技术资料

Off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication. To identify such compounds,we conducted 2 independent cell-based chemical screens and identified the antimicrobial ciclopirox olamine (CPX) in both screens. CPX decreased cell growth and viability of malignant leukemia,myeloma,and solid tumor cell lines as well as primary AML patient samples at low-micromolar concentrations that appear pharmacologically achievable. Furthermore,oral CPX decreased tumor weight and volume in 3 mouse models of leukemia by up to 65% compared with control without evidence of weight loss or gross organ toxicity. In addition,oral CPX prevented the engraftment of primary AML cells in nonobese diabetic/severe combined immunodeficiency mouse models,thereby establishing its ability to target leukemia stem cells. Mechanistically,CPX bound intracellular iron,and this intracellular iron chelation was functionally important for its cytotoxicity. By electron paramagnetic resonance,CPX inhibited the iron-dependent enzyme ribonucleotide reductase at concentrations associated with cell death. Thus,in summary,CPX has previously unrecognized anticancer activity at concentrations that are pharmacologically achievable. Therefore,CPX could be rapidly repurposed for the treatment of malignancies,including leukemia and myeloma. View Publication

Acute myelogenous leukemia is driven by leukemic stem cells (LSCs) generated by mutations that confer (or maintain) self-renewal potential coupled to an aberrant differentiation program. Using retroviral mutagenesis,we identified genes that generate LSCs in collaboration with genetic disruption of the gene encoding interferon response factor 8 (Irf8),which induces a myeloproliferation in vivo. Among the targeted genes,we identified Mef2c,encoding a MCM1-agamous-deficiens-serum response factor transcription factor,and confirmed that overexpression induced a myelomonocytic leukemia in cooperation with Irf8 deficiency. Strikingly,several of the genes identified in our screen have been reported to be up-regulated in the mixed-lineage leukemia (MLL) subtype. High MEF2C expression levels were confirmed in acute myelogenous leukemia patient samples with MLL gene disruptions,prompting an investigation of the causal interplay. Using a conditional mouse strain,we demonstrated that Mef2c deficiency does not impair the establishment or maintenance of LSCs generated in vitro by MLL/ENL fusion proteins; however,its loss led to compromised homing and invasiveness of the tumor cells. Mef2c-dependent targets included several genes encoding matrix metalloproteinases and chemokine ligands and receptors,providing a mechanistic link to increased homing and motility. Thus,MEF2C up-regulation may be responsible for the aggressive nature of this leukemia subtype. View Publication

The role of miRNAs in regulating megakaryocyte differentiation was examined using bipotent K562 human leukemia cells. miR-34a is strongly up-regulated during phorbol ester-induced megakaryocyte differentiation,but not during hemin-induced erythrocyte differentiation. Enforced expression of miR-34a in K562 cells inhibits cell proliferation,induces cell-cycle arrest in G(1) phase,and promotes megakaryocyte differentiation as measured by CD41 induction. miR-34a expression is also up-regulated during thrombopoietin-induced differentiation of CD34(+) hematopoietic precursors,and its enforced expression in these cells significantly increases the number of megakaryocyte colonies. miR-34a directly regulates expression of MYB,facilitating megakaryocyte differentiation,and of CDK4 and CDK6,to inhibit the G(1)/S transition. However,these miR-34a target genes are down-regulated rapidly after inducing megakaryocyte differentiation before miR-34a is induced. This suggests that miR-34a is not responsible for the initial down-regulation but may contribute to maintaining their suppression later on. Previous studies have implicated miR-34a as a tumor suppressor gene whose transcription is activated by p53. However,in p53-null K562 cells,phorbol esters induce miR-34a expression independently of p53 by activating an alternative phorbol ester-responsive promoter to produce a longer pri-miR-34a transcript. View Publication

RNA-binding motif protein 15 (RBM15) is involved in the RBM15-megakaryoblastic leukemia 1 fusion in acute megakaryoblastic leukemia. Although Rbm15 has been reported to be required for B-cell differentiation and to inhibit myeloid and megakaryocytic expansion,it is not clear what the normal functions of Rbm15 are in the regulation of hematopoietic stem cell (HSC) and megakaryocyte development. In this study,we report that Rbm15 may function in part through regulation of expression of the proto-oncogene c-Myc. Similar to c-Myc knockout (c-Myc-KO) mice,long-term (LT) HSCs are significantly increased in Rbm15-KO mice due to an apparent LT-HSC to short-term HSC differentiation defect associated with abnormal HSC-niche interactions caused by increased N-cadherin and beta(1) integrin expression on mutant HSCs. Both serial transplantation and competitive reconstitution capabilities of Rbm15-KO LT-HSCs are greatly compromised. Rbm15-KO and c-Myc-KO mice also share related abnormalities in megakaryocyte development,with mutant progenitors producing increased,abnormally small low-ploidy megakaryocytes. Consistent with a possible functional interplay between Rbm15 and c-Myc,the megakaryocyte increase in Rbm15-KO mice could be partially reversed by ectopic c-Myc. Thus,Rbm15 appears to be required for normal HSC-niche interactions,for the ability of HSCs to contribute normally to adult hematopoiesis,and for normal megakaryocyte development; these effects of Rbm15 on hematopoiesis may be mediated at least in part by c-Myc. View Publication

After DNA replication,sister chromatids must be untangled,or decatenated,before mitosis so that chromatids do not tear during anaphase. Topoisomerase IIalpha (Topo IIalpha) is the major decatenating enzyme. Topo IIalpha inhibitors prevent decatenation,causing cells to arrest during mitosis. Here we report that acute myeloid leukemia cells fail to arrest at the mitotic decatenation checkpoint,and their progression through this checkpoint is regulated by the DNA repair component Metnase (also termed SETMAR). Metnase contains a SET histone methylase and transposase nuclease domain,and is a component of the nonhomologous end-joining DNA double-strand break repair pathway. Metnase interacts with Topo IIalpha and enhances its decatenation activity. Here we show that multiple types of acute leukemia cells have an attenuated mitotic arrest when decatenation is inhibited and that in an acute myeloid leukemia (AML) cell line this is mediated by Metnase. Of further importance,Metnase permits continued proliferation of these AML cells even in the presence of the clinical Topo IIalpha inhibitor VP-16. In vitro,purified Metnase prevents VP-16 inhibition of Topo IIalpha decatenation of tangled DNA. Thus,Metnase expression levels may predict AML resistance to Topo IIalpha inhibitors,and Metnase is a potential therapeutic target for small molecule interference. View Publication

Immunotherapy with the EGFR-specific mAb cetuximab is clinically effective in 10-20% of patients with squamous cell carcinoma of the head and neck (SCCHN). Little information is available about the mechanism(s) underlying patients' differential clinical response to cetuximab-based immunotherapy,although this information may contribute to optimizing the design of cetuximab-based immunotherapy. Our understanding of these mechanisms would benefit from the characterization of the variables which influence the extent of cell dependent-lysis of SCCHN cells incubated with cetuximab in vitro. Therefore,in this study we have investigated the role of FcgammaR IIIa-158 genotype expressed by effector NK cells,cetuximab concentration,and EGFR expression level by SCCHN cells in the extent of their in vitro lysis and in the degree of NK cell activation. PBMC or purified CD56+ NK cells genotyped at IIIa codon 158 and SCCHN cell lines expressing different levels of EGFR have been used as effectors and targets,respectively,in antibody dependent cellular cytotoxicity (ADCC) assays. Furthermore,supernatants from ADCC assays were analyzed for cytokine and chemokine levels using multiplexed ELISA. We found that the extent of lysis of SCCHN cells was influenced by the EGFR expression level,cetuximab concentration,and FcgammaR polymorphism. Effector cells expressing the FcgammaR IIIa-158 VV allele were significantly (P textless 0.0001) more effective than those expressing FcgammaR IIIa VF and FF [corrected] alleles in mediating lysis of SCCHN cells expressed higher levels of the activation markers CD69 and CD107a,and secreted significantly (P textless 0.05) larger amounts of inflammatory cytokines and chemokines. IL-2 or IL-15 treatment increased cetuximab-mediated ADCC by poor binding FcgammaR IIIa 158 FF expressing NK cells. The importance of the FcgammaR IIIa-158 polymorphism in cytotoxicity of SCCHN cells by NK cells supports a potential role for immune activation and may explain patient variability of cetuximab mediated clinical responses. Cellular and secreted immune profiles and FcgammaR genotypes from patients' lymphocytes may provide clinically useful biomarkers of immune activation in cetuximab treated patients. View Publication

Recurrent/metastatic head and neck cancer remains a devastating disease with insufficient treatment options. We investigated the MET receptor tyrosine kinase as a novel target for the treatment of head and neck squamous cell carcinoma (HNSCC). MET/phosphorylated MET and HGF expression was analyzed in 121 tissues (HNSCC/normal) by immunohistochemistry,and in 20 HNSCC cell lines by immunoblotting. The effects of MET inhibition using small interfering RNA/two small-molecule inhibitors (SU11274/PF-2341066) on signaling,migration,viability,and angiogenesis were determined. The complete MET gene was sequenced in 66 head and neck cancer tissue samples and eight cell lines. MET gene copy number was determined in 14 cell lines and 23 tumor tissues. Drug combinations of SU11274 with cisplatin or erlotinib were tested in SCC35/HN5 cell lines. Eighty-four percent of the HNSCC samples showed MET overexpression,whereas 18 of 20 HNSCC cell lines (90%) expressed MET. HGF overexpression was present in 45% of HNSCC. MET inhibition with SU11274/PF-2341066 abrogated MET signaling,cell viability,motility/migration in vitro,and tumor angiogenesis in vivo. Mutational analysis of 66 tumor tissues and 8 cell lines identified novel mutations in the semaphorin (T230M/E168D/N375S),juxtamembrane (T1010I/R988C),and tyrosine kinase (T1275I/V1333I) domains (incidence: 13.5%). Increased MET gene copy number was present with textgreater10 copies in 3 of 23 (13%) tumor tissues. A greater-than-additive inhibition of cell growth was observed when combining a MET inhibitor with cisplatin or erlotinib and synergy may be mediated via erbB3/AKT signaling. MET is functionally important in HNSCC with prominent overexpression,increased gene copy number,and mutations. MET inhibition abrogated MET functions,including proliferation,migration/motility,and angiogenesis. MET is a promising,novel target for HNSCC and combination approaches with cisplatin or EGFR inhibitors should be explored. View Publication

Histone deacetylase inhibitors (HdI) could potentially improve the differentiation of leukemic dendritic cells (DC). Therefore,bone marrow samples from 100 children with acute lymphoblastic leukemia (ALL) were cultured in the cytokines TNF-alpha,GM-CSF,c-kit ligand,and fetal liver tyrosine kinase 3 ligand,with or without IL-3 and -4 and after administration of HdI valproic acid (VAL),suberoylanilide hydroxamic acid (SAHA),isobutyramid,or trichostatin A. Among the tested samples,25 were positive for the chromosomal translocation t(12;21),encoding the fusion gene translocation ETS-like leukemia/acute myeloid leukemia 1 (TEL/AML1). SAHA increased CD83 expression of TEL/AML1-positive blasts in conditions without ILs,and SAHA and VAL increased the number of CD86(+)80(-) cells in the presence of ILs. VAL and isobutyramid supported the allostimulatory capacities of TEL/AML1-positive,leukemic DC; VAL and SAHA reduced those of TEL/AML1-negative DC. Cytotoxic T cells sensitized with leukemic DC produced more IFN-gamma and TNF-alpha upon presentation of the TEL/AML1 peptide. They also induced the cytotoxic lysis of nondifferentiated blasts,which was enhanced when TEL/AML1-positive DC had developed after addition of VAL or SAHA. Therefore,the use of HdI in the differentiation of leukemic DC from patients with TEL/AML1-positive ALL is recommended. View Publication

Throughout its history,chronic myeloid leukemia (CML) has set precedents for cancer research and therapy. These range from the identification of the first specific chromosomal abnormality associated with cancer to the development of imatinib as a specific,targeted therapy for the disease. The successful development of imatinib as a therapeutic agent for CML can be attributed directly to decades of scientific discoveries. These discoveries determined that the BCR-ABL tyrosine kinase is the critical pathogenetic event in CML and an ideal target for therapy. This was confirmed in clinical trials of imatinib,with imatinib significantly improving the long-term survival of patients with CML. Continuing in this tradition of scientific discoveries leading to improved therapies,the understanding of resistance to imatinib has rapidly led to strategies to circumvent resistance. Continued studies of hematologic malignancies will allow this paradigm of targeting molecular pathogenetic events to be applied to many additional hematologic cancers. View Publication

Recent efforts in our study of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) have led to the identification of CD133 as a prominent HCC CSC marker. Findings were based on experiments done on cell lines and xenograft tumors where expression of CD133 was detected at levels as high as 65%. Based on the CSC theory,CSCs are believed to represent only a minority number of the tumor mass. This is indicative that our previously characterized CD133(+) HCC CSC population is still heterogeneous,consisting of perhaps subsets of cells with differing tumorigenic potential. We hypothesized that it is possible to further enrich the CSC population by means of additional differentially expressed markers. Using a two-dimensional PAGE approach,we compared protein profiles between CD133(+) and CD133(-) subpopulations isolated from Huh7 and PLC8024 and identified aldehyde dehydrogenase 1A1 as one of the proteins that are preferentially expressed in the CD133(+) subfraction. Analysis of the expression of several different ALDH isoforms and ALDH enzymatic activity in liver cell lines found ALDH to be positively correlated with CD133 expression. Dual-color flow cytometry analysis found the majority of ALDH(+) to be CD133(+),yet not all CD133(+) HCC cells were ALDH(+). Subsequent studies on purified subpopulations found CD133(+)ALDH(+) cells to be significantly more tumorigenic than their CD133(-)ALDH(+) or CD133(-)ALDH(-) counterparts,both in vitro and in vivo. These data,combined with those from our previous work,reveal the existence of a hierarchical organization in HCC bearing tumorigenic potential in the order of CD133(+)ALDH(+) textgreater CD133(+)ALDH(-) textgreater CD133(-)ALDH(-). ALDH,expressed along CD133,can more specifically characterize the tumorigenic liver CSC population. View Publication

Systemic hormones are key regulators of postnatal mammary gland development and play an important role in the etiology and treatment of breast cancer. Mammary ductal morphogenesis is controlled by circulating hormones,and these same hormones are also critical mediators of mammary stem cell fate decisions. Recent studies have helped further our understanding of the origin,specification,and fate of mammary stem cells during postnatal development. Here we review recent studies on the involvement of hormone receptors and several transcription factors in mammary stem/progenitor cell differentiation and lineage commitment. View Publication

Many agents are active in multiple myeloma,but the majority of patients relapse. This clinical pattern suggests most cancer cells are eliminated,but cells with the clonogenic potential to mediate tumor regrowth are relatively chemoresistant. Our previous data suggested that CD138(+) multiple myeloma plasma cells cannot undergo long-term proliferation but rather arise from clonogenic CD138(neg) B cells. We compared the relative sensitivity of these distinct cell types to clinical antimyeloma agents and found that dexamethasone,lenadilomide,bortezomib,and 4-hydroxycyclophosphamide inhibited CD138(+) multiple myeloma plasma cells but had little effect on CD138(neg) precursors in vitro. We further characterized clonogenic multiple myeloma cells and stained cell lines using the Hoechst side population and Aldefluor assays. Each assay identified CD138(neg) cells suggesting that they possess high drug efflux capacity and intracellular drug detoxification activity. We also found that multiple myeloma cells expressing the memory B-cell markers CD20 and CD27 could give rise to clonogenic multiple myeloma growth in vitro and engraft immunodeficient nonobese diabetes/severe combined immunodeficient mice during both primary and secondary transplantation. Furthermore,both the side population and Aldefluor assays were capable of identifying circulating clonotypic memory B-cell populations within the peripheral blood of multiple myeloma patients. Our results suggest that circulating clonotypic B-cell populations represent multiple myeloma stem cells,and the relative drug resistance of these cells is mediated by processes that protect normal stem cells from toxic injury. View Publication