技术资料

The RAS proteins undergo farnesylation of a carboxyl-terminal cysteine (the C" of the carboxyl-terminal CaaX motif). After farnesylation� View Publication

Canonical Wnt signaling has emerged as a critical regulatory pathway for stem cells. The association between ectopic activation of Wnt signaling and many different types of human cancer suggests that Wnt ligands can initiate tumor formation through altered regulation of stem cell populations. Here we have shown that mice deficient for the Wnt co-receptor Lrp5 are resistant to Wnt1-induced mammary tumors,which have been shown to be derived from the mammary stem/progenitor cell population. These mice exhibit a profound delay in tumorigenesis that is associated with reduced Wnt1-induced accumulation of mammary progenitor cells. In addition to the tumor resistance phenotype,loss of Lrp5 delays normal mammary development. The ductal trees of 5-week-old Lrp5-/- females have fewer terminal end buds,which are structures critical for juvenile ductal extension presumed to be rich in stem/progenitor cells. Consequently,the mature ductal tree is hypomorphic and does not completely fill the fat pad. Furthermore,Lrp5-/- ductal cells from mature females exhibit little to no stem cell activity in limiting dilution transplants. Finally,we have shown that Lrp5-/- embryos exhibit substantially impaired canonical Wnt signaling in the primitive stem cell compartment of the mammary placodes. These findings suggest that Lrp5-mediated canonical signaling is required for mammary ductal stem cell activity and for tumor development in response to oncogenic Wnt effectors. View Publication

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

Therapeutic mAbs that target tumor-associated Ags on the surface of malignant cells have proven to be an effective and specific option for the treatment of certain cancers. However,many of these protein markers of carcinogenesis are not expressed on the cells' surface. Instead these tumor-associated Ags are processed into peptides that are presented at the cell surface,in the context of MHC class I molecules,where they become targets for T cells. To tap this vast source of tumor Ags,we generated a murine IgG2a mAb,3.2G1,endowed with TCR-like binding specificity for peptide-HLA-A*0201 (HLA-A2) complex and designated this class of Ab as TCR mimics (TCRm). The 3.2G1 TCRm recognizes the GVL peptide (GVLPALPQV) from human chorionic gonadotropin beta presented by the peptide-HLA-A*0201 complex. When used in immunofluorescent staining reactions using GVL peptide-loaded T2 cells,the 3.2G1 TCRm specifically stained the cells in a peptide and Ab concentration-dependent manner. Staining intensity correlated with the extent of cell lysis by complement-dependent cytotoxicity (CDC),and a peptide concentration-dependent threshold level existed for the CDC reaction. Staining of human tumor lines demonstrated that 3.2G1 TCRm was able to recognize endogenously processed peptide and that the breast cancer cell line MDA-MB-231 highly expressed the target epitope. The 3.2G1 TCRm-mediated CDC and Ab-dependent cellular cytotoxicity of a human breast carcinoma line in vitro and inhibited in vivo tumor implantation and growth in nude mice. These results provide validation for the development of novel TCRm therapeutic reagents that specifically target and kill tumors via recognition and binding to MHC-peptide epitopes. View Publication

Multiple myeloma is characterized by the proliferation of clonal plasma cells that have a heterogeneous expression of various cell surface markers,precluding successful use of monoclonal antibodies for therapeutic targeting of the tumor cell. Thymoglobulin (rabbit-derived polyclonal anti-thymocyte globulin),by virtue of its method of preparation,contains antibodies against several B-cell and plasma cell antigens and offers an attractive option for immunotherapy of myeloma. Here,we demonstrate potent anti-myeloma activity of the rabbit anti-thymocyte globulin preparation Thymoglobulin in vitro and in vivo in an animal model of myeloma. Thymoglobulin was able to induce dose- and time-dependent apoptosis of several myeloma cell lines,including those resistant to conventional anti-myeloma agents. Importantly,the anti-myeloma activity was preserved even when myeloma cells were grown with different cytokines demonstrating the ability to overcome microenvironment-mediated resistance. Thymoglobulin induced apoptosis of freshly isolated primary myeloma cells from patients. Using a competitive flow cytometric analysis,we were able to identify the potential antigen targets for Thymoglobulin preparation. Finally,in a plasmacytoma mouse model of myeloma,Thymoglobulin delayed the tumor growth in a dose-dependent manner providing convincing evidence for continued evaluation of this agent in the clinic in patients with myeloma,either alone or in combination with other agents. 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