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1.
Mol Syst Biol ; 10: 772, 2014 Dec 23.
Article in English | MEDLINE | ID: mdl-25538140

ABSTRACT

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down-regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.


Subject(s)
Melanoma/genetics , Proto-Oncogene Proteins B-raf/metabolism , rho-Associated Kinases/metabolism , Cell Line, Tumor , Chromatography, Liquid , Down-Regulation , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Humans , Indazoles/pharmacology , Indoles/pharmacology , Molecular Targeted Therapy , Mutation , Piperazines/pharmacology , Proteomics , Proto-Oncogene Proteins B-raf/genetics , RNA Interference , RNA, Small Interfering/genetics , Receptor, ErbB-3/genetics , Receptor, ErbB-3/metabolism , Sulfonamides/pharmacology , Tandem Mass Spectrometry , Vemurafenib , rho-Associated Kinases/genetics
2.
Immunity ; 32(5): 628-41, 2010 May 28.
Article in English | MEDLINE | ID: mdl-20510871

ABSTRACT

The transcription factor PU.1 plays multiple context and concentration dependent roles in lymphoid and myeloid cell development. Here we showed that PU.1 (encoded by Sfpi1) was essential for dendritic cell (DC) development in vivo and that conditional ablation of PU.1 in defined precursors, including the common DC progenitor, blocked Flt3 ligand-induced DC generation in vitro. PU.1 was also required for the parallel granulocyte-macrophage colony stimulating factor-induced DC pathway from early hematopoietic progenitors. Molecular studies demonstrated that PU.1 directly regulated Flt3 in a concentration-dependent manner, as Sfpi1(+/-) cells displayed reduced expression of Flt3 and impaired DC formation. These studies identify PU.1 as a critical regulator of both conventional and plasmacytoid DC development and provide one mechanism how altered PU.1 concentration can have profound functional consequences for hematopoietic cell development.


Subject(s)
Dendritic Cells/immunology , Membrane Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Animals , Base Sequence , Cell Differentiation , Cells, Cultured , Dendritic Cells/drug effects , Dose-Response Relationship, Drug , Flow Cytometry , Membrane Proteins/drug effects , Membrane Proteins/genetics , Mice , Mice, Transgenic , Models, Immunological , Molecular Sequence Data , Polymerase Chain Reaction , Promoter Regions, Genetic , Proto-Oncogene Proteins/pharmacology , Trans-Activators/pharmacology
3.
Blood ; 115(14): 2796-805, 2010 Apr 08.
Article in English | MEDLINE | ID: mdl-20130238

ABSTRACT

c-Myb is a transcription factor with functions in many hematopoietic lineages. c-Myb-deficient mice display reduced numbers of B cells; however, it is unknown what role c-Myb plays in B lymphopoiesis because no critical target genes have been identified in the B-cell lineage. We demonstrate that conditional deletion of c-Myb in B-cell progenitors completely abolishes B-cell development. c-Myb is required for lymphoid progenitors to respond to the cytokines interleukin-7 and thymic stromal lymphopoietin; in the absence of sufficient c-Myb activity, mice display a B lymphopenia that closely resembles that observed in interleukin-7 receptor alpha-deficient animals. Analysis of the multipotent progenitor compartment indicates that c-Myb is also required for up-regulation of multiple lymphoid-associated genes, including Il7r, and for the subsequent development of the common lymphoid progenitor population. These data show that c-Myb plays a critical role in the regulatory pathways governing lymphoid specification and early B-cell differentiation.


Subject(s)
B-Lymphocytes/metabolism , Cell Differentiation/physiology , Lymphoid Progenitor Cells/metabolism , Lymphopoiesis/physiology , Multipotent Stem Cells/metabolism , Proto-Oncogene Proteins c-myb/metabolism , Animals , B-Lymphocytes/cytology , Cytokines/genetics , Cytokines/metabolism , Gene Deletion , Interleukin-7/genetics , Interleukin-7/metabolism , Lymphoid Progenitor Cells/cytology , Lymphopenia/genetics , Lymphopenia/metabolism , Mice , Mice, Transgenic , Multipotent Stem Cells/cytology , Proto-Oncogene Proteins c-myb/genetics , Receptors, Interleukin-7/biosynthesis , Receptors, Interleukin-7/genetics , Up-Regulation/physiology , Thymic Stromal Lymphopoietin
4.
Blood ; 112(8): 3148-53, 2008 Oct 15.
Article in English | MEDLINE | ID: mdl-18684867

ABSTRACT

In an N-nitroso-N-ethylurea (ENU) mutagenesis screen using Mpl(-/-) mice, we isolated a semidominant suppressor of thrombocytopenia, termed Plt6. The gene mutated in Plt6 mice encodes the transcriptional coregulator p300, and the mutation, a tyrosine to asparagine substitution at amino acid 630 (Y630N), disrupts the interaction between p300 and c-Myb. Mpl(-/-) p300(Plt6/+) mice displayed elevated platelet counts relative to Mpl(-/-) p300(+/+) controls, whereas mice homozygous for the Plt6 mutation produced supraphysiological levels of circulating platelets. On a wild-type genetic background, mice homozygous for the p300(Plt6) mutation, or recipients of Mpl(+/+) p300(Plt6/Plt6) bone marrow, also exhibited thrombocytosis as well as deficiencies in B-lymphoid cells. Increased platelet numbers in Plt6 mutant mice were accompanied by significant increases in megakaryocyte progenitor cells within the bone marrow and spleen with concomitantly elevated numbers of megakaryocytes. The expansion of megakaryocytopoiesis and suppression of Mpl(-/-) thrombocytopenia in Plt6 mutants is highly reminiscent of that observed in mice with mutations affecting the p300 partner protein c-Myb, suggesting an indispensable repressive role for the c-Myb/p300 transcriptional regulatory complex in megakaryocyte development, the inhibition of which allows substantial thrombopoietin (TPO)-independent platelet production.


Subject(s)
Blood Platelets/metabolism , E1A-Associated p300 Protein/physiology , Mutation , Point Mutation , Proto-Oncogene Proteins c-myb/physiology , Receptors, Thrombopoietin/genetics , Thrombocytopenia/genetics , Animals , Base Sequence , E1A-Associated p300 Protein/genetics , Homozygote , Megakaryocytes/cytology , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Proto-Oncogene Proteins c-myb/metabolism , Thrombopoietin/metabolism
5.
Semin Immunol ; 20(4): 247-56, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18585056

ABSTRACT

While it has long been known that the transcription factor c-Myb is an essential regulator of hematopoiesis, its precise molecular targets have remained elusive. Cell line studies suggest that c-Myb promotes proliferation and at the same time inhibits differentiation, however the early lethality of c-Myb deficient embryos precluded analysis of its role in adult hematopoiesis. Here we review insights derived from recently developed mouse models of c-Myb deficiency that are viable as adults. These studies reveal a complex array of functions for c-Myb in multiple hematopoietic cell types that will redefine our understanding of this crucial transcription factor.


Subject(s)
Hematopoiesis/physiology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Proto-Oncogene Proteins c-myb/physiology , Animals , Humans , Mice , Models, Biological
6.
J Immunol ; 179(11): 7514-22, 2007 Dec 01.
Article in English | MEDLINE | ID: mdl-18025196

ABSTRACT

The noncanonical NF-kappaB pathway regulates the development and function of multiple organs and cell lineages. We have generated mice harboring a novel mutation in Nfkb2 that prevents the processing of the inhibitory precursor, p100, into the active subunit, p52. Mutant mice express a complex phenotype with abnormalities in a variety of tissues, and with a spectrum that is more severe than in mice carrying a targeted deletion of Nfkb2. Signaling through the noncanonical pathway is ablated due to the absence of p52, resulting in disorganized splenic architecture and disrupted B cell development. The inhibitory precursor form of NF-kappaB2 interacts with RelA, preventing activation of RelA dimers in response to both canonical and noncanonical stimuli, which in combination with p52 deficiency, results in defective lymph node formation and bone homeostasis. These findings demonstrate a key role for NF-kappaB2 in the regulation of RelA activation and suggest overlap in the function of NF-kappaB members in canonical and noncanonical pathway signaling.


Subject(s)
NF-kappa B p52 Subunit/physiology , Animals , B-Lymphocytes/immunology , Female , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mutation , NF-kappa B p52 Subunit/genetics , Osteoclasts/immunology , Pedigree , T-Lymphocytes/immunology , Transcription Factor RelA/physiology
7.
Mol Cell Biol ; 27(16): 5849-59, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17548465

ABSTRACT

Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity.


Subject(s)
Embryonic Development , Hematopoiesis , Phosphoglucomutase/metabolism , Uridine Diphosphate N-Acetylglucosamine/biosynthesis , Alleles , Animals , Base Sequence , Female , Glycosylation , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Sequence Data , Mutation/genetics , Pancreas/abnormalities , Phosphoglucomutase/genetics , RNA Splicing/genetics , Salivary Glands/abnormalities , Spermatogenesis
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