Selection of down-regulated sequences along the monocytic differentiation of leukemic HL60 cells

In order to dissect the molecular mechanisms of monocytic differentiation we have developed a subtractive hybridisation method based on a simplified 'representational difference analysis'. We have selected 16 sequences and confirmed their down-regulation along the TPA-induced monocytic differentiation of HL60 cells. Among these sequences we have identified the alpha-tubulin, the TaxREB protein and two ribosomal protein sequences which had not been previously described as differentially expressed. These results add to our knowledge about the molecules implicated along the monocytic differentiation and growth arrest of leukemic cells and provide a first step in the study of their respective roles

APOBEC signature mutation generates an oncogenic enhancer that drives LMO1 expression in T-ALL

Oncogenic driver mutations are those that provide a proliferative or survival advantage to neoplastic cells, resulting in clonal selection. Although most cancer-causing mutations have been detected in the protein-coding regions of the cancer genome; driver mutations have recently also been discovered within noncoding genomic sequences. Thus, a current challenge is to gain precise understanding of how these unique genomic elements function in cancer pathogenesis, while clarifying mechanisms of gene regulation and identifying new targets for therapeutic intervention. Here we report a C-to-T single nucleotide transition that occurs as a somatic mutation in noncoding sequences 4 kb upstream of the transcriptional start site of the LMO1 oncogene in primary samples from patients with T-cell acute lymphoblastic leukaemia. This single nucleotide alteration conforms to an APOBEC-like cytidine deaminase mutational signature, and generates a new binding site for the MYB transcription factor, leading to the formation of an aberrant transcriptional enhancer complex that drives high levels of expression of the LMO1 oncogene. Since APOBEC-signature mutations are common in a broad spectrum of human cancers, we suggest that noncoding nucleotide transitions such as the one described here may activate potent oncogenic enhancers not only in T-lymphoid cells but in other cell lineages as well

Les pseudomonas et autres bacilles à gram négatif dans les bioglées thermales

Les boues thermales sont préparées en mettant en contact des substrats alluviaux ou forestiers avec des eaux minérales, pendant des temps variables selon les saisons et les préparations locales. Afin d'édicter des recommandations pour les paramètres bactériologiques destinés au contrôle sanitaire, les bacilles à Gram négatif hétérotrophes ont été dénombrés dans les bioglées formées à l'interface substrat-eau minérale lors de la maturation des boues thermales. Les dilutions de bioglées ont été ensemencées en bouillon Cétrimide et l'évaluation quantitative a été réalisée par la méthode du Nombre le Plus Probable. Les Pseudomonas ont été trouvés dans 57 % des échantillons, à des taux allant de 10 à 104 ufc/g, et parmi eux l'espèce Pseudomonas aeruginosa représente 14 % des souches isolées, à des taux inférieurs à 100 ufc/g. Les autres espèces trouvées sont : Pseudomonas alcaligenes, Pseudomonas pseudoalcaligenes, Pseudomonas stutzeri, plus rarement Stenotrophomonas (anciennement Pseudomonas) maltophilia, Pseudomonas fluorescens, Burkholderia (anciennement Pseudomonas) cepacia, Pseudomonas vesicularis, Pseudomonas putida, et Pseudomonas picketii. Les autres bacilles à Gram négatif hétérotrophes sont Aeromonas sobria, Aeromonas hydrophila et Alcaligenes faecalis. L'impact sanitaire de ces bactéries semble peu important car elles sont, pour la plupart, non pathogènes pour l'homme, et les autres sont seulement des opportunistes. P. aeruginosa est sans danger pour la peau saine, et pourrait être toléré en petite quantité dans les boues thermales, à condition que cette bactérie ne puisse pas se multiplier dans ce biotope

The SCL complex regulates c-kit expression in hematopoietic cells through functional interaction with Sp1.

The combinatorial interaction among transcription factors is believed to determine hematopoietic cell fate. Stem cell leukemia (SCL, also known as TAL1 [T-cell acute lymphoblastic leukemia 1]) is a tissue-specific basic helix-loop-helix (bHLH) factor that plays a central function in hematopoietic development; however, its target genes and molecular mode of action remain to be elucidated. Here we show that SCL and the c-Kit receptor are coexpressed in hematopoietic progenitors at the single-cell level and that SCL induces c-kit in chromatin, as ectopic SCL expression in transgenic mice sustains c-kit transcription in developing B lymphocytes, in which both genes are normally down-regulated. Through transient transfection assays and coimmunoprecipitation of endogenous proteins, we define the role of SCL as a nucleation factor for a multifactorial complex (SCL complex) that specifically enhances c-kit promoter activity without affecting the activity of myelomonocytic promoters. This complex, containing hematopoietic-specific (SCL, Lim-only 2 (LMO2), GATA-1/GATA-2) and ubiquitous (E2A, LIM- domain binding protein 1 [Ldb-1]) factors, is tethered to DNA via a specificity protein 1 (Sp1) motif, through direct interactions between elements of the SCL complex and the Sp1 zinc finger protein. Furthermore, we demonstrate by chromatin immunoprecipitation that SCL, E2A, and Sp1 specifically co-occupy the c-kit promoter in vivo. We therefore conclude that c-kit is a direct target of the SCL complex. Proper activation of the c-kit promoter depends on the combinatorial interaction of all members of the complex. Since SCL is down-regulated in maturing cells while its partners remain expressed, our observations suggest that loss of SCL inactivates the SCL complex, which may be an important event in the differentiation of pluripotent hematopoietic cells

Modeling T-cell acute lymphoblastic leukemia induced by the SCL and LMO1 oncogenes

Deciphering molecular events required for full transformation of normal cells into cancer cells remains a challenge. In T-cell acute lymphoblastic leukemia (T-ALL), the genes encoding the TAL1/SCL and LMO1/2 transcription factors are recurring targets of chromosomal translocations, whereas NOTCH1 is activated in >50% of samples. Here we show that the SCL and LMO1 oncogenes collaborate to expand primitive thymocyte progenitors and inhibit later stages of differentiation. Together with pre-T-cell antigen receptor (pre-TCR) signaling, these oncogenes provide a favorable context for the acquisition of activating Notch1 mutations and the emergence of self-renewing leukemia-initiating cells in T-ALL. All tumor cells harness identical and specific Notch1 mutations and Tcrβ clonal signature, indicative of clonal dominance and concurring with the observation that Notch1 gain of function confers a selective advantage to SCL-LMO1 transgenic thymocytes. Accordingly, a hyperactive Notch1 allele accelerates leukemia onset induced by SCL-LMO1 and bypasses the requirement for pre-TCR signaling. Finally, the time to leukemia induced by the three transgenes corresponds to the time required for clonal expansion from a single leukemic stem cell, suggesting that SCL, LMO1, and Notch1 gain of function, together with an active pre-TCR, might represent the minimum set of complementing events for the transformation of susceptible thymocytes

Selection of down-regulated sequences along the monocytic differentiation of leukemic HL60 cells

In order to dissect the molecular mechanisms of monocytic differentiation we have developed a subtractive hybridisation method based on a simplified 'representational difference analysis'. We have selected 16 sequences and confirmed their down-regulation along the TPA-induced monocytic differentiation of HL60 cells. Among these sequences we have identified the alpha-tubulin, the TaxREB protein and two ribosomal protein sequences which had not been previously described as differentially expressed. These results add to our knowledge about the molecules implicated along the monocytic differentiation and growth arrest of leukemic cells and provide a first step in the study of their respective roles

<i>SCL</i>, <i>LMO1</i> and <i>Notch1</i> Reprogram Thymocytes into Self-Renewing Cells

<div><p>The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive <i>NOTCH1</i> allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive <i>NOTCH1</i> cannot reprogram thymocytes on its own, despite the fact that <i>NOTCH1</i> is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating <i>NOTCH1</i> triggers a parallel pathway involving <i>Hes1</i> and <i>Myc</i> that dramatically enhances the activity of <i>SCL-LMO1</i> We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, <i>LYL1</i> and <i>LMO1</i> or <i>LMO2</i> are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network described here may be relevant to a majority of human T-ALL.</p></div

Transcription activation driven by SCL-LMO1 interaction is critical for thymocyte reprogramming and T-ALL induction.

<p>(<b>A</b>) Generation of transgenic mice expressing the LMO1-binding defective mutant SCLm13. The sequence coding for wild type human SCL or human SCLm13 HLH domain mutant <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004768#pgen.1004768-Lecuyer1" target="_blank">[37]</a> were cloned into the VA h<i>CD2</i> cassette to generate transgenic mice. Shown are amino acids of the HLH region of SCL or SCLm13. (<b>B</b>) Immunofluorescence of human SCL (wt or m13) by flow cytometry. Thymocytes were stained with the monoclonal antibody against human SCL (BTL73). Control cells were stained with the second antibody only. (<b>C</b>) Expression of E protein target genes is inhibited both by <i>SCL-LMO1</i> and <i>SCLm13-LMO1</i> transgenes in DN3 thymocytes. mRNA levels in purified DN3 thymocytes from the indicated transgenic mice were determined by qRT-PCR and normalized to <i>β-Actin</i> (Mean +/- SD, n = 3). (<b>D</b>) Kaplan-Meier curves of the time to leukemia for <i>LMO1^tg</i>, <i>E2a^+/-LMO1^tg</i>, <i>SCL^tgLMO1^tg</i> and <i>SCLm13^tgLMO1^tg</i> mice. (<b>E</b>) The interaction between SCL and LMO1 is required to activate the transcription of the self-renewal genes <i>Lyl1</i>, <i>Hhex</i> and <i>Nfe2</i> in DN3 thymocytes. mRNA levels in purified DN3 thymocytes from the indicated transgenic mice were determined by qRT-PCR and normalized to <i>β-Actin</i> (Mean +/- SD, n = 3). (<b>F–G</b>) SCL but not the LMO1-binding defective SCL-m13 mutant collaborates with LMO1 to induce abnormal thymic reconstitution potential to thymocytes. Pre-leukemic thymocytes (1.5×10⁷ cells) from 3-week-old mice were transplanted. Recipient mice were analysed for thymic reconstitution (CD45.2⁺Thy1⁺) after 6 weeks (F) and the proportion of DP cells in engrafted CD45.2⁺Thy1⁺ thymocytes was assessed by FACS (G).</p