Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9.

Leukaemias and other cancers possess a rare population of cells capable of the limitless self-renewal necessary for cancer initiation and maintenance. Eradication of these cancer stem cells is probably a critical part of any successful anti-cancer therapy, and may explain why conventional cancer therapies are often effective in reducing tumour burden, but are only rarely curative. Given that both normal and cancer stem cells are capable of self-renewal, the extent to which cancer stem cells resemble normal tissue stem cells is a critical issue if targeted therapies are to be developed.

A method for high-throughput gene expression signature analysis.

Genome-wide transcriptional profiling has shown that different biologic states (for instance, disease and response to pharmacologic manipulation) can be recognized by the expression pattern of relatively small numbers of genes. However, the lack of a practical and cost-effective technology for detection of these gene expression 'signatures' in large numbers of samples has severely limited their exploitation in important medical and pharmaceutical discovery applications. Here, we describe a solution based on the combination of ligation-mediated amplification with an optically addressed microsphere and flow cytometric detection system.

MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.

Background: The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative.

Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma.

Our understanding of Ewing's sarcoma development mediated by the EWS/FLI fusion protein has been limited by a lack of knowledge regarding the tumor cell of origin. To circumvent this, we analyzed the function of EWS/FLI in Ewing's sarcoma itself. By combining retroviral-mediated RNA interference with reexpression studies, we show that ongoing EWS/FLI expression is required for the tumorigenic phenotype of Ewing's sarcoma.

Increased expression of genes converting adrenal androgens to testosterone in androgen-independent prostate cancer.

Androgen receptor (AR) plays a central role in prostate cancer, and most patients respond to androgen deprivation therapies, but they invariably relapse with a more aggressive prostate cancer that has been termed hormone refractory or androgen independent. To identify proteins that mediate this tumor progression, gene expression in 33 androgen-independent prostate cancer bone marrow metastases versus 22 laser capture-microdissected primary prostate cancers was compared using Affymetrix oligonucleotide microarrays. Multiple genes associated with aggressive behavior were increased in the androgen-independent metastatic tumors (MMP9, CKS2, LRRC15, WNT5A, EZH2, E2F3, SDC1, SKP2, and BIRC5), whereas a candidate tumor suppressor gene (KLF6) was decreased.

Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling.

Individuals with Down syndrome (DS) are predisposed to develop acute megakaryoblastic leukemia (AMKL), characterized by expression of truncated GATA1 transcription factor protein (GATA1s) due to somatic mutation. The treatment outcome for DS-AMKL is more favorable than for AMKL in non-DS patients. To gain insight into gene expression differences in AMKL, we compared 24 DS and 39 non-DS AMKL samples.

Respiratory failure due to differentiation arrest and expansion of alveolar cells following lung-specific loss of the transcription factor C/EBPalpha in mice.

The leucine zipper family transcription factor CCAAT enhancer binding protein alpha (C/EBPalpha) inhibits proliferation and promotes differentiation in various cell types. In this study, we show, using a lung-specific conditional mouse model of C/EBPalpha deletion, that loss of C/EBPalpha in the respiratory epithelium leads to respiratory failure at birth due to an arrest in the type II alveolar cell differentiation program. This differentiation arrest results in the lack of type I alveolar cells and differentiated surfactant-secreting type II alveolar cells.

Application of oligonucleotide microarrays to assess the biological effects of neoadjuvant imatinib mesylate treatment for localized prostate cancer.

Purpose: Neoadjuvant administration of antineoplastic therapies is used to rapidly assess the clinical and biological activity of novel systemic treatments. To assess the feasibility of using microarrays to assess molecular end points following targeted treatment in a heterogeneous tumor, we measured global gene expression in localized prostate cancer before and following neoadjuvant treatment with imatinib mesylate.

Patients And Methods: Patients with intermediate-risk to high-risk prostate cancer were treated for 6 weeks with 200 to 300 mg of oral imatinib mesylate.

Epstein-Barr-virus-encoded LMP2A induces primary epithelial cell migration and invasion: possible role in nasopharyngeal carcinoma metastasis.

Nonkeratinizing nasopharyngeal carcinomas (NPC) are >95% associated with the expression of the Epstein-Barr virus (EBV) LMP2A latent protein. However, the role of EBV, in particular, LMP2A, in tumor progression is not well understood. Using Affymetrix chips and a pattern-matching computational technique (neighborhood analysis), we show that the level of LMP2A expression in NPC biopsy samples correlates with that of a cellular protein, integrin-alpha-6 (ITGalpha6), that is associated with cellular migration in vitro and metastasis in vivo.

BRAF mutation predicts sensitivity to MEK inhibition.

The kinase pathway comprising RAS, RAF, mitogen-activated protein kinase kinase (MEK) and extracellular signal regulated kinase (ERK) is activated in most human tumours, often through gain-of-function mutations of RAS and RAF family members. Using small-molecule inhibitors of MEK and an integrated genetic and pharmacologic analysis, we find that mutation of BRAF is associated with enhanced and selective sensitivity to MEK inhibition when compared to either 'wild-type' cells or cells harbouring a RAS mutation. This MEK dependency was observed in BRAF mutant cells regardless of tissue lineage, and correlated with both downregulation of cyclin D1 protein expression and the induction of G1 arrest.

Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.

Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation.

A zebrafish bmyb mutation causes genome instability and increased cancer susceptibility.

A major goal of cancer research has been to identify genes that contribute to cancer formation. The similar pathology between zebrafish and human tumors, as well as the past success of large-scale genetic screens in uncovering human disease genes, makes zebrafish an ideal system in which to find such new genes. Here, we show that a zebrafish forward genetic screen uncovered multiple cell proliferation mutants including one mutant, crash&burn (crb), that represents a loss-of-function mutation in bmyb, a transcriptional regulator and member of a putative proto-oncogene family.

Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma.

Systematic analyses of cancer genomes promise to unveil patterns of genetic alterations linked to the genesis and spread of human cancers. High-density single-nucleotide polymorphism (SNP) arrays enable detailed and genome-wide identification of both loss-of-heterozygosity events and copy-number alterations in cancer. Here, by integrating SNP array-based genetic maps with gene expression signatures derived from NCI60 cell lines, we identified the melanocyte master regulator MITF (microphthalmia-associated transcription factor) as the target of a novel melanoma amplification.

Gefitinib induces myeloid differentiation of acute myeloid leukemia.

Cure rates for patients with acute myeloid leukemia (AML) remain low despite ever-increasing dose intensity of cytotoxic therapy. In an effort to identify novel approaches to AML therapy, we recently reported a new method of chemical screening based on the modulation of a gene expression signature of interest. We applied this approach to the discovery of AML-differentiation-promoting compounds.

MicroRNA expression profiles classify human cancers.

Recent work has revealed the existence of a class of small non-coding RNA species, known as microRNAs (miRNAs), which have critical functions across various biological processes. Here we use a new, bead-based flow cytometric miRNA expression profiling method to present a systematic expression analysis of 217 mammalian miRNAs from 334 samples, including multiple human cancers. The miRNA profiles are surprisingly informative, reflecting the developmental lineage and differentiation state of the tumours.

NFkappaB activity, function, and target-gene signatures in primary mediastinal large B-cell lymphoma and diffuse large B-cell lymphoma subtypes.

Primary mediastinal large B-cell lymphoma (MLBCL) shares important clinical and molecular features with classic Hodgkin lymphoma, including nuclear localization of the nuclear factor kappaB (NFkappaB) subunit c-REL (reticuloendotheliosis viral oncogene homolog) in a pilot series. Herein, we analyzed c-REL subcellular localization in additional primary MLBCLs and characterized NFkappaB activity and function in a MLBCL cell line. The new primary MLBCLs had prominent c-REL nuclear staining, and the MLBCL cell line exhibited high levels of NFkappaB binding activity.

Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis.

Polycythemia vera (PV), essential thrombocythemia (ET), and myeloid metaplasia with myelofibrosis (MMM) are clonal disorders arising from hematopoietic progenitors. An internet-based protocol was used to collect clinical information and biological specimens from patients with these diseases. High-throughput DNA resequencing identified a recurrent somatic missense mutation JAK2V617F in granulocyte DNA samples of 121 of 164 PV patients, of which 41 had homozygous and 80 had heterozygous mutations.

Androgen mediated regulation and functional implications of fkbp51 expression in prostate cancer.

Purpose: Androgen ablation continues to be the most effective therapy for metastatic prostate cancer, although the biologically active androgen receptor (AR) target genes remain largely unknown. Because AR signaling continues in hormone refractory disease, effector AR target genes may have therapeutic import.

Materials And Methods: We used oligonucleotide microarrays to identify genes with expression induced by androgen and associated with androgen independent growth.

PI(4,5)P2-dependent microdomain assemblies capture microtubules to promote and control leading edge motility.

The lipid second messenger PI(4,5)P(2) modulates actin dynamics, and its local accumulation at plasmalemmal microdomains (rafts) might mediate regulation of protrusive motility. However, how PI(4,5)P(2)-rich rafts regulate surface motility is not well understood. Here, we show that upon signals promoting cell surface motility, PI(4,5)P(2) directs the assembly of dynamic raft-rich plasmalemmal patches, which promote and sustain protrusive motility.

An RNA interference model of RPS19 deficiency in Diamond-Blackfan anemia recapitulates defective hematopoiesis and rescue by dexamethasone: identification of dexamethasone-responsive genes by microarray.

Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia, is a model disease for the study of erythroid differentiation but is poorly understood. RPS19 is the only gene yet to have been associated with DBA, but its relevance to erythroid differentiation is unclear. The molecular basis for the stimulation of erythropoiesis by glucocorticoids in patients with DBA has not been identified.

Systematic discovery of regulatory motifs in human promoters and 3' UTRs by comparison of several mammals.

Comprehensive identification of all functional elements encoded in the human genome is a fundamental need in biomedical research. Here, we present a comparative analysis of the human, mouse, rat and dog genomes to create a systematic catalogue of common regulatory motifs in promoters and 3' untranslated regions (3' UTRs). The promoter analysis yields 174 candidate motifs, including most previously known transcription-factor binding sites and 105 new motifs.

Spatial control of actin-based motility through plasmalemmal PtdIns(4,5)P2-rich raft assemblies.

The interactions of cells with their environment involve regulated actin-based motility at defined positions along the cell surface. Sphingolipid- and cholesterol-dependent microdomains (rafts) order proteins at biological membranes, and have been implicated in most signalling processes at the cell surface. Many membrane-bound components that regulate actin cytoskeleton dynamics and cell-surface motility associate with PtdIns(4,5)P(2)-rich lipid rafts.

Conditional MLL-CBP targets GMP and models therapy-related myeloproliferative disease.

Chromosomal translocations that fuse the mixed lineage leukemia (MLL) gene with multiple partners typify acute leukemias of infancy as well as therapy-related leukemias. We utilized a conditional knockin strategy to bypass the embryonic lethality caused by MLL-CBP expression and to assess the immediate effects of induced MLL-CBP expression on hematopoiesis. Within days of activating MLL-CBP, the fusion protein selectively expanded granulocyte/macrophage progenitors (GMP) and enhanced their self-renewal/proliferation.

An oncogenic KRAS2 expression signature identified by cross-species gene-expression analysis.

Using advanced gene targeting methods, generating mouse models of cancer that accurately reproduce the genetic alterations present in human tumors is now relatively straightforward. The challenge is to determine to what extent such models faithfully mimic human disease with respect to the underlying molecular mechanisms that accompany tumor progression. Here we describe a method for comparing mouse models of cancer with human tumors using gene-expression profiling.

Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF.

The genomic organization of the CDK2 gene, which overlaps the melanocyte-specific gene SILV/PMEL17, poses an interesting regulatory challenge. We show that, despite its ubiquitous expression, CDK2 exhibits tissue-specific regulation by the essential melanocyte lineage transcription factor MITF. In addition, functional studies revealed this regulation to be critical for maintaining CDK2 kinase activity and growth of melanoma cells.