技术资料

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors,very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel,aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally,array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes,thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis,potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4,a GTPase regulator located in the commonly deleted 7q31 region,was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets,providing further validation of our findings. Finally,DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis,thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region. View Publication

Chromosomal translocations of the mixed lineage leukemia (MLL) gene are a common cause of acute leukemias. The oncogenic function of MLL fusion proteins is,in part,mediated through aberrant activation of Hoxa genes and Meis1,among others. Here we demonstrate using a tamoxifen-inducible Cre-mediated loss of function mouse model that DOT1L,an H3K79 methyltransferase,is required for both initiation and maintenance of MLL-AF9-induced leukemogenesis in vitro and in vivo. Through gene expression and chromatin immunoprecipitation analysis we demonstrate that mistargeting of DOT1L,subsequent H3K79 methylation,and up-regulation of Hoxa and Meis1 genes underlie the molecular mechanism of how DOT1L contributes to MLL-AF9-mediated leukemogenesis. Our study not only provides the first in vivo evidence for the function of DOT1L in leukemia,but also reveals the molecular mechanism for DOT1L in MLL-AF9 mediated leukemia. Thus,DOT1L may serve as a potential therapeutic target for the treatment of leukemia caused by MLL translocations. View Publication

Oncogene-induced senescence (OIS) is a barrier for tumor development. Oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and,accordingly,ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes,however,inhibit ARF/p53 and only infrequently select for ARF or p53 mutations,suggesting the involvement of other tumor-suppressive pathways. We report that NPM-ALK,the initiating oncogene of anaplastic large cell lymphomas (ALCLs),induces DNA damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is caused by activation of the p16INK4a/pRb tumor-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in premalignant lesions and decreased tumor latency in the absence of p16INK4a. Accordingly,human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a,which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence,and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumors. View Publication

PURPOSE: To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma (GBM) therapy that will be effective in glioblastoma stem cells (GSC),an important and untargeted component of GBM. One approach to enhance oHSV efficacy is by combination with other therapeutic modalities. EXPERIMENTAL DESIGN: MG18L,containing a U(S)3 deletion and an inactivating LacZ insertion in U(L)39,was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection in HSV-susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared with other oHSVs,alone or in combination with phosphoinositide-3-kinase (PI3K)/Akt inhibitors (LY294002,triciribine,GDC-0941,and BEZ235). Cytotoxic interactions between MG18L and PI3K/Akt inhibitors were determined using Chou-Talalay analysis. In vivo efficacy studies were conducted using a clinically relevant mouse model of GSC-derived GBM. RESULTS: MG18L was severely neuroattenuated in mice,replicated well in GSCs,and had anti-GBM activity in vivo. PI3K/Akt inhibitors displayed significant but variable antiproliferative activities in GSCs,whereas their combination with MG18L synergized in killing GSCs and glioma cell lines,but not human astrocytes,through enhanced induction of apoptosis. Importantly,synergy was independent of inhibitor sensitivity. In vivo,the combination of MG18L and LY294002 significantly prolonged survival of mice,as compared with either agent alone,achieving 50% long-term survival in GBM-bearing mice. CONCLUSIONS: This study establishes a novel therapeutic strategy: oHSV manipulation of critical oncogenic pathways to sensitize cancer cells to molecularly targeted drugs. MG18L is a promising agent for the treatment of GBM,being especially effective when combined with PI3K/Akt pathway-targeted agents. View Publication

Markers that reliably identify cancer stem cells (CSC) in ovarian cancer could assist prognosis and improve strategies for therapy. CD133 is a reported marker of ovarian CSC. Aldehyde dehydrogenase (ALDH) activity is a reported CSC marker in several solid tumors,but it has not been studied in ovarian CSC. Here we report that dual positivity of CD133 and ALDH defines a compelling marker set in ovarian CSC. All human ovarian tumors and cell lines displayed ALDH activity. ALDH(+) cells isolated from ovarian cancer cell lines were chemoresistant and preferentially grew tumors,compared with ALDH(-) cells,validating ALDH as a marker of ovarian CSC in cell lines. Notably,as few as 1,000 ALDH(+) cells isolated directly from CD133(-) human ovarian tumors were sufficient to generate tumors in immunocompromised mice,whereas 50,000 ALDH(-) cells were unable to initiate tumors. Using ALDH in combination with CD133 to analyze ovarian cancer cell lines,we observed even greater growth in the ALDH(+)CD133(+) cells compared with ALDH(+)CD133(-) cells,suggesting a further enrichment of ovarian CSC in ALDH(+)CD133(+) cells. Strikingly,as few as 11 ALDH(+)CD133(+) cells isolated directly from human tumors were sufficient to initiate tumors in mice. Like other CSC,ovarian CSC exhibited increased angiogenic capacity compared with bulk tumor cells. Finally,the presence of ALDH(+)CD133(+) cells in debulked primary tumor specimens correlated with reduced disease-free and overall survival in ovarian cancer patients. Taken together,our findings define ALDH and CD133 as a functionally significant set of markers to identify ovarian CSCs. View Publication

To investigate whether aldehyde dehydrogenase-1 (ALDH-1) in human lung cancer can be used as a sorting marker for stem cells in targeted therapies against human lung cancer. Spheres were induced by incubating cancer cells in a serum-free medium and formed with epidermal growth factor and fibroblast growth factor-10 (FGF10). Spheroid cells were combined with flow cytometry using the Aldefluor reagent to separate the SSCloALDEbr (ALDH-1-positive) cells. Cancer stem cells (CSCs) were characterized by their proliferation,colony formation,and tumorigenesis in nude mice and using phenotypic analysis. Float-growing spheres (pulmospheres") were developed after SPC-A1 cells were cultured in a serum-free medium. The resultant sphere-forming cells included ALDH-1-positive cells as high as 15.13%. ALDH-1-positive CSCs have high proliferative ability� View Publication

Identification of cancer stem cells is crucial for advancing cancer biology and therapy. Several markers including CD24,CD44,CD117,CD133,the G subfamily of ATP-binding cassette transporters (ABCG),epithelial specific antigen (ESA) and aldehyde dehydrogenase (ALDH) are used to identify and investigate human epithelial cancer stem cells in the literature. We have now systemically analyzed and compared the expression of these markers in fresh ovarian epithelial carcinomas. Although the expression levels of these markers were unexpectedly variable and partially overlapping in fresh ovarian cancer cells from different donors,we reliably detected important levels of CD133 and ALDH in the majority of fresh ovarian cancer. Furthermore,most of these stem cell markers including CD133 and ALDH were gradually lost following in vitro passage of primary tumor cells. However,the expression of ALDH and CD133,but not CD24,CD44 and CD117,could be partially rescued by the in vitro serum-free and sphere cultures and by the in vivo passage in the immune-deficient xenografts. ALDH+ and CD133+ cells formed three-dimensional spheres more efficiently than their negative counterparts. These sphere-forming cells expressed high levels of stem cell core gene transcripts and could be expanded and form additional spheres in long-term culture. ALDH+,CD133+ and ALDH+ CD133+ cells from fresh tumors developed larger tumors more rapidly than their negative counterparts. This property was preserved in the xenografted tumors. Altogether,the data suggest that ALDH+ and CD133+ cells are enriched with ovarian cancer-initiating (stem) cells and that ALDH and CD133 may be widely used as reliable markers to investigate ovarian cancer stem cell biology. View Publication

Acute basophilic leukemia (ABL) is a rare subtype of acute leukemia with clinical features and symptoms related to hyperhistaminemia because of excessive growth of basophils. No known recurrent cytogenetic abnormality is associated with this leukemia. Rare cases of t(X;6)(p11;q23) translocation have been described but these were sporadic. We report here 4 cases of ABL with a t(X;6)(p11;q23) translocation occurring in male infants. Because of its location on chromosome 6q23,MYB was a good candidate gene. Our molecular investigations,based on fluorescence in situ hybridization and rapid amplification of cDNA ends,revealed that the translocation generated a MYB-GATA1 fusion gene. Expression of MYB-GATA1 in mouse lineage-negative cells committed them to the granulocyte lineage and blocked at an early stage of differentiation. Taken together,these results establish,for the first time,a link between a recurrent chromosomal translocation and the development of this particular subtype of infant leukemia. View Publication

We studied leukemic stem cells (LSCs) in a Smad4(-/-) mouse model of acute myelogenous leukemia (AML) induced either by the HOXA9 gene or by the fusion oncogene NUP98-HOXA9. Although Hoxa9-Smad4 complexes accumulate in the cytoplasm of normal hematopoietic stem cells and progenitor cells (HSPCs) transduced with these oncogenes,there is no cytoplasmic stabilization of HOXA9 in Smad4(-/-) HSPCs,and as a consequence increased levels of Hoxa9 is observed in the nucleus leading to increased immortalization in vitro. Loss of Smad4 accelerates the development of leukemia in vivo because of an increase in transformation of HSPCs. Therefore,the cytoplasmic binding of Hoxa9 by Smad4 is a mechanism to protect Hoxa9-induced transformation of normal HSPCs. Because Smad4 is a potent tumor suppressor involved in growth control,we developed a strategy to modify the subcellular distribution of Smad4. We successfully disrupted the interaction between Hoxa9 and Smad4 to activate the TGF-β pathway and apoptosis,leading to a loss of LSCs. Together,these findings reveal a major role for Smad4 in the negative regulation of leukemia initiation and maintenance induced by HOXA9/NUP98-HOXA9 and provide strong evidence that antagonizing Smad4 stabilization by these oncoproteins might be a promising novel therapeutic approach in leukemia. View Publication

Mammary cancer stem cells (MaCSCs) have been identified as a rare population of cells capable of self-renewal to drive mammary tumorigenesis and metastasis. Nevertheless,relatively little is known about the intracellular signaling pathways regulating self-renewal and metastatic activities of MaCSCs in vivo. Using a recently developed breast cancer mouse model with focal adhesion kinase (FAK) deletion in mammary tumor cells (MFCKO-MT mice),here we present evidence suggesting a compensatory function of Pyk2,a FAK-related kinase,in the regulation of MaCSCs and metastasis in these mice. Increased expression of Pyk2 was found selectively in pulmonary metastatic nodules of MFCKO-MT mice,and its inhibition significantly reduced mammary tumor development and metastasis in these mice. Consistent with the idea of metastasis driven by MaCSCs,we detected selective up-regulation of Pyk2 in MaCSCs,but not bulk mammary tumor cells,of primary tumors developed in MFCKO-MT mice. We further showed that inhibition of Pyk2 in FAK-null MaCSCs significantly decreased their tumorsphere formation and migration in vitro as well as self-renewal,tumorigenicity,and metastatic activity in vivo. Last,we identified PI3K/Akt signaling as a major mediator of FAK regulation of MaCSCs as well as a target for the compensatory function of Pyk2 in FAK-null MaCSCs. Together,these results further advance our understanding of FAK and its related tyrosine kinase Pyk2 in regulation of MaCSCs in breast cancer and suggest that pharmaceutically targeting these kinases may hold promise as a novel treatment for the disease by targeting and eradicating MaCSCs. View Publication

The phosphatidylinositol-3,4,5-triphosphate (PIP3) binding function of pleckstrin homology (PH) domain is essential for the activation of oncogenic Akt/PKB kinase. Following the PIP3-mediated activation at the membrane,the activated Akt is subjected to other regulatory events,including ubiquitination-mediated deactivation. Here,by identifying and characterizing an allosteric inhibitor,SC66,we show that the facilitated ubiquitination effectively terminates Akt signaling. Mechanistically,SC66 manifests a dual inhibitory activity that directly interferes with the PH domain binding to PIP3 and facilitates Akt ubiquitination. A known PH domain-dependent allosteric inhibitor,which stabilizes Akt,prevents the SC66-induced Akt ubiquitination. A cancer-relevant Akt1 (e17k) mutant is unstable,making it intrinsically sensitive to functional inhibition by SC66 in cellular contexts in which the PI3K inhibition has little inhibitory effect. As a result of its dual inhibitory activity,SC66 manifests a more effective growth suppression of transformed cells that contain a high level of Akt signaling,compared with other inhibitors of PIP3/Akt pathway. Finally,we show the anticancer activity of SC66 by using a soft agar assay as well as a mouse xenograft tumor model. In conclusion,in this study,we not only identify a dual-function Akt inhibitor,but also demonstrate that Akt ubiquitination could be chemically exploited to effectively facilitate its deactivation,thus identifying an avenue for pharmacological intervention in Akt signaling. View Publication

Recently,vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However,it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here,we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted,WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery,T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells,whereas T cells specific for a non-self-viral antigen retained a CD44(low) naive phenotype. Only the WT1-specific T cells,but not the virus-specific T cells,displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells,the animals did not develop autoimmunity,and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor-associated self-antigen may drive differentiation of functionally competent memory T cells. View Publication