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New series of 8-nitroquinolone based aromatic heterocyclic acyl hydrazones have been synthesized and characterized through various spectroscopic techniques. They were theoretically examined for molecular docking with various proteins related with...

Xing et al demonstrate the requirements for Tle transcriptional corepressors in CD8+ T cell development. Tle proteins are differentially partitioned to the Runx and Tcf/Lef complexes to promote CD8+ lineage choice and establish CD8+ T cell identity, respectively., Tle/Groucho proteins are transcriptional corepressors interacting with Tcf/Lef and Runx transcription factors, but their physiological roles in T cell development remain unknown. Conditional targeting of Tle1, Tle3 and Tle4 revealed gene dose–dependent requirements for Tle proteins in CD8+ lineage cells. Upon ablating all three Tle proteins, generation of CD8+ T cells was greatly diminished, largely owing to redirection of MHC-I–selected thymocytes to CD4+ lineage; the remaining CD8-positive T cells showed aberrant up-regulation of CD4+ lineage-associated genes including Cd4, Thpok, St8sia6, and Foxp3. Mechanistically, Tle3 bound to Runx-occupied Thpok silencer, in post-selection double-positive thymocytes to prevent excessive ThPOK induction and in mature CD8+ T cells to silence Thpok expression. Tle3 also bound to Tcf1-occupied sites in a few CD4+ lineage-associated genes, including Cd4 silencer and St8sia6 introns, to repress their expression in mature CD8+ T cells. These findings indicate that Tle corepressors are differentially partitioned to Runx and Tcf/Lef complexes to instruct CD8+ lineage choice and cooperatively establish CD8+ T cell identity, respectively., Graphical Abstract

Tle1 (transducin-like enhancer of split 1) is a corepressor that interacts with a variety of DNA-binding transcription factors and has been implicated in many cellular functions; however, physiological studies are limited. Tle1-deficient (Tle1(Δ/Δ)) mice, although grossly normal at birth, exhibit skin defects, lung hypoplasia, severe runting, poor body condition, and early mortality. Tle1(Δ/Δ) mice display a chronic inflammatory phenotype with increased expression of inflammatory cytokines and chemokines in the skin, lung, and intestine and increased circulatory IL-6 and G-CSF, along with a hematopoietic shift toward granulocyte macrophage progenitor and myeloid cells. Tle1(Δ/Δ) macrophages produce increased inflammatory cytokines in response to Toll-like receptor (TLR) agonists and lipopolysaccharides (LPS), and Tle1(Δ/Δ) mice display an enhanced inflammatory response to ear skin 12-O-tetradecanoylphorbol-13-acetate treatment. Loss of Tle1 not only results in increased phosphorylation and activation of proinflammatory NF-κB but also results in decreased Hes1 (hairy and enhancer of split-1), a negative regulator of inflammation in macrophages. Furthermore, Tle1(Δ/Δ) mice exhibit accelerated growth of B6-F10 melanoma xenografts. Our work provides the first in vivo evidence, to our knowledge, that TLE1 is a major counterregulator of inflammation with potential roles in a variety of inflammatory diseases and in cancer progression.

The MUC1 heterodimeric transmembrane glycoprotein is aberrantly overexpressed by diverse human carcinomas. Galectin-3 is a beta-galactoside binding protein that has also been associated with the development of human cancers. The present results demonstrate that MUC1 induces galectin-3 expression by a posttranscriptional mechanism. We show that the MUC1 C-terminal subunit is glycosylated on Asn-36 and that this modification is necessary for upregulation of galectin-3. N-glycosylated MUC1-C increases galectin-3 mRNA levels by suppressing expression of the microRNA miR-322 and thereby stabilizing galectin-3 transcripts. The results show that, in turn, galectin-3 binds to MUC1-C at the glycosylated Asn-36 site. The significance of the MUC1-C-galectin-3 interaction is supported by the demonstration that galectin-3 forms a bridge between MUC1 and the epidermal growth factor receptor (EGFR) and that galectin-3 is essential for EGF-mediated interactions between MUC1 and EGFR. These findings indicate that MUC1 and galectin-3 function as part of a miR-322-dependent regulatory loop.

The mucin (MUC) family consists of secreted and membrane-bound forms. The transmembrane mucin 1 (MUC1) is a heterodimer that is aberrantly overexpressed by diverse human carcinomas and certain hematologic malignancies. The MUC1 N-terminal (MUC1-N) and C-terminal (MUC1-C) subunits are generated by autocleavage within a SEA domain. The MUC1 cytoplasmic domain (MUC1-CD) located downstream of the SEA domain is sufficient for the induction of anchorage-independent growth and tumorigenicity; however, no information is available regarding the origin of these transforming sequences. Previous work demonstrated that, except for the SEA domain, MUC1 has no sequence homology with other membrane-bound mucins. The present results demonstrate that MUC1-CD evolved from repeat regions in the MUC5B secreted mucin. We also show that MUC1 sequences upstream to the SEA domain emerged from MUC5B. These findings indicate that both the MUC1-N and MUC1-C subunits evolved from secreted gel-forming mucins and that the MUC1-CD oncogenic function emerged by diversification after evolution from MUC5B.

Abstract 1197 TLE1 belongs to the Groucho/TLE family of co-repressors that act as master regulators during development affecting segmentation, neurogenesis, myogenesis, and multiple cell fate decisions. TLE1 modulate several major signaling pathways including Wnt and Notch, and specifically interacts with multiple transcription factors involved in hematopoiesis such TCF/LEF, HES1, RUNX1/AML. TLE1 has also been implicated in Crohn's disease via its interaction with NOD2, a regulator of NFkB. Our laboratory identified TLE1 as a likely AML tumor suppressor gene, commonly deleted in subgroups of AML, and others have shown its role as a tumor suppressor gene in myeloid and other hematopoietic malignancies. To better understand the role of TLE1 in hematopoiesis and leukemogenesis we created a line of Tle1 null mice. Tle1 null mice are born normally, but become progressively growth retarded by 3 days of life, with only 50% survival by 4 weeks as compared to heterozygous and wild type littermates. Abnormalities are observed in several organs systems including the hematopoietic system. We characterized the hematopoietic system in Tle1 knock out mice between two and 12 weeks of age. The bone marrow cellularity in the Tle1 knock out mice is comparable to the wild type mice at all time points examined. However, frequency of granulocyte macrophage progenitors in bone marrow mononuclear cells is significantly higher in the Tle1 knockout bone marrow compared to heterozygous and wild type mice. The proportion and number of myeloid cells as evidenced by Gr1, Mac1 expression are significantly higher in the bone marrow, spleen and blood of these knockout mice. There were significantly lower B-cells (B220+cells) in the Tle1 knockout mice compared to heterozygous and wild type. In colony forming assays there was a trend towards higher number of CFU-GM (7.66 vs 5), p=0.07) and CFU-M (27.16 vs 12.5, p=0.05) colonies from Tle1 null bone marrow as compared to wild type bone marrow. The spleens from four week and 17 months old Tle1 knockout mice had higher frequency of Gr1-negative, Mac1-positive and F4/80 positive macrophages. We also observed a significantly higher production of the inflammatory cytokines IL6 and TNFafrom peritoneal macrophages harvested from Tle1 null mice as compared to those from wild type mice in response to TLR ligand stimulation. To investigate the potential mechanism of this inhibitory effect of TLE1 on inflammation we demonstrated that TLE1 expression is able to block the nuclear translocation of NFkB in THP1 cells in response to LPS-K12 (p In summary this work demonstrates that the lack of Tle1 expression biases hematopoiesis towards myeloid differentiation, a finding of potential relevance given the inactivation of TLE1 seen in subsets of myeloid malignancies. We further show that inactivation of Tle1 leads to an increase in macrophages primed to release increased inflammatory cytokines. This is notable given the recent observation that TLE1 may modulate the effects of NOD2 in the pathogenesis of Crohn's disease. These Tle1 null mice will allow the investigation of the potential role of TLE1 as a modulator of a variety of other inflammatory diseases. Disclosures: No relevant conflicts of interest to declare.

Abstract 2473 The Groucho/TLE family of corepressors has been described as master regulatory genes during development, affecting multiple cell fate decisions. These proteins bind to a variety of transcription factors and recruit inhibitory proteins to repress transcription. We previously identified TLE1 as a novel tumor suppressor gene that is deleted or methylated in subgroups of acute myeloid leukemia (AML) and other hematological malignancies. We find the loss of Tle1 alone is insufficient to induce leukemia in mice and apparently requires cooperation with additional oncogenes. Our studies, and those from other groups, have shown that over-expression of TLE1 in leukemia cells slows cell cycle progression, colony formation and tumor growth in xenografts, while silencing results in increased cell proliferation. The pathways by which TLE1 affects oncogenesis is unclear, but this gene family is capable of interacting with effectors of Myc, Wnt, Notch, TGFB signaling–prominent pathways dysregulated in malignancies. Myc is important for hematopoietic stem cell proliferation, survival and differentiation and is over-expressed in most AML samples. The TLE homologue Groucho binds and represses Drosophila Myc expression of target genes, thus we postulated that TLE1 could be an important regulator of Myc activity in leukemia. Using hematopoietic progenitor cells from Tle1 knockout and wild-type fetal livers we found that the loss of Tle1 dramatically increased proliferation and serial replating efficiency. Expression of N-Myc by itself in wild type fetal liver cells triggered significant cell death and apoptosis. However, when N-Myc expression was combined with the additional loss of Tle1, not only was N-Myc induced apoptosis inhibited, but a dramatic cell proliferation, well in excess of that seen with Tle1 loss by itself, was seen. Furthermore, mice transplanted with N-Myc transduced hematopoietic cells from Tle1 knockout mice fetal liver developed a more aggressive leukemia, compared to N-Myc transfected wild type mice fetal liver hematopoietic cells, with increased proliferation of leukemic cells as demonstrated by in vitro colony assays and higher secondary transplantability. We extended these studies to a zebrafish model of Rag2-Myc mediated T-ALL. Using these zebrafish we demonstrated over-expression of the TLE homologue, Groucho, completely blocked the initiation and progression of Myc induced leukemia development. Expression of a truncated version of Groucho reduced the initiation of T-ALL and prolonged the survival of fish developing leukemia. These studies demonstrate TLE1 can inhibit the oncogenicity of Myc, and suggests modulation of expression of this gene family may be of importance for a variety of malignancies. Disclosures: No relevant conflicts of interest to declare.

Transducin-like enhancer of split-1 (TLE1) is a novel tumor suppressor gene that is deleted or methylated in subgroups of acute myeloid leukemia (AML) and other hematological malignancies. TLE1 belongs to a family of Gro/TLE proteins that function as corepressors that can bind and potentially inhibit numerous transcription factors known to be involved in development and carcinogenesis including myc, beta-catenin, NF-kB, and TGF-B. In Drosophila Groucho is recognized as a master regulatory gene in development. Our studies and those from other groups have shown that over-expression of TLE1 in leukemia cells slows cell cycle progression, colony formation and tumor growth in xenografts, while silencing results in increased cell proliferation. To further evaluate the role of TLE1 in development and oncogenesis we have knocked out TLE1 in mice. These mice are viable and do not spontaneously develop malignancies in the absence of additional cooperating oncogenes. In Drosophila the TLE homologue Groucho binds and represses Drosophila Myc expression of target genes. N-Myc is important for hematopoietic stem cell proliferation, survival and differentiation and is over-expressed in most AML samples. We postulated that TLE 1 might be an important regulator of N-Myc activity in leukemia and the loss of TLE may accelerate the leukemia development in cooperation with N-Myc. We demonstrate here that the loss of Tle1 cooperates with N-Myc to dramatically increase the serial replating and proliferation of hematopoietic progenitor cells from fetal livers. Mice transplanted with N-Myc transduced hematopoietic cells from the fetal liver of Tle1 knockout mice developed leukemia with a much shorter latency as compared with fetal liver cells from wild type mice. We are currently investigating the mechanisms of this cooperation. This is the first demonstration that Tle1 is a potent modulator of Myc induced tumorigenesis and may have implications in a broader spectrum of malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3113.