Friend Leukemia Virus Integration Research Articles

Analysis of expression of FLI1 and MMP1 in American cutaneous leishmaniasis caused by Leishmania braziliensis infection

FLI1 (Friend leukemia virus integration 1) and IL6 (interleukin 6; IL-6) are associated with Leishmania braziliensis susceptibility. Cutaneous lesions show exaggerated matrix metalloproteinase 1 (MMP1). In other skin diseases, FLI1 promoter methylation reduces FLI1 expression, and low FLI1 down-regulates MMP1. IL-6 increases FLI1 expression. We hypothesized that epigenetic regulation of FLI1 in cutaneous leishmaniasis, together with IL-6, might determine MMP1 expression. While generally low (<10%), percent FLI1 promoter methylation was lower (P=0.001) in lesion biopsies than normal skin. Contrary to expectation, a strong positive correlation occurred between FLI1 methylation and gene expression in lesions (r=0.98, P=0.0005) and in IL-6-treated L. braziliensis-infected macrophages (r=0.99, P=0.0004). In silico analysis of the FLI1 promoter revealed co-occurring active H3K27ac and repressive DNA methylation marks to enhance gene expression. FLI1 expression was enhanced between 3 and 24hour post infection in untreated (P=0.0002) and IL-6-treated (P=0.028) macrophages. MMP1 was enhanced in lesion biopsies (P=0.0002), induced (P=0.007) in infected macrophages, but strongly inhibited by IL-6. No correlations occurred between FLI1 and MMP1 expression in lesions or infected macrophages (with/without IL-6). We conclude that MMP1 is regulated by factors other than FLI1, and that the influence of IL-6 on MMP1 was independent of its effect on FLI1.

Prenatal Exposure to Lipopolysaccharide Alters Renal DNA Methyltransferase Expression in Rat Offspring.

Prenatal exposure to inflammation results in hypertension during adulthood but the mechanisms are not well understood. Maternal exposure to lipopolysaccharide (LPS) alters interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels in the fetal environment. As reported in many recent studies, IL-6 regulates DNA methyltransferases (DNMTs) through the transcription factor friend leukemia virus integration 1 (Fli-1). The present study explores the role of intrarenal DNMTs during development of hypertension induced by prenatal exposure to LPS. Pregnant rats were randomly divided into four treatment groups: control, LPS, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor), and the combination of LPS and PDTC. Expression of IL-6, Fli-1, TNF-α, DNMT1 and DNMT3B was significantly increased in the offspring of LPS-treated rats. Global DNA methylation level of renal cortex also increased dramatically in rat offspring of the LPS group. Prenatal PDTC administration reversed the increases in gene expression and global DNA methylation level. These findings suggest that prenatal exposure to LPS may result in changes of intrarenal DNMTs through the IL-6/Fli-1 pathway and TNF-α, which probably involves hypertension in offspring due to maternal exposure to inflammation.

Why Patients with 5q- Syndrome Have Thrombocythemia?

Introduction. Fli1 (Friend leukemia virus integration 1) together with other transcription factors induces the megakaryocytic differentiation of MEP (megakarycytic and erythroid progenitor). Refractory anemia and thrombocythemia is typical for 5q- syndrome. We found increased mRNA level of Fli1 in mononuclear bone marrow cells of 5q- syndrome patients in comparison with healthy controls (Neuwirtova et al., Ann Hematol 2013). The reason of the elevated Fli1 in 5q- syndrome is haploinsufficiency of microRNA-145, which targets Fli1 mRNA (Kumar et al., Blood 2011). Due to haploinsufficiency of RPS14 in 5q- syndrome non-consumed ribosomal proteins cause ribosomal stress and inactivate HDM2 in erythroblasts. E3 ubiquitin ligase HDM2 regulates p53 level by p53 degradation in proteasome. Inactivated HDM2 in erythroid precursors of 5q- syndrome leads to apoptosis of erythroblasts and to anemia. Why ribosomal stress does not cause thrombocytopenia and ineffective megakaryopoiesis as well? Our previous results support significant role of Fli1 in this process. Fli1 binds to promoter of the HDM2 gene and increases its transcription (Truong et al., Oncogene 2005). The increased activity of HDM2 in megakaryocytes inspite of ribosomal stress maintains p53 regulation and its degradation in proteasome. Megakaryopoiesis remains effective. Why it is not the case in erythroid precursors? To answer this question it was necessary to detect Fli1 as the protein and to determine in which cells Fli1 is present.Material and Methods. Twenty-three control representative bone marrow trephine biopsies of patients from controls (8 negative staging biopsies in lymphoma) and of patients with various hematological diagnoses (7 MDS with normal chromosome 5, 4 MPN, 3 AML and 1 RARS-T) and from 15 patients with 5q- syndrome were examined. In 13 patients with 5q- syndrome, samples taken before and 6 months after lenalidomide (Revlimid) therapy were available. The expression of Fli1 protein was investigated by immunohistochemistry (IHC). Expression of Fli1 on erythroid precursors was studied by double staining IHC procedure utilizing antibodies against Fli1 and either glycophorin A or E-cadherin known as reliable markers for erythroid precursors.Results. Nuclear expression of Fli1 was demonstrated in normal as well as in dysplastic megakaryocytes, in most cells of granulocytic series and lymphocytes. No staining for Fli1 was seen in erythroblasts and proerythroblasts visualized by expression of either glycophorin A or E-cadherin both in 5q- syndrome and controls. There were no significant differences in Fli1 expression between samples taken before and after lenalidomide treatment.The used IHC technique does not permit quantitative analysis of Fli1 protein levels. This fact could explain why we did not find any difference in Fli1 protein labeling in megakaryocytes before and after lenalidomide treatment while Fli1 mRNA level was decreased in majority of 5q- syndrome patients after six months of this therapy.Conclusion. Fli1 expression was found in normal as well as in dysplastic megakaryocytes. However, no Fli1 positivity was found in erythroid precursors in both 5q- syndrome and controls. Negativity of Fli1 expression in erythroid precursors in 5q- syndrome support our hypothesis of protective role of Fli1 against apoptosis under ribosomal stress in megakaryocytes in contrast to erythroblasts lacking Fli1. This protective role of Fli1 in megakaryocytes consists in Fli1 potentiation of expression of the E3 ubiquitine ligase HDM2 (Truong et al., Oncogene 2005). The presence of increased Fli1 in megakaryocytes helps to explain effective megakaryopoiesis in 5q- syndrome and is the answer to the question in the title of our abstract.Supported by Ministry of Health, Czech Republic-conceptual development of research organization Institute of Hematology and Blood Transfusion 00023736, RVO-VFN64165 and PRVOUK P-27/LF1/2. DisclosuresNo relevant conflicts of interest to declare.

Myeloid Fli1 deficiency induces tissue fibrosis, vasculopathy, and immune abnormalities recapitulating systemic sclerosis

Systemic sclerosis (SSc) is a multisystem chronic disease characterized by immune abnormalities, vasculopathy, and tissue fibrosis. In SSc lesional skin, transcription factor Friend leukemia virus integration 1 (Fli1) is constitutively down-regulated in various cell types, especially by an epigenetic mechanism in dermal fibroblasts, and Fli1 deficiency in dermal fibroblasts and endothelial cells induces an SSc-like phenotype in each cell type. However, it has still remained to be elucidated whether Fli1 deficiency in immune cells has an impact on the pathogenesis of SSc. As a part of studies to address this issue, we focused our interests on myeloid cells in this study. We generated myeloid cell-specific Fli1 deficient (Fli1 Mye-KO) mice (LysM-Cre+/−Fli1flox/flox) and examined the phenotypic changes in these mice. Fli1 Mye-KO mice showed increased dermal thickness and collagen production through activation of dermal fibroblasts. As for vasculopathy, Fli1 Mye-KO mice showed stenosis, dilatation, and regression of dermal blood vessels as visualized by FITC-conjugated dextran injection. Increased permeability of dermal blood vessels was also confirmed by leakage of Evans blue dye into interstitial spaces in Fli1 Mye-KO mice. Histopathological analysis of the skin of Fli1 Mye-KO mice showed decreased pericyte coverage in dermal blood vessels, leading to endothelial hyperplasia, vascular dilatation, and decreased stability of blood vessels. Regarding immune dysfunction, Fli1 Mye-KO mice showed production of anti-nuclear antibodies. Thus, Fli1 deficiency in myeloid cells may contribute to the induction of three cardinal pathological features of SSc, including dermal fibrosis, vascular injury, and immune abnormalities.

MiR-145 promotes osteosarcoma growth by reducing expression of the transcription factor friend leukemia virus integration 1.

Osteosarcoma (OS) is the most common malignant bone tumor in children and young adults. miR-145 is a microRNA highly expressed in vascularized tissues and has been widely studied in cancers. In this study, we explored the expression and function of miR-145 in OS. We found that miR-145 was consistently under-expressed in OS tissues and cell lines as compared to normal bone tissues and osteoblast cells. Ectopic expression of miR-145 in OS cells inhibited their proliferation and migration and induced apoptosis. miR-145 targets a putative microRNA regulatory element (MRE) in the 3′-UTR of friend leukemia virus integration 1 gene (FLI-1), and its abundance was inversely related to FLI-1 expression in OS tissues and cell lines. miR-145 decreased expression FLI-1 protein and mRNA, but mutation of the miR-145 MRE sequence in the FLI-1 3′-UTR abolished the activity of miR-145 in a reporter assay. Restored expression of FLI-1 diminished miR-145-mediated suppression of tumor progression. These results suggest that miR-145 acts as a tumor suppressor by directly reducing expression of FLI-1, and that the miR-145/FLI-1 pathway is important for tumor progression in OS.

Cooperation between somatic mutations and germline susceptibility variants in tumorigenesis – a dangerous liaison

ABSTRACTHigh-throughput genotyping and sequencing generate comprehensive catalogs of inherited genetic variations and acquired somatic mutations. However, their possible interactions and roles in tumorigenesis remain largely unexplored. We recently reported cooperation between the EWSR1-FLI1 (Ewing sarcoma breakpoint region 1 – Friend leukemia virus integration 1) fusion oncogene and a germline variant that regulates the EGR2 (early growth response 2) Ewing sarcoma susceptibility gene via a GGAA-microsatellite.

FLI1 Levels Impact CXCR3 Expression and Renal Infiltration of T Cells and Renal Glycosphingolipid Metabolism in the MRL/lpr Lupus Mouse Strain.

The ETS factor Friend leukemia virus integration 1 (FLI1) is a key modulator of lupus disease expression. Overexpressing FLI1 in healthy mice results in the development of an autoimmune kidney disease similar to that observed in lupus. Lowering the global levels of FLI1 in two lupus strains (Fli1(+/-)) significantly improved kidney disease and prolonged survival. T cells from MRL/lpr Fli1(+/-) lupus mice have reduced activation and IL-4 production, neuraminidase 1 expression, and the levels of the glycosphingolipid lactosylceramide. In this study, we demonstrate that MRL/lpr Fli1(+/-) mice have significantly decreased renal neuraminidase 1 and lactosylceramide levels. This corresponds with a significant decrease in the number of total CD3(+) cells, as well as CD4(+) and CD44(+)CD62L(-) T cell subsets in the kidney of MRL/lpr Fli1(+/-) mice compared with the Fli1(+/+) nephritic mice. We further demonstrate that the percentage of CXCR3(+) T cells and Cxcr3 message levels in T cells are significantly decreased and correspond with a decrease in renal CXCR3(+) cells and in Cxcl9 and Cxcl10 expression in the MRL/lpr Fli1(+/-) compared with the Fli1(+/+) nephritic mice. Our results suggest that reducing the levels of FLI1 in MRL/lpr mice may be protective against development of nephritis in part through downregulation of CXCR3, reducing renal T cell infiltration and glycosphingolipid levels.

MN1-Fli1 oncofusion transforms murine hematopoietic progenitor cells into acute megakaryoblastic leukemia cells.

Long-term outcome of acute megakaryoblastic leukemia (AMKL) patients without Down's syndrome remains poor. Founding mutations and chimeric oncogenes characterize various AMKL subtypes. However, for around one third of all cases the underlying mechanisms of AMKL leukemogenesis are still largely unknown. Recently, an in-frame fusion of meningeoma 1–friend leukemia virus integration 1 (MN1–Fli1) gene was detected in a child with AMKL. We intended to investigate the potential role of this oncofusion in leukemogenesis of acute myeloid leukemia. Strikingly, expression of MN1–Fli1 in murine hematopoietic progenitor cells was sufficient to induce leukemic transformation generating immature myeloid cells with cytomorphology and expression of surface markers typical for AMKL. Systematic structure function analyses revealed FLS and 3′ETS domains of Fli1 as decisive domains for the AMKL phenotype. Our data highlight an important role of MN1–Fli1 in AMKL leukemogenesis and provide a basis for research assessing the value of this oncofusion as a future diagnostic marker and/or therapeutic target in AMKL patients.

Overexpression of Fli-1 is Associated with Adverse Prognosis of Endometrial Cancer

This study aimed to investigate the expression of Friend leukemia virus integration 1 (Fli-1) and its correlation with the prognosis of endometrial cancer (EC). Thirty-two EC tissue samples were evaluated for Fli-1 expression using immunohistochemistry. Fli-1 showed significantly high expression in EC cells, followed by hyperplasia cells, and was negative in adjacent normal tissues. The high expression of Fli-1 was significantly associated with a high differentiation grade, mutated P53 expression, and histological subtype (p < .05). Downregulation of Fli-1 in AN3CN cells using RNA interference inhibited cell clone formation and proliferation but did not affect apoptosis and migration of the cells. This study provides the first evidence that Fli-1 expression gradually increases in parallel with disease progression, and its overexpression might predict poor prognosis in EC.

Friend leukemia virus integration 1 activates the Rho GTPase pathway and is associated with metastasis in breast cancer.

Breast cancer is the most prevalent malignant disease in women worldwide. In patients with breast cancer, metastasis to distant sites directly determines the survival outcome. However, the molecular mechanism underlying metastasis in breast cancer remains to be defined. In this report, we found that Friend leukemia virus integration 1 (FLI1) proto-oncogene was differentially expressed between the aggressive MDA-MB231 and the non-aggressive MCF-7 breast cancer cells. Congruently, immunohistochemical staining of clinical samples revealed that FLI1 was overexpressed in breast cancers as compared with the adjacent tissues. The abundance of FLI1 protein was strongly correlated with the advanced stage, poor differentiation, and lymph node metastasis in breast cancer patients. Knockdown of FLI1 with small interfering RNAs significantly attenuated the potential of migration and invasion in highly metastatic human breast cancer cells. FLI1 oncoprotein activated the Rho GTPase pathway that is known to play a role in tumor metastasis. This study for the first time identifies FLI1 as a clinically and functionally important target gene of metastasis, providing a rationale for developing FLI1 inhibitors in the treatment of breast cancer.

Wound healing genes and susceptibility to cutaneous leishmaniasis in Brazil: Role of COL1A1

Previous studies have demonstrated a role for wound healing genes in resolution of cutaneous lesions caused by Leishmania spp. in both mice and humans, including the gene FLI1 encoding Friend leukemia virus integration 1. Reduction of Fli1 expression in mice has been shown to result in up-regulation of collagen type I alpha 1 (Col1a1) and alpha 2 (Col1a2) genes and, conversely, in down-regulation of the matrix metalloproteinase 1 (Mmp1) gene, suggesting that Fli1 suppression is involved in activation of the profibrotic gene program. Here we examined single nucleotide polymorphisms (SNPs) in these genes as risk factors for cutaneous (CL) and mucosal leishmaniasis (ML), and leishmaniasis per se, caused by L. braziliensis in humans. SNPs were genotyped in 168 nuclear families (250 CL; 87 ML cases) and replicated in 157 families (402 CL; 39 ML cases). Family-based association tests (FBAT) showed the strongest association between SNPs rs1061237 (combined P=0.002) and rs2586488 (combined P=0.027) at COL1A1 and CL disease. This contributes to our further understanding of the role of wound healing in the resolution of CL disease, providing potential for therapies modulating COL1A1 via drugs acting on FLI1.

Friend Leukemia Virus Integration 1 Expression Has Prognostic Significance in Nasopharyngeal Carcinoma

This study aimed to investigate the expression pattern and prognostic value of friend leukemia virus integration 1 (FLI-1) in nasopharyngeal carcinoma (NPC). Immunohistochemistry (IHC) staining of FLI-1 was performed in specimens from 198 untreated NPC patients. Ninety-nine patients were randomly assigned to the training set to analyze the prognostic value of FLI-1 and other clinicopathological characteristics, while the others were assigned to the testing set for validation. Clinicopathological data were compared using the Pearson chi-square test. Univariate and multivariate analyses were performed using the Cox proportional hazards model to test independent prognostic factors and calculate the hazard ratio (HR) and 95% confidence interval (CI). Cytoplasmic FLI-1 expression positively correlated with N stage, distant metastasis and death (P<0.05) and also predicted poorer overall survival (OS) (P=0.014), distant metastasis-free survival (DMFS) (P=0.010), progression-free survival (PFS) (P=0.031). In multivariate analysis, FLI-1 expression and clinical stage were both independent prognostic factors of poor OS and DMFS. Prognoses of patients in the training set, the testing set, and the entire set were clearly divided into four risk subgroups by supplementing FLI-1 with clinical stage. These results indicate that FLI-1 expression is an independent prognostic factor for NPC patients and suggest that supplementing FLI-1 with clinical stage could be helpful for more accurate risk definition.

Primary Subcutaneous Spindle Cell Ewing Sarcoma with Strong S100 Expression and EWSR1-FLI1 Fusion: A Case Report

Ewing sarcoma is described classically as a small, round cell tumor of bone and soft tissue in children and young adults. Ewing sarcoma most often is characterized by a fusion of the Ewing sarcoma breakpoint region 1 (EWSR1) and the Friend leukemia virus integration 1 (FLI1) genes, forming an EWSR1-FLI1 fusion transcript. We report an exceptional case of primary subcutaneous Ewing sarcoma in a 16-year-old female composed entirely of spindle cells with focal fascicular growth and exhibiting strong, diffuse immunohistochemical reactivity for S100, unlike classic Ewing sarcoma. However, reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed the presence of a rare variant of the EWSR1-FLI1 fusion transcript, featuring fusion of EWSR1 exon 10 to FLI1 exon 6. To our knowledge, the combined histologic, molecular, and clinical features have not been reported previously in Ewing sarcoma, and raise a broad differential diagnosis emphasizing the importance of molecular techniques in the diagnosis of this tumor.

Mouse model of Ewing's sarcoma with cell-specific expression of a transgenic Ewing sarcoma breakpoint region 1 (EWSR1; EWS)-Friend leukemia virus integration 1 (FLI1) fusion protein

Mouse model of Ewing's sarcoma with cell-specific expression of a transgenic Ewing sarcoma breakpoint region 1 (EWSR1; EWS)-Friend leukemia virus integration 1 (FLI1) fusion protein

The Friend leukaemia virus integration 1 (Fli-1) transcription factor affects lupus nephritis development by regulating inflammatory cell infiltration into the kidney

The transcription factor Friend leukaemia virus integration 1 (Fli-1) is implicated in the pathogenesis of systemic lupus erythematosus in both human patients and murine models of lupus. Murphy Roths large (MRL)/lpr mice and New Zealand mixed (NZM)2410 mice, murine models of lupus, with decreased expression of Fli-1 had significantly prolonged survival and reduced nephritis. Lupus nephritis is a major cause of mortality and morbidity in patients, and inflammatory cell infiltration plays a key role in the development of the disease. To study how the expression of Fli-1 affects the infiltration of inflammatory cells into the kidneys, we generated congenic enhanced green fluorescent protein (GFP) transgenic MRL/lpr mice. A significantly increased number of GFP-expressing inflammatory cells infiltrated the kidneys of wild-type MRL/lpr mice compared to Fli-1 heterozygous (Fli-1(+/-)) MRL/lpr mice after injection of GFP(+) cells. Expression of inflammatory chemokine mRNA, including chemokine (C-C motif) ligand (CCL)2, CCL3, CCL4 and CCL5, was significantly lower in the kidneys from Fli-1(+/-) MRL/lpr mice compared to wild-type littermates. Numbers of infiltrated cells into the kidneys correlate with expression levels of CCL2, CCL4 and CCL5, but not the titres of anti-dsDNA autoantibodies in these mice. Significantly increased inflammatory cells from wild-type MRL/lpr mice infiltrated into kidneys compared to the cells from Fli-1(+/-) MRL/lpr mice. The chemotaxis of inflammatory cells from Fli-1(+/-) MRL/lpr mice towards each chemokine was decreased significantly compared to inflammatory cells from wild-type MRL/lpr mice in the transwell migration assay in vitro. Our results indicate that Fli-1 affects lupus nephritis development by regulating the expression of chemokines in the kidney and the migration of inflammatory cells.

RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4gene expression in combination with ETS family proteins

Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt-related transcription factor 1 (RUNX1) of FPD patients. To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1-mediated induction of the FPD phenotype. We assessed whether RUNX1 and its mutants, in combination with E26 transformation-specific-1 (ETS-1), Core-binding factor subunit beta (CBFβ), and Friend leukemia virus integration 1 (FLI-1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real-time RT-PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos-7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos-7 cells. We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS-1 and CBFβ, or FLI-1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA-binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS-1, CBFβ, and FLI-1. RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.

EWS represses cofilin 1 expression by inducing nuclear retention of cofilin 1 mRNA

In Ewing's sarcoma family tumors (ESFTs), the proto-oncogene EWS that encodes an RNA-binding protein is fused by chromosomal translocation to the gene encoding one of the E-twenty six (ETS) family of transcription factors, most commonly friend leukemia virus integration 1 (FLI-1). Although EWS/FLI-1 chimeric proteins are necessary for carcinogenesis, additional events seem to be required for transformation to occur. We have previously reported that a protein product of an EWS mRNA target, whose expression is negatively regulated by EWS but not by EWS/FLI-1, contributes to ESFT development. However, the mechanism by which EWS represses protein expression remains to be elucidated. Here, we report that overexpression of full-length EWS repressed protein expression and induced nuclear retention of reporter mRNAs in a tethering assay. In contrast, when a mutant lacking the EWS C-terminal nuclear localization signal (classified as a PY-NLS) was expressed, reporter protein expression was upregulated, and the number of cells exporting reporter mRNA to the cytoplasm increased. EWS binds to the 3'-untranslated region in another mRNA target, cofilin 1 (CFL1), and negatively regulates the expression of CFL1. Overexpression of EWS induced nuclear retention of CFL1 mRNA. Furthermore, ESFT cell proliferation and metastatic potential were suppressed by small interfering RNA-mediated CFL1 knockdown. Together, our findings suggest that EWS induces nuclear retention of CFL1 mRNA, thereby suppressing expression of CFL1, and that CFL1 promotes development of ESFT. Targeting CFL1 might therefore provide another novel approach for treatment of this aggressive disease.

Expression of FLI-1 and the proliferation and survival of small cell lung cancer cells.

e18550 Background: Small cell lung cancer (SCLC) exhibits aggressive behavior, rapid growth, early spread to distant sites. Over the last few decades, prognosis for patients with SCLC has changed little and there remains an critical need for evaluating novel agents. Here, we report that Fli-1 (Friend leukemia virus integration 1), a transcription factor and member of the Ets family, has an essential role in SCLC progression and may be a promising target for therapy. Methods: Immunohistochemical staining was used to investigate the expression level of Fli-1 in 25 patients with SCLC, including 12 patients in limited stage and 13 patients in extensive stage. The growth, apoptosis assay and molecular mechanism of Fli-1 in SCLC cell line (NCI-H446) was studied by down-regulating Fli-1 expression through RNA interference. Results: It was shown that expression level of Fli-1 was significantly up-regulated in SCLC tissues compared with adjacent tissues (p=0.0002). Its staining was found specifically in the nuclei of cancer cells. Expression level of Fli-1 was higher in extensive stage disease than that in limited stage disease (p=0.0045). Down-regulation of Fli-1 expression by RNA interference in NCI-H446 was found to strongly decrease cell growth, promote cell apoptosis and reduce metastasis. Importantly, when Fli-1 was down-regulated in NCI-H446, epidermal growth factor (EGF)-induced Akt activation was dramatically decreased. It was implied that Fli-1 was involved in the Akt regulation passway. It was also confirmed that the apoptosis induced by decrease of Fli-1 expression via down-regulation of its target regulation gene, bcl-2. Conclusions: The novel mechanistic insight into a critical role of Fli-1 in SCLC progression was shown in this study. Fli-1 overexpression promotes SCLC aggression partly via activating Akt phosphorylation. We propose that Fli-1 is a new powerful predictor for SCLC progression, and is also a new potential target for SCLC treatment.

TGF-β1 Supports Leukemia Cell Survival Via Negative Regulation of FLI-1 Transcription Factor, ERK Inactivation and MMP-1 Secretion

Abstract 3543Transforming growth factor β (TGF-β) is an essential regulator of cell proliferation, survival, and apoptosis, depending on the cellular context. We have previously reported pro-survival effects of TGF-β1 in myelo-monocytic leukemia cells (Xu et al., Br J Haematol.2008) and the anti-leukemic effects of action of TGF-β neutralizing antibody under TGF-β abundant and hypoxic bone marrow (BM) microenvironment (ASH, 2012). Friend leukemia virus integration 1 (FLI-1), a member of Ets transcriptional factors, plays a pivotal role in the regulation of extracellular matrix (ECM) genes, and is known to be negatively regulated through TGF-β1-dependent acetylation. We have recently reported that abnormal (high or low) expression of FLI-1 protein analyzed by reverse phase protein arrays is associated with inferior remission duration and reduced survival (Kornblau et al., Blood, 2011). Notably, among 195 proteins tested, FLI-1 expression correlated most with SMAD4, the common mediator in a family of SMAD proteins involved in TGFβ signaling. In this study, we investigated the molecular interactions between TGF-β1 and FLI-1 in AML cells and its functional role in TGF-β-mediated survival. In four AML cell lines, MV4;11, U937, NB4, and OCI-AML3, recombinant TGF-β1 (2ng/mL) induced the TGF-β downstream signaling targets plasminogen activator inhibitor-1 (PAI-1, mRNA) and/or Smad2 phosphorylation, which was reversed by anti-TGF-β neutralizing antibody 1D11 (Genzyme). No consistent change of Smad4 expression was observed in TGF-β1 treated cells. Treatment with rhTGF-β1 inhibited serum starvation-induced apoptosis in MV4;11, U937 and NB4, but not in OCI-AML3 cells. The anti-apoptotic effect of TGF-β1 was associated with G0/G1 cell cycle arrest, which was effectively reversed by anti-TGF-β antibody 1D11.In MV4;11, U937 and NB4 cells, in which rhTGF-β1 promoted cell survival, rhTGF-β1 downregulated expression levels of FLI-1 mRNA and/or protein. However, FLI-1 was upregulated by rhTGF-β1 in OCI-AML3 cells. Since FLI-1 activation is known to cause cell proliferation associated with Ras pathway activation, we investigated MAPK signaling downstream of Ras. Changes in ERK phosphorylation levels after rhTGF-β1 treatment were fully concordant with FLI-1, whereby phospho-ERK was downregulated in MV4;11, U937, and NB4 cells, and upregulated in OCI-AML3 cells. These effects were reversed by anti-TGF-β antibody 1D11. In turn, rhTGF-β1 induced Matrix metalloproteinase-1 (MMP-1) mRNA which inversely correlated with FLI-1 expression. (U937; 6.9 fold increase, OCI-AML3; 3.1 fold decrease). It has been reported that ERK signaling upregulates MMP-1 expression, and that FLI-1 downregulates MMP-1 promoter activity in human fibroblasts. MMP-1, being responsible for degradation of collagenous proteins of ECM, correlates with poor prognosis in leukemia. We also observed that rhTGF-β1 induced significant upregulation of anti-apoptotic Bcl-2 in MV4;11, U937, and NB4 cells, but not in OCI-AML3 cells.In summary, TGF-β-induced FLI-1 downregulation and ERK inactivation may be implicated in pathological matrix remodeling via oncogenic MMP-1 transcription in TGF-β abundant BM microenvironment. These findings suggest that FLI-1 and MMP-1 contribute to chemoresistance and poor outcomes in AML and represent potentially targetable molecular aberrations in AML. Disclosures:No relevant conflicts of interest to declare.

Targeting HRASV12G Expression to the Zebrafish Early Hemogenic Progenitors Induces a Myeloproliferative Disorder by Repressing the Notch Pathway

AbstractAbstract 4676 IntroductionMyeloproliferative diseases (MPDs) are a group of haematological disorders characterized by the hyper proliferation of different blood cells in peripheral blood and other hematopoietic organs. The clinical heterogenity of these neoplasms reflects the different gene pathways involved, most of which are only partially known. Given the genetic homology and the physiological similarity to mammals, zebrafish has emerged as an ideal model to study human normal and malignant haematopoiesis. In the last decade several oncogenes involved in the development of hematopoietic neoplasms have been used to model leukemia in zebrafish with the aim to discovery new molecular pathways involved in malignant transformation. Despite the first encouraging results these experimental models failed to fully recapitulate human myeloproliferative disorders. MethodsWe took advantage of the Gal4/UAS binary system to induce the expression of human oncogenic HRASV12G in the zebrafish hematopoietic compartment. We used a specific transgenic line that drives oncogene expression in zebrafish early hematopoietic progenitors under control of the FLI.1 (Friend Leukemia virus Integration 1) promoter. ResultsWe observed the development of a myelo-erythroid proliferative disease in few days in zebrafish transgenic larva. The pathological phenotype is characterized by the expansion of the hematopoietic tissue, an increased expression of myelo-erythroid specific genes (PU.1, gata1, mpx, c-mpl) associated with a slight increase of staminality markers (lmo2, scl, c-myb, runx.1), and a higher number of l-plastin expressing cells. Moreover blood smear of pathological larva displayed leukemic blasts and the arrest of erythrocyte differentiation whereas kidney marrow of juvenile fish displayed abnormal myelopoiesis characterized by the increase of erythro-myeloid progenitors.We found that the pathological phenotype is associated with a down regulation of the Notch pathway as shown by the decreased gene expression of notch pathways target genes (notch1, notch3, her6). Furthermore we discovered a novel set of genes involved in neoplastic transformation induced by HRASV12 expression through RNA-Seq analysis of pathological larva. ConclusionsThe expansion of the zebrafish hematopoietic compartment characterized by the hyper-proliferation of the myelo-erythroid progenitors that we found in this model reproduces some of the pathological features of human myeloproliferative disorders. This study showed that forcing oncogene expression in the hemogenic endothelial cells induces the transdifferentiation of the early hemogenic pluripotent stem cells into abnormal myeloerythoid progenitors by repressing the Notch pathways. Transcriptome analysis identified a number of potential effectors of this transformation. Disclosures:No relevant conflicts of interest to declare.