Targeting FOXM1 Transcription Factor In T-Cell Acute Lymphoblastic Leukemia

The Forkhead box protein M1(FoxM1) is an important  transcriptional factor that takes play in regulation of cell cyle, proliferation, DNA repair, apoptosis, and angiogenesis. FoxM1 overexpression has been reported to be related with many types of cancer. Since many studies have reported that FOXM1 is an important target for cancer therapy, many researchers are studying on the identification of FOXM1 inhibitors. Siomycin A, a thiazol antibiotic, is known to inhibit FoxM1 transcriptional activity. Dexamethasone is a glucocorticoid  that is very important in treatment of acute lymphoblastic leukemia (ALL) and is known to be more potent compared to other steroids in the treatment of T-cell ALL. In this study, our aims were to determine the gene expression levels of FoxM1 in Jurkat cells (T-ALL cell line), to find out the possible synergistic and apoptotic effects of siomycin A and dexamethasone on this cell line and to investigate the changes in expression profiles of some important genes that have vital roles in cellular processes by targeting FoxM1 with siomycin A and dexamethasone on Jurkat cells.

The gene expression levels of FoxM1 were studied with reverse transcriptase polymerase chain reaction (RT-PCR). The cytotoxic effects of siomycin  A and dexamethasone on Jurkat cells were assesed by MTT cell proliferation test.  The possible synergistic, additive, neutral, and antagonistic effect of combination of  dexamethasone and siomycin A was determined with isobologram analysis.  The apoptotic effects of these two agents were evaluated by  Caspase-3 activity, loss of mitochondrial membrane potential and localisation of phosphatidilserine on plasma membrane. For this purpose, Caspase-3 calorimetric assay kit, JC-1 mitochondrial membrane potential assay kit, and Annexin V-FITC apoptosis detection kit were used, respectively. For cell cycle analysis, Jurkat cells treated with siomycin A alone or in combination with dexamethasone were stained by propidium iodide and then analyzed by flow cytometry. Expression profiles of Jurkat cells treated with siomycin A alone or in combination with dexamethasone were determined by Cancer Pathway Finder PCR Array.

We found that FoxM1 gene is overexpressed in T-ALL cell line and dexamethasone and siomycin A caused a reduction in gene expression levels of FoxM1 in Jurkat cells. 8% to 13% decrease in proliferation of Jurkat cells were observed when these cells were treated with 1 and 10 µM doses of dexamethasone for 72 hours, respectively. The same doses of dexamethasone combined with siomycin A caused 74% and 75% decrease in proliferation of Jurkat cells. Isobologram analysis revealed very strong synergy between dexamethasone and siomycin A. Apoptotic tests showed no apoptotic activity of dexamethasone and siomycin A on Jurkat cells. Cell cycle analysis demonstrated that, reduction of FOXM1 expression by combination of dexamethasone and siomycin A in Jurkat cells inhibited cell proliferation through induction of G1 phase arrest. PCR Array results showed that apoptotic CASPASE-2, CASPASE-7, and CASPASE-9 genes and XIAP and CYCLIN D3 genes were upregulated in response to the treatment. ETS2 gene, which is known as a protooncogene and shown to be involved in regulation of telomerase, was downregulated in response to siomycin A and dexamethosone alone and in combined treatment. TERF1 gene, which inhibits telomerase activity, was upregulated by the treatment. Combination of Siomycin A and dexamethasone downregulated the MCM-2 gene, which is a key component of the pre-replication complex and involved in the formation of DNA replication fork. Moreover, combined treatment resulted in the downregulation of MKI67 gene encoding a nuclear protein associated with  cellular proliferation. WEE1 gene, which inhibits G2/M phase transition in cell cycle, was also upregulated.

These data indicate that FoxM1 gene is strongly overexpressed in T-ALL cell line and targeting FoxM1 by siomycin A and dexamethasone causes a significant decrease in T-ALL cell proliferation through induction of  G1 cell cycle arrest. Importantly, PCR array analyses also showed that siomycin A and dexamethasone treatment affects Jurkat cells via upregulating or downregulating the key genes of cell cycle, apoptosis, cell proliferation, telomere, and telomerase function. All these findings suggest a possible role for FoxM1 in T-ALL pathogenesis and represent FoxM1 as an attractive target for T-ALL therapy.