Abstract
Oncogenic mutations leading to persistent kinase activities are associated with malignancies. Therefore, deciphering the signaling networks downstream of these oncogenic stimuli remains a challenge to gather insights into targeted therapy. To elucidate the biochemical networks connecting the Kit mutant to leukemogenesis, in the present study, we performed a global profiling of tyrosine-phosphorylated proteins from mutant Kit-driven murine leukemia proerythroblasts and identified Shp2 and Stat5 as proximal effectors of Kit. Shp2 or Stat5 gene depletion by sh-RNA, combined with pharmacologic inhibition of PI3kinase or Mek/Erk activities, revealed 2 distinct and independent signaling pathways contributing to malignancy. We demonstrate that cell survival is driven by the Kit/Shp2/Ras/Mek/Erk1/2 pathway, whereas the G(1)/S transition during the cell cycle is accelerated by both the Kit/Stat5 and Kit/PI3K/Akt pathways. The combined use of the clinically relevant drugs NVP-BEZ235, which targets the cell cycle, and Obatoclax, which targets survival, demonstrated synergistic effects to inhibit leukemia cell growth. This synergy was confirmed with a human mast leukemia cell line (HMC-1.2) that expresses mutant Kit. The results of the present study using liquid chromatography/tandem mass spectrometry analysis have elucidated signaling networks downstream of an oncogenic kinase, providing a molecular rationale for pathway-targeted therapy to treat cancer cells refractory to tyrosine kinase inhibitors.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenylate Kinase / antagonists & inhibitors
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Adenylate Kinase / physiology
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Animals
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Antineoplastic Agents / pharmacology
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Cell Cycle / drug effects
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Cell Line, Tumor / metabolism
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Cell Survival / drug effects
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Drug Resistance, Neoplasm / drug effects*
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Female
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Humans
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Imidazoles / pharmacology
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Indoles
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Leukemia, Mast-Cell / pathology
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Mice
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Mice, Nude
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Mice, Transgenic
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Neoplasm Proteins / antagonists & inhibitors*
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Phosphatidylinositol 3-Kinases / physiology
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Phosphoinositide-3 Kinase Inhibitors
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Phosphorylation / drug effects
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Phosphotyrosine / analysis
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Protein Kinase Inhibitors / pharmacology*
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Protein Processing, Post-Translational / drug effects
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Protein Tyrosine Phosphatase, Non-Receptor Type 11 / antagonists & inhibitors
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Protein Tyrosine Phosphatase, Non-Receptor Type 11 / genetics
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Protein Tyrosine Phosphatase, Non-Receptor Type 11 / physiology
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Proto-Oncogene Proteins c-kit / antagonists & inhibitors*
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Pyrroles / pharmacology
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Quinolines / pharmacology
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RNA Interference
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RNA, Small Interfering / pharmacology
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STAT5 Transcription Factor / antagonists & inhibitors
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STAT5 Transcription Factor / genetics
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STAT5 Transcription Factor / physiology
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Signal Transduction / drug effects*
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Tumor Stem Cell Assay
Substances
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Antineoplastic Agents
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Imidazoles
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Indoles
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Neoplasm Proteins
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Phosphoinositide-3 Kinase Inhibitors
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Protein Kinase Inhibitors
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Pyrroles
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Quinolines
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RNA, Small Interfering
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STAT5 Transcription Factor
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Phosphotyrosine
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Proto-Oncogene Proteins c-kit
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Adenylate Kinase
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adenylate kinase 1
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Protein Tyrosine Phosphatase, Non-Receptor Type 11
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Ptpn11 protein, mouse
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obatoclax
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dactolisib