Identification of novel proto-oncogenes in murine myeloid leukemias by retroviral insertional mutagenesis : the peripheral cannabinoid receptor

Dynamic and complex processes of cell proliferation, differentiation, maturation, apoptosis, and survival maintain homeostasis in bone marrow and perTheral blood. In steady state, the turnover of blood cells is approximately I x 10' cells per day. During stress conditions, such as infections or bleeding, blood cell formation is properly adjusted by the production of terminally differentiated functional cells. Leukemia is a progressive neoplastic disease of the blood cell forming system, in which non-functional blood cells accumulate in the bone marrow, thereby interfering with normal blood cell formation. The lIlain objectives of the experimental work described in this thesis lVere the identification of !lovel prolo-oncogenes or tumor suppressor genes involved ill lIlyeloid leukelllia developlllelll by using retroviral insertionallllutagenesis and the characterization of these novel genes

Retroviral insertions in Evi12, a novel common virus integration site upstream of Tra1/Grp94, frequently coincide with insertions in the gene encoding the peripheral cannabinoid receptor Cnr2

The common virus integration site (VIS) Evi11 was recently identified within the gene encoding the hematopoietic G-protein-coupled peripheral cannabinoid receptor Cnr2 (also refer

Hematopoietic cells expressing the peripheral cannabinoid receptor migrate in response to the endocannabinoid 2-arachidonoylglycerol

Cb2 is a novel protooncogene encoding the peripheral cannabinoid receptor. Previous studies demonstrated that 2 distinct noncoding first exons exist: exon-1A and exon-1B, which both splice to protein-coding exon-2. We demonstrate that in retrovirally induced murine myeloid leukemia cells with proviral insertion in Cb2, exon-1B/exon-2 Cb2 messenger RNA levels have been increased, resulting in high receptor numbers. In myeloid leukemia cells without virus insertion in this locus, low levels of only exon-1A/exon-2 Cb2 transcripts were present and receptors could not be detected. To elucidate the function of Cb2 in myeloid leukemia cells, a set of in vitro experiments was carried out using 32D/G-CSF-R (granulocyte colony-stimulating factor receptor) cells transfected with exon-1B/exon-2 Cb2 complementary DNA and a myeloid cell line carrying a virus insertion in Cb2 (ie, NFS 78). We demonstrate that a major function of the Cb2 receptor is stimulation of migration as determined in a transwell assay. Exposure of Cb2-expressing cells to different cannabinoids showed that the true ligand for Cb2 is 2-arachidonoylglycerol (2-AG), which may act as chemoattractant and as a chemokinetic agent. Furthermore, we observed a significant synergistic activity between 2-AG and interleukin-3 or G-CSF, suggesting cross-talk between the different receptor systems. Radioactive-ligand binding studies revealed significant numbers of Cb2 receptors in normal spleen. Transwell experiments carried out with normal mouse spleen cells showed 2-AG-induced migration of B220-, CD19-, immunoglobulin M-, and immunoglobulin D-expressing B lymphocytes. Our study demonstrates that a major function of Cb2 receptor expressed on myeloid leukemia cells or normal splenocytes is stimulation of migration

A 4-gene expression score associated with high levels of Wilms Tumor-1 (WT1) expression is an adverse prognostic factor in acute myeloid leukaemia

Wilms Tumor-1 (WT1) expression level is implicated in the prognosis of acute myeloid leukaemia (AML). We hypothesized that a gene expression profile associated with WT1 expression levels might be a good surrogate marker. We identified high WT1 gene sets by comparing the gene expression profiles in the highest and lowest quartiles of WT1 expression in two large AML studies. Two high WT1 gene sets were found to be highly correlated in terms of the altered genes and expression profiles. We identified a 17-probe set signature of the high WT1 set as the optimal prognostic predictor in the first AML set, and showed that it was able to predict prognosis in the second AML series after adjustment for Europe

Prognostically useful gene-expression profiles in acute myeloid leukemia

BACKGROUND: In patients with acute myeloid leukemia (AML) a combination of methods must be used to classify the disease, make therapeutic decisions, and determine the prognosis. However, this combined approach provides correct therapeutic and prognostic information in only 50 percent of cases. METHODS: We determined the gene-expression profiles in samples of peripheral blood or bone marrow from 285 patients with AML using Affymetrix U133A GeneChips containing approximately 13,000 unique genes or expression-signature tags. Data analyses were carried out with Omniviz, significance analysis of microarrays, and prediction analysis of microarrays software. Statistical analyses were performed to determine the prognostic significance of cases of AML with specific molecular signatures. RESULTS: Unsupervised cluster analyses identified 16 groups of patients with AML on the basis of molecular signatures. We identified the genes that defined these clusters and determined the minimal numbers of genes needed to identify prognostically important clusters with a high degree of accuracy. The clustering was driven by the presence of chromosomal lesions (e.g., t(8;21), t(15;17), and inv(16)), particular genetic mutations (CEBPA), and abnormal oncogene expression (EVI1). We identified several novel clusters, some consisting of specimens with normal karyotypes. A unique cluster with a distinctive gene-expression signature included cases of AML with a poor treatment outcome. CONCLUSIONS: Gene-expression profiling allows a comprehensive classification of AML that includes previously identified genetically defined subgroups and a novel cluster with an adverse prognosis

High INDO (indoleamine 2,3-dioxygenase) mRNA level in blasts of acute myeloid leukemic patients predicts poor clinical outcome

Indoleamine 2,3-dioxygenase degrades the amino acid tryptophan which is essential for T cells. Tryptophan depletion causes T-cell cycle arrest and solid tumors that express high levels of indoleamine 2,3-dioxygenase can create immune suppression. Recently, blasts of patients with acute myeloid leukemia were shown to express indoleamine 2,3-dioxygenase. We determined INDO (encoding gene for indoleamine 2,3-dioxygenase) mRNA expression in leukemic blasts of 286 patients with acute myeloid leukemia by gene-expression profiling. Results were validated by quantitative polymerase chain reaction analysis in blasts of an independent cohort of 71 patients. High INDO expression was correlated to significantly shortened overall and relapse-free survival. Correlation of INDO expression to relevant known prognostic factors and survival identified high INDO expression as a strong negative independent predicting variable for overall and relapse-free survival. Inhibition of indoleamine 2,3-dioxygenase expressed by myeloid leukemic blasts may result in breaking immune tolerance and offers new therapeutic options for patients with acute myeloid leukemia

High INDO (indoleamine 2,3-dioxygenase) mRNA level in blasts of acute myeloid leukemic patients predicts poor clinical outcome

Indoleamine 2,3-dioxygenase degrades the amino acid tryptophan which is essential for T cells. Tryptophan depletion causes T-cell cycle arrest and solid tumors that express high levels of indoleamine 2,3-dioxygenase can create immune suppression. Recently, blasts of patients with acute myeloid leukemia were shown to express indoleamine 2,3-dioxygenase. We determined INDO (encoding gene for indoleamine 2,3-dioxygenase) mRNA expression in leukemic blasts of 286 patients with acute myeloid leukemia by gene-expression profiling. Results were validated by quantitative polymerase chain reaction analysis in blasts of an independent cohort of 71 patients. High INDO expression was correlated to significantly shortened overall and relapse-free survival. Correlation of INDO expression to relevant known prognostic factors and survival identified high INDO expression as a strong negative independent predicting variable for overall and relapse-free survival. Inhibition of indoleamine 2,3-dioxygenase expressed by myeloid leukemic blasts may result in breaking immune tolerance and offers new therapeutic options for patients with acute myeloid leukemia

Integrated genome-wide genotyping and gene expression profiling reveals BCL11B as a putative oncogene in acute myeloid leukemia with 14q32 aberrations

Acute myeloid leukemia is a neoplasm characterized by recurrent molecular aberrations traditionally demonstrated by cytogenetic analyses. We used high density genome-wide genotyping and gene expression profiling to reveal acquired cryptic abnormalities in acute myeloid leukemia. By genome-wide genotyping of 137 cases of primary acute myeloid leukemia, we disclosed a recurrent focal amplification on chromosome 14q32, which included the genes BCL11B, CCNK, C14orf177 and SETD3, in two cases. In the affected cases, the BCL11B gene showed consistently high mRNA expression, whereas the expression of the other genes was unperturbed. Flu

Stem cell factor receptor (c-KIT) codon 816 mutations predict development of bilateral testicular germ-cell tumors

Testicular germ-cell tumors (TGCTs) of adolescents and adults originate from intratubular germ cell neoplasia (ITGCN), which is composed of the malignant counterparts of embryonal germ cells. ITGCN cells are characterized, among others, by the presence of stem cell factor receptor c-KIT. Once established, ITGCN will always progress to invasiveness. Approximately 2.5-5% of patients with a TGCT will develop bilateral disease and require complete castration, resulting in infertility, a need for lifelong androgen replacement, and psychological stress. To date, the only way to predict a contralateral tumor is surgical biopsy of the contralateral testis to demonstrate ITGCN. We did a retrospective study of 224 unilateral and 61 proven bilateral TGCTs (from 46 patients, in three independently collected series in Europe) for the presence of activating c-KIT codon 816 mutations. A c-KIT codon 816 mutation was found in three unilateral TGCT (1.3%), and in 57 bilateral TGCTs (93%; P < 0.0001). In the two wild-type bilateral tumors for which ITGCN was available, the preinvasive cells contained the mutation. The mutations were somatic in origin and identical in both tumors. We conclude that somatic activating codon 816 c-KIT mutations are associated with development of bilateral TGCT. Detection of c-KIT codon 816 mutations in unilateral TGCT identifies patients at risk for bilateral disease. These patients may undergo tailored treatment to prevent the development of bilateral disease, with retention of testicular hormonal function