技术资料

Defining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers,including myeloid malignancies. We investigated the effects of an oncogenic Kras(G12D) allele on phosphorylated signaling molecules in primary c-kit(+) lin(-/low) hematopoietic stem/progenitor cells. Comparison of wild-type and Kras(G12D) c-kit(+) lin(-/low) cells shows that K-Ras(G12D) expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5,ERK,and S6 under basal and stimulated conditions. Whereas Kras(G12D) cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor,we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell-targeted interventions. View Publication

The epithelium of the mammary gland exists in a highly dynamic state,undergoing dramatic morphogenetic changes during puberty,pregnancy,lactation,and regression. The recent identification of stem and progenitor populations in mouse and human mammary tissue has provided evidence that the mammary epithelium is organized in a hierarchical manner. Characterization of these normal epithelial subtypes is an important step toward understanding which cells are predisposed to oncogenesis. This review summarizes progress in the field toward defining constituent cells and key molecular regulators of the mammary epithelial hierarchy. Potential relationships between normal epithelial populations and breast tumor subtypes are discussed,with implications for understanding the cellular etiology underpinning breast tumor heterogeneity. 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

Myelodysplastic syndromes (MDSs) are a group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral blood cytopenias. Lenalidomide has dramatic therapeutic effects in patients with low-risk MDS and a chromosome 5q31 deletion,resulting in complete cytogenetic remission in textgreater60% of patients. The molecular basis of this remarkable drug response is unknown. To gain insight into the molecular targets of lenalidomide we investigated its in vitro effects on growth,maturation,and global gene expression in isolated erythroblast cultures from MDS patients with del(5)(q31). Lenalidomide inhibited growth of differentiating del(5q) erythroblasts but did not affect cytogenetically normal cells. Moreover,lenalidomide significantly influenced the pattern of gene expression in del(5q) intermediate erythroblasts,with the VSIG4,PPIC,TPBG,activin A,and SPARC genes up-regulated by textgreater2-fold in all samples and many genes involved in erythropoiesis,including HBA2,GYPA,and KLF1,down-regulated in most samples. Activin A,one of the most significant differentially expressed genes between lenalidomide-treated cells from MDS patients and healthy controls,has pleiotropic functions,including apoptosis of hematopoietic cells. Up-regulation and increased protein expression of the tumor suppressor gene SPARC is of particular interest because it is antiproliferative,antiadhesive,and antiangiogenic and is located at 5q31-q32,within the commonly deleted region in MDS 5q- syndrome. We conclude that lenalidomide inhibits growth of del(5q) erythroid progenitors and that the up-regulation of SPARC and activin A may underlie the potent effects of lenalidomide in MDS with del(5)(q31). SPARC may play a role in the pathogenesis of the 5q- syndrome. View Publication

BACKGROUND AND OBJECTIVES: Leptin receptors can be expressed by acute myelogenous leukemia (AML) cells,but the functional effects of leptin on native AML blasts have not been characterized in detail. We investigated systemic leptin levels in AML patients and in vitro effects of leptin on cultured AML blasts. DESIGN AND METHODS: Serum leptin levels were compared for patients with untreated AML and healthy controls. Native AML blasts were derived from a large group of consecutive patients,and effects of leptin on proliferation (suspension cultures and colony formation),constitutive cytokine secretion,differentiation and apoptosis regulation were assayed in vitro. RESULTS: Systemic leptin levels were decreased in patients with untreated AML,and leptin levels in acute leukemia patients were not altered during severe chemotherapy-induced cytopenia and complicating febrile neutropenia. In vitro studies demonstrated that leptin increased AML blast release of interleukin (IL) 1beta,IL6,tumor necrosis factor (TNF) alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF). This enhancing effect showed no correlation with CD34 expression and was not dependent on the presence of serum,induction of differentiation or alteration of caspase 3 activity with decreased in vitro apoptosis. Leptin also increased spontaneous AML blast proliferation,whereas divergent effects on blast proliferation were observed in the presence of exogenous cytokines. The in vitro effects were usually observed at concentrations exceeding the systemic levels. INTERPRETATION AND CONCLUSIONS: Our results suggest that systemic leptin levels alone do not have a major influence on native AML blasts,but the systemic levels in combination with local leptin release in the bone marrow may affect the functional characteristics of these cells. View Publication

The ability of acute myeloid leukemic (AML) blasts to differentiate into leukemic dendritic cells (DC) thus acquiring the potential to present known and unknown leukemic antigens efficiently,holds promise as a possible new treatment for AML patients with minimal residual disease. Recent advances in culture methods have made the clinical use of leukemic DC feasible. However,additional measures appear to be essential in order to potentiate vaccines and to overcome the intrinsic tolerant state of the patients immune system. This review describes ways to improve AML-DC vaccines and discusses critical aspects concerning the development of clinical vaccination protocols. View Publication

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA,encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast,no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines,mice,dogs,nonhuman primates,and human subjects. Here,we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose,and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells,such hits were overrepresented in leukemic clones,pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases,indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings,we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery. 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

PTPN11,which encodes the tyrosine phosphatase SHP2,is mutated in approximately 35% of patients with juvenile myelomonocytic leukemia (JMML) and at a lower incidence in other neoplasms. To model JMML pathogenesis,we generated knockin mice that conditionally express the leukemia-associated mutant Ptpn11(D61Y). Expression of Ptpn11(D61Y) in all hematopoietic cells evokes a fatal myeloproliferative disorder (MPD),featuring leukocytosis,anemia,hepatosplenomegaly,and factor-independent colony formation by bone marrow (BM) and spleen cells. The Lin(-)Sca1(+)cKit(+) (LSK) compartment is expanded and right-shifted� View Publication

Recognition of tumor-associated Ags (TAAs) on tumor cells by CTLs and the subsequent tumor cell death are assumed to be dependent on TAA protein expression and to correlate directly with the level of peptide displayed in the binding site of the HLA class I molecule. In this study we evaluated whether the levels of Her-2/neu protein expression on human tumor cell lines directly correlate with HLA-A*0201/Her2/neu peptide presentation and CTL recognition. We developed a TCR mimic (TCRm) mAb designated 1B8 that specifically recognizes the HLA-A2.1/Her2/neu peptide (369-377) (Her2(369)-A2) complex. TCRm mAb staining intensity varied for the five human tumor cell lines analyzed,suggesting quantitative differences in levels of the Her2(369)-A2 complex on these cells. Analysis of tumor cell lines pretreated with IFN-gamma and TNF-alpha for Her2/neu protein and HLA-A2 molecule expression did not reveal a direct correlation between the levels of Her2/neu Ag,HLA-A2 molecule,and Her2(369)-A2 complex expression. However,compared with untreated cells,cytokine-treated cell lines showed an increase in Her2(369)-A2 epitope density that directly correlated with enhanced tumor cell death (p = 0.05). Although a trend was observed between tumor cell lysis and the level of the Her2(369)-A2 complex for untreated cells,the association was not significant. These findings suggest that tumor cell susceptibility to CTL-mediated lysis may be predicted based on the level of specific peptide-MHC class I expression rather than on the total level of TAA expression. Further,these studies demonstrate the potential of the TCRm mAb for validation of endogenous HLA-peptide epitopes on tumor cells. View Publication

PURPOSE: Aberrant activation of the Notch signaling pathway is commonly observed in human pancreatic cancer,although the mechanism(s) for this activation has not been elucidated. EXPERIMENTAL DESIGN: A panel of 20 human pancreatic cancer cell lines was profiled for the expression of Notch pathway-related ligands,receptors,and target genes. Disruption of intracellular Notch signaling,either genetically by RNA interference targeting NOTCH1 or pharmacologically by means of the gamma-secretase inhibitor GSI-18,was used for assessing requirement of Notch signaling in pancreatic cancer initiation and maintenance. RESULTS: Striking overexpression of Notch ligand transcripts was detectable in the vast majority of pancreatic cancer cell lines,most prominently JAGGED2 (18 of 20 cases,90%) and DLL4 (10 of 20 cases,50%). In two cell lines,genomic amplification of the DLL3 locus was observed,mirrored by overexpression of DLL3 transcripts. In contrast,coding region mutations of NOTCH1 or NOTCH2 were not observed. Genetic and pharmacologic inhibition of Notch signaling mitigated anchorage-independent growth in pancreatic cancer cells,confirming that sustained Notch activation is a requirement for pancreatic cancer maintenance. Further,transient pretreatment of pancreatic cancer cells with GSI-18 resulted in depletion in the proportion of tumor-initiating aldehyde dehydrogenase-expressing subpopulation and was associated with inhibition of colony formation in vitro and xenograft engraftment in vivo,underscoring a requirement for the Notch-dependent aldehyde dehydrogenase-expressing cells in pancreatic cancer initiation. CONCLUSIONS: Our studies confirm that Notch activation is almost always ligand dependent in pancreatic cancer,and inhibition of Notch signaling is a promising therapeutic strategy in this malignancy. View Publication