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The importance of cancer metabolism has been appreciated for many years,but the intricacies of how metabolic pathways interconnect with oncogenic signaling are not fully understood. With a clear understanding of how metabolism contributes to tumorigenesis,we will be better able to integrate the targeting of these fundamental biochemical pathways into patient care. The mevalonate (MVA) pathway,paced by its rate-limiting enzyme,hydroxymethylglutaryl coenzyme A reductase (HMGCR),is required for the generation of several fundamental end-products including cholesterol and isoprenoids. Despite years of extensive research from the perspective of cardiovascular disease,the contribution of a dysregulated MVA pathway to human cancer remains largely unexplored. We address this issue directly by showing that dysregulation of the MVA pathway,achieved by ectopic expression of either full-length HMGCR or its novel splice variant,promotes transformation. Ectopic HMGCR accentuates growth of transformed and nontransformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice and,importantly,cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts cells. We further explore whether the MVA pathway may play a role in the etiology of human cancers and show that high mRNA levels of HMGCR and additional MVA pathway genes correlate with poor prognosis in a meta-analysis of six microarray datasets of primary breast cancer. Taken together,our results suggest that HMGCR is a candidate metabolic oncogene and provide a molecular rationale for further exploring the statin family of HMGCR inhibitors as anticancer agents. View Publication

The Mediator complex forms the bridge between transcriptional activators and the RNA polymerase II. Med1 (also known as PBP or TRAP220) is a key component of Mediator that interacts with nuclear hormone receptors and GATA transcription factors. Here,we show dynamic recruitment of GATA-1,TFIIB,Mediator,and RNA polymerase II to the β-globin locus in induced mouse erythroid leukemia cells and in an erythropoietin-inducible hematopoietic progenitor cell line. Using Med1 conditional knockout mice,we demonstrate a specific block in erythroid development but not in myeloid or lymphoid development,highlighted by the complete absence of β-globin gene expression. Thus,Mediator subunit Med1 plays a pivotal role in erythroid development and in β-globin gene activation. View Publication

Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome,a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction,but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14,the ribosomal protein gene deleted in the 5q-syndrome,or RPS19,the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect,whereas nutlin-3,a compound that activates p53 through inhibition of HDM2,selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome,we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53,providing a basis for the failure of erythropoiesis in the 5q-syndrome,DBA,and perhaps other bone marrow failure syndromes. View Publication

Serum response factor (Srf) is a MADS-box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1,a cofactor of Srf,is part of the t(1;22) translocation in acute megakaryoblastic leukemia,and plays a critical role in megakaryopoiesis. To test the role of Srf in megakaryocyte development,we crossed Pf4-Cre mice,which express Cre recombinase in cells committed to the megakaryocytic lineage,to Srf(F/F) mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/Srf(F/F) knockout (KO) mice are born with normal Mendelian frequency,but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast,the BM has increased number and percentage of CD41(+) megakaryocytes (WT: 0.41% ± 0.06%; KO: 1.92% ± 0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation,and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are down-regulated in KO megakaryocytes. Thus,Srf is required for normal megakaryocyte maturation and platelet production partly because of regulation of cytoskeletal genes. View Publication

Two critical concerns in clinical cord blood transplantation are the initial time to engraftment and the subsequent restoration of immune function. These studies measured the impact of progenitor cell dose on both the pace and strength of hematopoietic reconstitution by transplanting nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-gamma-null (NSγ) mice with lineage-depleted aldehyde dehydrogenase-bright CD34(+) human cord blood progenitors. The progress of each transplant was monitored over an extended time course by repeatedly analyzing the peripheral blood for human hematopoietic cells. In vivo human hematopoietic development was complete. After long-term transplantation assays (≥ 19 weeks),human T-cell development was documented within multiple tissues in 16 of 32 NSγ mice. Human T-cell differentiation was active within NSγ thymuses,as documented by the presence of CD4(+) CD8(+) T-cell progenitors as well as T-cell receptor excision circles. It is important to note that although myeloid and B-cell engraftment was detected as early as 4 weeks after transplantation,human T-cell development was exclusively late onset. High progenitor cell doses were associated with a robust human hematopoietic chimerism that accelerated both initial time to engraftment and subsequent T-cell development. At lower progenitor cell doses,the chimerism was weak and the human hematopoietic lineage development was frequently incomplete. View Publication

Hematopoiesis is tightly controlled by transcription regulatory networks,but how and when specific transcription factors control lineage commitment are still largely unknown. Within the hematopoietic stem cell (Lin(-)Sca-1(+)c-Kit(+)) compartment these lineage-specific transcription factors are expressed at low levels but are up-regulated with the process of lineage specification. CCAAT/enhancer binding protein α (C/EBPα) represents one of these factors and is involved in myeloid development and indispensable for formation of granulocytes. To track the cellular fate of stem and progenitor cells,which express C/EBPα,we developed a mouse model expressing Cre recombinase from the Cebpa promoter and a conditional EYFP allele. We show that Cebpa/EYFP(+) cells represent a significant subset of multipotent hematopoietic progenitors,which predominantly give rise to myeloid cells in steady-state hematopoiesis. C/EBPα induced a strong myeloid gene expression signature and down-regulated E2A-induced regulators of early lymphoid development. In addition,Cebpa/EYFP(+) cells compose a fraction of early thymic progenitors with robust myeloid potential. However,Cebpa/EYFP(+) multipotent hematopoietic progenitors and early thymic progenitors retained the ability to develop into erythroid and T-lymphoid lineages,respectively. These findings support an instructive but argue against a lineage-restrictive role of C/EBPα in multipotent hematopoietic and thymic progenitors. View Publication

RATIONALE: Fibrocytes are progenitor cells characterized by the simultaneous expression of mesenchymal,monocyte,and hematopoietic stem cell markers. We previously documented their presence in lungs of patients with idiopathic pulmonary fibrosis. However,the mechanisms involved in their migration,subsequent homing,and local role remain unclear. Matrix metalloproteinases (MMPs) facilitate cell migration and have been implicated in the pathogenesis of pulmonary fibrosis. OBJECTIVES: To evaluate the expression and role of matrix metalloproteinases in human fibrocytes. METHODS: Fibrocytes were purified from CD14(+) monocytes and cultured for 8 days; purity of fibrocyte cultures was 95% or greater as determined by flow cytometry. Conditioned media and total RNA were collected and the expression of MMP-1,MMP-2,MMP-7,MMP-8,and MMP-9 was evaluated by real-time polymerase chain reaction. Protein synthesis was examined using a Multiplex assay,Western blot,fluorescent immunocytochemistry,and confocal microscopy. MMP-2 and MMP-9 enzymatic activities were evaluated by gelatin zymography. Migration was assessed using collagen I-coated Boyden chambers. Stromal cell-derived factor-1α and platelet-derived growth factor-B were used as chemoattractant with or without a specific MMP-8 inhibitor. MEASUREMENTS AND MAIN RESULTS: Fibrocytes showed gene and protein expression of MMP-2,MMP-9,MMP-8,and MMP-7. MMP-2 and MMP-9 enzymatic activities were also demonstrated by gelatin zymography. Likewise,we found colocalization of MMP-8 and MMP-7 with type I collagen in fibrocytes. Fibrocyte migration toward platelet-derived growth factor-B or Stromal cell-derived factor-1α in collagen I-coated Boyden chambers was significantly reduced by a specific MMP-8 inhibitor. CONCLUSIONS: Our findings reveal that fibrocytes express a variety of MMPs and that MMP-8 actively participates in the process of fibrocyte migration. View Publication

In up to one-third of patients with acute myeloid leukemia,a C-terminal frame-shift mutation results in abnormal and abundant cytoplasmic accumulation of the usually nucleoli-bound protein nucleophosmin (NPM),and this is thought to function in cancer pathogenesis. Here,we demonstrate a gain-of-function role for cytoplasmic NPM in the inhibition of caspase signaling. The NPM mutant specifically inhibits the activities of the cell-death proteases,caspase-6 and -8,through direct interaction with their cleaved,active forms,but not the immature procaspases. The cytoplasmic NPM mutant not only affords protection from death ligand-induced cell death but also suppresses caspase-6/-8-mediated myeloid differentiation. Our data hence provide a potential explanation for the myeloid-specific involvement of cytoplasmic NPM in the leukemogenesis of a large subset of acute myeloid leukemia. View Publication

Retroviral overexpression of NF-Ya,the regulatory subunit of the transcription factor NF-Y,activates the transcription of multiple genes implicated in hematopoietic stem cell (HSC) self-renewal and differentiation and directs HSCs toward self-renewal. We asked whether TAT-NF-Ya fusion protein could be used to transduce human CD34(+) cells as a safer,more regulated alternative approach to gene therapy. Here we show that externally added recombinant protein was able to enter the cell nucleus and activate HOXB4,a target gene of NF-Ya,using real-time polymerase chain reaction RNA and luciferase-based protein assays. After TAT-NF-Ya transduction,the proliferation of human CD34(+) cells in the presence of myeloid cytokines was increased 4-fold. Moreover,TAT-NF-Ya-treated human primary bone marrow cells showed a 4-fold increase in the percentage of huCD45(+) cells recovered from the bone marrow of sublethally irradiated,transplanted NOD-Scid IL2Rγ(null) mice. These data demonstrate that TAT-peptide therapies are an alternative approach to retroviral stem cell therapies and suggest that NF-Ya peptide delivery should be further evaluated as a tool for HSC/progenitors ex vivo expansion and therapy. View Publication

SPARC is an extracellular matrix protein that exerts pleiotropic effects on extracellular matrix organization,growth factor availability,cell adhesion,differentiation,and immunity in cancer. Chronic myelogenous leukemia (CML) cells resistant to the BCR-ABL inhibitor imatinib (IM-R cells) were found to overexpress SPARC mRNA. In this study,we show that imatinib triggers SPARC accumulation in a variety of tyrosine kinase inhibitor (TKI)-resistant CML cell lines. SPARC silencing in IM-R cells restored imatinib sensitivity,whereas enforced SPARC expression in imatinib-sensitive cells promoted viability as well as protection against imatinib-mediated apoptosis. Notably,we found that the protective effect of SPARC required intracellular retention inside cells. Accordingly,SPARC was not secreted into the culture medium of IM-R cells. Increased SPARC expression was intimately linked to persistent activation of the Fyn/ERK kinase signaling axis. Pharmacologic inhibition of this pathway or siRNA-mediated knockdown of Fyn kinase resensitized IM-R cells to imatinib. In support of our findings,increased levels of SPARC mRNA were documented in blood cells from CML patients after 1 year of imatinib therapy compared with initial diagnosis. Taken together,our results highlight an important role for the Fyn/ERK signaling pathway in imatinib-resistant cells that is driven by accumulation of intracellular SPARC. View Publication

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment,we silenced c-myb in human CD34(+) hematopoietic stem/progenitor cells. Noteworthy,c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages,whereas erythroid differentiation was impaired,as demonstrated by clonogenic assay,morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb-silenced CD34(+) cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets,which can account for c-myb knockdown effects. Indeed,chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently,the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing,whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34(+) cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment,by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed,we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision. View Publication

Although practiced clinically for more than 40 years,the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative,StemRegenin 1 (SR1),that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy. View Publication