Forkhead Family Of Transcription Factors Research Articles

Identification of a FOXO gene and its roles in anti-WSSV infection through regulation of Dicers and Argos in Macrobrachium nipponense

Forkhead box O (FOXO) proteins are a subgroup of the forkhead family of transcription factors that play important roles in the immune response. In this study, we cloned and identified a FOXO gene named MnFOXO from Macrobrachium nipponense. The full-length cDNA of MnFOXO is 2086 bp and contains a 1302 bp open reading frame, which encodes 433 amino acids. MnFOXO consists of five low-complexity regions and a conserved DNA-binding domain (forkhead domain). Evolutionary analyses indicate that MnFOXO proteins cluster with FOXO proteins from crustaceans. Tissue distribution analysis showed that MnFOXO was expressed in all detected tissues, with relatively higher expression levels in the intestine, eyestalks, stomach, and hemocytes than in the hepatopancreas, gills, and heart. The expression levels of MnFOXO in the hepatopancreas and intestine were significantly up-regulated in M. nipponense infected with white spot syndrome virus (WSSV) at 24 and 48 h. Furthermore, knockdown of MnFOXO increased the expression of WSSV envelope protein VP28 during WSSV infection. Further studies showed that knockdown of the MnFOXO gene in M. nipponense inhibited the synthesis of Dicers (MnDicer1, MnDicer2) and Argonautes (MnArgo1, MnArgo2) during WSSV invasion. These findings suggest that MnFOXO positively regulates the expression of Dicers and Argos, and inhibits the expression of VP28. This study provides new evidence for understanding the role of FOXO in antiviral innate immunity in crustaceans.

Metformin Prevents Tumor Cell Growth and Invasion of Human Hormone Receptor-Positive Breast Cancer (HR+ BC) Cells via FOXA1 Inhibition.

Women with type 2 diabetes (T2D) have a higher risk of being diagnosed with breast cancer and have worse survival than non-diabetic women if they do develop breast cancer. However, more research is needed to elucidate the biological underpinnings of these relationships. Here, we found that forkhead box A1 (FOXA1), a forkhead family transcription factor, and metformin (1,1-dimethylbiguanide hydrochloride), a medication used to treat T2D, may impact hormone-receptor-positive (HR+) breast cancer (BC) tumor cell growth and metastasis. Indeed, fourteen diabetes-associated genes are highly expressed in only three HR+ breast cancer cell lines but not the other subtypes utilizing a 53,805 gene database obtained from NCBI GEO. Among the diabetes-related genes, FOXA1, MTA3, PAK4, FGFR3, and KIF22 were highly expressed in HR+ breast cancer from 4032 breast cancer patient tissue samples using the Breast Cancer Gene Expression Omnibus. Notably, elevated FOXA1 expression correlated with poorer overall survival in patients with estrogen-receptor-positive/progesterone-receptor-positive (ER+/PR+) breast cancer. Furthermore, experiments demonstrated that loss of the FOXA1 gene inhibited tumor proliferation and invasion in vitro using MCF-7 and T47D HR+ breast cancer cell lines. Metformin, an anti-diabetic medication, significantly suppressed tumor cell growth in MCF-7 cells. Additionally, either metformin treatment or FOXA1 gene deletion enhanced tamoxifen-induced tumor growth inhibition in HR+ breast cancer cell lines within an ex vivo three-dimensional (3D) organoid model. Therefore, the diabetes-related medicine metformin and FOXA1 gene inhibition might be a new treatment for patients with HR+ breast cancer when combined with tamoxifen, an endocrine therapy.

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.

Regulation of mitochondrial function by FOXOs in ischemic stroke and Alzheimer’s disease

Transcriptional control is a pivotal mechanism governing various cellular processes. FOXO proteins, a subgroup of the forkhead family of transcription factors, play a key role in determining cell fate. The localization and function of FOXO proteins are regulated by post-translational modifications to control target gene expression, with a pronounced impact on various aspects of mitochondrial function, including mitochondrial dynamics, biogenesis, and quality control. Mitochondria stand out as the primary target of FOXO transcription factors, which recruit downstream signaling factors to govern mitochondrial processes. Essential signaling pathways are modulated by FOXOs, exemplified by their regulation of mitochondrial biogenesis through SIRT1-Pgc1a and NRF1-TFAM, as well as their influence on mitochondrial dynamics involving Mfn1, Mfn2, Drp1, and Fis1. Furthermore, FOXOs demonstrate the ability to upregulate and downregulate genes that serve as modulators in oxidative and apoptosis cascades. The functional role of FOXO proteins is highly context-dependent, varying with cell type, organ, and specific FOXO isoform. Notably, FOXOs emerge as prominent players in various pathological conditions, including ischemic conditions, neurodegenerative diseases, cancer, and metabolic disorders. Unraveling the complex role of FOXOs in mammalian cell pathology positions them as promising therapeutic targets receptive to pharmacological treatment. This review aims to provide insights into the intricate roles of FOXOs in mitochondria, illuminating their potential as therapeutic targets amenable to pharmacological intervention in diverse pathological contexts, particularly in ischemic stroke and Alzheimer’s disease.

Forkhead box E1, frequently downregulted by promoter methylation, inhibits colorectal cancer cell growth and migration

Forkhead box E1 (FOXE1), also known as thyroid transcription factor 2 (TTF-2), belongs to a large family of forkhead transcription factors. It plays important roles in embryogenesis, cell growth, and differentiation. Cancer-specific FOXE1 hypermethylation events have been identified in several cancers. However, the expression and function of FOXE1 in the tumorigenesis of colorectal cancer remain still unknown. In this study, we examined FOXE1 expression and methylation in normal colon mucosa, colorectal cancer (CRC) cell lines, and primary tumors by immunohistochemistry, semi-quantitative RT-PCR, methylation-specific PCR, and bisulfite genomic sequencing. We found that FOXE1 was frequently methylated and silenced in CRC cell lines and was downregulated in CRC tissues compared with paired adjacent non-tumor tissues. Meanwhile, low FOXE1 expression was significantly correlated with lymph node metastasis and advanced TNM stages, indicating its potential as a tumor marker. Subsequently, we established colon cancer cell lines with stable FOXE1 expression to observe the biological effect on colorectal cancer, including cell growth, migration, actin cytoskeleton, and growth of human colorectal xenografts in nude mice. Ectopic expression of FOXE1 could suppress tumor cell growth and migration and affect the organization of the actin cytoskeleton together with suppressing tumorigenicity in vivo. FOXE1 methylation was frequently seen in association with a complete absence of or downregulated gene expression, and FOXE1 plays a suppressive role in the development and progression of colorectal cancer.

Exploring the Potential of Natural Products as FoxO1 Inhibitors: an In Silico Approach.

FoxO1, a member of the Forkhead transcription factor family subgroup O (FoxO), is expressed in a range of cell types and is crucial for various pathophysiological processes, such as apoptosis and inflammation. While FoxO1's roles in multiple diseases have been recognized, the target has remained largely unexplored due to the absence of cost-effective and efficient inhibitors. Therefore, there is a need for natural FoxO1 inhibitors with minimal adverse effects. In this study, docking, MMGBSA, and ADMET analyses were performed to identify natural compounds that exhibit strong binding affinity to FoxO1. The top candidates were then subjected to molecular dynamics (MD) simulations. A natural product library was screened for interaction with FoxO1 (PDB ID- 3CO6) using the Glide module of the Schrödinger suite. In silico ADMET profiling was conducted using SwissADME and pkCSM web servers. Binding free energies of the selected compounds were assessed with the Prime-MMGBSA module, while the dynamics of the top hits were analyzed using the Desmond module of the Schrödinger suite. Several natural products demonstrated high docking scores with FoxO1, indicating their potential as FoxO1 inhibitors. Specifically, the docking scores of neochlorogenic acid and fraxin were both below -6.0. These compounds also exhibit favorable drug-like properties, and a 25 ns MD study revealed a stable interaction between fraxin and FoxO1. Our findings highlight the potential of various natural products, particularly fraxin, as effective FoxO1 inhibitors with strong binding affinity, dynamic stability, and suitable ADMET profiles.

Variants in FOXC1 and FOXC2 identified in patients with conotruncal heart defects

Conotruncal heart defects (CTD), subtypes of congenital heart disease, result from abnormal cardiac outflow tract development (OFT). FOXC1 and FOXC2 are closely related members of the forkhead transcription factor family and play essential roles in the development of OFT. We confirmed their expression pattern in mouse and human embryos, identifying four variants in FOXC1 and three in FOXC2 by screening these two genes in 605 patients with sporadic CTD. Western blot demonstrated expression levels, while Dual-luciferase reporter assay revealed affected transcriptional abilities for TBX1 enhancer in two FOXC1 variants and three FOXC2 variants. This might result from the altered DNA-binding abilities of mutant proteins. These results indicate that functionally impaired FOXC1 and FOXC2 variants may contribute to the occurrence of CTD.

Abstract B057: Oncogenic Foxl2 directs enhancer reprogramming and changes in 3D genome structure in ovarian granulosa cell tumors

Abstract Background: Adult-type granulosa cell tumors (aGCTs) are a rare monogenic disease. Nearly all aGCTs carry a missense point mutation in the Forkhead domain containing FOXL2 (Foxl2 p.C134W) transcription factor, but the oncogenic mechanism of this mutation is unknown. Other Forkhead family transcription factors have well-described “pioneer” activity, binding to compacted, nucleosome-bound DNA and increasing accessibility for other regulatory proteins. Objectives: To investigate if oncogenic Foxl2-C134W has pioneering activity and if mutant Foxl2 operates its oncogenic activity through enhancer reprogramming. Methods: CRISPR/Cas9 editing was used to generate isogenic granulosa cell tumor cells lacking either the FOXL2 wild-type allele (single knock-out; SKO) or both the mutant and wild-type FOXL2 alleles (double knock-out; DKO). ATAC-Seq and endogenous Foxl2 ChIP-Seq were performed on these isogenic lines to determine the differential chromatin accessibility at Foxl2-bound regulatory regions across genotypes. Promoter capture HiC was performed using a bespoke genome-wide hybrid capture probe set, CHiCAGO was used to identify significant HiC interactions, and an activity-by-contact model was constructed to identify enhancer elements bound by endogenous Foxl2-C134W and determine their target promoter contact frequency (promoter capture HiC) and enhancer activity (geometric mean of H3K27ac and ATACseq signal. Results: Endogenous ChIP-seq of Foxl2-C134W from the SKO cell line identified 1147 high-confidence peaks. De novo motif discovery identified the canonical Foxl2 binding motif (TGTTTACATT, P = 1.4 × 10−7) in 44.9% of peaks, as well as a novel variant motif (TGTTTTGTCT, P = 6.7 × 10−15) in 68.5% of peaks. Median chromatin accessibility at Foxl2-C134W peak regions, as measured by ATAC-seq, was significantly decreased in the DKO cells compared to either SKO or parental cell lines (P < 2 × 10-16 for both comparisons). Decreased chromatin accessibility at Foxl2-C134W ChIP-seq peaks in the DKO cells was driven by peaks containing the novel variant Foxl2 binding motif. Promoter capture HiC through activity-by-contact model identified promoter-enhancer interactions lost in the DKO cell line involving several genes, including ADAMTS5, FOXF2, and PRICKLE1 (adjusted P < 0.05). Conclusion: Foxl2-C134W exhibits “pioneering” activity, increasing chromatin accessibility at key gene regulatory elements with associated re-programming of promoter-enhancer interactions. The oncogenic mechanism of Foxl2-C134W in granulosa cell tumors may involve changes in DNA binding specificity, re-directing this pioneering function to sites containing a novel variant Foxl2 binding motif. Citation Format: Veena K. Vuttaradhi, Eleonora Khlebus, Thomas Welte, Barrett Lawson, Robert Tyler Hillman. Oncogenic Foxl2 directs enhancer reprogramming and changes in 3D genome structure in ovarian granulosa cell tumors [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 B057.

Targeting Estrogen Receptor Beta Ameliorates Diminished Ovarian Reserve via Suppression of the FOXO3a/Autophagy Pathway.

Diminished ovarian reserve (DOR) refers to a decrease in the number and/or quality of oocytes, leading to infertility, poor ovarian response and adverse pregnancy outcomes. Currently, the pathogenesis of DOR is largely unknown, and the efficacy of existing therapeutic methods is limited. Therefore, in-depth exploration of the mechanism underlying DOR is highly important for identifying molecular therapeutic targets for DOR. Our study showed that estrogen receptor beta (ERβ) mRNA and protein expression was upregulated in granulosa cells (GCs) from patients with DOR and in the ovaries of DOR model mice. Mechanistically, elevated ERβ promotes forkhead transcription factor family 3a (FOXO3a) expression, which contributes to autophagic activation in GCs. Activation of FOXO3a/autophagy signalling leads to decreased cell proliferation and increased cell apoptosis and ultimately leads to DOR. In a cyclophosphamide (Cy)-induced DOR mouse model, treatment with PHTPP, a selective ERβ antagonist, rescued fertility by restoring normal sex hormone secretion, estrus cycle duration, follicle development, oocyte quality and litter size. Taken together, these findings reveal a pathological mechanism of DOR based on ERβ overexpression and identify PHTPP as a potential therapeutic agent for DOR.

Neuroprotective role of FOXA1 in Parkinson's disease: Involvements of NF1 transcription activation and MAPK signaling pathway inhibition

Forkhead box A1 (FOXA1), a member of the forkhead family of transcription factors, plays a crucial role in the development of various organ systems and exhibits neuroprotective properties. This study aims to investigate the effect of FOXA1 on Parkinson's disease (PD) and unravel the underlying mechanism. Transcriptome analysis of PD was conducted using three GEO datasets to identify aberrantly expressed genes. A mouse model of PD was generated by injecting neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), resulting in reduced FOXA1 expression. FOXA1 decline was also observed in 1-methyl-4-phenylpyridinium-treated SH-SY5Y cells. Artificial upregulation of FOXA1 improved motor abilities of mice according to rotarod and pole tests, and it mitigated tissue damage, cell loss, and neuronal damage in the mouse substantia nigra or in vitro. FOXA1 was found to bind to the neurofibromin 1 (NF1) promoter, thereby inducing its transcription and inactivating the mitogen-activated protein kinase (MAPK) signaling pathway. Further experimentation revealed that silencing NF1 in mice or SH-SY5Y cells counteracted the neuroprotective effects of FOXA1. In conclusion, this research suggests that FOXA1 activates NF1 transcription and inactivates the MAPK signaling pathway, ultimately ameliorating neuronal damage and motor disability in PD. The findings may offer novel ideas in the field of PD management.

FKBP3 aggravates the malignant phenotype of diffuse large B-cell lymphoma by PARK7-mediated activation of Wnt/β-catenin signalling.

Diffuse large B-cell lymphoma (DLBCL) is difficult to treat due to the high recurrence rate and therapy intolerance, so finding potential therapeutic targets for DLBCL is critical. FK506-binding protein 3 (FKBP3) contributes to the progression of various cancers and is highly expressed in DLBCL, but the role of FKBP3 in DLBCL and its mechanism are not clear. Our study demonstrated that FKBP3 aggravated the proliferation and stemness of DLBCL cells, and tumour growth in a xenograft mouse model. The interaction between FKBP3 and parkinsonism associated deglycase (PARK7) in DB cells was found using co-immunoprecipitation assay. Knockdown of FKBP3 enhanced the degradation of PARK7 through increasing its ubiquitination modification. Forkhead Box O3 (FOXO3) belongs to the forkhead family of transcription factors and inhibits DLBCL, but the underlying mechanism has not been reported. We found that FOXO3 bound the promoter of FKBP3 and then suppressed its transcription, eventually weakening DLBCL. Mechanically, FKBP3 activated Wnt/β-catenin signalling pathway mediated by PARK7. Together, FKBP3 increased PARK7 and then facilitated the malignant phenotype of DLBCL through activating Wnt/β-catenin pathway. These results indicated that FKBP3 might be a potential therapeutic target for the treatment of DLBCL.

The forkhead DNA-binding domain binds specific G2-rich RNA sequences

Abstract Transcription factors contain a DNA-binding domain ensuring specific recognition of DNA target sequences. The family of forkhead (FOX) transcription factors is composed of dozens of paralogs in mammals. The forkhead domain (FHD) is a segment of about 100 amino acids that binds an A-rich DNA sequence. Using DNA and RNA PCR-SELEX, we show that recombinant FOXL2 proteins, either wild-type or carrying the oncogenic variant C134W, recognize similar DNA-binding sites. This suggests that the oncogenic variant does not alter the intrinsic sequence-specificity of FOXL2. Most importantly, we show that FOXL2 binds G2-rich RNA sequences whereas it virtually fails to bind similar sequences in DNA chemistry. Interestingly, a statistically significant subset of genes responding to the knock-down of FOXL2/Foxl2 harbor such G2-rich sequences and are involved in crucial signaling pathways and cellular processes. In addition, we show that FOXA1, FOXO3a and chimeric FOXL2 proteins containing the FHD of the former are also able to interact with some of the preferred FOXL2-binding sequences. Our results point to an unexpected and novel characteristic of the forkhead domain, the biological relevance of which remains to be explored.

Foxi3GFP and Foxi3CreER mice allow identification and lineage labeling of pharyngeal arch ectoderm and endoderm, and tooth and hair placodes.

FOXI3 is a forkhead family transcription factor that is expressed in the progenitors of craniofacial placodes, epidermal placodes, and the ectoderm and endoderm of the pharyngeal arch region. Loss of Foxi3 in mice and pathogenic Foxi3 variants in dogs and humans cause a variety of craniofacial defects including absence of the inner ear, severe truncations of the jaw, loss or reduction in external and middle ear structures, and defects in teeth and hair. To allow for the identification, isolation, and lineage tracing of Foxi3-expressing cells in developing mice, we targeted the Foxi3 locus to create Foxi3GFP and Foxi3CreER mice. We show that Foxi3GFP mice faithfully recapitulate the expression pattern of Foxi3 mRNA at all ages examined, and Foxi3CreER mice can trace the derivatives of pharyngeal arch ectoderm and endoderm, the pharyngeal pouches and clefts that separate each arch, and the derivatives of hair and tooth placodes. Foxi3GFP and Foxi3CreER mice are new tools that will be of use in identifying and manipulating pharyngeal arch ectoderm and endoderm and hair and tooth placodes.

Epigenetic activation of secretory phenotypes in senescence by the FOXQ1-SIRT4-GDH signaling

Although metabolic reprogramming is characterized as a hallmark of aging, implications of the crucial glutamate dehydrogenase (GDH) in human senescence remain poorly understood. Here, we report that GDH activity is significantly increased in aged mice and senescent human diploid fibroblasts. This enzymatic potentiation is associated with de-repression of GDH from its functionally suppressive ADP-ribosylation modification catalyzed by NAD-dependent ADP-ribosyltransferase/deacetylase SIRT4. A series of transcription analyses led to the identification of FOXQ1, a forkhead family transcription factor (TF), responsible for the maintenance of SIRT4 expression levels in juvenile cells. However, this metabolically balanced FOXQ1-SIRT4-GDH axis, is shifted in senescence with gradually decreasing expressions of FOXQ1 and SIRT4 and elevated GDH activity. Importantly, pharmaceutical inhibition of GDH suppresses the aberrantly activated transcription of IL-6 and IL-8, two major players in senescence-associated secretory phenotype (SASP), and this action is mechanistically associated with erasure of the repressive H3K9me3 (trimethylation of lysine 9 on histone H3) marks at IL-6 and IL-8 promoters, owing to the requirement of α-ketoglutaric acid (α-KG) from GDH-mediated glutamate dehydrogenase reaction as a cofactor for histone demethylation. In supplement with the phenotypic evidence from FOXQ1/SIRT4/GDH manipulations, these data support the integration of metabolism alterations and epigenetic regulation in driving senescence progression and highlight the FOXQ1-SIRT4-GDH axis as a novel druggable target for improving human longevity.

Network Pharmacology-Based Strategy to Reveal the Mechanism of Action of Cistanche Against Cataracts

Cistanche is known to relieve cataracts; however, the active ingredients and mechanism of action of cistanche to relieve cataracts are unclear. At present, the active ingredients of cistanche were screened through the Traditional Chinese Medicine Systems Pharmacology website, and a total of six active ingredients were identified. Drug and disease targets were obtained through Swiss target prediction and the Genecard website, and a total of 172 common targets were identified. The biological functions and Kyoto Encyclopedia of Genes and Genomes pathway enrichment of the targets were found to be concentrated in the phosphatidylinositol 3-kinase/protein kinase B, forkhead family of transcription factors, and mitogen-activated protein kinases pathways. The protein–protein interaction network was established, and phosphatidylinositol 3-kinase and protein kinase B-related genes were found to be the key genes. Molecular docking was carried out between the active ingredient and the key target, and it was found that the combination of the active ingredient and the key target was stable. In summary, cistanche can reduce cataracts by regulating the key targets of the active ingredients and thus alleviating cataracts.

Supraphysiological FOXP3 expression in human CAR-Tregs results in improved stability, efficacy, and safety of CAR-Treg products for clinical application

The forkhead family transcription factor (FOXP3) is an essential regulator for the development of regulatory T cells (Tregs) and orchestrates both suppressive function and Treg lineage identity. Stable expression of FOXP3 enables Tregs to maintain immune homeostasis and prevent autoimmunity. However, under pro-inflammatory conditions, FOXP3 expression in Tregs can become unstable, leading to loss of suppressive function and conversion into pathogenic T effector cells. Therefore, the success of adoptive cell therapy with chimeric antigen receptor (CAR) Tregs is highly dependent on the stability of FOXP3 expression to ensure the safety of the cell product. To warrant the stable expression of FOXP3 in CAR-Treg products, we have developed an HLA-A2-specific CAR vector that co-expresses FOXP3. The transduction of isolated human Tregs with the FOXP3-CAR led to an increase in the safety and efficacy of the CAR-Treg product. In a hostile microenvironment, under pro-inflammatory and IL-2-deficient conditions, FOXP3-CAR-Tregs showed a stable expression of FOXP3 compared to Control-CAR-Tregs. Furthermore, additional exogenous expression of FOXP3 did not induce phenotypic alterations and dysfunctions such as cell exhaustion, loss of functional Treg characteristics or abnormal cytokine secretion. In a humanized mouse model, FOXP3-CAR-Tregs displayed an excellent ability to prevent allograft rejection. Furthermore, FOXP3-CAR-Tregs revealed coherent Treg niche-filling capabilities. Overexpression of FOXP3 in CAR-Tregs has thereby the potential to increase the efficacy and reliability of cellular products, promoting their clinical use in organ transplantation and autoimmune diseases.

Soluble monomeric human programmed cell death-ligand 1 inhibits the functions of activated T cells.

The presence of soluble human programmed cell death-ligand 1 (shPD-L1) in the blood of patients with cancer has been reported to be negatively correlated with disease prognosis. However, little information exists about the mechanisms underlying high levels of shPD-L1 for promoting disease progression. In this study, we first analyzed the correlations between shPD-L1 and apoptosis of T cells in patients with cancer, then tested the effect of shPD-L1 on T-cell functions and the production of regulatory T cells. We found that the apoptosis of human peripheral PD-1+CD4+ T cells was significantly elevated in patients with cancer compared with healthy donors and was positively correlated with circulating PD-L1 levels in patients with cancer. In vitro, monomeric shPD-L1 significantly inhibited the proliferation, cytokine secretion, and cancer cell-killing activity of peripheral blood mononuclear cells (PBMCs) activated by either agonist antibodies or HATac (high-affinity T cell activation core)-NYE (NY-ESO-1 antigen). It also promoted CD4+ T cells to express forkhead family transcription factor 3 (FoxP3) for the conversion of induced T regulatory cells, which was more significant than that mediated by soluble human PD-L1 fusion protein (shPD-L1-Fc). These results confirm that soluble PD-L1 could be a candidate for inhibiting the functions of activated T cells, promoting peripheral tolerance to tumor cells, and implicating in system tumor immune escape in addition to the tumor microenvironment. This is an important mechanism explaining the negative correlation between peripheral blood PD-L1 levels and cancer prognosis. Therefore, understanding the roles of hPD-L1 in peripheral blood will be helpful for the development of precision immunotherapy programs in treating various tumors.

Targeting SWI/SNF ATPases in H3.3K27M diffuse intrinsic pontine gliomas

Diffuse midline gliomas (DMGs) including diffuse intrinsic pontine gliomas (DIPGs) bearing lysine-to-methionine mutations in histone H3 at lysine 27 (H3K27M) are lethal childhood brain cancers. These tumors harbor a global reduction in the transcriptional repressive mark H3K27me3 accompanied by an increase in the transcriptional activation mark H3K27ac. We postulated that H3K27M mutations, in addition to altering H3K27 modifications, reprogram the master chromatin remodeling switch/sucrose nonfermentable (SWI/SNF) complex. The SWI/SNF complex can exist in two main forms termed BAF and PBAF that play central roles in neurodevelopment and cancer. Moreover, BAF antagonizes PRC2, the main enzyme catalyzing H3K27me3. We demonstrate that H3K27M gliomas show increased protein levels of the SWI/SNF complex ATPase subunits SMARCA4 and SMARCA2, and the PBAF component PBRM1. Additionally, knockdown of mutant H3K27M lowered SMARCA4 protein levels. The proteolysis targeting chimera (PROTAC) AU-15330 that simultaneously targets SMARCA4, SMARCA2, and PBRM1 for degradation exhibits cytotoxicity in H3.3K27M but not H3 wild-type cells. AU-15330 lowered chromatin accessibility measured by ATAC-Seq at nonpromoter regions and reduced global H3K27ac levels. Integrated analysis of gene expression, proteomics, and chromatin accessibility in AU-15330-treated cells demonstrated reduction in the levels of FOXO1, a key member of the forkhead family of transcription factors. Moreover, genetic or pharmacologic targeting of FOXO1 resulted in cell death in H3K27M cells. Overall, our results suggest that H3K27M up-regulates SMARCA4 levels and combined targeting of SWI/SNF ATPases in H3.3K27M can serve as a potent therapeutic strategy for these deadly childhood brain tumors.

Abstract 1462: Oncogenic Foxl2 is a chromatin-remodeling pioneer transcription factor in adult-type ovarian granulosa cell tumors

Abstract Background: A missense mutation in the Forkhead domain-containing FOXL2 (Foxl2 p.C134W) transcription factor is found in nearly all adult-type granulosa cell tumors, but the oncogenic mechanism of this mutation is not known. Other Forkhead family transcription factors have well-described “pioneer” activity, binding to compacted, nucleosome-bound DNA and increasing accessibility for other regulatory proteins. Objectives: To develop novel cell culture model systems and determine whether oncogenic Foxl2-C134W has pioneering activity. Methods: CRISPR/Cas9 editing was used to generate isogenic granulosa cell tumor cells lacking either the FOXL2 wild-type allele (single knock-out; SKO) or both the mutant and wild-type FOXL2 alleles (double knock-out; DKO). ATAC-Seq and endogenous Foxl2 ChIP-Seq were performed on these isogenic lines to determine the differential chromatin accessibility at Foxl2-bound regulatory regions across genotypes. ENCODE pipelines and data standards were used for analysis, and the irreproducible discovery rate was used to identify high-reliability ATAC-seq and ChIP-seq peaks. De novo motifs were identified with STREME. Promoter capture HiC was performed using a bespoke genome-wide hybrid capture probe set, and CHiCAGO was used to identify significant HiC interactions. Results: Endogenous ChIP-seq of Foxl2-C134W from the SKO cell line identified 1147 high-confidence peaks. De novo motif discovery identified the canonical Foxl2 binding motif (TGTTTACATT, P = 1.4 x 10-7) in 44.9% of peaks, as well as a novel variant motif (TGTTTTGTCT, P = 6.7 x 10-15) in 68.5% of peaks. Median chromatin accessibility at Foxl2-C134W peak regions, as measured by ATAC-seq, was significantly decreased in the DKO cells compared to either SKO or parental cell lines (P < 2 x 10-16 for both comparisons). No difference was observed between SKO and parental cell lines (P = 0.19). Decreased chromatin accessibility at Foxl2-C134W ChIP-seq peaks in the DKO cells was driven by peaks containing the novel variant Foxl2 binding motif. Promoter capture HiC identified promoter-enhancer interactions lost in the DKO cell line involving several genes, including HUNK, NOVA1, andSFRP2 (adjusted P < 0.05). Conclusion: Foxl2-C134W exhibits “pioneering” activity, increasing chromatin accessibility at key gene regulatory elements with associated re-programming of promoter-enhancer interactions. The oncogenic mechanism of Foxl2-C134W in granulosa cell tumors may involve changes in DNA binding specificity, re-directing this pioneering function to sites containing a novel variant Foxl2 binding motif. Citation Format: Veena Vuttaradhi, Eleonora Khlebus, Thomas Welte, Namrata Khurana, Barrett Lawson, Robert Tyler Hillman. Oncogenic Foxl2 is a chromatin-remodeling pioneer transcription factor in adult-type ovarian granulosa cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1462.

Abstract 453: Targeting FOXL2C402Gvulnerabilities in adult type ovarian granulosa cell tumors

Abstract Background: Nearly all adult-type granulosa cell tumors of the ovary (aGCTs) carry the same oncogenic mutation affecting the second winged helix domain of the Forkhead family transcription factor FOXL2 (c.C402G; p.C134W). There are no currently available therapies that exploit the high prevalence of FOXL2 mutations in this disease. Objectives: Exploit FOXL2-C402G oncogenic addiction in order to identify FDA-approved compounds with differential potency in KGN-FOXL2WT/C402G cells and isogenic KGN-FOXL2—/— cells. Methods: CRISPR/Cas9 was used to generate isogenic derivatives of KGN, a cell line derived from a recurrent aGCT. Parental cells (KGN-FOXL2WT/C402G) or isogenic cells lacking both FOXL2 alleles (KGN-FOXL2—/—) were functionally characterized and loss of Foxl2 protein was confirmed by immunoblot. Two drug libraries comprising a total of 2,416 compounds (majority FDA approved) were screened for effectiveness in KGN-FOXL2WT/C402G cells and KGN-FOXL2—/— cells. Our assay included staining with DAPI, followed by automated image analysis of each well to enumerate nuclei/cell numbers. Based on Fraction Affected (FA) statistics, compounds were ranked according to effectiveness in KGN-FOXL2WT/C402G cells. FA-area under the curve (FA-AUC) cutoff >0.1 yielded 152 drug candidates, categorized into 13 drug classes. 40 drug candidates, including the top 1-4 hits in each drug class were chosen for further validation. Validation rate in the confirmatory screen was at 95%. A novel primary tumor organoid culture system for aGCT was developed and validated using whole exome sequencing and immunophenotype. Selected candidate compounds were further investigated in aGCT organoid cultures from two relapsed tumors. Results: We identified classes of drugs such as the inhibitors of Bromodomain family proteins and Hydroxy-Camptothecin analogs, with enhanced activity in KGN-FOXL2WT/C402G cells relative to KGN-FOXL2—/— cells. Unexpectedly glucocorticoids as a class were found to stimulate proliferation of KGN-FOXL2WT/C402G cells whereas they lacked this activity in KGN-FOXL2—/— cells. For 36 glucocorticoids present in the screen, FA-AUC ranged from -0.3191 to -0.1342 (highest proliferation index among all test compounds) in KGN-FOXL2WT/C402G cells compared to -0.0353 to +0.1667 in KGN-FOXL2—/— cells. In recurrent aGCT organoids, the high-potency glucocorticoid dexamethasone induced cell proliferation and co-treatment with glucocorticoids increased the IC50 of paclitaxel in KGN-FOXL2WT/C402G cells from 1.43 nM to 4.04 nM. Conclusion: Mutant FOXL2 mediates a proliferative response to glucocorticoids in cultured cells and primary aGCT organoids. Paclitaxel is a standard of care chemotherapy in the treatment of aGCTs and is often administered with glucocorticoid pre-medication. The adverse effect of glucocorticoids on paclitaxel efficiency shown here should thus be taken into consideration in the clinic. Citation Format: Thomas Welte, Veena Vuttaradhi, Eleonora Khlebus, Barrett Lawson, Robert T. Hillman, Nghi Nguyen, Mary Sobieski, Reid T. Powell, Clifford Stephan. Targeting FOXL2C402Gvulnerabilities in adult type ovarian granulosa cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 453.