Identification of the major autophosphorylation site of the Met/hepatocyte growth factor receptor tyrosine kinase.

The MET proto-oncogene encodes a transmembrane tyrosine kinase receptor for HGF (p190MET). In this work, p190MET was immunoprecipitated, allowed to phosphorylate in the presence of [gamma-32P]ATP, and digested with trypsin. A major phosphopeptide was purified by reverse phase chromatography. The phosphorylated tyrosine was identified as residue 1235 (Tyr1235) by Edman covalent radiosequencing. A synthetic peptide derived from the corresponding MET sequence was phosphorylated by p190MET in an in vitro assay and coeluted in reverse phase chromatography. Tyr1235 lies within the tyrosine kinase domain of p190MET, within a canonical tyrosine autophosphorylation site that shares homology with the corresponding region of the insulin, CSF-1 and platelet-derived growth factor receptors, and of p60src and p130gag-fps. The p190MET kinase is constitutively phosphorylated on tryosine in a gastric carcinoma cell line (GTL16), due to the amplification and overexpression of the MET gene. Metabolic labeling of GTL-16 cells with [32P]orthophosphate followed by immunoprecipitation and tryptic phosphopeptide mapping of p190MET showed that Tyr1235 is a major site of tyrosine phosphorylation in vivo as well. Since phosphorylation activates p190MET kinase, we propose a regulatory role for Tyr1235

173. Insulin B9-23 LV-Driven Expression in Hepatocytes Combined With Suboptimal Dose of Anti-CD3 mAb Cures Type 1 Diabetes in NOD Mice

Type 1 diabetes (T1D) is an autoimmune disease resulting in complete destruction of insulin-producing pancreatic β cells. In T1D in human and in the non-obese diabetic (NOD) mouse, the spontaneous murine model of T1D, auto-reactive T cells target islet-associated antigens. Induction of antigen (Ag)-specific tolerance could cure Type 1 Diabetes (T1D) but it has not been achieved yet. We previously showed that lentiviral vector (LV)-mediated gene expression in hepatocytes induces active tolerance toward the encoded-Ag. Systemic administration of a single dose of Integrase competent (IC) or integrase defective (ID) LV.ET.InsB9-23.142T, enabling stable and transient expression of InsB9-23 in hepatocytes, respectively, arrests β cell destruction in NOD mice at advanced pre-diabetic stage by generating InsB9-23-specific FoxP3+ T regulatory cells (Tregs). In the present study we tested the efficacy of hepatocytes-directed LV.ET. InsB9-23.142T gene transfer in protecting from disease progression at later stages and in reversing T1D.Treatment with LV.ET.InsB9-23.142T in NOD mice with glucose levels ranging from 200mg/mL to 250mg/mL blocked T1D progression in only 27% of the mice. Co-expression of the late auto-Ags-derived epitopes GAD206-220 and IGRP195-214 in hepatocytes did not improve the efficacy of LV.ET.InsB9-23.142T treatment. LV.ET.InsB9-23.142T treatment in diabetic NOD mice with blood glucose levels ranging from 250mg/mL to 300mg/mL did not result in reversion to normoglycemic levels in any of the treated mice.We next combined InsB9-23 gene transfer with anti-CD3 monoclonal antibody (mAb) treatment. Treatment with anti-CD3 mAb at optimal doses is able per se to reverse T1D in NOD mice. Therefore, we tested decreasing doses of anti-CD3 mAb in diabetic NOD mice with blood glucose levels ranging from 250mg/mL to 300mg/mL to identify the sub-optimal dose unable to revert T1D. We found that a single administration of anti-CD3 mAb at 5μg instead of 10μg results was not effective. This sub-optimal dose of anti-CD3 mAb (1X 5μg) was administered together with LV.ET.InsB9-23.142T to NOD mice with blood glucose levels ranging from 250mg/mL to 300mg/mL. Results showed T1D reversal in 75% of ICLV-treated and 40% of the IDLV-treated mice. These data indicate that the LV.ET. InsB9-23.142T treatment combined with sub-optimal anti-CD3 mAb treatment is able to reverse overt diabetes

ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially

BACKGROUND: The mitogen-activated protein (MAP) kinases p44(ERK1 )and p42(ERK2 )are crucial components of the regulatory machinery underlying normal and malignant cell proliferation. A currently accepted model maintains that ERK1 and ERK2 are regulated similarly and contribute to intracellular signaling by phosphorylating a largely common subset of substrates, both in the cytosol and in the nucleus. RESULTS: Here, we show that ablation of ERK1 in mouse embryo fibroblasts and NIH 3T3 cells by gene targeting and RNA interference results in an enhancement of ERK2-dependent signaling and in a significant growth advantage. By contrast, knockdown of ERK2 almost completely abolishes normal and Ras-dependent cell proliferation. Ectopic expression of ERK1 but not of ERK2 in NIH 3T3 cells inhibits oncogenic Ras-mediated proliferation and colony formation. These phenotypes are independent of the kinase activity of ERK1, as expression of a catalytically inactive form of ERK1 is equally effective. Finally, ectopic expression of ERK1 but not ERK2 is sufficient to attenuate Ras-dependent tumor formation in nude mice. CONCLUSION: These results reveal an unexpected interplay between ERK1 and ERK2 in transducing Ras-dependent cell signaling and proliferation. Whereas ERK2 seems to have a positive role in controlling normal and Ras-dependent cell proliferation, ERK1 probably affects the overall signaling output of the cell by antagonizing ERK2 activity

690. Permanent Epigenetic Silencing of Human Genes With Artificial Transcriptional Repressors

There are several diseases whereby the goal of gene therapy is to silence rather than replace a gene function. Paradigmatic examples are diseases caused by a dominant negative mutation or those in which silencing of a host gene confers resistance to a pathogen or compensates the function of the missing gene. Yet, gene silencing can be used to enhance efficacy of cell therapy and for biotechnological applications. Until now, two technologies have been used to silence gene expression, namely RNA interference with short harping RNAs (shRNA) and gene disruption with Artificial Nucleases (ANs). Although some promising pre-clinical and clinical data have been already obtained, the low efficiency of knock-down with shRNA and of biallelic disruption with ANs may limit efficacy of these treatments, especially when residual gene activity can exert a biological function. To overcome this issue, we have developed a novel modality of gene silencing that exploits endogenous epigenetic mechanisms to convey robust and heritable states of repression at the desired target gene. We have generated Artificial Transcriptional Repressors (ATRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of a chromatinmodifying enzyme involved in silencing of Endogenous RetroViruses (ERVs). By performing iterative rounds of selection in human cell lines and primary cells engineered to report for synergistic activity of candidate effector domains, we identified a combination of 3 domains that, when transiently co-assembled on the promoter of the reporter cassette, fully abrogated transgene expression in up to 90% of treated cells. Importantly, silencing was maintained for more than 250 days in cultured cell lines, was resistant to in vitro differentiation or metabolic activation of primary cells, and was confined to the reporter cassette. Silencing was associated with high levels of de novo DNA methylation at the targeted locus and was dependent on this epigenetic mark for its propagation. Finally, transient transfection of 3 ATRs targeted to the promoter region of the Beta-2-microglobulin (B2M) gene resulted in the loss of surface expression of B2M and, consequently, of the MHC-I molecules in up to 80% of treated cells. This phenotype was associated with a switch in the epigenetic and transcriptional state of the constitutively active B2M gene, which became highly decorated with DNA methylation and deprived of RNA PolII and of its transcript. Of note, silencing was resistant to IFN-γ treatment, a potent B2M inducer. Overall, these data provide the first demonstration of efficient and stable silencing of an endogenous gene upon transient delivery of ATRs. This result was made possible by repurposing the machinery involved in silencing of ERVs, which instructs self-sustaining repressive epigenetic states on the gene of interest. While silencing of B2M might be used to generate universally transplantable allogeneic cells, our hit-and-run strategy provides a powerful new alternative to conventional gene silencing for the treatment of several diseases. (LN & AL co-authorship

62. Proteasome Activity Restricts Lentiviral Gene Transfer into Hematopoietic Stem Cells and Is Down-Regulated by Cytokines That Enhance Transduction

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Efficient Tet-dependent expression of human factor IX in vivo by a new self-regulating lentiviral vector.

Regulation of gene expression represents a long-sought goal of gene therapy. However, most viral vectors pose constraints on the incorporation of drug-dependent transcriptional regulatory systems. Here, by optimizing the design of self-regulating lentiviral vectors based on the tetracycline system, we have been able to overcome the limitations of previously reported constructs and to reach both robust expression and efficient regulation from a single vector. The improved performance allows us to report for the first time effective long-term in vivo regulation of a human clotting Factor IX (hF.IX) transgene upon systemic administration of a single vector to SCID mice. We showed that hF.IX expression in the plasma could be expressed to therapeutically significant concentrations, adjusted to different set levels by varying the tetracycline dose, rapidly turned off and on, and completely recovered after each treatment cycle. The new vector design was versatile, as it successfully incorporated a tissue-specific promoter that selectively targeted regulated expression to hepatocytes. Robust transgene expression in the systemic circulation coupled to the ability to switch off and even adjust the expression level may open the way to safer gene-based delivery of therapeutics

Intracellular calcium regulates the tyrosine kinase receptor encoded by the MET oncogene.

Previous work (Gandino, L., Di Renzo, M. F., Giordano, S., Bussolino, F., and Comoglio, P.M. (1990) Oncogene 5, 721-725) has shown that the tyrosine kinase activity of the receptor encoded by the MET protooncogene is negatively modulated by protein kinase C (PKC). We now show that an increase of intracellular Ca2+ has a similar inhibitory effect in vivo, via a PKC-independent mechanism. In GTL-16 cells the p145MET kinase is overexpressed and constitutively phosphorylated on tyrosine. A rapid and reversible decrease of p145MET tyrosine phosphorylation was induced by treatment with the calcium ionophores A23187 or ionomycin. Experiments performed with the ionophores in absence of extracellular calcium showed that a rise in cytoplasmic Ca2+ concentration to 450 nM (due to release from intracellular stores) resulted in a similar effect. These Ca2+ concentrations had no effect on p145MET autophosphorylation in an in vitro kinase assay. This suggests that the effect of Ca2+ on p145MET tyrosine phosphorylation is not direct but may be mediated by Ca(2+)-activated proteins(s). Involvement of Ca(2+)-dependent tyrosine phosphatases was ruled out by experiments carried out in presence of Na2VO4. In vivo labeling with [32P]orthophosphate showed that the rise of intracellular Ca2+ induces serine phosphorylation of p145MET on a specific phosphopeptide. This suggests that Ca2+ negatively modulates p145MET kinase through the phosphorylation of a critical serine residue by a Ca(2+)-activated serine kinase distinct from PKC

729 inheritable silencing of endogenous gene by hit and run targeted epigenetic editing

Gene silencing holds great promise for the treatment of several diseases and can be exploited to investigate gene function and activity of the regulatory genome. Here, we develop a novel modality of gene silencing that exploits epigenetics to achieve stable and highly efficient repression of target genes. To this end, we generated Artificial Transcriptional Repressors (ATRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of chromatin-modifying enzymes involved in silencing process of Endogenous RetroViruses (ERVs). By performing iterative rounds of selection in cells engineered to report for synergistic activity of candidate effector domains, we identified a combination of 3 domains (namely KRAB, DNMT3A and DNMT3L) that, when transiently co-assembled on the promoter of the reporter cassette, recreate a powerful embryonic-specific repressive complex capable of inducing full and long-term (>150 days) silencing of transgene expression in up to 90% of the cells. The ATR-induced silencing was cell type and locus independent, and resistant to metabolic activation of the cells. Importantly, these findings were holding true also for endogenous genes embedded in their natural chromatin context, as shown for the highly and ubiquitously expressed B2M gene. Here, transient co-delivery of TALE-based ATRs resulted in loss of surface expression of B2M and, consequently, of the MHC-I molecules in up to 80% of the cells. This phenotype was associated with a drastic switch in the epigenetic and transcriptional state of the constitutively active B2M promoter, which become highly decorated with de novo DNA methylation and deprived of RNAP II. Importantly, silencing was sharply confined to the targeted gene and resistant to INF-γ, a potent natural activator of B2M. We further extended these studies by showing that our silencing approach is portable to the CRISPR/dCas9 DNA binding technology. In this setting, comparable levels of B2M silencing (up to 80%) were achieved using either pools or even individual sgRNAs coupled to dCas9-based ATRs. Yet, adoption of this technology allowed performing simultaneous, highly efficient multiplex gene silencing within the same cell, as shown for B2M, IFNAR1 and VEGFA. Finally, we assessed resistance of the silenced gene to activity of potent artificial transcription activators and chromatin remodelers, and found that only targeted DNA demethylation was able to reawaken the silent gene. This allowed performing iterative cycles of silencing and reactivation of the same gene in the same cell population. Overall, these data provide the first demonstration of efficient and stable epigenetic silencing of endogenous genes upon transient delivery of ATRs. This was accomplished by repurposing the ERVs silencing machinery, which instructs self-sustaining repressive epigenetic states to the target gene. While silencing of B2M might be used to generate universally transplantable allogeneic cells, our hit-and-run strategy provides a powerful new alternative to conventional gene silencing for both basic and translational research