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963. MITF as a Potential Molecular Target for Gene Therapy for Human Melanoma; a Synergistic Inhibitory Effect of Inactivation of Both MITF and BRAFV600E on Melanoma Growth
CANCER-TARGETED GENE THERAPY: OTHER VIRUSES AND NEW APPROACHES 962. The Ras/Raf-1/MEK/ERK Signaling Pathway Dictates Host Cell Permissiveness to VSV Infection Josh A. Noser,1 Ryuta Sakuma,1 Patrick W. K. Lee,2 Yasuhiro Ikeda.1 1 Molecular Medicine, Mayo Clinic, Rochester, MN; 2Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada. Vesicular stomatitis virus (VSV) can replicate with greater efficiency in malignant cells compared with normal cells. Although this selective replication appears caused by the defects in the interferon (IFN) system in malignant cells, the mechanisms which render the cells less responsive to interferon remain to be determined. Here we demonstrate that Ras/Raf-1/Erk pathway plays a major role in the defects in the IFN pathway. NIH/3T3 or human primary fibroblast cells stably expressing Ras or Raf-1 were no longer protected by IFN-alpha treatment from VSV infection. Upon treatment with the MEK inhibitor U0126 the response to INF was restored in these cells, suggesting the involvement of the Ras/Raf-1/ MEK pathway. Importantly, most cancer cells examined showed elevated levels of phosphorylated Erk (p-Erk) and were less responsive to IFN-alpha treatment. Similar to the NIH/3T3 model, following treatment with U0126 the cells became far more protected by IFN-alpha treatment. Phosphorylated PKR and STAT1 were detectable in cancer cells following treatment with IFN-alpha regardless of treatment with U0126. The translation of viral proteins after treatment of IFN-alpha and U0126 was reduced to undetectable levels, suggesting that the elevated levels of p-Erk enhanced translation of viral proteins or the stability of viral mRNA under the presence of IFN-alpha. We will present our latest results on the relationship between p-Erk and the reduced INF response in cancer cells.
963. MITF as a Potential Molecular Target for Gene Therapy for Human Melanoma; a Synergistic Inhibitory Effect of Inactivation of Both MITF and BRAFV600E on Melanoma Growth Kenji Kido,1,2 Hidetoshi Sumimoto,1 Sakiyo Asada,1 Starlyn Okada,1 Toshiaki Saida,2 Yutaka Kawakami.1 1 Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; 2 Department of Dermatology, Shinshu University School of Medicine, Nagano, Japan. MITF (microphthalmia-associated transcription factor) is a family of transcriptional factors controlling melanocyte differentiation. However, the roles of MITF on the cell proliferation have been controversial. Previous works have suggested that MITF negatively regulates cell proliferation or apoptosis in melanocytes, which is mediated by induction of p16INK4a, p21 or Bcl-2. But a recent study showed gene amplification of MITF in melanoma tissues (10-21%) associated with increase of the MITF protein. Furthermore, MITF gene amplification was a prognostic factor for survival, and several in vitro studies suggested that the MITF protein is attributed to increased cell proliferation, suggesting that MITF is an oncogene. Presently, the role of MITF in melanoma and melanomagenesis is not clear. We have attempted to investigate the role of MITF in malignant phenotypes of melanoma cells by using HIV-mediated RNA interference (RNAi) in a panel of melanoma cell lines with different status of the MITF expression to clarify the significance of MITF as a target for gene therapy, and to find any factors affecting sensitivity for the MITF inactivation. Furthermore, we have attempted to evaluate any additional effects by simultaneous suppression of both MITF and BRAFV600E on the melanoma growth, since BRAFV600E, the most frequent form of human BRAF mutation, is attributed to the enhanced MAPK signaling and several malignant phenotypes of melanoma. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
We have constructed two short hairpin RNA (shRNA) HIV vectors for MITF, and one control shRNA HIV vector (GL3B; for firefly luciferase). An HIV vector expressing two shRNAs for MITF and BRAFV600E was also constructed by tandemly aligning two shRNA expression cassettes in one HIV vector. Seven melanoma cell lines were infected with these shRNA HIV vectors, and the effects of MITF RNAi on the cell growth and apoptosis were evaluated. In six of 7 melanoma cell lines with substantial expression of the MITF protein, the MITF RNAi induced significant inhibition of cell growth, associated with G1 arrest. One melanoma cell line, A375, without any detectable expression of MITF was resistant to the MITF RNAi. One melanoma cell line showed significant apoptotic changes represented by the increase of subG1 population (7.23-7.60%) and increase of cleaved caspase-3 and caspase-9, following the MITF RNAi. Interestingly, the MITF RNAi induced G1 arrest in most melanoma cell lines tested, however, no clear correlation was observed between the protein levels of cdk inhibitors and G1 arrest. Finally, three melanoma cell lines with the simultaneous inhibition of both MITF and BRAFV600E, showed synergistic inhibition of cell growth, which was associated with more G1 arrest. Our results suggest that MITF could be a molecular target for gene therapy for most human melanoma, and the synergistic therapeutic effects of MITF and BRAFV600E indicate the possibly more potent gene therapy for melanoma by simultaneous downregulation of these molecules.
964. Combinatorial Process for Engineering Specific Meganucleases with Tailored Specificities toward a Natural Target in the XPC Gene Christophe Perez, Sylvain Arnould, Julie Smith, Sylvestre Grizot, Agnes Gouble, Philippe Duchateau, Frederic Paques. 1 Research, CELLECTIS S.A., Romainville, France. Xeroderma pigmentosum (XP) is a rare disease transmitted in an autosomal recessive manner Patients have an extreme sensitivity to sunlight and develop serious sunburns with onset of poikilodermia in the light-exposed skin. Skin cancers already appear in childhood, and the disease can also be associated with neurological defects. XP results from defects in the Nucleotide Excision Repair (NER) pathway, a DNA maintenance system which removes UV induced DNA damage such as cyclobutane pyrimidine dimers. XP Patients were assigned to 7 complementation groups (XP-A to XP-G), each complementation group resulting from mutations in a distinct NER gene. There is no treatment, and the majority of patients die before reaching adulthood because of metastases. However, skin engraftment can be made locally, but with the general limitations of grafts. Thus gene and cell therapy represents a huge hope for this kind of disease. Meganucleases are at the basis of a new kind of gene therapy for inherited monogenic disease, based on gene correction instead of gene complementation. These sequence-specific endonucleases can stimulate homologous gene targeting by several orders of magnitude, thereby enabling the correction of mutated genes with significant efficiency. Recently, meganucleases have been used to induce targeted recombination events in mouse hepatocytes with high efficiency, paving the way for therapeutic applications. One of the major challenges is to tailor artificial meganucleases cleaving the gene of interest, while keeping high levels of specificity. We have used a semi-rational approach to produce meganucleases targeting the XPC gene. These novel meganucleases display high levels of activity and specificity. Such results identify modularity of different functional subdomains in the I-CreI DNA-binding scaffold that can be modified without altering the overall structure, and be combined to achieve novel specificities. S371
963. MITF as a Potential Molecular Target for Gene Therapy for Human Melanoma; a Synergistic Inhibitory Effect of Inactivation of Both MITF and BRAFV600E on Melanoma Growth Kenji Kido,1,2 Hidetoshi Sumimoto,1 Sakiyo Asada,1 Starlyn Okada,1 Toshiaki Saida,2 Yutaka Kawakami.1 1 Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; 2 Department of Dermatology, Shinshu University School of Medicine, Nagano, Japan. MITF (microphthalmia-associated transcription factor) is a family of transcriptional factors controlling melanocyte differentiation. However, the roles of MITF on the cell proliferation have been controversial. Previous works have suggested that MITF negatively regulates cell proliferation or apoptosis in melanocytes, which is mediated by induction of p16INK4a, p21 or Bcl-2. But a recent study showed gene amplification of MITF in melanoma tissues (10-21%) associated with increase of the MITF protein. Furthermore, MITF gene amplification was a prognostic factor for survival, and several in vitro studies suggested that the MITF protein is attributed to increased cell proliferation, suggesting that MITF is an oncogene. Presently, the role of MITF in melanoma and melanomagenesis is not clear. We have attempted to investigate the role of MITF in malignant phenotypes of melanoma cells by using HIV-mediated RNA interference (RNAi) in a panel of melanoma cell lines with different status of the MITF expression to clarify the significance of MITF as a target for gene therapy, and to find any factors affecting sensitivity for the MITF inactivation. Furthermore, we have attempted to evaluate any additional effects by simultaneous suppression of both MITF and BRAFV600E on the melanoma growth, since BRAFV600E, the most frequent form of human BRAF mutation, is attributed to the enhanced MAPK signaling and several malignant phenotypes of melanoma. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
We have constructed two short hairpin RNA (shRNA) HIV vectors for MITF, and one control shRNA HIV vector (GL3B; for firefly luciferase). An HIV vector expressing two shRNAs for MITF and BRAFV600E was also constructed by tandemly aligning two shRNA expression cassettes in one HIV vector. Seven melanoma cell lines were infected with these shRNA HIV vectors, and the effects of MITF RNAi on the cell growth and apoptosis were evaluated. In six of 7 melanoma cell lines with substantial expression of the MITF protein, the MITF RNAi induced significant inhibition of cell growth, associated with G1 arrest. One melanoma cell line, A375, without any detectable expression of MITF was resistant to the MITF RNAi. One melanoma cell line showed significant apoptotic changes represented by the increase of subG1 population (7.23-7.60%) and increase of cleaved caspase-3 and caspase-9, following the MITF RNAi. Interestingly, the MITF RNAi induced G1 arrest in most melanoma cell lines tested, however, no clear correlation was observed between the protein levels of cdk inhibitors and G1 arrest. Finally, three melanoma cell lines with the simultaneous inhibition of both MITF and BRAFV600E, showed synergistic inhibition of cell growth, which was associated with more G1 arrest. Our results suggest that MITF could be a molecular target for gene therapy for most human melanoma, and the synergistic therapeutic effects of MITF and BRAFV600E indicate the possibly more potent gene therapy for melanoma by simultaneous downregulation of these molecules.
964. Combinatorial Process for Engineering Specific Meganucleases with Tailored Specificities toward a Natural Target in the XPC Gene Christophe Perez, Sylvain Arnould, Julie Smith, Sylvestre Grizot, Agnes Gouble, Philippe Duchateau, Frederic Paques. 1 Research, CELLECTIS S.A., Romainville, France. Xeroderma pigmentosum (XP) is a rare disease transmitted in an autosomal recessive manner Patients have an extreme sensitivity to sunlight and develop serious sunburns with onset of poikilodermia in the light-exposed skin. Skin cancers already appear in childhood, and the disease can also be associated with neurological defects. XP results from defects in the Nucleotide Excision Repair (NER) pathway, a DNA maintenance system which removes UV induced DNA damage such as cyclobutane pyrimidine dimers. XP Patients were assigned to 7 complementation groups (XP-A to XP-G), each complementation group resulting from mutations in a distinct NER gene. There is no treatment, and the majority of patients die before reaching adulthood because of metastases. However, skin engraftment can be made locally, but with the general limitations of grafts. Thus gene and cell therapy represents a huge hope for this kind of disease. Meganucleases are at the basis of a new kind of gene therapy for inherited monogenic disease, based on gene correction instead of gene complementation. These sequence-specific endonucleases can stimulate homologous gene targeting by several orders of magnitude, thereby enabling the correction of mutated genes with significant efficiency. Recently, meganucleases have been used to induce targeted recombination events in mouse hepatocytes with high efficiency, paving the way for therapeutic applications. One of the major challenges is to tailor artificial meganucleases cleaving the gene of interest, while keeping high levels of specificity. We have used a semi-rational approach to produce meganucleases targeting the XPC gene. These novel meganucleases display high levels of activity and specificity. Such results identify modularity of different functional subdomains in the I-CreI DNA-binding scaffold that can be modified without altering the overall structure, and be combined to achieve novel specificities. S371