Acute myeloid leukemia (AML) patients with NPM1 mutations demonstrate a superior response to standard chemotherapy treatment. Our previous work has shown that these favorable outcomes are linked to ...the cytoplasmic relocalization and inactivation of FOXM1 driven by mutated NPM1. Here, we went on to confirm the important role of FOXM1 in increased chemoresistance in AML. A multiinstitution retrospective study was conducted to link FOXM1 expression to clinical outcomes in AML. We establish nuclear FOXM1 as an independent clinical predictor of chemotherapeutic resistance in intermediate-risk AML in a multivariate analysis incorporating standard clinicopathologic risk factors. Using colony assays, we show a dramatic decrease in colony size and numbers in AML cell lines with knockdown of FOXM1, suggesting an important role for FOXM1 in the clonogenic activity of AML cells. In order to further prove a potential role for FOXM1 in AML chemoresistance, we induced an FLT3-ITD-driven myeloid neoplasm in a FOXM1-overexpressing transgenic mouse model and demonstrated significantly higher residual disease after standard chemotherapy. This suggests that constitutive overexpression of FOXM1 in this model induces chemoresistance. Finally, we performed proof-of-principle experiments using a currently approved proteasome inhibitor, ixazomib, to target FOXM1 and demonstrated a therapeutic response in AML patient samples and animal models of AML that correlates with the suppression of FOXM1 and its transcriptional targets. Addition of low doses of ixazomib increases sensitization of AML cells to chemotherapy backbone drugs cytarabine and the hypomethylator 5-azacitidine. Our results underscore the importance of FOXM1 in AML progression and treatment, and they suggest that targeting it may have therapeutic benefit in combination with standard AML therapies.
Targeting FOXM1 in cancer Halasi, Marianna; Gartel, Andrei L.
Biochemical pharmacology,
03/2013, Letnik:
85, Številka:
5
Journal Article
Recenzirano
Oncogenic transcription factor FOXM1 is overexpressed in the majority of human cancers. In addition, FOXM1 has been implicated in cell migration, invasion, angiogenesis and metastasis. The important ...role of FOXM1 in cancer affirms its significance for therapeutic intervention. Current data suggest that targeting FOXM1 in mono- or combination therapy may have promising therapeutic benefits for the treatment of cancer. However, challenges with the delivery of anti-FOXM1 siRNA to tumors and the absence of small molecules, which specifically inhibit FOXM1, are delaying the development of FOXM1 inhibitors as feasible anticancer drugs. In this review, we describe and summarize the efforts that have been made to target FOXM1 in cancer and the consequences of FOXM1 suppression in human cancer cells.
Nanoparticle-encapsulated thiazole antibiotic, thiostrepton, has been shown to be an effective agent for inhibiting tumor growth in solid tumor models through the inhibition of proteasomal activity ...by the induction of apoptosis in cancer cells. Here, we show the efficacy of thiostrepton-micelles in inhibiting tumor growth in a DEN/PB-induced liver cancer model. We also demonstrate an enhanced anticancer effect of the combination treatment of thiostrepton with bortezomib, another proteasome inhibitor in this liver cancer model.
According to epidemiological research, skin autoimmune diseases are more prevalent among black Americans. We postulated that pigment-producing melanocytes may contribute to local immune regulation in ...the microenvironment. We examined murine epidermal melanocytes in vitro to determine the role of pigment production in immune responses mediated by dendritic cell (DC) activation. Our study revealed that darkly pigmented melanocytes produce more IL-3 and the pro-inflammatory cytokines, IL-6 and TNF-α, and consequently induce plasmacytoid DC (pDC) maturation. Additionally, we demonstrate that low pigment-associated fibromodulin (FMOD) interferes with cytokine secretion and subsequent pDC maturation.
Cancer cells are generally more sensitive to anticancer drugs than normal cells. This provides the rationale for using anticancer drugs specifically against tumor cells, but the explanation for the ...specificity is often elusive. In this study, we compared the sensitivity of normal BJ human fibroblasts, BJ fibroblasts with p53 knockdown and corresponding BJ immortal/oncogenic cell lines with inactivated p53 to anticancer drug-induced apoptosis. We found that only normal cells that have wild-type p53 were resistant to the thiazole antibiotic, thiostrepton, suggesting that p53 plays an antiapoptotic role in normal cells. Therefore, in this case p53 status, but not the transformation of cells per se determines their sensitivity to thiostrepton and possibly to other anticancer drugs. Since p53 is mutated in 50% of human cancers, thiostrepton may selectively kill cancer, but not normal cells. These data imply that wild-type p53 can protect normal cells from anticancer drug-induced cell death and its mutations may sensitize cancer cells to anti-neoplastic agents.
Skin pigmentation has been linked to the development, prevalence, and severity of several immune-mediated diseases such as SLE. Here, we asked whether fibromodulin (FMOD), which is highly expressed ...in skin with light complexion, can explain the known differences in the magnitude of inflammation. C57 mice with different levels of pigmentation and FMOD were injected with human lupus serum to induce skin inflammation. Histopathologic studies revealed that black C57 FMOD+/+ that produce low levels of FMOD and white C57 FMOD −/− mice develop more severe inflammation compared with white FMOD +/+ mice. This study also revealed that dark pigmentation and FMOD deletion correlates with the increased numbers of Langerhans cells. Altogether, we identify low pigmentation and FMOD are linked to low severity of inflammation and approaches to promote FMOD expression should offer clinical benefit.
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.
No relevant conflicts of interest to declare.
The oncogenic transcription factor FOXM1 is overexpressed in the majority of human cancers, and it is a potential target for anticancer therapy. We identified proteasome inhibitors as the first type ...of drugs that target FOXM1 in cancer cells. Here we found that HSP90 inhibitor PF-4942847 and heat shock also suppress FOXM1. The common effector, which was induced after treatment with proteasome and HSP90 inhibitors or heat shock, was the molecular chaperone HSP70. We show that HSP70 binds to FOXM1 following proteotoxic stress and that HSP70 inhibits FOXM1 DNA-binding ability. Inhibition of FOXM1 transcriptional autoregulation by HSP70 leads to the suppression of FOXM1 protein expression. In addition, HSP70 suppression elevates FOXM1 expression, and simultaneous inhibition of FOXM1 and HSP70 increases the sensitivity of human cancer cells to anticancer drug-induced apoptosis. Overall, we determined the unique and novel mechanism of FOXM1 suppression by proteasome inhibitors.
Tumor cells accumulate high level of reactive oxygen species (ROS) because they are metabolically more active than normal cells. Elevated ROS levels increase tumorigenecity but also render cancer ...cells more vulnerable to oxidative stress than normal cells. The oncogenic transcription factor Forkhead Box M1 (FOXM1), which is overexpressed in a wide range of human cancers, was reported to protect cancer cells from the adverse effects of oxidative stress by up regulating the expression of scavenger enzymes. We therefore hypothesized that the combination of FOXM1 ablation and ROS inducers could selectively eradicate cancer cells. We show that RNA interference–mediated knockdown of FOXM1 further elevates intracellular ROS levels and increases sensitivity of cancer cells to ROS-mediated cell death after treatment with ROS inducers. We also demonstrate that the combination of ROS inducers with FOXM1/proteasome inhibitors induces robust apoptosis in different human cancer cells. In addition, we show evidence that FOXM1/proteasome inhibitor bortezomib in combination with the ROS inducer β-phenylethyl isothiocyanate efficiently inhibits the growth of breast tumor xenografts in nude mice. We conclude that the combination of ROS inducers and FOXM1 inhibitors could be used as a therapeutic strategy to selectively eliminate cancer cells.
PDF
