Novel FOXM1 inhibitor STL001 sensitizes human cancers to a broad-spectrum of cancer therapies.

Forkhead box protein M1 (FOXM1) is often overexpressed in human cancers and strongly associated with therapy resistance and less good patient survival. The chemotherapy options for patients with the most aggressive types of solid cancers remain very limited because of the acquired drug resistance, making the therapy less effective. NPM1 mutation through the inactivation of FOXM1 via FOXM1 relocalization to the cytoplasm confers more favorable treatment outcomes for AML patients, confirming FOXM1 as a crucial target to overcome drug resistance. Pharmacological inhibition of FOXM1 could be a promising approach to sensitize therapy-resistant cancers. Here, we explore a novel FOXM1 inhibitor STL001, a first-generation modification drug of our previously reported FOXM1 inhibitor STL427944. STL001 preserves the mode of action of the STL427944; however, STL001 is up to 50 times more efficient in reducing FOXM1 activity in a variety of solid cancers. The most conventional cancer therapies studied here induce FOXM1 overexpression in solid cancers. The therapy-induced FOXM1 overexpression may explain the failure or reduced efficacy of these drugs in cancer patients. Interestingly, STL001 increased the sensitivity of cancer cells to conventional cancer therapies by suppressing both the high-endogenous and drug-induced FOXM1. Notably, STL001 does not provide further sensitization to FOXM1-KD cancer cells, suggesting that the sensitization effect is conveyed specifically through FOXM1 suppression. RNA-seq and gene set enrichment studies revealed prominent suppression of FOXM1-dependent pathways and gene ontologies. Also, gene regulation by STL001 showed extensive overlap with FOXM1-KD, suggesting a high selectivity of STL001 toward the FOXM1 regulatory network. A completely new activity of FOXM1, mediated through steroid/cholesterol biosynthetic process and protein secretion in cancer cells was also detected. Collectively, STL001 offers intriguing translational opportunities as combination therapies targeting FOXM1 activity in a variety of human cancers driven by FOXM1.

Regulation of HOX gene expression in AML.

As key developmental regulators, HOX cluster genes have varied and context-specific roles in normal and malignant hematopoiesis. A complex interaction of transcription factors, epigenetic regulators, long non-coding RNAs and chromatin structural changes orchestrate HOX expression in leukemia cells. In this review we summarize molecular mechanisms underlying HOX regulation in clinical subsets of AML, with a focus on NPM1 mutated (NPM1mut) AML comprising a third of all AML patients. While the leukemia initiating function of the NPM1 mutation is clearly dependent on HOX activity, the favorable treatment responses in these patients with upregulation of HOX cluster genes is a poorly understood paradoxical observation. Recent data confirm FOXM1 as a suppressor of HOX activity and a well-known binding partner of NPM suggesting that FOXM1 inactivation may mediate the effect of cytoplasmic NPM on HOX upregulation. Conversely the residual nuclear fraction of mutant NPM has also been recently shown to have chromatin modifying effects permissive to HOX expression. Recent identification of the menin-MLL interaction as a critical vulnerability of HOX-dependent AML has fueled the development of menin inhibitors that are clinically active in NPM1 and MLL rearranged AML despite inconsistent suppression of the HOX locus. Insights into context-specific regulation of HOX in AML may provide a solid foundation for targeting this common vulnerability across several major AML subtypes.

Small-molecule inhibitors targeting FOXM1: Current challenges and future perspectives in cancer treatments.

Forkhead box (FOX) protein M1 (FOXM1) is a critical proliferation-associated transcription factor (TF) that is aberrantly overexpressed in the majority of human cancers and has also been implicated in poor prognosis. A comprehensive understanding of various aspects of this molecule has revealed its role in, cell proliferation, cell migration, invasion, angiogenesis and metastasis. The FOXM1 as a TF directly or indirectly regulates the expression of several target genes whose dysregulation is associated with almost all hallmarks of cancer. Moreover, FOXM1 expression is associated with chemoresistance to different anti-cancer drugs. Several studies have confirmed that suppression of FOXM1 enhanced the drug sensitivity of various types of cancer cells. Current data suggest that small molecule inhibitors targeting FOXM1 in combination with anticancer drugs may represent a novel therapeutic strategy for chemo-resistant cancers. In this review, we discuss the clinical utility of FOXM1, further, we summarize and discuss small-molecule inhibitors targeting FOXM1 and categorize them according to their mechanisms of targeting FOXM1. Despite great progress, small-molecule inhibitors targeting FOXM1 face many challenges, and we present here all small-molecule FOXM1 inhibitors in different stages of development. We discuss the current challenges and provide insights on the future application of FOXM1 inhibition to the clinic.

FOXM1-AKT Positive Regulation Loop Provides Venetoclax Resistance in AML.

Forkhead box protein M1 (FOXM1) is a crucial regulator of cancer development and chemoresistance. It is often overexpressed in acute myeloid leukemia (AML) and is associated with poor survival and reduced efficacy of cytarabine therapy. Molecular mechanisms underlying high FOXM1 expression levels in malignant cells are still unclear. Here we demonstrate that AKT and FOXM1 constitute a positive autoregulatory loop in AML cells that sustains high activity of both pro-oncogenic regulators. Inactivation of either AKT or FOXM1 signaling results in disruption of whole loop, coordinated suppression of FOXM1 or AKT, respectively, and similar transcriptomic changes. AML cells with inhibited AKT activity or stable FOXM1 knockdown display increase in HOXA genes expression and BCL2L1 suppression that are associated with prominent sensitization to treatment with Bcl-2 inhibitor venetoclax. Taken together, our data indicate that AKT and FOXM1 in AML cells should not be evaluated as single independent regulators but as two parts of a common FOXM1-AKT positive feedback circuit. We also report for the first time that FOXM1 inactivation can overcome AML venetoclax resistance. Thus, targeting FOXM1-AKT loop may open new possibilities in overcoming AML drug resistance and improving outcomes for AML patients.

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.

Therapeutic Vulnerabilities of Transcription Factors in AML.

Acute myeloid leukemia (AML) is characterized by impaired myeloid lineage differentiation, uncontrolled proliferation, and inhibition of proapoptotic pathways. In spite of a relatively homogeneous clinical disease presentation, risk of long-term survival in AML varies from 20% to 80% depending on molecular disease characteristics. In recognition of the molecular heterogeneity of AML, the European Leukemia Net (ELN) and WHO classification systems now incorporate cytogenetics and increasing numbers of gene mutations into AML prognostication. Several of the genomic AML subsets are characterized by unique transcription factor alterations that are highlighted in this review. There are many mechanisms of transcriptional deregulation in leukemia. We broadly classify transcription factors based on mechanisms of transcriptional deregulation including direct involvement of transcription factors in recurrent translocations, loss-of-function mutations, and intracellular relocalization. Transcription factors, due to their pleiotropic effects, have been attractive but elusive targets. Indirect targeting approaches include inhibition of upstream kinases such as TAK1 for suppression of NFκB signaling and downstream effectors such as FGF signaling in HOXA-upregulated leukemia. Other strategies include targeting scaffolding proteins like BrD4 in the case of MYC or coactivators such as menin to suppress HOX expression; disrupting critical protein interactions in the case of ß-catenin:TCF/LEF, and preventing transcription factor binding to DNA as in the case of PU.1 or FOXM1. We comprehensively describe the mechanism of deregulation of transcription factors in genomic subsets of AML, consequent pathway addictions, and potential therapeutic strategies.

FOXM1: a potential therapeutic target in human solid cancers.

Introduction: FOXM1 is one of the most frequently overexpressed proteins in human solid cancers. Here, we discuss novel direct targets of FOXM1 as well as new pathways involving FOXM1, through which this protein exerts its oncogenic activity.Areas covered: We give a detailed review of FOXM1 transcriptional targets involved in 16 different types of human cancer as published in the literature in the last 5 years. We also discuss a novel positive feedback loop between FOXM1 and AKT - both well-established master regulators of cancer.Expert opinion: Despite the discovery of several FOXM1 inhibitors over the years (by our team and others), their therapeutic use is limited by their adverse off-target effects.Newly-discovered proteins regulated by FOXM1 present a promising alternative approach to target its pro-cancer activity. In addition, targeting regulating proteins that take part in the positive feedback loop between FOXM1/AKT has the double advantage of suppressing both, and can lead to developing novel anti-cancer drugs.

FOXM1 contributes to treatment failure in acute myeloid leukemia.

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.

Honokiol is a FOXM1 antagonist.

Honokiol is a natural product and an emerging drug for a wide variety of malignancies, including hematopoietic malignancies, sarcomas, and common epithelial tumors. The broad range of activity of honokiol against numerous malignancies with diverse genetic backgrounds suggests that honokiol is inhibiting an activity that is common to multiple malignancies. Oncogenic transcription factor FOXM1 is one of the most overexpressed oncoproteins in human cancer. Here we found that honokiol inhibits FOXM1-mediated transcription and FOXM1 protein expression. More importantly, we found that honokiol's inhibitory effect on FOXM1 is a result of binding of honokiol to FOXM1. This binding is specific to honokiol, a dimerized allylphenol, and was not observed in compounds that either were monomeric allylphenols or un-substituted dihydroxy phenols. This indicates that both substitution and dimerization of allylphenols are required for physical interaction with FOXM1. We thus demonstrate a novel and specific mechanism for FOXM1 inhibition by honokiol, which partially may explain its anticancer activity in cancer cells.

FOXM1 in Cancer: Interactions and Vulnerabilities.

FOXM1 is a transcription factor of the Forkhead family that is required for cell proliferation of normal cells. However, FOXM1 is repeatedly overexpressed in a variety of human cancers, and it has been implicated in all major hallmarks of cancer delineated by Hanahan and Weinberg. It has been postulated that the oncogenic potential of FOXM1 is determined by its capacity to transactivate target genes that are implicated in different phases of cancer development. However, FOXM1 may also play an oncogenic role by interacting with other proteins, such as ß-catenin or SMAD3 to induce oncogenic WNT and TGFß signaling pathways, respectively. In this review, I will discuss the protein-protein interactions of FOXM1 that are critical for cancer development and may represent novel targets for anticancer drugs. Cancer Res; 77(12); 3135-9. ©2017 AACR.

A Novel Function of Molecular Chaperone HSP70: SUPPRESSION OF ONCOGENIC FOXM1 AFTER PROTEOTOXIC STRESS.

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.

Mutual Regulation of FOXM1, NPM and ARF Proteins.

ARF, NPM and FOXM1 proteins interact with each other in mammalian cells. We showed previously that proteasome inhibitors suppress not only FOXM1 expression, but also the expression of ARF and NPM proteins. Using RNA interference we found that the depletion of each of these proteins by RNAi in human cancer HeLa cells leads to down-regulation of the two other partners, suggesting that these proteins stabilize each other in human cancer cells. Since the suppression of FOXM1 is one of hallmarks of proteasome inhibition, suppression of ARF and NPM by proteasome inhibitors may be explained in part as a secondary effect of downregulation of FOXM1 that modulate stability of ARF and NPM1 proteins.

Proteasome inhibitors suppress the protein expression of mutant p53.

Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53.

Suppression of the Oncogenic Transcription Factor FOXM1 by Proteasome Inhibitors.

The oncogenic transcription factor FOXM1 is one of the key regulators of tumorigenesis. We found that FOXM1 upregulates its own transcription and its protein stability depends on its interaction with the chaperone nucleophosmin. We also determined that FOXM1 is negatively regulated by the tumor suppressor p53. We identified the thiazole antibiotics Siomycin A and thiostrepton as inhibitors of transcriptional activity and FOXM1 expression via proteasome inhibition. In addition, we found that all tested proteasome inhibitors target FOXM1. We showed synergy between thiostrepton and bortezomib in different human cancer cell lines and in vivo. We generated isogenic human cancer cell lines of different origin with wild-type p53 or p53 knockdown and we demonstrated that proteasome inhibitors induce p53-independent apoptosis in these cells. Using RNA-interference or proteasome inhibitors to inhibit FOXM1 we found that suppression of FOXM1 sensitized human cancer cells to apoptosis induced by DNA-damaging agents or oxidative stress. We encapsulated thiostrepton into micelle-nanoparticles and after injection we detected accumulation of nanoparticles in tumors and in the livers of treated mice. This treatment led to inhibition of human xenograft tumor growth in nude mice. Our data indicate that targeting FOXM1 increases apoptosis and inhibits tumor growth.

ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors.

NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H2O2. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.

Combination of oxidative stress and FOXM1 inhibitors induces apoptosis in cancer cells and inhibits xenograft tumor growth.

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 ß-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.