ABSTRACT
Overexpression of HOXB4 in hematopoietic stem cells (HSCs) leads to increased self-renewal without causing hematopoietic malignancies in transplanted mice. The molecular basis of HOXB4-mediated benign HSC expansion in vivo is not well understood. To gain further insight into the molecular events underlying HOXB4-mediated HSC expansion, we analyzed gene expression changes at multiple time points in Lin(-)Sca1(+)c-kit(+) cells from mice transplanted with bone marrow cells transduced with a MSCV-HOXB4-ires-YFP vector. A distinct HOXB4 transcriptional program was reproducibly induced and stabilized by 12 weeks after transplant. Dynamic expression changes were observed in genes critical for HSC self-renewal as well as in genes involved in myeloid and B-cell differentiation. Prdm16, a transcription factor associated with human acute myeloid leukemia, was markedly repressed by HOXB4 but upregulated by HOXA9 and HOXA10, suggesting that Prdm16 downregulation was involved in preventing leukemia in HOXB4 transplanted mice. Functional evidence to support this mechanism was obtained by enforcing coexpression of sPrdm16 and HOXB4, which led to enhanced self-renewal, myeloid expansion, and leukemia. Altogether, these studies define the transcriptional pathways involved in HOXB4 HSC expansion in vivo and identify repression of Prdm16 transcription as a mechanism by which expanding HSCs avoid leukemic transformation.
Subject(s)
DNA-Binding Proteins/metabolism , Down-Regulation/physiology , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/metabolism , RNA, Messenger/biosynthesis , Transcription Factors/metabolism , Transcription, Genetic/physiology , Animals , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Cell Differentiation/physiology , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , DNA-Binding Proteins/genetics , Female , Gene Expression Regulation, Leukemic/genetics , Hematopoietic Stem Cells/cytology , Homeobox A10 Proteins , Homeodomain Proteins/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mice , Myeloid Cells/cytology , Myeloid Cells/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , RNA, Messenger/genetics , Transcription Factors/genetics , Transduction, GeneticABSTRACT
Lysins are bacteriophage-derived enzymes that degrade bacterial peptidoglycans. Lysin CF-301 is being developed to treat Staphylococcus aureus because of its potent, specific, and rapid bacteriolytic effects. It also demonstrates activity on drug-resistant strains, has a low resistance profile, eradicates biofilms, and acts synergistically with antibiotics. CF-301 was bacteriolytic against 250 S. aureus strains tested including 120 methicillin-resistant S. aureus (MRSA) isolates. In time-kill studies with 62 strains, CF-301 reduced S. aureus by 3-log10 within 30 minutes compared to 6-12 hours required by antibiotics. In bacteremia, CF-301 increased survival by reducing blood MRSA 100-fold within 1 hour. Combinations of CF-301 with vancomycin or daptomycin synergized in vitro and increased survival significantly in staphylococcal-induced bacteremia compared to treatment with antibiotics alone (P < .0001). Superiority of CF-301 combinations with antibiotics was confirmed in 26 independent bacteremia studies. Combinations including CF-301 and antibiotics represent an attractive alternative to antibiotic monotherapies currently used to treat S. aureus bacteremia.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteremia/drug therapy , Methicillin-Resistant Staphylococcus aureus/drug effects , Mucoproteins/pharmacology , Staphylococcal Infections/drug therapy , Amino Acid Sequence , Animals , Anti-Bacterial Agents/pharmacokinetics , Bacteremia/microbiology , Biofilms , Drug Synergism , Female , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Models, Molecular , Molecular Sequence Data , Mucoproteins/chemistry , Prophages/enzymology , Prophages/genetics , Staphylococcal Infections/microbiology , Viral Proteins/pharmacologyABSTRACT
Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell self-renewal and expansion ex vivo and in vivo. To investigate the downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line EML. Two genome-wide analytical techniques were used: RNA expression profiling using microarrays and chromatin immunoprecipitation (ChIP)-chip. RNA expression profiling revealed that 465 gene transcripts were differentially expressed in KLS (c-Kit(+), Lin(-), Sca-1(+))-EML cells that overexpressed HOXB4 (KLS-EML-HOXB4) compared with control KLS-EML cells that were transduced with vector alone. In particular, erythroid-specific gene transcripts were observed to be highly down-regulated in KLS-EML-HOXB4 cells. ChIP-chip analysis revealed that the promoter region for 1910 genes, such as CD34, Sox4, and B220, were occupied by HOXB4 in KLS-EML-HOXB4 cells. Side-by-side comparison of the ChIP-chip and RNA expression profiling datasets provided correlative information and identified Gp49a and Laptm4b as candidate "stemness-related" genes. Both genes were highly ranked in both dataset lists and have been previously shown to be preferentially expressed in hematopoietic stem cells and down-regulated in mature hematopoietic cells, thus making them attractive candidates for future functional studies in hematopoietic cells.
