技术资料

Normal stem cells and cancer stem cells (CSCs) share similar properties,in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields,there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently,it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells,and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition,emerging evidence suggests that ALDH1 is not only a marker for stem cells,but may also play important functional roles related to self-protection,differentiation,and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs,with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed. View Publication

Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However,the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1,the only known histone H3 lysine 79 (H3K79) methyltransferase,has been shown to interact with multiple MLL fusion partners including AF9,ENL,AF10,and AF17. In this study,we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9,we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis,suggesting the involvement of Dot1 in survival pathways. In summary,our data point to a pivotal requirement for Dot1 in MLL fusion protein-mediated leukemogenesis and implicate Dot1 as a potential therapeutic target. 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

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular,primitive leukemia cells,often termed leukemia stem cells,are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins,presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation,CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise,we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly,these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism,which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1),as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism,an intrinsic property of primary human AML cells. View Publication

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The mechanisms underlying deregulation of HOX gene expression in AML are poorly understood. The ParaHox gene CDX2 was shown to act as positive upstream regulator of several HOX genes. In this study,constitutive expression of Cdx2 caused perturbation of leukemogenic Hox genes such as Hoxa10 and Hoxb8 in murine hematopoietic progenitors. Deletion of the N-terminal domain of Cdx2 abrogated its ability to perturb Hox gene expression and to cause acute myeloid leukemia (AML) in mice. In contrast inactivation of the putative Pbx interacting site of Cdx2 did not change the leukemogenic potential of the gene. In an analysis of 115 patients with AML,expression levels of CDX2 were closely correlated with deregulated HOX gene expression. Patients with normal karyotype showed a 14-fold higher expression of CDX2 and deregulated HOX gene expression compared with patients with chromosomal translocations such as t(8:21) or t(15;17). All patients with AML with normal karyotype tested were negative for CDX1 and CDX4 expression. These data link the leukemogenic potential of Cdx2 to its ability to dysregulate Hox genes. They furthermore correlate the level of CDX2 expression with HOX gene expression in human AML and support a potential role of CDX2 in the development of human AML with aberrant Hox gene expression. View Publication

Infection with a recombinant murine-feline gammaretrovirus,MoFe2,or with the parent virus,Moloney murine leukemia virus,caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective,in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state. View Publication

Mutations in the RUNX1 gene are found at high frequencies in minimally differentiated acute myelogenous leukemia. In addition to null mutations,many of the mutations generate Runx1 DNA-binding (RDB) mutants. To determine if these mutants antagonize wild-type protein activity,cDNAs were transduced into murine bone marrow or human cord blood cells using retroviral vectors. Significantly,the RDB mutants did not act in a transdominant fashion in vivo to disrupt Runx1 activity in either T-cell or platelet development,which are highly sensitive to Runx1 dosage. However,RDB mutant expression impaired expansion and differentiation of the erythroid compartment in which Runx1 expression is normally down-regulated,showing that a RDB-independent function is incompatible with erythroid differentiation. Significantly,both bone marrow progenitors expressing RDB mutants or deficient for Runx1 showed increased replating efficiencies in vitro,accompanied by the accumulation of myeloblasts and dysplastic progenitors,but the effect was more pronounced in RDB cultures. Disruption of the interface that binds CBFbeta,an important cofactor of Runx1,did not impair RDB mutant replating activity,arguing against inactivation of Runx1 function by CBFbeta sequestration. We propose that RDB mutants antagonize Runx1 function in early progenitors by disrupting a critical balance between DNA-binding-independent and DNA-binding-dependent signaling. 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

Chromosomal rearrangements of the 11p15 locus have been identified in hematopoietic malignancies,resulting in translocations involving the N-terminal portion of the nucleoporin gene NUP98. Fifteen different fusion partner genes have been identified for NUP98,and more than one half of these are homeobox transcription factors. By contrast,the NUP98 fusion partner in t(11;20) is Topoisomerase I (TOP1),a catalytic enzyme recognized for its key role in relaxing supercoiled DNA. We now show that retrovirally engineered expression of NUP98-TOP1 in murine bone marrow confers a potent in vitro growth advantage and a block in differentiation in hematopoietic precursors,evidenced by a competitive growth advantage in liquid culture,increased replating efficient of colony-forming cells (CFCs),and a marked increase in spleen colony-forming cell output. Moreover,in a murine bone marrow transplantation model,NUP98-TOP1 expression led to a lethal,transplantable leukemia characterized by extremely high white cell counts,splenomegaly,and mild anemia. Strikingly,a mutation to a TOP1 site to inactivate the isomerase activity essentially left unaltered the growth-promoting and leukemogenic effects of NUP98-TOP1. These findings,together with similar biologic effects reported for NUP98-HOX fusions,suggest unexpected,overlapping functions of NUP98 fusion genes,perhaps related to common DNA binding properties. View Publication

The erythroid defect in Diamond Blackfan anemia (DBA) is known to be intrinsic to the stem cell,but its molecular pathophysiology remains obscure. Using a 2-phase liquid erythroid culture system,we have demonstrated a consistent defect in DBA,regardless of clinical severity,including 3 first-degree relatives with normal hemoglobin levels but increased erythrocyte adenosine deaminase activity. DBA cultures were indistinguishable from controls until the end of erythropoietin (Epo)-free phase 1,but failed to demonstrate the normal synchronized wave of erythroid expansion and terminal differentiation on exposure to Epo. Dexamethasone increased Epo sensitivity of erythroid progenitor cells,and enhanced erythroid expansion in phase 2 in both normal and DBA cultures. In DBA cultures treated with dexamethasone,Epo sensitivity was comparable to normal,but erythroid expansion remained subnormal. In clonogenic phase 2 cultures,the number of colonies did not significantly differ between normal cultures and DBA,in the presence or absence of dexamethasone,and at both low and high Epo concentrations. However,colonies were markedly smaller in DBA under all conditions. This suggests that the Epo-triggered onset of terminal maturation is intact in DBA,and the defect lies down-stream of the Epo receptor,influencing survival and/or proliferation of erythroid progenitors. View Publication