number of proteins including p53 and PTEN in CLL cells, and has been demonstrated to influence sub-cellular localisation of FOXO family members in melanoma. To define the relationship between FOXO1 and USP7, we performed co-immunoprecipitation assays and demonstrated a direct interaction between FOXO1 and USP7, an interaction that was increased upon BCR crosslinking. To elucidate a role for USP7 in CLL cells, we performed lentiviral-shRNA technology to selectively knockdown (KD) USP7 in MEC1, achieving 68% reduction in USP7 expression. Cells targeted with sh-USP7 showed downregulation of p-FOXO1T24 and total FOXO1 at protein levels, and significant increase in the gene expression of FOXO1. Furthermore, the expression of FOXO1 gene targets were altered, including a significant upregulation of Sestrin3 (SESN3), Bcl-2-like protein 11 (BCL2L11) and cyclin-dependent kinase inhibitor 1A (CDKN1A), and a significant downregulation of cyclin D2 (CCND2) and cyclin G2 (CCNG2). These data suggest that silencing USP7 increases FOXO1 functions, indicating that USP7 is responsible for the reduction of FOXO1 activities. As cellular localisation of FOXO1 dictates its DNA binding activity, we performed cellular fractionation on MEC1 and primary CLL cells in the presence and absence of ibrutinib and/or DUBi, and sh-USP7 KD MEC1 cells. These studies revealed a significant upregulation of FOXO1 in the nuclear fraction and upon ibrutinib treatment, which was further enhanced in the USP7 KD cells, or the combination with DUBi. Indeed, USP7 KD sensitised MEC1 cells to ibrutinib treatment leading to increase in FOXO1 nuclear localisation. Additionally, FOXO1 activity studies demonstrated significant activation of FOXO1 upon ibrutinib treatment, which was further enhanced in the USP7 KD and combination with USP7i. These studies suggest that selective DUBs play a role in BCR-mediated signalling to aid in the promotion of CLL cell survival and chemo-resistance through FOXO1 inhibition, and targeting these DUBs enhance FOXO1 activity leading to cell cycle arrest and apoptosis.