技术资料

Genes that are strongly repressed after B-cell activation are candidates for being inactivated,mutated,or repressed in B-cell malignancies. Krüppel-like factor 4 (Klf4),a gene down-regulated in activated murine B cells,is expressed at low levels in several types of human B-cell lineage lymphomas and leukemias. The human KLF4 gene has been identified as a tumor suppressor gene in colon and gastric cancer; in concordance with this,overexpression of KLF4 can suppress proliferation in several epithelial cell types. Here we investigate the effects of KLF4 on pro/pre-B-cell transformation by v-Abl and BCR-ABL,oncogenes that cause leukemia in mice and humans. We show that overexpression of KLF4 induces arrest and apoptosis in the G1 phase of the cell cycle. KLF4-mediated death,but not cell-cycle arrest,can be rescued by Bcl-XL overexpression. Transformed pro/pre-B cells expressing KLF4 display increased expression of p21CIP and decreased expression of c-Myc and cyclin D2. Tetracycline-inducible expression of KLF4 in B-cell progenitors of transgenic mice blocks transformation by BCR-ABL and depletes leukemic pre-B cells in vivo. Collectively,our work identifies KLF4 as a putative tumor suppressor in B-cell malignancies. View Publication

Activating mutations of c-KIT lead to ligand-independent growth. Internal tandem duplications (ITDs) of exon 11,which encodes the juxtamembrane domain (JMD),are constitutively activating mutations found in 7% of gastrointestinal stromal tumors (GISTs) but have not been described in childhood acute myeloid leukemia (AML). DNA and cDNA from 60 children with AML were screened by polymerase chain reaction (PCR) for mutations of the JMD. A complex ITD (kit cITD) involving exon 11 and exon 12 was identified with a relative frequency of 7% (4/60). The human kit cITDs were inserted into the murine c-Kit backbone and expressed in Ba/F3 cells. KIT cITD induced factorindependent growth and apoptosis resistance,and exhibited constitutive autophosphorylation. KIT cITD constitutively activated the PI3K/AKT pathway and phosphorylated STAT1,STAT3,STAT5,and SHP-2. Imatinib (IM) or rapamycin (Rap) led to complete inhibition of growth,with IC50 values at nanomolar levels. IM and Rap synergistically inhibited growth and surmounted KIT cITD-induced apoptosis resistance. IM but not LY294002 inhibited phosphorylation of STAT3 and STAT5,suggesting aberrant cross talk between PI3K- and STAT-activating pathways. The findings presented may have immediate therapeutic impact for a subgroup of childhood AML-expressing c-KIT mutations. View Publication

Retroviral vectors with long terminal repeats (LTRs),which contain strong enhancer/promoter sequences at both ends of their genome,are widely used for stable gene transfer into hematopoietic cells. However,recent clinical data and mouse models point to insertional activation of cellular proto-oncogenes as a dose-limiting side effect of retroviral gene delivery that potentially induces leukemia. Self-inactivating (SIN) retroviral vectors do not contain the terminal repetition of the enhancer/promoter,theoretically attenuating the interaction with neighboring cellular genes. With a new assay based on in vitro expansion of primary murine hematopoietic cells and selection in limiting dilution,we showed that SIN vectors using a strong internal retroviral enhancer/promoter may also transform cells by insertional mutagenesis. Most transformed clones,including those obtained after dose escalation of SIN vectors,showed insertions upstream of the third exon of Evi1 and in reverse orientation to its transcriptional orientation. Normalizing for the vector copy number,we found the transforming capacity of SIN vectors to be significantly reduced when compared with corresponding LTR vectors. Additional modifications of SIN vectors may further increase safety. Improved cell-culture assays will likely play an important role in the evaluation of insertional mutagenesis. View Publication

NUP98-HOXA9,the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation,is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. We examined its effect on differentiation,proliferation,and gene expression in primary human CD34+ hematopoietic cells. Colony-forming cell (CFC) assays in semisolid medium combined with morphologic examination and flow cytometric immunophenotyping revealed that NUP98-HOXA9 increased the numbers of erythroid precursors and impaired both myeloid and erythroid differentiation. In continuous liquid culture,cells transduced with NUP98-HOXA9 exhibited a biphasic growth curve with initial growth inhibition followed by enhanced long-term proliferation,suggesting an increase in the numbers of primitive self-renewing cells. This was confirmed by a dramatic increase in the numbers of long-term culture-initiating cells,the most primitive hematopoietic cells detectable in vitro. To understand the molecular mechanisms underlying the effects of NUP98-HOXA9 on hematopoietic cell proliferation and differentiation,oligonucleotide microarray analysis was done at several time points over 16 days,starting at 6 hours posttransduction. The early growth suppression was preceded by up-regulation of IFNbeta1 and accompanied by marked up-regulation of IFN-induced genes,peaking at 3 days posttransduction. In contrast,oncogenes such as homeobox transcription factors,FLT3,KIT,and WT1 peaked at 8 days or beyond,coinciding with increased proliferation. In addition,several putative tumor suppressors and genes associated with hematopoietic differentiation were repressed at later time points. These findings provide a comprehensive picture of the changes in proliferation,differentiation,and global gene expression that underlie the leukemic transformation of human hematopoietic cells by NUP98-HOXA9. View Publication

Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia associated with a poor prognosis. However,there are relatively few insights into the genetic etiology of AMKL. We developed a screening assay for mutations that cause AMKL,based on the hypothesis that constitutive activation of STAT5 would be a biochemical indicator of mutation in an upstream effector tyrosine kinase. We screened human AMKL cell lines for constitutive STAT5 activation,and then used an approach combining mass spectrometry identification of tyrosine phosphorylated proteins and growth inhibition in the presence of selective small molecule tyrosine kinase inhibitors that would inform DNA sequence analysis of candidate tyrosine kinases. Using this strategy,we identified a new JAK2T875N mutation in the AMKL cell line CHRF-288-11. JAK2T875N is a constitutively activated tyrosine kinase that activates downstream effectors including STAT5 in hematopoietic cells in vitro. In a murine transplant model,JAK2T875N induced a myeloproliferative disease characterized by features of AMKL,including megakaryocytic hyperplasia in the spleen; impaired megakaryocyte polyploidization; and increased reticulin fibrosis of the bone marrow and spleen. These findings provide new insights into pathways and therapeutic targets that contribute to the pathogenesis of AMKL. View Publication

SALL4,a human homolog to Drosophila spalt,is a novel zinc finger transcriptional factor essential for development. We cloned SALL4 and its isoforms (SALL4A and SALL4B). Through immunohistochemistry and real-time reverse-transcription-polymerase chain reaction (RT-PCR),we demonstrated that SALL4 was constitutively expressed in human primary acute myeloid leukemia (AML,n = 81),and directly tested the leukemogenic potential of constitutive expression of SALL4 in a murine model. SALL4B transgenic mice developed myelodysplastic syndrome (MDS)-like features and subsequently AML that was transplantable. Increased apoptosis associated with dysmyelopoiesis was evident in transgenic mouse marrow and colony-formation (CFU) assays. Both isoforms could bind to beta-catenin and synergistically enhanced the Wnt/beta-catenin signaling pathway. Our data suggest that the constitutive expression of SALL4 causes MDS/AML,most likely through the Wnt/beta-catenin pathway. Our murine model provides a useful platform to study human MDS/AML transformation,as well as the Wnt/beta-catenin pathway's role in the pathogenesis of leukemia stem cells. View Publication

Diamond-Blackfan anemia (DBA) is a broad developmental disease characterized by anemia,bone marrow (BM) erythroblastopenia,and an increased incidence of malignancy. Mutations in ribosomal protein gene S19 (RPS19) are found in approximately 25% of DBA patients; however,the role of RPS19 in the pathogenesis of DBA remains unknown. Using global gene expression analysis,we compared highly purified multipotential,erythroid,and myeloid BM progenitors from RPS19 mutated and control individuals. We found several ribosomal protein genes downregulated in all DBA progenitors. Apoptosis genes,such as TNFRSF10B and FAS,transcriptional control genes,including the erythropoietic transcription factor MYB (encoding c-myb),and translational genes were greatly dysregulated,mostly in diseased erythroid cells. Cancer-related genes,including RAS family oncogenes and tumor suppressor genes,were significantly dysregulated in all diseased progenitors. In addition,our results provide evidence that RPS19 mutations lead to codownregulation of multiple ribosomal protein genes,as well as downregulation of genes involved in translation in DBA cells. In conclusion,the altered expression of cancer-related genes suggests a molecular basis for malignancy in DBA. Downregulation of c-myb expression,which causes complete failure of fetal liver erythropoiesis in knockout mice,suggests a link between RPS19 mutations and reduced erythropoiesis in DBA. View Publication

Somatic activation of a conditional targeted Kras(G12D) allele induces a fatal myeloproliferative disease in mice that closely models juvenile and chronic myelomonocytic leukemia. These mice consistently develop severe and progressive anemia despite adequate numbers of clonogenic erythroid progenitors in the bone marrow and expanded splenic hematopoiesis. Ineffective erythropoiesis is characterized by impaired differentiation. These results demonstrate that endogenous levels of oncogenic Ras have cell lineage-specific effects and support efforts to modulate Ras signaling for therapy of anemia in patients with myelodysplastic syndromes and myeloproliferative disorders. View Publication

Most patients with acute promyelocytic leukemia (APL) express PML-RAR alpha,the fusion product of t(15;17)(q22;q11.2). Transgenic mice expressing PML-RAR alpha develop APL with long latency,low penetrance,and acquired cytogenetic abnormalities. Based on observations that 4% to 10% of APL patients harbor oncogenic ras mutations,we coexpressed oncogenic K-ras from its endogenous promoter with PML-RAR alpha to generate a short-latency,highly penetrant mouse model of APL. The APL disease was characterized by splenomegaly,leukocytosis,extramedullary hematopoiesis (EMH) in spleen and liver with an increased proportion of immature myeloperoxidase-expressing myeloid forms; transplantability to secondary recipients; and lack of cytogenetic abnormalities. Bone marrow cells showed enhanced self-renewal in vitro. This model establishes a role for oncogenic ras in leukemia pathogenesis and thus validates the oncogenic RAS signaling pathway as a potential target for therapeutic inhibition in leukemia patients. This mouse model should be useful for investigating signaling pathways that promote self-renewal in APL and for testing the in vivo efficacy of RAS signaling pathway inhibitors in conjunction with other targeted therapies such as ATRA (all trans retinoic acid) and arsenic trioxide. View Publication

The t(16:16) and inv(16) are associated with FAB M4Eo myeloid leukemias and result in fusion of the CBFB gene to the MYH11 gene (encoding smooth muscle myosin heavy chain [SMMHC]). Knockout of CBFbeta causes embryonic lethality due to lack of definitive hematopoiesis. Although knock-in of CBFB-MYH11 is not sufficient to cause disease,expression increases the incidence of leukemia when combined with cooperating events. Although mouse models are valuable tools in the study of leukemogenesis,little is known about the contribution of CBFbeta-SMMHC to human hematopoietic stem and progenitor cell self-renewal. We introduced the CBFbeta-MYH11 cDNA into human CD34+ cells via retroviral transduction. Transduced cells displayed an initial repression of progenitor activity but eventually dominated the culture,resulting in the proliferation of clonal populations for up to 7 months. Long-term cultures displayed a myelomonocytic morphology while retaining multilineage progenitor activity and engraftment in NOD/SCID-B2M-/- mice. Progenitor cells from long-term cultures showed altered expression of genes defining inv(16) identified in microarray studies of human patient samples. This system will be useful in examining the effects of CBFbeta-SMMHC on gene expression in the human preleukemic cell,in characterizing the effect of this oncogene on human stem cell biology,and in defining its contribution to the development of leukemia. View Publication

A JAK2(V617F) mutation is frequently found in several BCR/ABL-negative myeloproliferative disorders. To address the contribution of this mutant to the pathogenesis of these different myeloproliferative disorders,we used an adoptive transfer of marrow cells transduced with a retrovirus expressing JAK2(V617F) in recipient irradiated mice. Hosts were analyzed during the 6 months after transplantation. For a period of 3 months,mice developed polycythemia,macrocytosis and usually peripheral blood granulocytosis. Transient thrombocytosis was only observed in a low-expresser group. All mice displayed trilineage hyperplasia in marrow and spleen along with an amplification of myeloid and erythroid progenitor cells and a formation of endogenous erythroid colonies. After 3 to 4 months,polycythemia regressed,abnormally shaped red blood cells and platelets were seen in circulation,and a deposition of reticulin fibers was observed in marrow and spleen. Development of fibrosis was associated with anemia,thrombocytopenia,high neutrophilia,and massive splenomegaly. These features mimic human polycythemia vera and its evolution toward myelofibrosis. This work demonstrates that JAK2(V617F) is sufficient for polycythemia and fibrosis development and offers an in vivo model to assess novel therapeutic approaches for JAK2(V617F)-positive pathologies. Questions remain regarding the exact contribution of JAK2(V617F) in other myeloproliferative disorders. View Publication

Chronic phase-to-blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPalpha,a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region-Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPalpha activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation,inhibited proliferation in vitro and in mice,and suppressed leukemogenesis. Moreover,activation of C/EBPalpha eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells,respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis,as indicated by the effects of p42 C/EBPalpha,which were more potent than those of K298E C/EBPalpha,a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPalpha in blast cells from 4 patients with CML-BC,including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation,also induced granulocyte differentiation. Thus,these data indicate that C/EBPalpha has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors,and they portend the development of anti-leukemia therapies that rely on C/EBPalpha activation. View Publication