FoxM1-Mediated Signaling Promotes the Progression of 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 (Wang et al., NEJM, 2013). 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 have previously found this gene to be associated with IBN resistance in our gene expression analysis; therefore, we investigated the prognostic significance of FoxM1 and its potential as a new MCL therapeutic target.

Methods

We assessed the anti-MCL effects of targeting FoxM1 in both ibrutinib-sensitive and -resistant MCL cell lines and clinical specimens. Cell viability assays were conducted targeting FoxM1 with thiostrepton, a published FoxM1 inhibitor. 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 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. Furthermore, we investigated the importance of FoxM1 signaling in tumor migration and adhesion in Jeko-1 WT and BTK KD (generated from Jeko-1 using CRISPR/Cas9) cells. Jeko-1 WT and two BTK KD variants were transiently transfected with control siRNA and siRNA against FoxM1 (siFoxM1) for 24 and 48 hours. Afterwards, cells were loaded into transwell migration inserts in the presence or absence of human stromal cells. Migration was evaluated by counting migrated cells and normalizing the migrated cells to migration in the absence of stromal cells. The in vivo efficacy of thiostrepton was evaluated in two cell line Jeko-1 and Jeko-1 BTK KD mouse xenograft models. Upon engraftment, thiostrepton was administered intravenously five consecutive days a week at 50 mg/kg, and tumor burden was assessed via the measurement of circulating human β2M levels and tumor volume. FoxM1 plasma levels in the xenografted mice were also evaluated using ELISA at 0, 10, 20, and 30 days.

Results

Inhibition of FoxM1 with thiostrepton reduced the cell viability of both ibrutinib-sensitive (Jeko-1; SP-49; PT-1; PT-2) cell lines and patient samples as well as ibrutinib-resistant MCL cells (Maver-1; Z138; Jeko-BTK KD 1 and 2; PTs 3-6) at half-maximal inhibitory concentration (IC₅₀) in the micromolar range (IC₅₀ = 1-3 μM) for the majority of tested cells and patient samples. siFoxM1 significantly reduced (P < 0.05) stromal cell-mediated migration of both Jeko-1 WT and BTK KD cells compared with the cells transfected with control siRNA. Moreover, siFoxM1-treated cells showed reduced levels of the migration- and adhesion-related proteins snail, vimentin, and N-cadherin compared with the control cells. Additionally, in comparison to vehicle-treated control mice, thiostrepton treatment significantly reduced tumor volume (36% and 17%, respectively) and β2M levels (71% and 79%, respectively) in Jeko-1 and Jeko-1 BTK KO xenografted mice at Day 30 of treatment regardless of BTK status. Lastly, thiostrepton treatment significantly suppressed the plasma levels of FoxM1 in both Jeko-1 (14%) and Jeko-1 BTK KO (11%) xenografted mice at Day 30 of treatment.

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 vivo studies. Therefore, exploring the role of FoxM1 in MCL disease progression and therapeutic resistance may lead to novel therapeutic breakthroughs to improve patient clinical outcomes.