FoxM1: A New Therapeutic Target for Mantle Cell Lymphoma

Introduction

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma subtype with elevated B-cell receptor activity. Ibrutinib (IBN), the Bruton's tyrosine kinase (BTK) inhibitor, has been shown to have an overall response rate of 68% in relapsed or refractory MCL patients. However, with the emergence of IBN resistance, novel therapies to thwart resistance are urgently needed. FOXM1 (Forkhead box M1) is a proliferation-associated transcription factor that stimulates cell proliferation and exhibits a proliferation-specific expression pattern. FOXM1 has recently been classified as a human proto-oncogene and we previously found this gene to be associated with IBN resistance in our gene expression analysis; therefore, the prognostic significance of FOXM1 and its potential as a new MCL therapeutic target was investigated.

Methods

Bone marrow aspirates and peripheral blood clinical specimens were obtained from MCL patients. FOXM1 gene expression was assessed in 50 primary MCL patient samples using Quantitative real-time PCR (Q-PCR). To conduct dose-dependent cell viability assays, MCL cell lines (ATCC) were cultured in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (Millipore). The drug screening was performed in a 96-well format in which MCL cells were seeded at 10,000 cells per well and were treated with the FOXM1 inhibitor thiostrepton or ibrutinib at the following concentrations: 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 uM. Cell viability was tested using the CellTiter-Glo luminescent cell viability assay (Promega) after a 72-hour incubation period. To determine the effects of inhibiting FOXM1 on apoptosis, MCL cells (200,000 cells) were incubated with IBN or thiostrepton at varying doses (1, 3, 5 uM) for 24 hours, and apoptosis was detected using the Annexin V-binding assay. Furthermore, caspase-3/7 activity was assessed in MCL cells with the Caspase-Glo-3/7 Assay (Promega) after 24 hours of treatment with thiostrepton or IBN.

Results

To evaluate the possibility that FOXM1 is important for MCL, we conducted an analysis of FOXM1 expression in MCL patient samples at the time of diagnosis using Q-PCR. FOXM1 expression was detected in 50 MCL patient samples, and FOXM1 expression levels were significantly higher (82.3%, fold change=2) than the levels observed in peripheral blood mononuclear cells (PBMCs). The association between FOXM1 expression in MCL and clinical pathology was also analyzed by examining the relationship between FOXM1 and Ki-67 expression (a cell-proliferation marker) in 40 diagnosed MCL patient samples, and a significant correlation was noted between the two markers (p<0.05).

The effects of inhibiting FOXM1 on MCL cell lines (Jeko-1, Jeko-1-BTK-KO-#1, Jeko-1-BTK-KO-#2, Mino, Maver-1, Z-138, JVM-2, and JVM-13) in vitro were examined by transfection of siRNA targeting FOXM1 or treatment with the FOXM1 inhibitor thiostrepton. Thiostrepton reduced the cell viability of the MCL cell lines in a dose-dependent manner compared with the DMSO control. Likewise, siFOXM1-transfected MCL cell lines showed reduced cell viability compared with the control siRNA-transfected cells (P < 0.01). Annexin V/PI dual staining showed that thiostrepton induced apoptosis in the MCL cell lines in a dose-dependent manner. Furthermore, activated-caspase3-7 expression was increased in a dose-dependent manner in MCL cell lines after treatment with thiostrepton.

Conclusion

We have shown that FOXM1 inhibition may be a potential candidate treatment for MCL based on the results of our clinicopathological assessment and in vitro studies. Clinical studies are warranted to further validate FOXM1 as a potential therapeutic target in high-risk MCL.