Targetable vulnerability of deregulated FOXM1/PLK1 signaling axis in diffuse large B cell lymphoma

Abstract

FOXM1 is a transcription factor that controls cell cycle regulation, cell proliferation, and differentiation. Overexpression of FOXM1 has been implicated in various cancer types. However, the activation status and functional significance of FOXM1 in diffuse large B cell lymphoma (DLBCL) have not been well investigated. Using proteomic approaches, we discovered that the protein expression levels of FOXM1 and PLK1 were positively correlated in DLBCL cell lines and primary DLBCL. Expression levels of FOXM1 and PLK1 mRNAs were also significantly higher in DLBCL than in normal human B cells and could predict poor prognosis of DLBCL, particularly in patients with germinal center B cell-like (GCB) DLBCL. Furthermore, proteomic studies defined a FOXM1-PLK1 signature that consisted of proteins upstream and downstream of that axis involved in the p38-MAPK-AKT pathway, cell cycle, and DNA damage/repair. Further studies demonstrated a mechanistic function of the FOXM1/PLK1 axis in connection with the DNA damage response pathways regulating the S/G2 checkpoint of the cell cycle. Therapeutic targeting of FOXM1/PLK1 using a FOXM1 or PLK1 inhibitor, as well as other clinically relevant small-molecule inhibitors targeting ATR-CHK1, was highly effective in DLBCL in vitro models. These findings are instrumental for lymphoma drug discovery aiming at the FOXM1/PLK1/ATR/CHK1 axis.

Keywords: DLBCL; FOXM1; PLK1; therapeutic potential.

Figure 1

FOXM1 and PLK1 expression levels are positively correlated and highly expressed in DLBCL. A. FOXM1 and PLK1 protein expression levels in DLBCL cell lines (n=38) were measured by RPPA analysis, and quantitated protein expression levels were plotted. B. Linear regression analysis of FOXM1 and PLK1 protein expression in representative DLBCL cell lines. C. In three separate DLBCL cohorts extracted from the Oncomine database [30-33], the mRNA expression levels of FOXM1 and PLK1 were also positively correlated. D. In the same cohorts, the mRNA levels of FOXM1 (top) and PLK1 (bottom) were significantly higher in primary DLBCL cells compared to normal B cells.

Figure 2

Prognostic analysis of FOXM1 and PLK1 expression in de novo DLBCL treated with standard R-CHOP regimen. FOXM1 (left) or PLK1 (right) mRNA expressions were assessed in association with poorer overall survival in overall DLBCL patients (A), germinal center B cell-like (GCB) DLBCL patients (B), activated B cell-like (ABC) DLBCL patients (C), and Type 3 DLBCL patients (D). The cutoffs for FOXM1 and PLK1 mRNA mRNA are 2.4 and 2.0 for the normalized log2 values of the microarray data, respectively. The values above and below these are considered high and low, respectively.

Figure 3

Downstream signaling pathways involved in FOXM1/PLK1 axis. A. Venn diagram showing proteins negatively or positively associated with FOXM1 or PLK1 and proteins shared between FOXM1 and PLK1 on RPPA analysis. B. Linear regression analysis of expression levels of FOXM1 and cell cycle-related protein CDK1 and cyclin B1 (CCNB1) was plotted in 38 DLBCL cell lines, and the Pearson correlation coefficient and P value were determined. C. Model depicting the proposed FOXM1/PLK1 pathway and potential mechanism of action, based on the RPPA data of 38 representative DLBCL cell lines.

Figure 4

Thiostrepton downregulates the expression of PLK1 and other downstream targets in DLBCL cells. Two representative DLBCL cell lines, SUDHL5 and MZ, were treated with thiostrepton (Thio) at various doses (A) and schedules (B), and protein expression levels of FOXM1, PLK1, c-Myc, cyclin B1, CHK1, p-p38, p38, and β-actin (loading control) were assessed by Western blotting. For the varying durations of thiostrepton treatment, a 4 µM concentration was used. (C) Multi-guide sgFOXM1-transfected cells showed lower abundance of FOXM1, PLK1 and cyclin B1 by Western blotting. (D) Knock-down of FOXM1 by multi-guide sRNA inhibited DLBCL cell proliferation in vitro. (E) Linear regression analysis of RPPA protein expression profiles of DLBCL cell lines (n=38) and thiostrepton IC50 values. FOXM1 protein expression showed the highest correlation with thiostrepton treatment (r=-0.4914; P<0.0023). (F) IC50 of GCB or ABC subtypes of DLBCL with thiostrepton treatment (P=0.0310).

Figure 5

Thiostrepton inhibits cell growth and induces apoptosis and cell cycle arrest in DLBCL. A. Two representative DLBCL cell lines were treated with thiostrepton in a dose-dependent manner for 24 and 48 h, and apoptosis was measured using annexin V/PI staining and subsequently analyzed by flow cytometry. B. Two representative DLBCL cell lines were treated with thiostrepton in a dose-dependent manner for 24 h, and protein extracts were analyzed by Western blotting for apoptotic biomarkers caspase 3 and PARP cleavage and DNA damage biomarker pH2AX. C. Four representative DLBCL cell lines (SUDHL5, MZ, RC, and McA) were treated with thiostrepton in a dose-dependent manner (0, 0.25, 0.5, 0.75, and 1 µM) for 24 h, and cell cycle analysis was performed using PI staining and flow cytometry analysis.

Figure 6

The FOXM1 inhibitor thiostrepton synergizes with key therapies targeting the FOXM1/PLK1 signaling axis. A. SUDHL5 and MZ cells were treated with a FOXM1 inhibitor thiostrepton (T; 0.5 µM), p38 inhibitor BIRB-796 (B; 25 µM), or the combination of both (T+B) for 24 h, protein extracts were used to detect for p-p38, p38, and actin (loading control) by Western blotting. B. Four-day CellTiter-Glo viability assays were performed in SUDHL5 and MZ cells treated with the FOXM1 inhibitor thiostrepton (T; 0.62 µM), p38 inhibitor BIRB-796 (B; 25 µM), or the combination of both. C. SUDHL5 and MZ cells were treated with thiostrepton (T; 0.5 µM), PLK1 inhibitor volasertib (V; 100 nM), or the combination of both (T+V) for 24 h, protein extracts were used to detect for FOXM1, PLK1, and actin (loading control) by Western blotting. D. Four-day CellTiter-Glo viability assays were performed in SUDHL5 and MZ cells treated with the FOXM1 inhibitor thiostrepton, PLK1 inhibitor volasertib, or the combination of both, at varying doses.