title: Structural Determinants of the Gain-of-Function Phenotype of Human Leukemia-associated Mutant CBL Oncogene.
abstract: Mutations of the tyrosine kinase-directed ubiquitin ligase CBL cause myeloid leukemias, but the molecular determinants of the dominant leukemogenic activity of mutant CBL oncogenes are unclear. Here, we first define a gain-of-function attribute of the most common leukemia-associated CBL mutant, Y371H, by demonstrating its ability to increase proliferation of hematopoietic stem/progenitor cells (HSPCs) derived from CBL-null and CBL/CBL-B-null mice. Next, we express second-site point/deletion mutants of CBL-Y371H in CBL/CBL-B-null HSPCs or the cytokine-dependent human leukemic cell line TF-1 to show that individual or combined Tyr → Phe mutations of established phosphotyrosine residues (Tyr-700, Tyr-731, and Tyr-774) had little impact on the activity of the CBL-Y371H mutant in HSPCs, and the triple Tyr → Phe mutant was only modestly impaired in TF-1 cells. In contrast, intact tyrosine kinase-binding (TKB) domain and proline-rich region (PRR) were critical in both cell models. PRR deletion reduced the stem cell factor (SCF)-induced hyper-phosphorylation of the CBL-Y371H mutant and the c-KIT receptor and eliminated the sustained p-ERK1/2 and p-AKT induction by SCF. GST fusion protein pulldowns followed by phospho-specific antibody array analysis identified distinct CBL TKB domains or PRR-binding proteins that are phosphorylated in CBL-Y371H-expressing TF-1 cells. Our results support a model of mutant CBL gain-of-function in which mutant CBL proteins effectively compete with the remaining wild type CBL-B and juxtapose TKB domain-associated PTKs with PRR-associated signaling proteins to hyper-activate signaling downstream of hematopoietic growth factor receptors. Elucidation of mutant CBL domains required for leukemogenesis should facilitate targeted therapy approaches for patients with mutant CBL-driven leukemias.
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases.|the Departments of Genetics, Cell Biology and Anatomy.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Nadeau
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases.|the Departments of Genetics, Cell Biology and Anatomy.
authors.forename: Wei
authors.initials: W
authors.lastname: An
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases.|Biochemistry and Molecular Biology.
authors.forename: Bhopal C
authors.initials: BC
authors.lastname: Mohapatra
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases.|Pathology and Microbiology, College of Medicine, and.
authors.forename: Insha
authors.initials: I
authors.lastname: Mushtaq
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Bielecki
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases.|the Departments of Genetics, Cell Biology and Anatomy.
authors.forename: Haitao
authors.initials: H
authors.lastname: Luan
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases.|Pathology and Microbiology, College of Medicine, and.
authors.forename: Neha
authors.initials: N
authors.lastname: Zutshi
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases.
authors.forename: Gulzar
authors.initials: G
authors.lastname: Ahmad
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Storck
authors.affiliation: the Department of Pathology and Tumor Biology, Kyoto University, Yoshida-Konoe-Cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Sanada
authors.affiliation: the Department of Pathology and Tumor Biology, Kyoto University, Yoshida-Konoe-Cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Seishi
authors.initials: S
authors.lastname: Ogawa
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases.|the Departments of Genetics, Cell Biology and Anatomy.|the Fred and Pamela Buffet Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198 and.
authors.forename: Vimla
authors.initials: V
authors.lastname: Band
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases, hband@unmc.edu.|the Departments of Genetics, Cell Biology and Anatomy.|Biochemistry and Molecular Biology.|Pathology and Microbiology, College of Medicine, and.|the Fred and Pamela Buffet Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198 and.
authors.forename: Hamid
authors.identifier: 0000-0002-4996-9002
authors.initials: H
authors.lastname: Band
mesh_terms: D000818:Animals; D001665:Binding Sites; D045744:Cell Line, Tumor; D002469:Cell Separation; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry; D015973:Gene Expression Regulation, Leukemic; D005982:Glutathione Transferase / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D009857:Oncogenes; D010641:Phenotype; D010649:Phenylalanine / Q000737:chemistry; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry; D000072417:Protein Domains; D050721:Proto-Oncogene Proteins c-cbl / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry
chemical_list: D016207:Cytokines; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D010649:Phenylalanine; D011392:Proline; D050721:Proto-Oncogene Proteins c-cbl; D005982:Glutathione Transferase; C067419:CBL protein, human; C493849:Cbl protein, mouse
keywords: CBL; E3 ubiquitin ligase; leukemia; mutagenesis; oncogene; proline-Rich region; receptor tyrosine kinase; tyrosine kinase binding domain