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Background: We previously described a t(2;11)(p21;q23) chromosomal translocation found in patients with myelodysplasia or acute myeloid leukemia that leads to over-expression of the microRNA miR-125b, and we showed that transplantation of mice with murine stem/progenitor cells overexpressing miR-125b is able to induce leukemia. In this study, we investigated the mechanism of myeloid transformation by miR-125b. Design and Methods: To investigate the consequences of miR-125b over-expression on myeloid differentiation, apoptosis and proliferation, we used the NB4 and HL60 human promyelocytic cell lines and the 32Dclone3 murine promyelocytic cell line. To test whether miR-125b is able to transform myeloid cells, we used the non-tumorigenic and interleukin-3-dependent 32Dclone3 cell line over-expressing miR-125b, in xenograft experiments in nude mice and in conditions of interleukin-3 deprivation. To identify new miR-125b targets, we compared, by RNA-sequencing, the transcriptome of cell lines that do or do not over-express miR-125b. Results: We showed that miR-125b over-expression blocks apoptosis and myeloid differentiation and enhances proliferation in both species. More importantly, we demonstrated that miR-125b is able to transform the 32Dclone3 cell line by conferring growth independence from interleukin-3; xenograft experiments showed that these cells form tumors in nude mice. Using RNA-sequencing and quantitative real-time polymerase chain reaction experiments, we identified multiple miR-125b targets. We demonstrated that ABTB1, an anti-proliferative factor, is a new direct target of miR-125b and we confirmed that CBFB, a transcription factor involved in hematopoiesis, is also targeted by miR-125b. MiR-125b controls apoptosis by down-regulating genes involved in the p53 pathway including BAK1 and TP53INP1. Conclusions: This study demonstrates that in a myeloid context, miR-125b is an oncomiR able to transform cell lines. miR-125b blocks myeloid differentiation in, Leukemia & Lymphoma Society of America, National Institutes of Health (U.S.) (NIH grant DK068348), National Institutes of Health (U.S.) (NIH grant 5P01 HL066105)

available in PMC 2011 October 10., Leukemia is a complex disease with many different types and subtypes caused by a huge diversity of genetic and epigenetic aberrations. Until recently, alterations of protein-coding genes were thought to be the sole cause of tumorigenesis. With the recent discovery of multiple types of non-coding RNAs, it has become evident that mutations in these also contribute to the development of cancer. Among the non-coding RNAs, microRNAs play a crucial role in cancer owing to their involvement in fundamental processes such as apoptosis, differentiation and proliferation. MicroRNAs are small noncoding RNAs (approximately 19–25 nucleotides in length) that bind to and downregulate multiple mRNA targets; in mammals, the production of over a third of all proteins is regulated by microRNAs [3]. Several studies demonstrated that microRNAs are involved in leukemia progression but their role as the primary cause or a determinant of progression in leukemia has been unclear. Some have been identified as oncogenes or tumor suppressor genes, which suggests that they are playing a central role in tumorigenesis, while others appear to be associated with a specific stage in disease progression. Deciphering the exact role of microRNAs in oncogenesis is important in order to improve the diagnosis and treatment of leukemia patients., National Institutes of Health (U.S.) (NIH grant DK068348), National Institutes of Health (U.S.) (NIH Grant 5P01 HL066105), Leukemia & Lymphoma Society of America (Recherche sur le Cancer (ARC) fellowship)

MicroRNA miR-125b has been implicated in several kinds of leukemia. The chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b of up to 90-fold normal. Moreover, miR-125b is also up-regulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative neoplasm, suggesting an important role for miR-125b in early hematopoiesis. Furthermore, coexpression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice, compared with control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus, we show that overexpression of miR-125b is sufficient both to shorten the latency of BCR-ABL–induced leukemia and to independently induce leukemia in a mouse model., National Institutes of Health (U.S.) (Grant DK068348), National Institutes of Health (U.S.) (grant 5P01 HL066105)

PAX5 is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). We analyzed 153 adult and child B-ALL harboring karyotypic abnormalities at chromosome 9p, to determine the frequency and the nature of PAX5 alterations. We found PAX5 internal rearrangements in 21% of the cases. To isolate fusion partners, we used classic and innovative techniques (rolling circle amplification-rapid amplification of cDNA ends) and single nucleotide polymorphism-comparative genomic hybridization arrays. Recurrent and novel fusion partners were identified, including NCoR1, DACH2, GOLGA6, and TAOK1 genes showing the high variability of the partners. We noted that half the fusion genes can give rise to truncated PAX5 proteins. Furthermore, malignant cells carrying PAX5 fusion genes displayed a simple karyotype. These data strongly suggest that PAX5 fusion genes are early players in leukemogenesis. In addition, PAX5 deletion was observed in 60% of B-ALL with 9p alterations. Contrary to cases with PAX5 fusions, deletions were associated with complex karyotypes and common recurrent translocations. This supports the hypothesis of the secondary nature of the deletion. Our data shed more light on the high variability of PAX5 alterations in B-ALL. Therefore, it is probable that gene fusions occur early, whereas deletions should be regarded as a late/secondary event. (Blood. 2010;115(15):3089-3097)

Les translocations chromosomiques sont fréquemment associées aux hémopathies malignes. On distingue des anomalies qualitatives pouvant conduire à la création d'une protéine de fusion et des anomalies quantitatives qui altèrent le taux de transcription des gènes. L'ideintification et la caractérisation des translocations chromosomiques dans les hémopathies malignes sont essentielles au diagnostic, au pronostic, à la recherche de la maladie résiduelle et à l'orientation thérapeutique. Ainsi, nous avons caractérisé trois nouvelles translocations chromosomques. La translocation t(8;9)(p22;p24) retrouvée chez des patients atteints de leucémie myéloïde chronique atypique juxtapose les gènes PCM1 (Pericentriolar Material 1) et JAK2 (Janus kinase 2). La protéine de fusion obtenue conserve les domaines d'oligomérisation de PCM1 et le domaine kinase de JAK2. Nous pouvons supposer que PCM1-JAK2 permet l'activation constitutive de JAK2 comme dans le modèle BCR-ABL. La translocation t(7;9)(q11;p13) retrouvée chez des patients atteints de leucémie aiguë lymphoblastique B fusionne le facteur de transcription PAX5 à l'élastine, protéine de la matrice extracellulaire. Nous avons démontré que la chimère PAX5-ELN était localisée au niveau du noyau et qu'elle était capable de se lier aux gènes cibles de PAX5. Nous avons également démontré que la chimère agissait en dominant négatif sur PAX5 sauvage. Enfin, l'étude de la translocation t(2;11)(p21;q23) retrouvée chez 14 patients atteints de myélodysplasie ou de leucémie aiguë myéloblastique a permis de décrire pour la première fois la surexpression d'un microARN associée à une translocation dans des hémopathies malignes. En effet, nous avons démontré que miR-125b était surexprimé (x10 à x90) chez les patients porteurs de la t(2;11)(p21;q23) et que cette surexpression aboutissait à un blocage de différenciation myélomonocytaire in vitro et probablement in vivo. En conclusion, l'identification et la caractérisation de translocations chromo, Chromosomal translocations are frequently implicated in hematological malignancies and involve oncogenes which are either up-regulated (quantitative abnormality) or form part of new chimeric genes (qualitative abnormality). The characterization of chromosomal translocations in hematological malignancies is essential for the diagnosis, the prognosis, the detection of residual disease and therapeutic choices. During my thesis, I characterized three new chromosomal translocations. The t(8;9)(p22;p24) translocation fused the PCM1 (Pericentriolar Material 1) gene to the JAK2 (Janus kinase 2) gene in cases of atypical chronic myeloid leukemia. The fusion protein conserved the oligomerization domains of PCM1 and the tyrosine kinase domain of JAK2. We can suppose that PCM1-JAK2 allows constitutive activation of JAK2 as in the BCR-ABL model. The t(7;9)(q11;p13) translocation was found in cases of B-acute lymphoblastic leukemia and fused the transcription factor PAX5 (Paired box gene) to the elastin, an extracellular matrix protein. We demonstrated that PAX5-ELN was localized in the nucleus and was able to bind promoters of several PAX5 targets. In fact, PAX5-ELN acts as a dominant negative on PAX5, accounting for the block of differentiation in leukemic cells. The translocation t(2;11)(p21;q23) found in 14 patients with myelodysplasia or acute myeloid leukemia.is the first translocation that leads to an up-regulation of a microRNA in hematological malignancies. Indeed, we showed that miR-125b was overexpressed (x10 to x90) among patients carrying the t(2;11)(p21;q23) and that this up-regulation leads to a block of myelomonocytic differentiation in vitro. In conclusion, the characterization of chromosomal translocations, even rare, allows deciphering new oncogeneic pathways in hematological malignancies.