Novel FOXM1 inhibitor identified via gene network analysis induces autophagic FOXM1 degradation to overcome chemoresistance of human cancer cells

FOXM1 transcription factor is an oncogene and a master regulator of chemoresistance in multiple cancers. Pharmacological inhibition of FOXM1 is a promising approach but has proven to be challenging. We performed a network-centric transcriptomic analysis to identify a novel compound STL427944 that selectively suppresses FOXM1 by inducing the relocalization of nuclear FOXM1 protein to the cytoplasm and promoting its subsequent degradation by autophagosomes. Human cancer cells treated with STL427944 exhibit increased sensitivity to cytotoxic effects of conventional chemotherapeutic treatments (platinum-based agents, 5-fluorouracil, and taxanes). RNA-seq analysis of STL427944-induced gene expression changes revealed prominent suppression of gene signatures characteristic for FOXM1 and its downstream targets but no significant changes in other important regulatory pathways, thereby suggesting high selectivity of STL427944 toward the FOXM1 pathway. Collectively, the novel autophagy-dependent mode of FOXM1 suppression by STL427944 validates a unique pathway to overcome tumor chemoresistance and improve the efficacy of treatment with conventional cancer drugs.

Exosomes of Adipose-derived Stem Cells Conditioned Media Promotes Retinoblastoma and Forkhead-Box M1 Protein Expression

BACKGROUND: In the senescence process, the retinoblastoma (Rb) protein binds to E2F in hypophosphorylated conditions, preventing the cell to enter the S-phase in the cell cycle. Human Forkhead Box M1 (FOXM1) protein, key regulator G1/S and G2/M phases, decreases in the senescence process. Many studies have been carried out to reverse this system, one of which used exosomes of adipose-derived stem c ells conditioned media (ADSC-CM). These exosomes contain a variety of specific proteins which have pro-proliferation properties, however, little is known on the role of these exosomes toward the change of phosphorylated Rb and FOXM1. AIM: This study aims to find out the involvement of exosomes of ADSC-CM on these two proteins on senescence human dermal fibroblasts (HDFs). METHODS: In vitro experiment was undergone randomization sample and non-blinded pre-/post-test control group. The primary culture of senescent HDFs was transfected with exosomes of ADSC-CM; then, its effect on migration and senescence reversal was observed through analyzing Sa-β-gal, Rb, and FOXM1 protein expression. RESULTS: The expression of Sa-β-gal was higher in the control group. Our result demonstrated the exosome of ADSC-CM significantly induced the expression of Rb and FOXM1 protein in senescent HDFs (p < 0.05). CONCLUSION: It proved that exosomes of ADSC-CM could shift the senescent fibroblast into metabolically active cells.

Destabilization of FoxM1 and Inhibition of Topoisomerase I Contribute to Cytotoxicity of Prenylated Xanthones Isolated from Metaxya rostrata

We recently isolated the prenylated xanthones 2-deprenyl-rheediaxanthone B (XB) and 2-deprenyl-7-hydroxy-rheediaxanthone B (OH-XB) from the South American tree fern Metaxya rostrata. This study explores the mechanisms underlying the FoxM1 downregulation induced by both xanthones. Analysis of cell viability and cell-death induction in SW480, HCT116, Caco-2, DLD1 and HT29 exposed to xanthones found cell-loss and activation of caspase in all cell lines except HT29 that do not have high FoxM1 protein levels. To determine the cellular mechanism of xanthone-induced FoxM1 loss, protein stability was analyzed by cycloheximide-chase experiments and showed reduction of FoxM1 stability by XB but not OH-XB. Destabilization was prevented by inhibiting proteasome activity using MG-132 and moderately by the lysosomal inhibitor bafilomycin A1 (baf A1). OH-XB had a stronger impact than XB on FoxM1 mRNA expression by qRT-PCR, and MG-132 positively affected FoxM1 protein level in OH-XB exposed cells even though no decrease in protein abundance had been induced by the xanthone. Additionally, the compound inhibited topoisomerase I causing DNA DSB and early cell cycle arrest. This may reduce FoxM1 gene expression, which may in turn compromise DNA repair and enhance xanthone-induced cell death. With regard to xanthone-induced cell death, MG-132 protected cultures from cell loss induced by both compounds, and baf A1 was active against these XB-induced effects. In summary, both destabilization of FoxM1 protein and topoisomerase I inhibition contribute to both XB and OH-XB cytotoxic activity albeit at different ratios.

Suppression of FOXM1 activities and breast cancer growth in vitro and in vivo by a new class of compounds

The transcription factor FOXM1 is upregulated and overexpressed in aggressive, therapy-resistant forms of hormone receptor-positive and triple negative breast cancers, and is associated with less good patient survival. FOXM1 signaling is also a key driver in many other cancers. Here, we identify a new class of compounds effective in suppressing FOXM1 activity in breast cancers, and displaying good potency for antitumor efficacy. The compounds bind directly to FOXM1 and alter its proteolytic sensitivity, reduce the cellular level of FOXM1 protein by a proteasome- dependent process, and suppress breast cancer cell proliferation and cell cycle progression and increase apoptosis. RNA-seq and gene set enrichment analyses indicate that the compounds decrease expression of FOXM1-regulated genes and suppress gene ontologies under FOXM1 regulation. Several compounds have favorable pharmacokinetic properties and show good tumor suppression in preclinical breast tumor models. These compounds may be suitable for further clinical evaluation in targeting aggressive breast cancers driven by FOXM1.

Inhibition of FOXM1 By Ixazomib Confers Chemosensitivity in NPM1-Wild Type Acute Myeloid Leukemia

Background: FOXM1 represents an attractive therapeutic target owing to selective up-regulation in dividing cells. Proteasome inhibitors target FOXM1 by inhibiting FOXM1 through the stabilization of HSP70, which is the negative regulator of FOXM1 {Halasi. J Biol Chem. 2016}. Ixazomib has previously been shown to induce cell death in NPM1-mutant AML cells by oxidative stress {Garcia, Clin Cancer Research 2016} and is currently being tested in NPM1-mutant AML patients as a single agent. We have shown that FOXM1 is inactive in NPM1- mutant AML (Bhat UG. J Bio; Chem 2012) supporting an alternative mechanism of action such as reactive oxygen species induction. In this abstract we propose a FOXM1 dependent mechanism of action in NPM1-wild type AML. We demonstrate that the novel proteasome inhibitor ixazomib is an inhibitor of FOXM1 and can enhance sensitivity to standard chemotherapy drug cytarabine in AML. With its favorable toxicity profile and oral route of administration, this is an attractive option to study as a chemosensitizing drug in leukemia. Materials and Methods: Ixazomib was purchased from Selleck Chemicals. The doxycycline-inducible U2OS-derived C3-luc cell line expressing FOXM1-dependent firefly luciferase was developed in Dr. Gartel's lab (Radhakrishnan. Cancer Res. 2006). KG-1, MV4-11 and HL60 cell lines were purchased from ATCC. Patient samples were collected at the University of Illinois Cancer Center after informed consent using an IRB approved protocol. Results: Using the C3-luc cell line, we studied the effect of ixazomib on FOXM1 transcriptional activity. Cells were treated with 1μg/ml doxycycline and ixazomib doses from 300-1000 nM overnight. The luciferase activity was determined by the Luciferase Assay System (Promega). We demonstrate significant dose-dependent inhibition of FOXM1 transcriptional activity at doses as low as 300nM. Inhibition of FOXM1 protein expression was then confirmed in NPM1-wild type AML cells. We analyzed KG-1, MV4-11 and HL60 cell lines by immunoblotting for FOXM1 protein after 24 hour incubation with ixazomib at doses ranging from 10-1000 nM. Given the well-studied role of FOXM1 in mediating chemoresistance, we studied the effect of ixazomib on increasing sensitivity of AML cells to the chemotherapeutic agent cytarabine. The MTS assay was used to calculate the IC50 of ixazomib in KG-1 and MV4-11 cells. The IC50 for KG1 cells was 38 nM (95% CI: 18-78nM) and for MV4-11 cells it was 27nM (95% CI: 16-45nM). We then used a range of doses of ixazomib (0-100nM) in combination with cytarabine at a fixed ratio in AML cell lines to assess cell proliferation using the MTS assay. A combination index (CI) was calculated using CompuSyn and the combination was synergistic in inhibiting proliferation in KG-1 and MV4-11 cells at all doses tested. Inhibition of FOXM1 expression with combination therapy was confirmed by immunoblotting and was accompanied by increased caspase-3 cleavage after 24 hour exposure to the drug. Finally, to establish that this anti-leukemic effect is dependent on proteasome inhibition, we show a rescue effect with N-Acetyl L-Cysteine, which antagonizes the activity of proteasome inhibitors (Halasi. Biochemical J. 2013). In primary NPM1-wild type AML mononuclear cells, treatment with ixazomib ex vivo for 24 hours in liquid culture resulted in dose dependent cell death measured by flow cytometry and caspase-3 cleavage. We are currently investigating FOXM1 expression and transcriptional activity in primary AML cells treated with ixazomib. Conclusion: Ixazomib demonstrates anti-neoplastic activity against NPM1-wild type AML and potentiates the effect of standard chemotherapy cytarabine. We present a novel mechanism of action whereby ixazomib, through its effect as a proteasome inhibitor, suppresses FOXM1 an oncogenic transcription factor that confers chemoresistance in AML. We are performing animal experiments to confirm the in vitro data and link the anti-leukemic activity of ixazomib with suppression of FOXM1. Our findings lay the groundwork for a target driven trial in AML. Using a novel, well-tolerated oral proteasome inhibitor to target FOXM1 we would expect increased efficacy with lower doses of the chemotherapy drug cytarabine in the treatment of NPM1-wild type AML. Disclosures No relevant conflicts of interest to declare.