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Assaying pharmacodynamic endpoints with targeted therapy: Flavopiridol and 17AAG induced dephosphorylation of histone H1.5 in acute myeloid leukemia

Authors

  • Liwen Wang,

    1. Department of Chemistry, The Ohio State University, Columbus OH, USA
    2. Comprehensive Cancer Center, The Ohio State University, Columbus OH, USA
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  • Sean W. Harshman,

    1. Comprehensive Cancer Center, The Ohio State University, Columbus OH, USA
    2. Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus OH, USA
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  • Shujun Liu,

    1. Comprehensive Cancer Center, The Ohio State University, Columbus OH, USA
    2. Department of Internal Medicine, The Ohio State University, Columbus OH, USA
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  • Chen Ren,

    1. Department of Chemistry, The Ohio State University, Columbus OH, USA
    2. Comprehensive Cancer Center, The Ohio State University, Columbus OH, USA
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  • Hua Xu,

    1. Comprehensive Cancer Center, The Ohio State University, Columbus OH, USA
    2. Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus OH, USA
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  • Larry Sallans,

    1. Mass Spectrometry Facility, University of Cincinnati, Cincinnati OH, USA
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  • Michael Grever,

    1. Comprehensive Cancer Center, The Ohio State University, Columbus OH, USA
    2. Department of Internal Medicine, The Ohio State University, Columbus OH, USA
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  • John C. Byrd,

    1. Comprehensive Cancer Center, The Ohio State University, Columbus OH, USA
    2. Department of Internal Medicine, The Ohio State University, Columbus OH, USA
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  • Guido Marcucci,

    1. Comprehensive Cancer Center, The Ohio State University, Columbus OH, USA
    2. Department of Internal Medicine, The Ohio State University, Columbus OH, USA
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  • Michael A. Freitas

    Corresponding author
    1. Comprehensive Cancer Center, The Ohio State University, Columbus OH, USA
    2. Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus OH, USA
    • The Ohio State University Medical Center, 460 West 12th Avenue, Columbus, OH 43210, USA Fax: +1-614-688-8675
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Abstract

Histone H1 is commonly used to assay kinase activity in vitro. As many promising targeted therapies affect kinase activity of specific enzymes involved in cancer transformation, H1 phosphorylation can serve as potential pharmacodynamic marker for drug activity within the cell. In this study we utilized a phosphoproteomic workflow to characterize histone H1 phosphorylation changes associated with two targeted therapies in the Kasumi-1 acute myeloid leukemia cell line. The phosphoproteomic workflow was first validated with standard casein phosphoproteins and then applied to the direct analysis of histone H1 from Kasumi-1 nuclear lysates. Ten H1 phosphorylation sites were identified on the H1 variants, H1.2, H1.3, H1.4, H1.5 and H1.x. LC MS profiling of intact H1s demonstrated global dephosphorylation of H1.5 associated with therapy by the cyclin-dependent kinase inhibitor, flavopiridol and the Heat Shock Protein 90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin. In contrast, independent treatments with a nucleotide analog, proteosome inhibitor and histone deacetylase inhibitor did not exhibit decreased H1.5 phosphorylation. The data presented herein demonstrate that potential of histones to assess the cellular response of reagents that have direct and indirect effects on kinase activity that alters histone phosphorylation. As such, this approach may be a highly informative marker for response to targeted therapies influencing histone phosphorylation.

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