In vivo CD8+ T cell CRISPR screening reveals control by Fli1 in infection and cancer.

Improving effector activity of antigen-specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TFs), the transcriptional coordination of TEFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a key mechanism restraining TEFF biology through the ETS family TF, Fli1. Genetic deletion of Fli1 enhanced TEFF responses without compromising memory or exhaustion precursors. Fli1 restrained TEFF lineage differentiation by binding to cis-regulatory elements of effector-associated genes. Loss of Fli1 increased chromatin accessibility at ETS:RUNX motifs, allowing more efficient Runx3-driven TEFF biology. CD8+ T cells lacking Fli1 provided substantially better protection against multiple infections and tumors. These data indicate that Fli1 safeguards the developing CD8+ T cell transcriptional landscape from excessive ETS:RUNX-driven TEFF cell differentiation. Moreover, genetic deletion of Fli1 improves TEFF differentiation and protective immunity in infections and cancer.

Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain.

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription.

Gene activation by mammalian transcription factors (TFs) requires multivalent interactions of their low-complexity domains (LCDs), but how such interactions regulate transcription remains unclear. It has been proposed that extensive LCD-LCD interactions culminating in liquid-liquid phase separation (LLPS) of TFs is the dominant mechanism underlying transactivation. Here, we investigated how tuning the amount and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. Quantitative single-cell and single-molecule imaging reveals that the oncogenic TF EWS::FLI1 requires a narrow optimum of LCD-LCD interactions to activate its target genes associated with GGAA microsatellites. Increasing LCD-LCD interactions toward putative LLPS represses transcription of these genes in patient-derived cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS::FLI1 into a well-documented LLPS compartment, the nucleolus, inhibits EWS::FLI1-driven transcription and oncogenic transformation. Our findings show how altering the balance of LCD-LCD interactions can influence transcriptional regulation and suggest a potential therapeutic strategy for targeting disease-causing TFs.

Targeted long-read sequencing of the Ewing sarcoma 6p25.1 susceptibility locus identifies germline-somatic interactions with EWSR1-FLI1 binding.

Ewing sarcoma (EwS) is a rare bone and soft tissue malignancy driven by chromosomal translocations encoding chimeric transcription factors, such as EWSR1-FLI1, that bind GGAA motifs forming novel enhancers that alter nearby expression. We propose that germline microsatellite variation at the 6p25.1 EwS susceptibility locus could impact downstream gene expression and EwS biology. We performed targeted long-read sequencing of EwS blood DNA to characterize variation and genomic features important for EWSR1-FLI1 binding. We identified 50 microsatellite alleles at 6p25.1 and observed that EwS-affected individuals had longer alleles (>135 bp) with more GGAA repeats. The 6p25.1 GGAA microsatellite showed chromatin features of an EWSR1-FLI1 enhancer and regulated expression of RREB1, a transcription factor associated with RAS/MAPK signaling. RREB1 knockdown reduced proliferation and clonogenic potential and reduced expression of cell cycle and DNA replication genes. Our integrative analysis at 6p25.1 details increased binding of longer GGAA microsatellite alleles with acquired EWSR-FLI1 to promote Ewing sarcomagenesis by RREB1-mediated proliferation.

Microsatellite enhancers can be targeted to impair tumorigenesis.

Dysregulation of repetitive elements has been implicated in many cancers and other human diseases; however, the role of repetitive elements remains largely unexplored. In this issue of Genes & Development, Boulay and colleagues (pp. 1008-1019) explore the ability of GGAA repeats to act as alternative enhancers activated by EWS-FLI1 in Ewing sarcoma and contribute to tumorigenesis. Using CRISPR-mediated epigenome editing, repression of EWS-FLI1 targeted microsatellite enhancers halted aberrant gene expression and impaired the growth of Ewing sarcoma xenografts in vivo. The study reveals the regulatory capacity of repetitive elements in cancer and offers insight into therapeutic targets for Ewing sarcoma.

Epigenome editing of microsatellite repeats defines tumor-specific enhancer functions and dependencies.

Various types of repetitive sequences are dysregulated in cancer. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 induces chromatin features typical of active enhancers at GGAA microsatellite repeats, but the function of these sites has not been directly demonstrated. Here, by combining nascent transcription profiling with epigenome editing, we found that a subset of GGAA microsatellite repeats is transcriptionally active in Ewing sarcoma and that silencing individual repeats abolishes local nascent transcription and leads to markedly reduced expression of putative target genes. Epigenome silencing of these repeat sites does not affect gene expression in unrelated cells, can prevent the induction of gene expression by EWS-FLI1, and, in the case of a GGAA repeat that controls SOX2 expression from a distance of 470 kb, is sufficient to impair the growth of Ewing sarcoma xenografts. Using an experimental approach that is broadly applicable to testing different types of repetitive genomic elements, our study directly demonstrates that specific repeat microsatellites can have critical gene regulation functions in cancer and thus represent tumor-specific vulnerabilities that may be exploited to develop new therapies.

Integrated analysis reveals FLI1 regulates the tumor immune microenvironment via its cell-type-specific expression and transcriptional regulation of distinct target genes of immune cells in breast cancer.

BACKGROUND: Immunotherapy is a practical therapeutic approach in breast cancer (BRCA), and the role of FLI1 in immune regulation has gradually been unveiled. However, the specific role of FLI1 in BRCA was conflicted; thus, additional convincing evidence is needed. METHODS: We explored the upstream regulation of FLI1 expression via summary data-based Mendelian randomization (SMR) analysis and ncRNA network construction centering on FLI1 using BRCA genome-wide association study (GWAS) summary data with expression quantitative trait loci (eQTLs) and DNA methylation quantitative trait loci (mQTLs) from the blood and a series of in silico analyses, respectively. We illuminated the downstream function of FLI1 in immune regulation by integrating a series of analyses of single-cell RNA sequence data (scRNA-seq). RESULTS: We verified a causal pathway from FLI1 methylation to FLI1 gene expression to BRCA onset and demonstrated that FLI1 was downregulated in BRCA. FLI1, a transcription factor, served as myeloid and T cells' communication regulator by targeting immune-related ligands and receptor transcription in BRCA tissues. We constructed a ceRNA network centering on FLI1 that consisted of three LncRNAs (CKMT2-AS1, PSMA3-AS1, and DIO3OS) and a miRNA (hsa-miR-324-5p), and the expression of FLI1 was positively related to a series of immune-related markers, including immune cell infiltration, biomarkers of immune cells, and immune checkpoints. CONCLUSION: Low-methylation-induced or ncRNA-mediated downregulation of FLI1 is associated with poor prognosis, and FLI1 might regulate the tumor immune microenvironment via a cell-type-specific target genes manner in BRCA.

Superficial Neurocristic EWSR1::FLI1 Fusion Tumor: A Distinctive, Clinically Indolent, S100 Protein/SOX10-Positive Neoplasm.

It is now understood that identical gene fusions may be shared by different entities. We report a distinctive neoplasm of the skin and subcutis, harboring the Ewing sarcoma-associated EWSR1::FLI1 fusion but differing otherwise from Ewing sarcoma. Slides and blocks for 5 cutaneous neoplasms coded as other than Ewing sarcoma and harboring EWSR1::FLI1 were retrieved. Immunohistochemical and molecular genetic results were abstracted from reports. Methylation profiling was performed. Clinical information was obtained. The tumors occurred in 4 men and 1 woman (median: 25 years of age; range: 19-69 years) and involved the skin/subcutis of the back (2), thigh, buttock, and chest wall (median: 2.4 cm; range: 1-11 cm). Two tumors were present "years" before coming to clinical attention. The lesions were multinodular and circumscribed and consisted of nests of bland, round cells admixed with hyalinized collagenous bands containing spindled cells. Hemorrhage and cystic change were often present; necrosis was absent. All were diffusely S100 protein/SOX10-positive; 4 of 5 were CD99-negative. One tested case was strongly positive for NKX2.2. A variety of other tested markers were either focally positive (glial fibrillary acidic protein, p63) or negative. Molecular genetic results were as follows: EWSR1 exon 7::FLI1 exon 8, EWSR1 exon 11::FLI1 exon 5, EWSR1 exon 11::FLI1 exon 6, EWSR1 exon 7::FLI1 exon 6, and EWSR1 exon 10::FLI1 exon 6. Methylation profiling (3 cases) showed these to form a unique cluster, distinct from Ewing sarcoma. All patients underwent excision with negative margins; one received 1 cycle of chemotherapy. Clinical follow-up showed all patients to be alive without disease (median: 17 months; range: 11-62 months). Despite similar gene fusions, the morphologic, immunohistochemical, epigenetic, and clinical features of these unique EWSR1::FLI1-fused neoplasms of the skin and subcutis differ substantially from Ewing sarcoma. Interestingly, EWSR1 rearrangements involved exons 10 or 11, only rarely seen in Ewing sarcoma, in a majority of cases. Superficial neurocristic EWSR1::FLI1 fusion tumors should be rigorously distinguished from true cutaneous Ewing sarcomas.

FLI-1-driven regulation of endothelial cells in human diseases.

Endothelial cells (ECs) are widely distributed in the human body and play crucial roles in the circulatory and immune systems. ECs dysfunction contributes to the progression of various chronic cardiovascular, renal, and metabolic diseases. As a key transcription factor in ECs, FLI-1 is involved in the differentiation, migration, proliferation, angiogenesis and blood coagulation of ECs. Imbalanced FLI-1 expression in ECs can lead to various diseases. Low FLI-1 expression leads to systemic sclerosis by promoting fibrosis and vascular lesions, to pulmonary arterial hypertension by promoting a local inflammatory state and vascular lesions, and to tumour metastasis by promoting the EndMT process. High FLI-1 expression leads to lupus nephritis by promoting a local inflammatory state. Therefore, FLI-1 in ECs may be a good target for the treatment of the abovementioned diseases. This comprehensive review provides the first overview of FLI-1-mediated regulation of ECs processes, with a focus on its influence on the abovementioned diseases and existing FLI-1-targeted drugs. A better understanding of the role of FLI-1 in ECs may facilitate the design of more effective targeted therapies for clinical applications, particularly for tumour treatment.

Inherited thrombocytopenia associated with a variant in the FLI1 binding site in the 5' UTR of ANKRD26.

Variants in the 5' UTR of ANKRD26 are a common cause of inherited thrombocytopenia (ANKRD26-RT), and are associated with sustained ANKRD26 expression, which inhibits megakaryocyte maturation and proplatelet formation. ANKRD26 expression is controlled by the binding of a RUNX1/FLI1 complex to the 5' UTR. To date, all reported ANKRD26-RD associated variants have been within the RUNX1 binding site and a 22 base pair flanking region. Here, we report a novel variant in the 5' UTR of ANKRD26, c.-107C>T. This variant is in the FLI1 binding site, and is predicted to disrupt FLI1 binding due to loss of a hydrogen bond with FLI1. Differentiated PBMCs from affected family members showed impaired megakaryocyte maturation and proplatelet formation and sustained expression of ANKRD26, and platelets from affected family members had higher ANKRD26 expression than control platelets. The variant increased activity of the ANKRD26 promotor in a reporter assay. We also provide evidence that the previously reported c.-140C>G ANKRD26 5' UTR variant is benign and not associated with thrombocytopenia. Identification of the c.-107C>T variant extends the range of the regulatory region in the 5' UTR of ANKRD26 that is associated with ANKRD26-RT.

FLI1 induces erythroleukemia through opposing effects on UBASH3A and UBASH3B expression.

BACKGROUND: FLI1 is an oncogenic transcription factor that promotes diverse malignancies through mechanisms that are not fully understood. Herein, FLI1 is shown to regulate the expression of Ubiquitin Associated and SH3 Domain Containing A/B (UBASH3A/B) genes. UBASH3B and UBASH3A are found to act as an oncogene and tumor suppressor, respectively, and their combined effect determines erythroleukemia progression downstream of FLI1. METHODS: Promoter analysis combined with luciferase assays and chromatin immunoprecipitation (ChIP) analysis were applied on the UBASH3A/B promoters. RNAseq analysis combined with bioinformatic was used to determine the effect of knocking-down UBASH3A and UBASH3B in leukemic cells. Downstream targets of UBASH3A/B were inhibited in leukemic cells either via lentivirus-shRNAs or small molecule inhibitors. Western blotting and RT-qPCR were used to determine transcription levels, MTT assays to assess proliferation rate, and flow cytometry to examine apoptotic index. RESULTS: Knockdown of FLI1 in erythroleukemic cells identified the UBASH3A/B genes as potential downstream targets. Herein, we show that FLI1 directly binds to the UBASH3B promoter, leading to its activation and leukemic cell proliferation. In contrast, FLI1 indirectly inhibits UBASH3A transcription via GATA2, thereby antagonizing leukemic growth. These results suggest oncogenic and tumor suppressor roles for UBASH3B and UBASH3A in erythroleukemia, respectively. Mechanistically, we show that UBASH3B indirectly inhibits AP1 (FOS and JUN) expression, and that its loss leads to inhibition of apoptosis and acceleration of proliferation. UBASH3B also positively regulates the SYK gene expression and its inhibition suppresses leukemia progression. High expression of UBASH3B in diverse tumors was associated with worse prognosis. In contrast, UBASH3A knockdown in erythroleukemic cells increased proliferation; and this was associated with a dramatic induction of the HSP70 gene, HSPA1B. Accordingly, knockdown of HSPA1B in erythroleukemia cells significantly accelerated leukemic cell proliferation. Accordingly, overexpression of UBASH3A in different cancers was predominantly associated with good prognosis. These results suggest for the first time that UBASH3A plays a tumor suppressor role in part through activation of HSPA1B. CONCLUSIONS: FLI1 promotes erythroleukemia progression in part by modulating expression of the oncogenic UBASH3B and tumor suppressor UBASH3A.

Angiogenic protein profiling, phytochemical screening and in silico anti-cancer targets validation of stem, leaves, fruit, and seeds of Calotropis procera in human liver and breast cancer cell lines.

The main focus of anticancer drug discovery is on developing medications that are gentle on normal cells and should have the ability to target multiple anti-cancer pathways. Liver cancer is becoming a worldwide epidemic due to the highest occurring and reoccurring rate in some countries. Calotropis procera is a xerophytic herbal plant growing wildly in Saudi Arabia. Due to its anti-angiogenic and anticancer capabilities, "C. procera" is a viable option for developing innovative anticancer medicines. However, no study has been done previously, to discover angiogenic and anti-cancer targets which are regulated by C. procera in liver cancer. In this study, leaves, stems, flowers, and seeds of C. procera were used to prepare crude extracts and were fractionated into four solvents of diverse polarities. These bioactivity-guided solvent fractions helped to identify useful compounds with minimal side effects. The phytoconstituents present in the leaves and stem were identified by GC-MS. In silico studies were done to predict the anti-cancer targets by major bioactive constituents present in leaves and stem extracts. A human angiogenesis antibody array was performed to profile novel angiogenic targets. The results from this study showed that C. procera extracts are an ideal anti-cancer remedy with minimum toxicity to normal cells as revealed by zebrafish in vivo toxicity screening assays. The novel antiangiogenic and anticancer targets identified in this study could be explored to design medication against liver cancer.

VGLL4 promotes vascular endothelium specification via TEAD1 in the vascular organoids and human pluripotent stem cells-derived endothelium model.

BACKGROUND: We have shown that Hippo-YAP signaling pathway plays an important role in endothelial cell differentiation. Vestigial-like family member 4 (VGLL4) has been identified as a YAP inhibitor. However, the exact function of VGLL4 in vascular endothelial cell development remains unclear. In this study, we investigated the role of VGLL4, in human endothelial lineage specification both in 3D vascular organoid and 2D endothelial cell differentiation. METHODS AND RESULTS: In this study, we found that VGLL4 was increased during 3D vascular organoids generation and directed differentiation of human embryonic stem cells H1 towards the endothelial lineage. Using inducible ectopic expression of VGLL4 based on the piggyBac system, we proved that overexpression of VGLL4 in H1 promoted vascular organoids generation and endothelial cells differentiation. In contrast, VGLL4 knockdown (heterozygous knockout) of H1 exhibited inhibitory effects. Using bioinformatics analysis and protein immunoprecipitation, we further found that VGLL4 binds to TEAD1 and facilitates the expression of endothelial master transcription factors, including FLI1, to promote endothelial lineage specification. Moreover, TEAD1 overexpression rescued VGLL4 knockdown-mediated negative effects. CONCLUSIONS: In summary, VGLL4 promotes EC lineage specification both in 3D vascular organoid and 2D EC differentiation from pluripotent stem cell, VGLL4 interacts with TEAD1 and facilitates EC key transcription factor, including FLI1, to enhance EC lineage specification.

Antibody to Endogenous Cardiotonic Steroid Reverses Vascular Fibrosis and Restores Vasorelaxation in Chronic Kidney Disease.

Marinobufagenin (MBG) is implicated in chronic kidney disease, where it removes Fli1-induced inhibition of the collagen-1. We hypothesized that (i) in nephrectomized rats, aortic fibrosis develops due to elevated plasma MBG and inhibited Fli1, and (ii) that the antibody to MBG reduces collagen-1 and improves vasodilatation. A partial nephrectomy was performed in male Sprague-Dawley rats. Sham-operated animals comprised the control group. At 5 weeks following nephrectomy, rats were administered the vehicle (n = 8), or the anti-MBG antibody (n = 8). Isolated aortic rings were tested for their responsiveness to sodium nitroprusside following endothelin-1-induced constriction. In nephrectomized rats, there was an increase in the intensity of collagen staining in the aortic wall vs. the controls. In antibody-treated rats, the structure of bundles of collagen fibers had ordered organization. Western blots of the aorta had lower levels of Fli1 (arbitrary units, 1 ± 0.05 vs. 0.2 ± 0.01; p < 0.001) and greater collagen-1 (arbitrary units, 1 ± 0.01 vs. 9 ± 0.4; p < 0.001) vs. the control group. Administration of the MBG antibody to rats reversed the effect of the nephrectomy on Fli1 and collagen-1 proteins. Aortic rings pretreated with endothelin-1 exhibited 50% relaxation following the addition of sodium nitroprusside (EC50 = 0.28 µmol/L). The responsiveness of the aortic rings obtained from nephrectomized rats was markedly reduced (EC50 = 3.5 mol/L) compared to the control rings. Treatment of rats with the antibody restored vasorelaxation. Thus, the anti-MBG antibody counteracts the Fli1-collagen-1 system and reduces aortic fibrosis.

EWS-FLI1 and Activator Protein-1 (AP-1) Reciprocally Regulate Extracellular-Matrix Proteins in Ewing sarcoma Cells.

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis of deoxyribonucleotides and the target of multiple chemotherapy drugs, including gemcitabine. We previously identified that inhibition of RNR in Ewing sarcoma tumors upregulates the expression levels of multiple members of the activator protein-1 (AP-1) transcription factor family, including c-Jun and c-Fos, and downregulates the expression of c-Myc. However, the broader functions and downstream targets of AP-1, which are highly context- and cell-dependent, are unknown in Ewing sarcoma tumors. Consequently, in this work, we used genetically defined models, transcriptome profiling, and gene-set -enrichment analysis to identify that AP-1 and EWS-FLI1, the driver oncogene in most Ewing sarcoma tumors, reciprocally regulate the expression of multiple extracellular-matrix proteins, including fibronectins, integrins, and collagens. AP-1 expression in Ewing sarcoma cells also drives, concurrent with these perturbations in gene and protein expression, changes in cell morphology and phenotype. We also identified that EWS-FLI1 dysregulates the expression of multiple AP-1 proteins, aligning with previous reports demonstrating genetic and physical interactions between EWS-FLI1 and AP-1. Overall, these results provide novel insights into the distinct, EWS-FLI1-dependent features of Ewing sarcoma tumors and identify a novel, reciprocal regulation of extracellular-matrix components by EWS-FLI1 and AP-1.

FLI1 regulates radiotherapy resistance in nasopharyngeal carcinoma through TIE1-mediated PI3K/AKT signaling pathway.

BACKGROUND: Radiotherapy resistance is the main cause of treatment failure in nasopharyngeal carcinoma (NPC), which leads to poor prognosis. It is urgent to elucidate the molecular mechanisms underlying radiotherapy resistance. METHODS: RNA-seq analysis was applied to five paired progressive disease (PD) and complete response (CR) NPC tissues. Loss-and gain-of-function assays were used for oncogenic function of FLI1 both in vitro and in vivo. RNA-seq analysis, ChIP assays and dual luciferase reporter assays were performed to explore the interaction between FLI1 and TIE1. Gene expression with clinical information from tissue microarray of NPC were analyzed for associations between FLI1/TIE1 expression and NPC prognosis. RESULTS: FLI1 is a potential radiosensitivity regulator which was dramatically overexpressed in the patients with PD to radiotherapy compared to those with CR. FLI1 induced radiotherapy resistance and enhanced the ability of DNA damage repair in vitro, and promoted radiotherapy resistance in vivo. Mechanistic investigations showed that FLI1 upregulated the transcription of TIE1 by binding to its promoter, thus activated the PI3K/AKT signaling pathway. A decrease in TIE1 expression restored radiosensitivity of NPC cells. Furthermore, NPC patients with high levels of FLI1 and TIE1 were correlated with poor prognosis. CONCLUSION: Our study has revealed that FLI1 regulates radiotherapy resistance of NPC through TIE1-mediated PI3K/AKT signaling pathway, suggesting that targeting the FLI1/TIE1 signaling pathway could be a potential therapeutic strategy to enhance the efficacy of radiotherapy in NPC.

FLI-1/Melan-A dual stain is an alternative to PRAME in differentiating metastatic melanoma from nodal nevus: A monocentric retrospective study.

Melanocytic nevi existing in lymph nodes create a diagnostic challenge by mimicking metastases. PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemical (IHC) stain can differentiate one from another. FLI-1 IHC expression has been shown in malignant melanoma with variable sensitivity while melanocytic nevi were reported to be negative. We hypothesized that FLI-1/Melan-A dual IHC staining may be used in the distinction of metastatic melanoma from nodal nevi and can be an alternative and/or complementary to PRAME. In this study, we examined 13 lymph nodes with metastatic melanoma and 13 lymph nodes with benign deposits. We stained all of the lymph nodes with FLI-1, FLI-1/Melan-A dual, and PRAME IHC stains. In addition, we stained paired skin samples of the metastatic lymph nodes with FLI-1 and PRAME. In primary cutaneous melanomas, 11 of 13 were positive for FLI-1 and PRAME expression (85%). Malignant cells in 12 and 13 lymph nodes showed positive expression of PRAME and FLI-1, respectively. Only one case with a nevic cell deposit was weakly positive for FLI-1 and the remaining benign cases were negative for both FLI-1 and PRAME. Our results show that FLI-1/Melan-A dual stain is as sensitive and specific as PRAME in distinguishing lymph nodes with metastatic melanoma from nodal nevi. Further studies with larger case numbers are needed to support our significant results.

Suppression of Fli-1 protects against pericyte loss and cognitive deficits in Alzheimer's disease.

Brain pericytes regulate cerebral blood flow, maintain the integrity of the blood-brain barrier (BBB), and facilitate the removal of amyloid ß (Aß), which is critical to healthy brain activity. Pericyte loss has been observed in brains from patients with Alzheimer's disease (AD) and animal models. Our previous data demonstrated that friend leukemia virus integration 1 (Fli-1), an erythroblast transformation-specific (ETS) transcription factor, governs pericyte viability in murine sepsis; however, the role of Fli-1 and its impact on pericyte loss in AD remain unknown. Here, we demonstrated that Fli-1 expression was up-regulated in postmortem brains from a cohort of human AD donors and in 5xFAD mice, which corresponded with a decreased pericyte number, elevated inflammatory mediators, and increased Aß accumulation compared with cognitively normal individuals and wild-type (WT) mice. Antisense oligonucleotide Fli-1 Gapmer administered via intrahippocampal injection decelerated pericyte loss, decreased inflammatory response, ameliorated cognitive deficits, improved BBB dysfunction, and reduced Aß deposition in 5xFAD mice. Fli-1 Gapmer-mediated inhibition of Fli-1 protected against Aß accumulation-induced human brain pericyte apoptosis in vitro. Overall, these studies indicate that Fli-1 contributes to pericyte loss, inflammatory response, Aß deposition, vascular dysfunction, and cognitive decline, and suggest that inhibition of Fli-1 may represent novel therapeutic strategies for AD.

Current insights into the role of Fli-1 in hematopoiesis and malignant transformation.

Fli-1, a member of the ETS family of transcription factors, was discovered in 1991 through retroviral insertional mutagenesis as a driver of mouse erythroleukemias. In the past 30 years, nearly 2000 papers have defined its biology and impact on normal development and cancer. In the hematopoietic system, Fli-1 controls self-renewal of stem cells and their differentiation into diverse mature blood cells. Fli-1 also controls endothelial survival and vasculogenesis, and high and low levels of Fli-1 are implicated in the auto-immune diseases systemic lupus erythematosus and systemic sclerosis, respectively. In addition, aberrant Fli-1 expression is observed in, and is essential for, the growth of multiple hematological malignancies and solid cancers. Here, we review the historical context and latest research on Fli-1, focusing on its role in hematopoiesis, immune response, and malignant transformation. The importance of identifying Fli-1 modulators (both agonists and antagonists) and their potential clinical applications is discussed.

An inhibitor of endothelial ETS transcription factors promotes physiologic and therapeutic vessel regression.

During the progression of ocular diseases such as retinopathy of prematurity and diabetic retinopathy, overgrowth of retinal blood vessels results in the formation of pathological neovascular tufts that impair vision. Current therapeutic options for treating these diseases include antiangiogenic strategies that can lead to the undesirable inhibition of normal vascular development. Therefore, strategies that eliminate pathological neovascular tufts while sparing normal blood vessels are needed. In this study we exploited the hyaloid vascular network in murine eyes, which naturally undergoes regression after birth, to gain mechanistic insights that could be therapeutically adapted for driving neovessel regression in ocular diseases. We found that endothelial cells of regressing hyaloid vessels underwent down-regulation of two structurally related E-26 transformation-specific (ETS) transcription factors, ETS-related gene (ERG) and Friend leukemia integration 1 (FLI1), prior to apoptosis. Moreover, the small molecule YK-4-279, which inhibits the transcriptional and biological activity of ETS factors, enhanced hyaloid regression in vivo and drove Human Umbilical Vein Endothelial Cells (HUVEC) tube regression and apoptosis in vitro. Importantly, exposure of HUVECs to sheer stress inhibited YK-4-279-induced apoptosis, indicating that low-flow vessels may be uniquely susceptible to YK-4-279-mediated regression. We tested this hypothesis by administering YK-4-279 to mice in an oxygen-induced retinopathy model that generates disorganized and poorly perfused neovascular tufts that mimic human ocular diseases. YK-4-279 treatment significantly reduced neovascular tufts while sparing healthy retinal vessels, thereby demonstrating the therapeutic potential of this inhibitor.