FoxM1 Inhibitor Research Articles

Novel benzothiazole/benzothiazole thiazolidine-2,4-dione derivatives as potential FOXM1 inhibitors: In silico, synthesis, and in vitro studies.

The oncogenic transcription factor FOXM1 overexpressed in breast and other solid cancers, is a key driver of tumor growth and progression through complex interactions, making it an attractive molecular target for the development of targeted therapies. Despite the availability of small-molecule inhibitors, their limited specificity, potency, and efficacy hinder clinical translation. To identify effective FOXM1 inhibitors, we synthesized novel benzothiazole derivatives (KC10-KC13) and benzothiazole hybrids with thiazolidine-2,4-dione (KC21-KC36). These compounds were evaluated for FOXM1 inhibition. Molecular docking and molecular dynamics simulation analysis revealed their binding patterns and affinities for the FOXM1-DNA binding domain. The interactions with key amino acids such as Asn283, His287, and Arg286, crucial for FOXM1 inhibition, have been determined with the synthesized compounds. Additionally, the molecular modeling study indicated that KC12, KC21, and KC30 aligned structurally and interacted similarly to the reference compound FDI-6. In vitro studies with the MDA-MB-231 breast cancer cell line demonstrated that KC12, KC21, and KC30 significantly inhibited FOXM1, showing greater potency than FDI-6, with IC50 values of 6.13, 10.77, and 12.86 µM, respectively, versus 20.79 µM for FDI-6. Our findings suggest that KC12, KC21, and KC30 exhibit strong activity as FOXM1 inhibitors and may be suitable for in vivo animal studies.

Senescent lung-resident mesenchymal stem cells drive pulmonary fibrogenesis through FGF-4/FOXM1 axis

BackgroundIdiopathic pulmonary fibrosis (IPF) is an age-related disease featured with abnormal fibrotic response and compromised lung function. Cellular senescence is now considered as an essential driving mechanism for IPF. Given the poor knowledge of the mechanisms underpinning IPF progression, understanding the cellular processes and molecular pathways is critical for developing effective therapies of IPF.MethodsLung fibrosis was induced using bleomycin in C57BL/6 mice. Cellular senescence was measured by immunofluorescence. The effects of FGF-4 on fibroblast activation markers and signaling molecules were assessed with western blot and qPCR.ResultsWe demonstrated elevated abundance of senescent mesenchymal stem cells (MSCs) in IPF lung tissues, which was tightly correlated with the severity of pulmonary fibrosis in vivo. In addition, senescent MSCs could effectively induce the phenotype of pulmonary fibrosis both in vitro and in vivo. To further confirm how senescent MSCs regulate IPF progression, we demonstrate that FGF-4 is significantly elevated in senescent MSCs, which can induce the activation of pulmonary fibroblasts. In vitro, FGF-4 can activate Wnt signaling in a FOXM1-dependent manner. Inhibition of FOXM1 via thiostrepton effectively impairs FGF-4-induced activation of pulmonary fibroblast and dramatically suppresses the development of pulmonary fibrosis.ConclusionThese findings reveal that FGF-4 plays a crucial role in senescent MSCs-mediated pulmonary fibrogenesis, and suggests that strategies aimed at deletion of senescent MSCs or blocking the FGF-4/FOXM1 axis could be effective in the therapy of IPF.

Cystathionine γ lyase Attenuates Vascular Smooth Muscle Cell Senescence via Foxm1-Gas1 Pathway to Mediate Arterial Stiffness.

Aims Arterial stiffness, a hallmark of vascular aging, significantly contributes to hypertension and impaired organ perfusion. Vascular smooth muscle cell (VSMC) dysfunction, particularly VSMC senescence and its interaction with stiffness, is crucial in the pathogenesis of arterial stiffness. Although hydrogen sulfide (H2S) and its key enzyme cystathionine γ-lyase (CSE) are known to play roles in cardiovascular diseases, their effects on arterial stiffness are not well understood. Methods & Results First, we observed a downregulation of CSE/H2S in the aortic media during biological aging and Angiotensin II (AngII)-induced aging. The VSMC-specific CSE knockout mice were created by loxp-cre (Tagln-cre) system, and which exacerbated AngII-induced aortic aging and stiffness in vivo and VSMC senescence and stiffness in vitro. Conversely, the CSE agonist norswertianolin mitigated these effects. Next, we identified growth arrest-specific 1 (Gas1) as a crucial target of CSE/H2S and found it to be a downstream target gene of forkhead box protein M1 (Foxm1). siRNA knockdown Foxm1 increased Gas1 transcription and reduced the protective effects of H2S on VSMC senescence and stiffness. Finally, we demonstrated that CSE/H2S sulfhydrates Foxm1 at the C210 site, regulating its nuclear translocation and activity, thus reducing VSMC senescence and stiffness. Innovation Our findings highlight the protective role of CSE/H2S in arterial stiffness, emphasizing the novel contributions of CSE, Gas1, and Foxm1 to VSMC senescence and stiffness. Conclusion Endogenous CSE/H2S in VSMCs reduces VSMC senescence and stiffness, thereby attenuating arterial stiffness and aging, partly through sulfhydration-mediated activation of Foxm1 and subsequent inhibition of Gas1 signaling pathways.

Interplay Between FoxM1 and Dab2 Promotes Endothelial Cell Responses in Diabetic Wound Healing.

Diabetes mellitus can cause impaired and delayed wound healing, leading to lower extremity amputations; however, the mechanisms underlying the regulation of vascular endothelial growth factor (VEGF)-dependent angiogenesis remain uncertain and could reveal new therapeutic targets. In our study, the molecular underpinnings of endothelial dysfunction in diabetes were investigated, focusing on the roles of Disabled-2 (Dab2) and Forkhead Box M1 (FoxM1) in VEGF receptor 2 (VEGFR2) signaling and endothelial cell (EC) function. Bulk RNA-sequencing analysis identified significant downregulation of Dab2 in high concentrations glucose treated primary mouse skin ECs, simulating hyperglycemic conditions in diabetes mellitus. In diabetic mice with a genetic EC deficiency of Dab2 angiogenesis was reduced in vivo and in vitro when compared with wild-type mice. Restoration of Dab2 expression by injected mRNA-containing lipid nanoparticles rescued impaired angiogenesis and wound healing in diabetic mice. At the same time, FoxM1 was downregulated in skin ECs subjected to high glucose conditions as determined by RNA-sequencing analysis. FoxM1 was found to bind to the Dab2 promoter, regulating its expression and influencing VEGFR2 signaling. The FoxM1 inhibitor FDI-6 reduced Dab2 expression and phosphorylation of VEGFR2. These findings indicate that restoring Dab2 expression through targeted therapies can enhance angiogenesis and wound repair in diabetes. To explore this therapeutic potential, we tested LyP-1-conjugated lipid nanoparticles (LNPs) containing Dab2 or control mRNAs to target ECs and found the former significantly improved wound healing and angiogenesis in diabetic mice. This study provides evidence of the crucial roles of Dab2 and FoxM1 in diabetic endothelial dysfunction and establishes targeted delivery as a promising treatment for diabetic vascular complications.

FOXM1 affects oxidative stress, mitochondrial function, and the DNA damage response by regulating p21 in aging oocytes

Fertilization capacity and embryo survival rate are decreased in postovulatory aging oocytes, which results in a reduced reproductive rate in female animals. However, the key regulatory genes and related regulatory mechanisms involved in the process of postovulatory aging in oocytes remain unclear. In this study, RNA-Seq revealed that 3237 genes were differentially expressed in porcine oocytes between the MII and aging stages (MII + 24 h). The expression level of FOXM1 was increased at the aging stage, and FOXM1 was also observed to be enriched in many key biological processes, such as cell senescence, response to oxidative stress, and transcription, during porcine oocyte aging. Previous studies have shown that FOXM1 is involved in the regulation of various biological processes, such as oxidative stress, DNA damage repair, mitochondrial function, and cellular senescence, which suggests that FOXM1 may play a crucial role in the process of postovulatory aging. Therefore, in this study, we investigated the effects and mechanisms of FOXM1 on oxidative stress, mitochondrial function, DNA damage, and apoptosis during oocyte aging. Our study revealed that aging oocytes exhibited significantly increased ROS levels and significantly decreased GSH, SOD, T-AOC, and CAT levels than did oocytes at the MII stage and that FOXM1 inhibition exacerbated the changes in these levels in aging oocytes. In addition, FOXM1 inhibition increased the levels of DNA damage, apoptosis, and cell senescence in aging oocytes. A p21 inhibitor alleviated the effects of FOXM1 inhibition on oxidative stress, mitochondrial function, and DNA damage and thus alleviated the degree of senescence in aging oocytes. These results indicate that FOXM1 plays a crucial role in porcine oocyte aging. This study contributes to the understanding of the function and mechanism of FOXM1 during porcine oocyte aging and provides a theoretical basis for preventing oocyte aging and optimizing conditions for the in vitro culture of oocytes.

Abstract We023: P53 Activation Promotes Maturational Characteristics of Pluripotent Stem Cell-derived Cardiomyocytes in 3D Suspension Culture via FOXO-FOXM1 Regulation

Background: Current protocols can generate highly-pure populations of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate key characteristics of mature in vivo cardiomyocytes. Yet, there exists a risk of arrhythmias when hiPSC-CMs are injected into large animal models. This prompts further investigation into the mechanisms of hiPSC-CM maturation to facilitate clinical translation. Hypothesis: The forkhead box (FOX) family of transcription factors can regulate maturation in neonatal cardiomyocytes through a balance between FOXO and FOXM1. We also previously found that p53 activation could enhance hiPSC-CM maturation. Therefore, we hypothesized that p53 activation increases FOXO and decreases FOXM1 to promote hiPSC-CM maturation in three-dimensional (3D) suspension culture. Methodology: 3D cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator), LOM612 (FOXO activator), AS1842856 (FOXO inhibitor), or RCM-1 (FOXM1 inhibitor), starting 2 days after onset of beating. The hiPSC-CMs were assessed for maturation in metabolic, contractile, and electrophysiological properties, by Seahorse mito stress test, Multi electrode array, and VALA kinetic image cytometer respectively. Results: P53 activation promoted FOXO upregulation and FOXM1 downregulation in hiPSC-CMs, measured by RT-qPCR and immunostaining. Alongside this, p53 activation also promoted hiPSC-CM metabolic and contractile maturational characteristics, seen by increase in oxygen consumption and beat amplitude respectively. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2 and eliminated spontaneous beating. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics of hiPSC-CMs such as increase in contractility, oxygen consumption, and voltage peak maximum upstroke velocity. Further, by single-cell RNAseq, FOXO activated groups showed increase in cardiac maturational pathways compared against DMSO control groups. Conclusions: These results show that p53 activation promotes FOXO and suppresses FOXM1, which modulate hiPSC-CM maturation in 3D suspension. Our study expands current understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3D culture system.

Resistance to FOXM1 inhibitors in breast cancer is accompanied by impeding ferroptosis and apoptotic cell death

PurposeCancer treatments often become ineffective because of acquired drug resistance. To characterize changes in breast cancer cells accompanying development of resistance to inhibitors of the oncogenic transcription factor, FOXM1, we investigated the suppression of cell death pathways, especially ferroptosis, in FOXM1 inhibitor-resistant cells. We also explored whether ferroptosis activators can synergize with FOXM1 inhibitors and can overcome FOXM1 inhibitor resistance.MethodsIn estrogen receptor-positive and triple-negative breast cancer cells treated with FOXM1 inhibitor NB73 and ferroptosis activators dihydroartemisinin and JKE1674, alone and in combination, we measured suppression of cell viability, motility, and colony formation, and monitored changes in gene and protein pathway expressions and mitochondrial integrity.ResultsGrowth suppression of breast cancer cells by FOXM1 inhibitors is accompanied by increased cell death and alterations in mitochondrial morphology and metabolic activity. Low doses of FOXM1 inhibitor strongly synergize with ferroptosis inducers to reduce cell viability, migration, colony formation, and expression of proliferation-related genes, and increase intracellular Fe+2 and lipid peroxidation, markers of ferroptosis. Acquired resistance to FOXM1 inhibition is associated with increased expression of cancer stem-cell markers and proteins that repress ferroptosis, enabling cell survival and drug resistance. Notably, resistant cells are still sensitive to growth suppression by low doses of ferroptosis activators, effectively overcoming the acquired resistance.ConclusionDelineating changes in viability and cell death pathways that can overcome drug resistance should be helpful in determining approaches that might best prevent or reverse resistance to therapeutic targeting of FOXM1 and ultimately improve patient clinical outcomes.

P53 Activation Promotes Maturational Characteristics of Pluripotent Stem Cell-Derived Cardiomyocytes in 3-Dimensional Suspension Culture Via FOXO-FOXM1 Regulation.

Current protocols generate highly pure human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) invitro that recapitulate characteristics of mature invivo cardiomyocytes. Yet, a risk of arrhythmias exists when hiPSC-CMs are injected into large animal models. Thus, understanding hiPSC-CM maturational mechanisms is crucial for clinical translation. Forkhead box (FOX) transcription factors regulate postnatal cardiomyocyte maturation through a balance between FOXO and FOXM1. We also previously demonstrated that p53 activation enhances hiPSC-CM maturation. Here, we investigate whether p53 activation modulates the FOXO/FOXM1 balance to promote hiPSC-CM maturation in 3-dimensional suspension culture. Three-dimensional cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator, 10 μM), LOM612 (FOXO relocator, 5 μM), AS1842856 (FOXO inhibitor, 1 μM), or RCM-1 (FOXM1 inhibitor, 1 μM), starting 2 days after onset of beating, with dimethyl sulfoxide (0.2% vehicle) as control. P53 activation promoted hiPSC-CM metabolic and electrophysiological maturation alongside FOXO upregulation and FOXM1 downregulation, in n=3 to 6 per group for all assays. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics such as increased contractility, oxygen consumption, and voltage peak maximum upstroke velocity, in n=3 to 6 per group for all assays. Further, by single-cell RNA sequencing of n=2 LOM612-treated cells compared with dimethyl sulfoxide, LOM612-mediated FOXO activation promoted expression of cardiac maturational pathways. We show that p53 activation promotes FOXO and suppresses FOXM1 during 3-dimensional hiPSC-CM maturation. These results expand our understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3-dimensional culture system.

A drug repurposing study identifies novel FOXM1 inhibitors with in vitro activity against breast cancer cells

FOXM1, a proto-oncogenic transcription factor, plays a critical role in cancer development and treatment resistance in cancers, particularly in breast cancer. Thus, this study aimed to identify potential FOXM1 inhibitors through computational screening of drug databases, followed by in vitro validation of their inhibitory activity against breast cancer cells. In silico studies involved pharmacophore modeling using the FOXM1 inhibitor, FDI-6, followed by virtual screening of DrugBank and Selleckchem databases. The selected drugs were prepared for molecular docking, and the crystal structure of FOXM1 was pre-processed for docking simulations. In vitro studies included MTT assays to assess cytotoxicity, and Western blot analysis to evaluate protein expression levels. Our study identified Pantoprazole and Rabeprazole as potential FOXM1 inhibitors through in silico screening and molecular docking. Molecular dynamics simulations confirmed stable interactions of these drugs with FOXM1. In vitro experiments showed both Pantoprazole and Rabeprazole exhibited strong FOXM1 inhibition at effective concentrations and that showed inhibition of cell proliferation. Rabeprazole showed the inhibitor activity at 10 µM in BT-20 and MCF-7 cell lines. Pantoprazole exhibited FOXM1 inhibition at 30 µM and in BT-20 cells and at 70 µM in MCF-7 cells, respectively. Our current study provides the first evidence that Rabeprazole and Pantoprazole can bind to FOXM1 and inhibit its activity and downstream signaling, including eEF2K and pEF2, in breast cancer cells. These findings indicate that rabeprazole and pantoprazole inhibit FOXM1 and breast cancer cell proliferation, and they can be used for FOXM1-targeted therapy in breast or other cancers driven by FOXM1.Graphical

Expression of Forkhead Box M1 and Anticancer Effects of FOXM1 Inhibition in Epithelioid Sarcoma

Epithelioid sarcoma (ES) is a rare aggressive sarcoma that, unlike most soft-tissue sarcomas, shows a tendency toward local recurrence and lymph node metastasis. Novel antitumor agents are needed for ES patients. Forkhead box transcription factor 1 (FOXM1) is a member of the Forkhead transcription factor family and is associated with multiple oncogenic functions; FOXM1 is known to be overexpressed and correlated with pathogenesis in various malignancies. In this study, we immunohistochemically analyzed FOXM1 expression levels and their clinical, clinicopathologic, and prognostic significance in 38 ES specimens. In addition, to investigate potential correlations between FOXM1 downregulation and oncologic characteristics, we treated ES cell lines with thiostrepton, a naturally occurring antibiotic that inhibits both small interfering RNA (siRNA) and FOXM1. In the analyses using ES samples, all 38 specimens were diagnosed as positive for FOXM1 by immunohistochemistry. We separated specimens into high (n = 19) and low (n = 19) FOXM1–protein expression groups by staining index score, and into large (n = 12), small (n = 25), and unknown (n = 1) tumor-size groups using a cutoff of 5 cm maximum diameter. Although there were significantly more samples with high FOXM1 expression in the large tumor group (P = .013), there were no significant differences with respect to age (P = 1.00), sex (P = .51), primary site of origin (P = .74), histologic subtypes (P = 1.00), depth (P = .74), or survival rate (P = .288) between the high and low FOXM1–protein expression groups. In the in vitro experiments using ES cell lines, FOXM1 siRNA and thiostrepton successfully downregulated FOXM1 mRNA and protein expression. Furthermore, downregulation of FOXM1 inhibited cell proliferation, drug resistance against chemotherapeutic agents, migration, and invasion and caused cell cycle arrest in the ES cell lines. Finally, cDNA microarray analysis data showed that FOXM1 regulated cIAP2, which is one of the apoptosis inhibitors activated by the TNFα-mediated NF-κB pathway. In conclusion, the FOXM1 gene may be a promising therapeutic target for ES.

Long non-coding RNA (CMR) involved in autoprotection in S. aureus mastitis in dairy cows by regulating miR-877/FOXM1

Long non-coding RNAs (LncRNAs) are dysregulated in a variety of human diseases and are highly involved in the development and progression of tumors. Studies on lncRNAs associated with cow mastitis have been lagging behind compared to humans or model animals, therefore, the aim of this study was to explore the mechanism of LncRNAs (CMR) involved in autoprotection against S. aureus mastitis in Bovine Mammary Epithelial Cells (BMECs). First, qRT-PCR was used to examine the relative expression of CMR in a S. aureus mastitis model of BMECs. Then, cell proliferation and apoptosis were detected by EdU and apoptosis assay. Finally, the targeting relationship between miRNAs and mRNA/LncRNAs was determined by dual luciferase reporter gene, qRT-PCR and western blotting techniques. The results showed that CMR was upregulated in the S. aureus mastitis model of BMECs and promoted the expression of inflammatory factors, and SiRNA-mediated CMR inhibited the proliferation of mammary epithelial cells and induced apoptosis. Mechanistically, CMR acts as a competitive endogenous RNA (ceRNA) sponge miR-877, leading to upregulation of FOXM1, a target of miR-877. Importantly, either miR-877 overexpression or FOXM1 inhibition abrogated CMR knockdown-induced apoptosis promoting cell proliferation and reducing inflammatory factor expression levels. In summary, CMR is involved in the regulation of autoprotection against S. aureus mastitis through the miR-877/FOXM1 axis in BMECs and induces immune responses in mammary tissues and cells of dairy cows, providing an important reference for subsequent prevention and control of cow mastitis and the development of targeted drugs.

Structure-Based Virtual Screening Identifying Novel FOXM1 Inhibitors as the Lead Compounds for Glioblastoma.

Glioblastoma multiforme (GBM) is a highly heterogeneous brain tumor with limited treatment options and a poor prognosis. Cancer stem cells (CSCs) have emerged as a critical factor in GBM resistance and management, contributing to tumor growth, heterogeneity, and immunosuppression. The transcription factor FOXM1 has been identified as a key player in the progression, spread, and therapy resistance of various cancers, including GBM. In this research, the objective was to perform structure-based in silico screening with the aim of identifying natural compounds proficient in targeting the DNA-binding domain (DBD) of the FOXM1 protein. In this study, in silico tools were employed for screening a hundred naturally occurring compounds capable of targeting the FOXM1 protein. Through molecular docking analysis and pharmacokinetic profiling, five compounds were found to be promising candidates for extensive interaction with the FOXM1 protein. Further, these compounds were validated for the stability of the FOXM1-natural compound complex using molecular dynamics (MD) simulations. Four compounds, such as Withaferin A, Bryophyllin A, Silybin B, Sanguinarine and Troglitazone (control compound), emerged as promising candidates with substantial interactions with FOXM1, suggesting their potential as a protein inhibitor based on molecular docking investigations. After MD simulation analysis, the FOXM1- Bryophyllin A complex was found to maintain the highest stability, and the other three ligands had moderate but comparable binding affinities over a period of 100 ns. This study provides valuable insights into four promising FOXM1 inhibitors that have the ability to induce senescence in GBM stem cells. These findings contribute to the development of structure-based designing strategies for FOXM1 inhibitors and innovative therapeutic approaches for the treatment of Glioblastoma.

Activation of the FOXM1/ASF1B/PRDX3 axis confers hyperproliferative and antioxidative stress reactivity to gastric cancer

Nucleosome assembly during DNA replication is dependent on histone chaperones. Recent studies suggest that dysregulated histone chaperones contribute to cancer progression, including gastric cancer (GC). Further studies are required to explore the prognostic and therapeutic implications of histone chaperones and their mechanisms of action in GC progression. Here we identified histone chaperone ASF1B as a potential biomarker for GC proliferation and prognosis. ASF1B was significantly upregulated in GC, which was associated with poor prognosis. In vitro and in vivo experiments demonstrated that the inhibition of ASF1B suppressed the malignant characteristics of GC, while overexpression of ASF1B had the opposite effect. Mechanistically, transcription factor FOXM1 directly bound to the ASF1B-promoter region, thereby regulating its transcription. Treatment with thiostrepton, a FOXM1 inhibitor, not only suppressed ASF1B expression, but also inhibited GC progression. Furthermore, ASF1B regulated the mitochondrial protein peroxiredoxin 3 (PRDX3) transcription in a FOXM1-dependent manner. The crucial role of ASF1B-regulated PRDX3 in GC cell proliferation and oxidative stress balance was also elucidated. In summary, our study suggests that the FOXM1-ASF1B-PRDX3 axis is a potential therapeutic target for treating GC.

Abstract 4611: The co-targeting of PLK1 and FoxM1 contributes to synergistic antitumor effects in PTC

Abstract Polo-like Kinase 1 (PLK1) plays a key role in cell proliferation with vital regulatory functions in mitosis and cell survival. We investigated the expression of PLK1 protein in a large cohort of PTC patient’s samples and explored its functional role in PTC cell lines in vitro and in vivo. PLK1 overexpression was observed in 54% of all PTC and was significantly associated with aggressive clinico-pathological parameter and it was found to be independent prognostic marker for recurrence-free survival. PLK1 inhibition indeed inhibited cell proliferation and induced cell cycle arrest and apoptosis in PTC cell lines. Importantly, silencing of PLK1 decreases the self-renewal ability of spheroids generated from PTC cells. Immunoprecipitation analysis shows that PLK1 binds to FoxM1 and vice versa in vitro. Mechanistically, PLK1 knockdown suppresses FoxM1 expression, whereas inhibition of FoxM1 does not affect PLK1 expression, which suggests that PLK1 acts through the FoxM1 pathway. The combined inhibition of PLK1 with volasertib and FoxM1 with thiostrepton synergistically attenuated PTC cell growth in vitro and in vivo. Our findings suggest that co-targeting of PLK1 and FoxM1 could be a viable therapeutic strategy for treating patients with an aggressive subtype of PTC. Citation Format: Pratheeshkumar Poyil, Abdul K. Siraj, Divya Padmaja, Sandeep Kumar Parvathareddy, Saravanan Thangavel, Saif S. Al-Sobhi, Fouad Al-Dayel, Khawla S. Al-Kuraya. The co-targeting of PLK1 and FoxM1 contributes to synergistic antitumor effects in PTC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4611.

Abstract 642: Flavopiridol inhibits cell proliferation, migration, and invasion via downregulation of FOXM1 in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) represent 15-20% of all breast cancers and is the subtype of breast cancer that is not responsive to targeted breast cancer treatments such as anti-estrogens (tamoxifen) or HER2 targeted antibodies due to the lack of ER, PR, and HER2 receptors. TNBC is associated with poor prognosis, early relapse, and distant metastasis and drug resistance. With the use of standard chemotherapeutics in the neoadjuvant setting, unfortunately the majority of patients (~60%) with TNBC do not achieve complete remission and 40% of the patients with regional (Stage II) and 90% of the patients with distant metastasis (Stage IV) die within 5-years. Currently, there is no effective targeted therapy for TNBC patients for targeting oncogenic pathways. Although recently the FDA approved sacituzumab (govitecan), a Trop-2-receptor directed antibody conjugated with chemotherapeutic agent topoisomerase inhibitor in metastatic TNBC, and response rate was 30% and the median duration of response is 7.7 months, and the majority of patients did not maintain response longer than 12 months. Therefore, identifying novel molecular targets and developing alternative therapeutic strategies are urgently needed. FOXM1 is a protooncogenic transcription factor suppressed by p53 and drives expression of genes, which play a crucial role in promoting cancer cell proliferation, metastasis, progression, tumorigenesis of TNBC. We previously demonstrated for the first time that in vivo targeting of FOXM1 suppresses TNBC tumor growth in mice. Here, analyzing TCGA patient database we found that FOXM1 expression correlates with shorter patient survival and prognosis in TNBC patients. Overall data suggest that FOXM1 is a potential molecular target. However, currently, there is no FDA approved inhibitor of FOXM1. To identify potential inhibitors, utilizing silico docking and molecular dynamics studies we screened the FDA-approved compounds found that flavopiridol interacts with FOXM1. Flavopiridol is a small molecule inhibitor for CDKs and is approved by the FDA for the treatment of Acute Myeloid Leukemia. We demonstrated that flavodilol inhibits FOXM1 expression at nanomolar concentration and cell proliferation, migration, invasion, and induces apoptosis in TNBC cells. We are currently testing favopridol in MDA-MB-231 (human) and 4T1 mouse mammary) TNBC tumor models. In conclusion, our studies suggest that flavopiridol is a highly potent FOXM1 inhibitor and is a promising agent for repurposing to target FOXM1 and for the treatment of TNBC. Citation Format: Sayra Dilmac, Nermin Kahraman, Ferah Comert Onder, Zuhal Hamurcu, Bulent Ozpolat. Flavopiridol inhibits cell proliferation, migration, and invasion via downregulation of FOXM1 in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 642.

Abstract B068: FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models

Abstract The unfavorable prognosis of high-grade serous ovarian cancer, HGSOC, may be driven by a small subset of ovarian cancer stem cells (OCSC), which are endowed with tumor-initiating ability and are intrinsically resistant to cytotoxic treatments. These characteristics are sustained by signals coming from the tumor microenvironment (TME). As a common site of HGSOC presentation is the omentum, we partially recapitulated the architecture of human omentum in vitro using 3D co-cultures of primary human omental fibroblasts and mesothelial cells. Patient-derived HGSOC cells were then integrated in these organotypic models, either as bulk cancer cells or as OCSC from the same patients. After co-culture in this model, malignant cells were re-isolated by FACS sorting and subjected to whole-transcriptome RNA sequencing, comparing them to control cells that have not been in contact with the in vitro TME. Our data revealed transcriptional reprogramming induced by the in vitro TME in HGSOC cells. We found a number of pathways differentially regulated in the bulk population of OC cells vs OCSC, shedding light on the impact of the TME on OC stemness. Moreover, we identified a panel of transcription factors (TFs) specifically altered by the in vitro TME in OCSC. These TF-regulated gene networks represent OCSC vulnerabilities and, hence, potential targets in relapsing tumors. Among these, we focused on FOXM1, a TF with pleiotropic functions in cancer cells, whose activity is altered in 84% of ovarian cancer patients. The FOXM1 pathway was strongly induced by the in vitro TME specifically in OCSC. Mechanistically, the contact with the in vitro TME triggered a FAK/YAP axis which, in turn, accounted for the induction of FOXM1 and its downstream target genes. Moreover, the pharmacological inhibition of FOXM1 significantly impaired OCSC fitness, supporting, on one hand, the key role of TME in orchestrating OC stemness and suggesting, on the other, a new approach for the eradication of OCSC from the omental niche. Overall, our patient-derived organotypic platform offers the opportunity to unravel the impact of the TME on OCSC at the molecular level in experimental models with clinical relevance and can be exploited to design innovative HGSOC-eradicating treatments. Citation Format: Chiara Battistini, Hilary A. Kenny, Valentina Nieddu, Valentina Melocchi, Alessandra Decio, Beatrice Malacrida, Alessia Gatto, Fabrizio Bianchi, Raffaella Giavazzi, Nicoletta Colombo, Frances R. Balkwill, Ernst Lengyel, Ugo Cavallaro. FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B068.

Abstract B010: Development of folate receptor alpha targeted FOXM1 inhibitors for ovarian cancer

Abstract FOXM1 is a transcription factor that plays a canonical role in cell cycle progression. In addition, FOXM1 contributes to diverse oncogenic phenotypes including cell invasion and metastasis, metabolic reprogramming, and cancer stemness. Notably, FOXM1 is the top biomarker of poor prognosis in human pan-cancer, is associated with poor prognosis in high-grade serous ovarian cancer (HGSOC) and is the most frequently activated oncogenic pathway in HGSOC. Together, these data implicate FOXM1 as a potential high impact therapeutic target in HGSOC and other cancers. In this context, several small molecule inhibitors (SMI) of FOXM1 have been described, and these include compounds that disrupt FOXM1 DNA binding and compounds that promote FOXM1 proteolytic degradation. Recently, we have identified and described a series of novel diarylethene-based compounds (i.e., NB compounds) that fall into the second class of molecules (FOXM1 degraders) and have shown that NB compounds are potent inhibitors of FOXM1 in cancer cells. The aims of the present study were two-fold: 1) investigate the activity of NB compounds using HGSOC cell models, and 2) develop methods to specifically deliver NB compounds to HGSOC cells, thus increasing drug efficacy and reducing potential toxicities. To address the first aim, we used gene and protein expression analyses and cell biological measurements. Our resulting data revealed that NB compounds are potent and efficacious FOXM1 inhibitors in HGSOC cells, leading to FOXM1 pathway suppression and robust induction of apoptosis. In addition, NB compounds displayed specificity to FOXM1 over other FOX family members and can be used in synergistic combinations with other relevant SMI. To address the second aim, we are developing nanomedicine approaches for the HGSOC-specific delivery of NB compounds. Specifically, we are generating NB compound-loaded polymersomes capable of targeting folate receptor alpha (FRα), which is highly expressed on the surface of HGSOC cells. The necessary polymers needed for the proposed nanoconstruct have been synthesized and characterized by nuclear magnetic resonance. Polymersomes have been formulated through emulsification and were characterized by dynamic light scattering. Drug loading, drug release, polymersome stability, transmission electron microscopy, and fluorescence spectroscopy studies are currently ongoing. Planned biological assays include cellular uptake, drug release, IC50, target engagement, and cytotoxicity studies in HGSOC cells with variable FRα expression. In summary, we provide data supporting the utilitity of NB compound FOXM1 inhibitors as HGSOC therapeutics and are optimizing NB compound use by directing the inhibitors to HGSOC cells using FRα-targeted nanomedicines. Citation Format: Adam R. Karpf, Makenzie Vorderbruggen, Pedram Rikhtechi, Catalina Muñoz-Trujillo, Cassie Liu, Larry H Matherly, Sung Hoon Kim, Benita S. Katzenellenbogen, John A. Katzenellenbogen, Jered C. Garrison. Development of folate receptor alpha targeted FOXM1 inhibitors for ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B010.

FOXM1 promotes the progression of non-small cell lung cancer by inhibiting miR-509-5p expression via binding to the miR-509-5p promoter region

BackgroundForkhead box M1 (FOXM1) functions as a transcription factor and is consistently overexpressed in various cancers, including non-small-cell lung-, breast-, cervical-, and colorectal cancer. Its overexpression is associated with poor prognosis in patients with non-small-cell lung cancer, although the detailed mechanisms by which FOXM1 promotes the development of non-small-cell lung cancer remain unclear. ObjectiveThe mechanism of FOXM1 in migration, invasion, apoptosis, and viability of lung cancer cells was investigated. MethodsTranswell assay, scratch test, and flow cytometry were employed to study the effects of FOXM1 on migration, invasion, and apoptosis in A549 cells. A quantitative polymerase chain reaction was used to determine the impact of FOXM1 on miR-509-5p expression in A549 cells. Dual-luciferase reporter gene assay and chromatin immunoprecipitation were adopted to investigate the molecular mechanisms of FOXM1 on miR-509-5p expression. ResultsFDI-6 (a FOXM1 inhibitor) reduced the protein abundance of FOXM1, thereby increasing the expression of miR-509-5p in A549 cells. Moreover, FDI-6 treatment significantly reduced migration, invasion, and viability of A549 cells while promoting cell apoptosis. Furthermore, miR-509-5p inhibitor obviously alleviated the biological effects of FDI-6 on A549 cells, suggesting that FOXM1 primarily exerted its cancer promoting effect by regulating miR-509-5p. Mechanistically, FOXM1 directly bound to the miR-509-5p promoter to inhibit miR-509-5p expression. ConclusionFOXM1 directly binds to the promoter region of miR-509-5p to form a negative feedback loop, thereby inhibiting miR-509-5p expression and promoting the development of non-small-cell lung cancer. This study is expected to complement research on the pathogenesis of non-small-cell lung cancer and promote the development of novel therapeutic targets for this disease.

PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti-tumor effects.

Polo-like kinase 1 (PLK1; also known as serine/threonine-protein kinase PLK1) serves as a central player in cell proliferation, exerting critical regulatory roles in mitotic processes and cell survival. We conducted an analysis of PLK1 protein expression in a large cohort of samples from papillary thyroid carcinoma (PTC) patients and examined its functional significance in PTC cell lines, both in vitro and in vivo. PLK1 overexpression was noted in 54.2% of all PTC and was significantly associated with aggressive clinicopathological parameters; it was also found to be an independent prognostic marker for shorter recurrence-free survival. Given the significant association between PLK1 and forkhead box protein M1 (FoxM1), and their concomitant overexpression in a large proportion of PTC samples, we explored their correlation and their combined inhibitions in PTC in vitro and in vivo. Inhibition of PLK1 expression indeed suppressed cell proliferation, leading to cell cycle arrest and apoptosis in PTC cell lines. Significantly, the downregulation of PLK1 reduced the self-renewal capability of spheroids formed from PTC cells. Immunoprecipitation analysis shows that PLK1 binds to FoxM1 and vice versa in vitro. Mechanistically, PLK1 knockdown suppresses FoxM1 expression, whereas inhibition of FoxM1 does not affect PLK1 expression, which suggests that PLK1 acts through the FoxM1 pathway. The combined treatment of a PLK1 inhibitor (volasertib) and a FoxM1 inhibitor (thiostrepton) demonstrated a synergistic effect in reducing PTC cell growth in vitro and delaying tumor growth in vivo. This study highlights the important role of PLK1 in PTC tumorigenesis and prognosis. It also highlights the synergistic therapeutic potential of dual-targeting PLK1 and FoxM1 in PTC, unveiling a potential innovative therapeutic strategy for managing aggressive forms of PTC.

Genomic and Transcriptomic Landscape of Hyper-Apobec Multiple Myeloma

Introduction APOBEC mutational signatures are one of the most important mutational signatures in newly diagnosed multiple myeloma (NDMM). APOBEC mutagenesis primarily works through two enzymes, APOBEC3A (A3A) and APOBEC3B (A3B). While these genes are expressed in most NDMM patients, their influence on APOBEC mutagenesis appear to be extremely heterogeneous, with some patients having no detectable mutations and others having high contribution (hyper-APOBEC). Methods To decipher the mechanism of APOBEC mutagenesis, we interrogated 752 low coverage long insert whole genomes, 723 whole exomes (WES) and 767 RNA sequencing (RNA-Seq) from NDMM patients enrolled in the CoMMpass study. Results APOBEC mutational activity was identified in 416/723 (57.5%) patients using WES. Of these, 41 (5.8%) had MAF/MAFB translocations. Overall, 48/723 (6.6%) patients were defined as hyper-APOBEC (13 without MAF/MAFB events). We used differential expression (DE) analysis between three groups: hyper-APOBEC with MAF/MAFB (HA_TRA: n=31), hyper-APOBEC without MAF/MAFB (HA_NORM: n=8), and non-hyper-APOBEC without MAF/MAFB (WT: n=510). 534 genes were significantly associated with hyper-APOBEC independently from MAF/MAFB events. Importantly, A3A and A3B expression were higher in hyper-APOBEC compared to other cases (logFC=2.38, logFC=1.58 respectively). Using Spearman's correlation, we identified 50 significantly correlated genes (r-squared>0.18; p<0.01) with A3B. Most of the genes belonged to the APOBEC-associated group defined by the DE and were enriched for cell cycle/proliferation activity, suggesting a link between highly proliferative disease and hyper-APOBEC. To validate this finding, we evaluated the link between these APOBEC-associated genes and hyper-APOBEC among ICGC breast cancers, a tumor known to have high prevalence of APOBEC mutagenesis. Overall, 35/50 of these genes showed similar correlation between hyper-APOBEC breasts and MM. Interestingly, most of these genes appear to be negatively controlled by E2F4 (DREAM complex), and positively controlled by E2F1, FOXM1, and MYBL2 (i.e. cell cycle genes). In line with mouse models (Roelofs et al., eLife, 2020), our data confirmed that highly proliferating tumors suppress the DREAM complex leading to induction of A3B. Overall, these data suggest a strong link between proliferation and A3B expression and mutagenesis. However, not all high proliferating tumors had high APOBEC mutagenesis. To better investigate this aspect, we compared 139 known MM genomic events to identify potential differences between the hyper-APOBEC group (HA) and the rest of the cohort (WT). HA_TRA and HA_NORM showed similarities, including a highly complex genomic profile with enrichment for 1q gain/amp (p < 0.02), 13q (p < 0.05) and 16q deletions (p<0.01). Interestingly, 5/13 (38.4%) of HA_NORM samples harbor CCND1 translocation. In terms of gene expression, both groups showed enrichment for GEP70 indicating a high-risk transcriptional profile apart from MAF/MAFB overexpression. Finally, HA_TRA and HA_NORM showed similar poor outcomes compared to WT. To further validate the association between APOBEC and complex genomic profile, we explored again the ICGC breast WGS, observing a strong correlation between homologous recombination deficiency (HRD) and hyper-APOBEC (p < 0.05). To validate the relationship between the DREAM complex and APOBEC, we pharmacologically inhibited FOXM1 (FDI6, 20uM) and E2F4/6 (HLM00074, 20uM) in 8226, U266 and MM1.S multiple myeloma cell lines and showed increased A3B levels upon 72h of E2F4/6 inhibition. Simultaneously, FOXM1 inhibition confirmed A3B downregulation. Conclusion Overall, our data support a model where, APOBEC genes are variably expressed in all NDMM, but only patients with high proliferation rate and high level of complexity acquire mutations (Figure 1).