Juliane Carvalho Cancer Biology Presentation
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Juliane Carvalho Cancer Biology Presentation

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Recent publication showing an interesting approach to identify potential cancer cells and related cell signalling inhibitors in development of anticancer drugs.

Recent publication showing an interesting approach to identify potential cancer cells and related cell signalling inhibitors in development of anticancer drugs.

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    Juliane Carvalho Cancer Biology Presentation Juliane Carvalho Cancer Biology Presentation Presentation Transcript

    • Genomic Alterations of Anaplastic Lymphoma Kinase May Sensitize Tumors to Anaplastic Lymphoma Kinase Inhibitors McDermott et. al. Cancer Research. 2008; 68 (9) 3389-3395 Juliane Carvalho Johns Hopkins University Cancer Biology: AS410_638_81_SP10 Prof: Elena Tilli Shiffert, PhD. Online presentation: February 16, 2009
    • Outline
      • Kinase inhibitors use in medical oncology
      • Research platform using human tumor cell lines against molecular inhibitors.
        • Selective Anaplastic Lymphoma Kinase (ALK) inhibitor and identification of genotype associated responses
      • Correlation of specific oncogene rearrangement and sensitivity to inhibitor treatment
      • Specific genetic alterations and potential use of it as markers for the development specific pharmacological cancer inhibitors of clinical significance
    • Anaplastic Lymphoma Kinase (ALK)
      • ALK is a receptor tyrosine kinase, originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases (RTKs).
      • Natural ligands: PTN (Pleiotrophin) and MK (Midkine)
      • ALK is restricted normal tissue distribution in adult human tissue mainly in the neural cells, pericytes and endothelial of the brain
      • Known for its potential oncogenic capability when chromosome rearrangements occur
      Biochemical Journal (2009) Volume 420 , 345-361
    • ALK cont’d
      • ALK gene alterations causing diseases:
        • Translocation: non-small-cell lung cancer and anaplastic large cell lymphomas
        • Gene amplification or point mutations: neuroblastomas
      • Approximately 50–60% of cases of Anaplastic Large Cell Lymphoma (ALCL) are associated with the t(2;5;)(p23;q35) chromosomal translocation, which generates a hybrid gene consisting of the intracellular domain of the ALK tyrosine kinase receptor juxtaposed with NPM (nucleophosmin): fusion partner.
        • Commonly observed is translocation: generation of fusion proteins increase constitutive activation of ALK: transformation > cancer
      • A group of human cells lines harboring ALK gene alteration are highly sensitive to ALK inhibitors
    • Materials and Methods
      • Human cancer cell lines
        • Commercially available, automated platform maintenance system, tested for cell viability
      • Kinase inhibitors
        • TAE684, BMS-536924, PF-2341066 and WZ-5-126 (small molecule inhibitors)
      • Protein detection
        • Immunoassay and SDS-PAGE
      • Antibodies:
        • Akt, ALK, Erk1-2 kinase, phospho-Erk1/2 (T202/Y204), phospho-ALK, STAT3 and phospho-STAT3 antibodies and poly (ADP ribose) polymerase antibody
      • Cell cycle analysis
        • Used anti-BrdUrd monoclonal antibody > FITC-conjugated anti-mouse IgG > propidium iodide > RNase A: two dimensional fluorescence cell sorting analysis
      • RNA interference
        • 2 shRNA targeting sequences downstream of ALK breakpoint, expressed from lentiviral vector to infect cells
      • Fluorescence in situ hybridization (FISH)
        • LSI ALK Dual Color, Break Apart Rearrangement Probe. (manufacturer protocol)
      • Polymerase Chain Reaction (PCR) detection of ALK fusion products
        • RNA extraction, reverse-transcription and PCR : Detection of ALK fusion products (manufacturer protocol)
      • DNA sequencing
        • Genomic DNA isolated. ALK gene amplicons (exons 1-29) amplified, PCR products purified and sequenced.
    • Figure 1. A, pie chart representation of the sensitivity of 602 human cancer cell lines to treatment with 200 nmol/L TAE684 Copyright ©2008 American Association for Cancer Research McDermott, U. et al. Cancer Res 2008;68:3389-3395
    • Copyright ©2008 American Association for Cancer Research McDermott, U. et al. Cancer Res 2008;68:3389-3395 Figure 2. A, FISH analysis of the BE(2)-C, KELLY, and NB-1 neuroblastoma cell lines using the LSI ALK Dual Color, Break Apart Rearrangement Probe
    • Copyright ©2008 American Association for Cancer Research McDermott, U. et al. Cancer Res 2008;68:3389-3395 Figure 3. A, pie chart representation of the sensitivity of 256 human cancer cell lines to 200 nmol/L of the IGF-IR inhibitor BMS-536924 following 72 h of treatment
    • Copyright ©2008 American Association for Cancer Research McDermott, U. et al. Cancer Res 2008;68:3389-3395 Figure 4. A, dose-response curves showing the effect of the ALK inhibitor TAE684 and the MET/ALK inhibitor PF-2341066 on cell viability 72 h after treatment in the SU-DHL-1 and Karpas-299 lymphoma cell lines and the NB-1 neuroblastoma cell line
    • Conclusions
      • ALK inhibition profile is similar across susceptible tumor cell lines and shows identical effect in downstream signal transduction effectors
      • Differential response to inhibitors seems to reflect differences in genetic background or predisposition acquired by cells
      • This research approach is a valuable strategy for evaluation of molecules that may play important role in pharmacological clinical response in cancer patients
      • Fusion proteins generated by genetic translocations can potentially be used as diagnostic markers in human cancer cells. But more work is needed to clarify its specific roles since fusion proteins may have normal functional properties in non tumor cells
      • The ALK genomic status is strong, but may not be the sole determinant of sensitivity to kinase inhibition. Genomic status of ALK must be studied in additional tumor cells sensitive to inhibitors other than TAE684
      • More research should be done using ALK si-RNA targeting ALK-fusion protein transcripts as potential therapeutic targets
      • DNA vaccine against human ALK: possible prevention/therapy alternative. Animals challenged with ALK-expressing lymphoma cells showed protection against the disease. Specific genotypes and immunological response must be considered.
      • Therapies for complete inhibition of ALK need to consider potential side effects in different animal developmental stages. Particular study may not be feasible in pregnant women
      • Future research (?) use of in vivo cancer cells in mice. Evaluate drug response against tumor grafts to predict clinical efficacy. Explore the combination of downstream effector inhibitors.
    • Questions
    • Literature consulted
      • McDermott, et. al. (2008). Genomic Alterations of Anaplastic Lymphoma Kinase May Sensitize Tumors to Anaplastic Lymphoma Kinase Inhibitors. Cancer Research. 68: (9).3389-3395.
      • Palmer, et. al. (2009). Anaplastic Lymphoma Kinase: Signaling in Development and Disease. Biochemical Journal. 420, 345-361.
      • Webb, T. et al. (2009). Anaplastic Lymphoma Kinase: Role in Cancer Pathogenesis and Small-molecule Inhibitor Development for Therapy. Expert Rev Anticancer Ther. Volume: 9, Issue: 3, Date: 2009 Mar , Pages: 331-56, 9(3), 331-356.
      • Chiarle, R. et. al. (2008). The Anaplastic Lymphoma Kinase is an effective oncoantigen for lymphoma vaccination. Nature Medicine 14, 676-680.
      • Stoica, G. E. et. al. (2001). Identification of Anaplastic Lymphoma Kinase as a Receptor for the Growth Factor Pleiotrophin. Journal of Biological Chemistry. Vol. 276, No 20, pp. 16772-16779.
      • Galkin A.V., et. al. (2007). Identification of NVP-TAE684, a potent, selective, and efficacious inhibitor of NPM-ALK. PNAS. 104: 270-5.
      • Weinberg, The Biology of Cancer . Chapter 4. 2007 by Garland Science, Taylor & Francis Group, LLC.