Friend Leukemia Virus Integration Research Articles

MiR-145a Regulation of Pericyte Dysfunction in a Murine Model of Sepsis.

Sepsis is a life-threatening systemic disease with severe microvascular dysfunction. Pericytes preserve vascular homeostasis. To our knowledge, the potential roles of microRNAs in sepsis-induced pericyte dysfunction have not been explored. We determined lung pericyte expression of miR-145a in cecal ligation and puncture (CLP)-induced sepsis. Mouse lung pericytes were isolated and transfected with a miR-145a mimic, followed by stimulation with lipopolysaccharide (LPS). We measured inflammatory cytokine levels. To assess the functions of miR-145a in vivo, we generated a pericyte-specific miR-145a-knockout mouse and determined sepsis-induced organ injury, lung and renal vascular leakage, and mouse survival rates. We used RNA sequencing and Western blotting to analyze the signaling pathways regulated by miR-145a. CLP led to decreased miR-145a expression in lung pericytes. The miR-145a mimic inhibited LPS-induced increases in cytokines. In CLP-induced sepsis, pericytes lacking miR-145a exhibited increased lung and kidney vascular leakage and reduced survival rates. We found that miR-145a could suppress LPS-induced NF-κB activation. In addition, we confirmed that the transcription factor Friend leukemia virus integration 1 (Fli-1) is a target of miR-145a and that Fli-1 activates NF-κB signaling. Our results demonstrated that pericyte miR-145a mediates sepsis-associated microvascular dysfunction, potentially by means of Fli-1-mediated modulation of NF-κB signaling.

MicroRNA-33b regulates hepatocellular carcinoma cell proliferation, apoptosis, and mobility via targeting Fli-1-mediated Notch1 pathway.

MicroRNAs (miRNAs) have been confirmed to play pivotal roles in hepatocellular carcinoma (HCC) carcinogenesis. However, the underlying function of microRNA-33b (miR-33b) in HCC remains unclear. Here, we found that miR-33b level was significantly reduced in both HCC tissues and tumor cell lines. Further, luciferase reporter assay and western blot analysis confirmed that Friend leukemia virus integration 1 (Fli-1) was a direct target of miR-33b. Overexpression of miR-33b dramatically suppressed HCC tumor cell proliferation and cell mobility, but facilitated tumor cell apoptosis in vitro. Besides, restoration of Fli-1 partially attenuated miR-33b-mediated inhibition of cell growth and metastasis via activating Notch1 signaling and its downstream effectors. Our findings demonstrate the important role of miR-33b/Fli-1 axis in HCC progression and provide novel therapeutic candidates for HCC clinical treatment.

Tocotrienol-rich fraction from annatto ameliorates expression of lysyl oxidase in human osteoblastic MG-63 cells.

Lysyl oxidase (LOX) is required for the formation of bone collagen cross-links. Inactivation of the LOX gene in osteoblasts by DNA methylation and JAK signaling has been reported to cause loss of cross-links and an increased risk of fractures. Tocotrienols (T3s) have proven benefits on bone strength, but their potential effects on LOX remain largely unknown. Thus, the present study investigates the in vitro effects of T3s on LOX expression in human osteoblastic MG-63 cells. Results indicated that Tocotrienol-Rich Fraction (TRF), the δ-T3 rich oil extracted from Annatto was the most effective and significantly increased LOX expression. TRF treatment decreased de-novo methyltransferases (DNMTs), DNMT3A and DNMT3B levels. In addition, TRF significantly inhibited JAK2 activation and decreased expression of Fli1, a transcription factor of DNMTs. We conclude that TRF induced an increase in LOX expression via inhibition of de-novo methylation and reduction of Fli1 expression by the inactivation of JAK2.Abbreviations: CpG: cytosine-guanine dinucleotide; DNMT: DNA methyltransferase; Fli1: friend leukemia virus integration 1; JAK: janus kinase; LOX: lysyl oxidase; PCR: polymerase chain reaction; STAT: signal transducers and activators of transcription; T3s: tocotrienols; TPs: tocopherols; TRF: Tocotrienol-Rich Fraction.

EWSR1 fusions: Ewing sarcoma and beyond.

EWSR1 fusions: Ewing sarcoma and beyond.

Fli-1 Regulates Multiple T-Cell Subsets during Inflammatory Responses and Experimental Graft-Versus-Host Disease

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a procedure undertaken to cure hematological malignancies, especially leukemia. Graft-Versus-Host Disease (GVHD) is a serious condition that often appears following allo-HCT. Friend Leukemia Virus Integration 1 (Fli-1) is a transcription factor highly expressed in cancers including erythroleukemia and acute myeloid leukemia while also implicated in pathogenesis of systemic lupus. We have interrogated the role of Fli-1 in T-cell responses by generating a novel T-cell specific conditional disruption of fli-1 where essential exons 3 and 4 of the gene are floxed and excised in the presence of CD4-Cre recombinase. Models of acute GVHD (aGVHD) and chronic GVHD (cGVHD) were tested utilizing hematopoietic cells from mice with a heterozygous (fli-1-/+ or Het) or homozygous (fli-1-/-or Null) disruption of the fli-1 gene, or from wild-type (fli-1+/+ or WT) littermate controls. At baseline, T cells among each of these three mouse strains showed no significant difference in CD44/CD62L expression or CD4+FoxP3+ (nTreg) frequencies. In the cGVHD model, BALB/c (H2Kd) recipients were infused with allogeneic B6 (H2Kb) genotype-respective bone marrow and splenocytes in order to induce cGVHD. Recipients that received fli-1-/+CD4Cre+ marrow and splenocytes demonstrated improved survival and mild cGVHD, whereas those that received fli-1-/-CD4Cre+ or WT donor cells developed serve cGVHD (Fig. 1 a-f). Cellular studies from lymphoid organs of cGVHD allo-HCT recipients revealed that disruption of fli-1 was associated with decreased frequencies of donor CD4+ cells expressing IL-17A, IFN-γ, T follicular-like (TFH-like) cell markers, and CD8+ cells expressing PD-1. In aGVHD settings, donor fli-1-/+CD4Cre+ T cells had a decreased ability to induce aGVHD compared to WT donor T cells and fli-1-/- donor T cells (Fig. 2 a-b). When investigating cellular mechanisms in aGVHD settings, we found that fli-1-/+CD4Cre+ T cells produced significantly lower IFN-γ early after T-cell activation in vivo compared to WT and fli-1-/-CD4Cre+ T cells (Fig. 2 c). To then investigate the role of Fli-1 in T cells beyond GVHD, we utilized a colitis model by transferring naïve CD4+ T cells (CD44-CD25-) into Rag2-/- syngeneic recipients. While fli-1+/+ T cells induced severe colitis as expected, fli-1-/- and fli--/+ T cells caused moderate and mild colitis, respectively. These results were consistent with those observed in GVHD models. To elucidate underlying mechanisms, we tested the effects of Fli-1 on antigen-specific T-cells using a MHC-II restricted TCR transgenic (Tg) mouse strain specific for HY-peptide. CD4+CD25- T cells from fli-1+/+, fli-1-/+, or fli-1-/- CD4Cre+ TCR-Tg mice were polarized in vitro with endogenous antigen presenting cells from spleen in presence of HY-peptide under iTreg- or Th17-polarizing conditions. Both fli-1-/+ and fli-1-/- TCR-tg T cells exhibited significant increases in iTreg (CD4+FoxP3+) frequencies and surface expression of iTreg functional markers (CD25, CD39, CD73, NRP-1), while also having significantly decreased frequencies of IL-17A producing T cells compared to WT-TCR-tg T cells. To further explore the molecular mechanisms, we retrieved fli-1+/+, fli-1-/+, or fli-1-/- donor T cells from recipient spleens after allo-HCT and did RNAseq analysis on these cells. RNAseq data reveals significant differences in mRNA expression within acute inflammatory response and positive regulation of the immune response enrichment pathways between fli-1-/+ T cells and littermate fli-1+/+ T cells, and to a lesser extent in fli-1-/- T cells (Fig. 2 d-f). Thus, reducing Fli-1 transcriptional activity represents a potential therapeutic concept toward ameliorating GVHD after allo-HCT, while simultaneously targeting cancers such as leukemia which typically overexpress Fli-1. Disclosures No relevant conflicts of interest to declare.

FLI1 Exonic Circular RNAs as a Novel Oncogenic Driver to Promote Tumor Metastasis in Small Cell Lung Cancer.

The aberrantly upregulated Friend leukemia virus integration 1 (FLI1) is closely correlated with the malignant phenotype of small cell lung cancer (SCLC). It is interesting to note that the CRISPR gene knockout by Cas9 gRNAs that target the FLI1 coding region and the posttranscriptional knockdown by shRNAs that target the 3' region of FLI1 mRNA yielded distinct antimetastasis effects in SCLC cells. This study attempts to examine if FLI1 exonic circular RNAs (FECR) function as a new malignant driver that determines the metastatic phenotype in SCLC. The clinical relevance of FECRs was examined in 56 primary SCLC tissues and 50 non-small cell lung cancer (NSCLC) tissues. The prognostic value of FECRs was examined by measuring serum exosomal FECRs in a longitudinal cohort of patients with SCLC. The oncogenic activity of FECRs was investigated in both SCLC cell lines and animal xenograft studies. Finally, we explored the molecular mechanisms underlying these noncoding RNAs as a malignant driver. Therapeutic comparison of CRISPR Cas9 knockout and shRNA knockdown of FLI1 identified FECRs as a new noncanonical malignant driver in SCLC. Using RNA FISH and quantitative PCR, we found that FECR1 (exons 4-2-3) and FECR2 (exons 5-2-3-4) were aberrantly upregulated in SCLC tissues (P < 0.0001), and was positively associated with lymph node metastasis (P < 0.01). Notably, serum exosomal FECR1 was associated with poor survival (P = 0.038) and clinical response to chemotherapy. Silencing of FECRs significantly inhibited the migration in two highly aggressive SCLC cell lines and reduced tumor metastasis in vivo. Mechanistically, we uncovered that FECRs sequestered and subsequently inactivated tumor suppressor miR584-3p, leading to the activation of the Rho Associated Coiled-Coil Containing Protein Kinase 1 gene (ROCK1). This study identifies FLI1 exonic circular RNAs as a new oncogenic driver that promotes tumor metastasis through the miR584-ROCK1 pathway. Importantly, serum exosomal FECR1 may serve as a promising biomarker to track disease progression of SCLC.

Fli-1 transcription factor regulates the expression of caspase-1 in lung pericytes

Our previous data demonstrated that Friend leukemia virus integration 1 (Fli-1), an ETS transcription factor, governs pericyte loss and vascular dysfunction in cecal ligation and puncture-induced murine sepsis by regulating essential pyroptosis markers including caspase-1. However, whether Fli-1 regulates caspase-1 expression levels in vitro and how Fli-1 regulates caspase-1 remain unknown. Our present work further demonstrated that overexpressed Fli-1 significantly increased caspase-1 and IL-18 expression levels in cultured mouse lung pericytes. Bacterial outer membrane vesicles (OMVs) have been found to induce cell pyroptosis through transferring LPS intracellularly. Using OMVs to induce an in vitro model of pyroptosis, we observed that OMVs significantly increased protein levels of Fli-1 in mouse lung pericytes. Furthermore, knockdown of Fli-1 by siRNA blocked OMVs-induced caspase-1, caspase-11 and IL-18 expression levels. As caspase-1 was predicted as a potential target of Fli-1, we cloned murine caspase-1 promoter into a luciferase construct. Our data demonstrate for the first time that Fli-1 regulates caspase-1 expression by directly binding to its promoter regions measured by chromatin immunoprecipitation (ChIP) assay and luciferase reporter system. In summary, our findings demonstrated a novel role and mechanism of Fli-1 in regulating caspase-1 expression in lung pericytes.

A potential contribution of trappin-2 to the development of vasculopathy in systemic sclerosis.

Trappin-2/pre-elafin is an endogenous inhibitor of human neutrophil elastase involved in inflammation, innate immunity and vascular remodelling, which consist of the complex pathological process of systemic sclerosis (SSc). To clarify the potential role of trappin-2 in SSc. Serum trappin-2 levels were determined by enzyme-linked immunosorbent assay in 51 SSc and 18 healthy subjects. Trappin-2 expression was evaluated in SSc lesional skin and cultured endothelial cells treated with FLI1 siRNA by immunohistochemistry, reverse transcription-real-time PCR and/or immunoblotting. Friend leukaemia virus integration 1 (Fli1) binding to the PI3 promoter was assessed by chromatin immunoprecipitation. Since serum trappin-2 levels inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction, SSc patients with normal renal function were analysed. Although serum trappin-2 levels were comparable between diffuse cutaneous SSc, limited cutaneous SSc and control subjects, the prevalence of digital ulcers or elevated right ventricular systolic pressure (RVSP) was significantly higher in SSc patients with elevated serum trappin-2 levels than in those with normal levels. Furthermore, serum trappin-2 levels were significantly increased in SSc patients with digital ulcers or elevated RVSP compared to those without. Moreover, serum trappin-2 levels positively correlated with RVSP values in SSc patients. Importantly, trappin-2 expression was enhanced in small vessels of SSc lesional skin. In cultured endothelial cells, trappin-2 expression was elevated by gene silencing of FLI1 at mRNA and protein levels and Fli1 occupied the PI3 promoter. Endothelial trappin-2 up-regulation partially due to Fli1 deficiency can be associated with the development of SSc vasculopathy.

Fli-1 Governs Pericyte Dysfunction in a Murine Model of Sepsis.

Pericytes are vascular mural cells and are embedded in the basement membrane of the microvasculature. Recent studies suggest a role for pericytes in lipopolysaccharide (LPS)-induced microvascular dysfunction and mortality, but the mechanisms of pericyte loss in sepsis are largely unknown. By using a cecal ligation and puncture (CLP)-induced murine model of sepsis, we observed that CLP led to lung and renal pericyte loss and reduced lung pericyte density and pericyte/endothelial cell (EC) coverage. Up-regulated Friend leukemia virus integration 1 (Fli-1) messenger ribonucleic acid (RNA) and protein levels were found in lung pericytes from CLP mice in vivo and in LPS-stimulated lung pericytes in vitro. Knockout of Fli-1 in Foxd1-derived pericytes prevented CLP-induced pericyte loss, vascular leak, and improved survival. Disrupted Fli-1 expression by small interfering RNA inhibited LPS-induced inflammatory cytokines and chemokines in cultured lung pericytes. Furthermore, CLP-induced pericyte pyroptosis was mitigated in pericyte Fli-1 knockout mice. Our findings suggest that Fli-1 is a potential therapeutic target in sepsis.

Abstract 3189: Targeting NPM1 for controlling growth of Ewing sarcoma

Abstract Ewing sarcoma is a rare cancer found in children and young adults. Long term survival rates are approximately 70% for patients who present without clinically overt metastases. However, 5 year survival is less than 20% for patients with recurrent tumors or metastasis. Thus, there is a need to improve local tumor control and to treat metastatic disease. Eighty five percent of these cancers are driven by the reciprocal chromosomal translocation t (11;22) (q24;q12), a consequence of a fusion between the 5' portion of Ewing sarcoma breakpoint region 1 on EWSR1 (chromosome 22) and the 3' portion of Friend leukemia virus integration site 1 on FLI1 (chromosome 11). Nucleophosmin1 (NPM1) is a chaperone protein involved in many cellular functions, including DNA repair. NPM1 has been shown to be a novel prognostic biomarker for patients with localized Ewing's sarcoma. Overall survival is significantly lower for patients whose tumors express high levels of NPM1. Besides promoting repair of DNA double strand breaks NPM1 directly interacts with and induces c-Myc-induced hyperproliferation and transformation. Consistent with this knowledge is the observation that relapsed Ewing's patients exhibit high levels of c-Myc expression compared to disease-free patients. Therefore, we wanted to ascertain whether NPM1 represented a molecular target for controlling growth of Ewing's sarcoma. CRISPR/Cas9-mediated targeting of NPM1 exons 3 and 4, as well as lentivirus-mediated shRNA targeting of NPM1 mRNA revealed that targeting of NPM1 expression reduced survival (colony formation) of TC32 and A673 cells (P&amp;lt;0.05). NPM shRNA also reduced TC32 sphere formation under non-adherent conditions. YTR107 and NSC348884 are small molecule inhibitors of NPM1 oligomerization, which is required for NPM1 activity. Both drugs reduced survival of TC32 and A673 Ewing's sarcoma cells in a dose-dependent fashion (P&amp;lt;0.05). We interpret these data to indicate that NPM1 represents a potential molecular target for controlling growth of Ewing's sarcoma. Supported in part by RO1CA140409. Citation Format: Geri Traver, Konjeti R. Sekhar, Narsimha R. Renthala, Peter A. Crooks, Maxwell Ofori, Michael L. Freeman. Targeting NPM1 for controlling growth of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3189.

Friend leukemia virus integration 1 is a predictor of poor prognosis of breast cancer and promotes metastasis and cancer stem cell properties of breast cancer cells.

Breast cancer is the most common cancer in women worldwide; despite the developments in diagnosis and therapy, recurrence and metastasis remain the main causes of death among patients with breast cancer. This study aimed to identify a promising biomarker for this disease. The study clarified (1) the association between Friend leukemia virus integration 1 (FLI‐1) and various molecular subtypes and (2) the prognostic value of FLI‐1 in breast cancer. To the best of our knowledge, this study is the first to report that FLI‐1 is a predictor of poor prognosis in patients with breast cancer and overexpressed in the triple negative breast cancer (TNBC) subtype. To further verify the effect of FLI‐1 in promoting the metastasis of TNBC, we performed a series of functional experiments in vitro and orthotopic xenograft experiments in the mammary fat pad of nude mice. FLI‐1, as a transcription factor, bound to the promoters of key EMT‐related genes (CDH1 and VIM), and regulated their expressions at the transcriptional level, thus induced epithelial‐mesenchymal transition (EMT). The overexpression of FLI‐1 significantly upregulated the expression of mesenchymal markers. After the modulation of FLI‐1, the changes in mammary stem cell markers (ALDH1A1 and CD133) and the capacity to form mammospheres were consistent with those of the EMT‐related markers. The orthotopic xenograft models further confirmed that the attenuation of stem cell traits after silencing FLI‐1 decreased the ability of tumorigenesis. These results indicate that FLI‐1 is a useful predictor of poor prognosis in patients with breast cancer. Furthermore, the preliminary exploration of metastatic mechanism in the patients with TNBC will provide a potential target to treat breast cancer in the near future.

FLI1 circular RNA as biomarkers for tracking disease progression and as potential therapeutic targets in small cell lung cancer.

e20573Background: Small cell lung cancer (SCLC) is ranked as the most devastative type of human lung malignancies. We have found Friend leukemia virus integration 1 (FLI1) circular RNA (cFLI1) is a...

What can we learn from Fli1-deficient mice, new animal models of systemic sclerosis?

Systemic sclerosis is a complex multifactorial disease characterized by autoimmunity, vasculopathy, and selective organ fibrosis. A series of genetic and epidemiological studies have demonstrated that environmental influences play a central role in the onset of systemic sclerosis, while genetic factors determine the susceptibility to and the severity of this disease. Therefore, the identification of predisposing factors related to environmental influences would provide us with an informative clue to better understand the pathological process of this disease. Based on this concept, the deficiency of transcription factor Friend leukemia virus integration 1, which is epigenetically suppressed in systemic sclerosis, seems to be a potential candidate acting as the predisposing factor of this disease. Indeed, Fli1-mutated mice serve as a set of useful disease models to disclose the complex pathology of systemic sclerosis. This article overviews the recent advancement in systemic sclerosis animal models associated with Friend leukemia virus integration 1 deficiency.

A mesenteric primary peripheral Ewing's sarcoma/primitive neuroectodermal tumor with molecular cytogenetic analysis: Report of a rare case and review of literature

Rare cases of Ewing's sarcoma/primitive neuroectodermal tumors (EWS/PNETs) arising from mesenteric tissue have been reported. This report describes an EWS/PNET in a 25-year-old woman who presented with abdominal pain lasting 3 days. Radiologic evaluation revealed a 9 cm × 6 cm homogeneous mass in the lower abdomen with homogeneous enhancement and invasion of the ileum. Surgical resection was completed during exploratory laparotomy. Immunohistochemically, the tumor cells revealed CD99, friend leukemia virus integration-1 and NKX2.2 (NK2 Homeobox 2, a protein coding gene) and subsequently showed EWSR1 rearrangement. The histological feature, immunohistochemical results and genetic fluorescence in situ hybridization analysis of this case were confirming the diagnosis of EWS/PNET. Adjuvant chemotherapy was suggested, but the patient was lost to follow-up.

Fli‑1 promotes metastasis by regulating MMP2 signaling in hepatocellular carcinoma.

Friend leukemia virus integration 1 (Fli‑1) is a newly identified ETS protein, and has critical roles in many malignancies. However, the physiological characters and potential mechanisms of Fli‑1 in hepatocellular carcinoma (HCC) progression remains unclear. In the present study, Fli‑1 was highly expressed in HCC samples and tumor cell lines. knockdown of Fli‑1 with small interfering (si)RNAs significantly reduced the colony formation and metastasis capacity of HCC cell lines invitro. Subsequent investigation identified that Fli‑1 functioned as an oncogene in HCC carcinogenesis and it exerted its promoting metastatic effect primarily by modulating the matrix metalloproteinase (MMP)2 signaling pathway. Collectively, these data provide a novel insight into the mechanism of Fli‑1/MMP2 signaling pathway in the pathogenesis of HCC, and Fli‑1 may serve as a novel therapeutic target for HCC.

The impact of transcription factor Fli1 deficiency on the regulation of angiogenesis.

The insufficiency of Friend leukaemia virus integration 1 (Fli1), a member of the Ets family transcription factors, is implicated in the pathogenesis of vasculopathy associated with systemic sclerosis (SSc). Fli1 deficiency accelerates early steps of angiogenesis, including detachment of pre-existing pericytes and extracellular matrix degradation by endothelial proteinases, but the impact of Fli1 deficiency on the other steps of angiogenesis has not been investigated. Therefore, we evaluated the effect of Fli1 deficiency on migration, proliferation, cell survival and tube formation of human dermal microvascular endothelial cells (HDMECs). HDMECs transfected with FLI1 siRNA exhibited a greater migratory property in scratch assay and transwell migration assay and a higher proliferation rate in BrdU assay than HDMECs transfected with non-silencing scrambled RNA. In flow cytometry-based apoptosis assay, FLI1 siRNA-transduced HDMECs revealed the decreased number of annexin and propidium iodide-double-positive apoptotic cells compared with control cells, reflecting the promotion of cell survival. On the other hand, tubulogenic activity on Matrigel was remarkably suppressed in Fli1-deficient HDMECs relative to control cells. These results indicate that Fli1 deficiency promotes migration, proliferation and cell survival, while abating tube formation of endothelial cells, suggesting that Fli1 deficiency is potentially attributable to the development of both proliferative obliterative vasculopathy (occlusion of arterioles and small arteries) and destructive vasculopathy (loss of small vessels) characteristic of SSc vasculopathy.

Abstract 521: Dual-targeting of IGF-1R and ErbB3 pathways in Ewing’s Sarcoma cellular models with istiratumab (MM-141), a bispecific, tetravalent monoclonal antibody

Abstract Ewing’s sarcoma family tumors (ES) are aggressive tumors that often present as metastatic in bone and soft tissue and predominantly affect adolescents and younger adults. Current treatment for ES includes surgical resection followed by loco-regional radiotherapy and chemotherapy. Survival rates for patients with metastatic disease continue to offer a particularly difficult clinical challenge, with a five-year survival rate of 20-30% for these patients. ES is primarily a genetic disease caused by fusion between the 5’ segment of the Ewing sarcoma breakpoint region 1 gene (EWSR1) on chromosome 22 and the 3’ portion of Friend leukemia virus integration site 1 (FLI1) on chromosome 11. Fusions of EWSR1 and other ETS transcription factors result in dis-regulated transcription factors which promote malignant progression of ES tumors. Recent studies have shown that most ES cell lines and clinical samples express IGF-1R. Importantly, an activated IGF-1R pathway appears to be a prerequisite for malignant transformation by the EWS-FLI1 translocation, presumably via activation of the PI3K-AKT and MAPK pathways. These findings have led to the preclinical and clinical evaluation of multiple IGF-1R-targeted therapeutics with varying results. Clinical experience with anti-IGF1R targeting therapies has demonstrated striking anticancer activity in minor subsets of patients with ES. Importantly, the paucity of a clinically useful biomarker to select patients continues to hinder IGF-1R drug development in ES. Istiratumab is an investigational, bi-specific, monoclonal antibody that acts as a tetravalent inhibitor of PI3K/AKT/mTOR, a major pro-survival pathway tumor cells use as a resistance mechanism to anticancer therapies. Istiratumab is designed to interfere with this pathway by blocking ligand-induced signaling through the IGF-1R and ErbB3 receptors, based on findings that ErbB3 activation mediates resistance to the IGF-1R blockade. We will present data in multiple ES models demonstrating the importance of both IGF-1R and ErbB3 in this disease as a mechanism of growth and resistance. Furthermore, preclinical xenograft studies demonstrate that the combination of istiratumab with an irinotecan-based chemotherapy regimen offers significant benefit over chemotherapy alone. These studies suggest that clinical evaluation of istiratumab in ES is warranted. Citation Format: Isabel Yannatos, Adam Camblin, Zhenhua Li, Michael Curley, Gege Tan, Chrystal U. Louis, Vasileios Askoxylakis, Greg Finn, Birgit Schoeberl, Rachel Nering. Dual-targeting of IGF-1R and ErbB3 pathways in Ewing’s Sarcoma cellular models with istiratumab (MM-141), a bispecific, tetravalent monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 521. doi:10.1158/1538-7445.AM2017-521

Fli1 Deficiency Induces CXCL6 Expression in Dermal Fibroblasts and Endothelial Cells, Contributing to the Development of Fibrosis and Vasculopathy in Systemic Sclerosis.

CXCL6, a chemokine with proangiogenic property, is reported to be involved in vasculopathy associated with systemic sclerosis (SSc). We investigated the contribution of CXCL6 to SSc development by focusing on the association of friend leukemia virus integration 1 (Fli1) deficiency, a potential predisposing factor of SSc, with CXCL6 expression and clinical correlation of serum CXCL6 levels. mRNA levels of target genes and the binding of Fli1 to the CXCL6 promoter were evaluated by quantitative reverse transcription-PCR and chromatin immunoprecipitation, respectively. Serum CXCL6 levels were determined by ELISA. FLI1 siRNA significantly enhanced CXCL6 mRNA expression in human dermal fibroblasts and human dermal microvascular endothelial cells, while Fli1 haploinsufficiency significantly suppressed CXCL6 mRNA expression in murine peritoneal macrophages stimulated with lipopolysaccharide. Supporting a critical role of Fli1 deficiency to induce SSc-like phenotypes, CXCL6 mRNA expression was higher in SSc dermal fibroblasts than in normal dermal fibroblasts. Importantly, Fli1 bound to the CXCL6 promoter in dermal fibroblasts, endothelial cells, and THP-1 cells. In patients with SSc, serum CXCL6 levels correlated positively with the severity of dermal and pulmonary fibrosis and were elevated in association with cardiac and pulmonary vascular involvement and cutaneous vascular symptoms, including Raynaud phenomenon, digital ulcers (DU)/pitting scars, and telangiectasia. Especially, serum CXCL6 levels were associated with DU/pitting scars and heart involvement by multiple regression analysis. CXCL6 expression is upregulated by Fli1 deficiency in fibroblasts and endothelial cells, potentially contributing to the development of fibrosis and vasculopathy in the skin, lung, and heart of SSc.

Friend leukemia virus integration 1 promotes tumorigenesis of small cell lung cancer cells by activating the miR-17-92 pathway.

Small cell lung cancer (SCLC) is regarded as the most devastative type of human lung malignancies. The rapid and disseminated growth pattern remains the primary cause of poor clinical prognosis in patients with SCLC. However, the molecular factors that drive rapid progression of SCLC remain unclear. Friend leukemia virus integration 1 (FLI1), an Ets transcription factor family member, has been previously reported to act as a major driver of hematological malignancies. In this study, we explored the potential role of FLI1 in SCLC. Using immunohistochemical staining, we found that FLI1 was significantly upregulated in SCLC tissues, compared to that in non-small cell lung cancer (NSCLC) and normal lung tissues (p < 0.01). The expression score of FLI1 oncoprotein was associated with the extensive stage of SCLC and the overexpressed Ki67. Knockdown of FLI1 with small interfering RNA (siRNA) or short hairpin RNA (shRNA) promoted apoptosis and induced repression of cell proliferation, tumor colony formation and in vivo tumorigenicity in highly aggressive SCLC cell lines. Importantly, we discovered that FLI1 promoted tumorigenesis by activating the miR-17-92 cluster family. This study uncovers FLI1 as an important driving factor that promotes tumor growth in SCLC through the miR-17-92 pathway. FLI1 may serve as an attractive target for therapeutic intervention of SCLC.

Epithelial Fli1 deficiency drives systemic autoimmunity and fibrosis: Possible roles in scleroderma.

Systemic sclerosis (SSc), or scleroderma, is a multisystem autoimmune disorder characterized by vasculopathy and fibrosis in the skin and internal organs, most frequently in the esophagus and lungs. Hitherto, studies on SSc pathogenesis centered on immune cells, vascular cells, and fibroblasts. Although dysregulated keratinocytes in SSc have been recently reported, the contribution of epithelial cells to pathogenesis remains unexplored. In this study, we demonstrated the induction of SSc-like molecular phenotype in keratinocytes by gene silencing of transcription factor Friend leukemia virus integration 1 (Fli1), the deficiency of which is implicated in SSc pathogenesis. Keratin 14-expressing epithelial cell-specific Fli1 knockout mice spontaneously developed dermal and esophageal fibrosis with epithelial activation. Furthermore, they developed remarkable autoimmunity with interstitial lung disease derived from thymic defects with down-regulation of autoimmune regulator (Aire). Importantly, Fli1 directly regulated Aire expression in epithelial cells. Collectively, epithelial Fli1 deficiency might be involved in the systemic autoimmunity and selective organ fibrosis in SSc. This study uncovers unidentified roles of dysregulated epithelial cells in SSc pathogenesis.