Clonal selection in therapy-related myelodysplastic syndromes and acute myeloid leukemia under azacitidine treatment.

INTRODUCTION: Therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/AML) are defined as complications of previous cytotoxic therapy. Azacitidine (AZA), a hypomethylating agent, has showed activity in t-MDS/AML. OBJECTIVES: We evaluated the clonal dynamics of AZA-treated t-MDS/AML. METHODS: We collected bone marrow samples, at diagnosis and during treatment, from AZA-treated t-MDS/AML patients. NGS on 19 myeloid genes was performed, and candidate mutations with a variant allele frequency >5% were selected. RESULTS: Seven t-AML and 12 t-MDS were included with median age of 71 (56-82) years old, median number of AZA cycles of 6 (1-15), and median overall survival (OS) of 14 (3-29) months. We observed correlation between AZA response and clonal selection. Decrease of TP53-mutated clone was correlated with response to AZA, confirming AZA efficacy in this subgroup. In some patients, emergence of mutations was correlated with progression or relapse without impact on OS. Clones with mutations in genes for DNA methylation regulation frequently occurred with other mutations and remained stable during AZA treatment, independent of AZA response. CONCLUSION: We confirmed that the molecular complexity of t-MNs and that the follow-up of clonal selection during AZA treatment could be useful to define treatment combination.

Molecular characterization and follow-up of five CML patients with new BCR-ABL1 fusion transcripts.

We report five chronic myeloid leukaemia (CML) patients in whom we identified and characterized undescribed BCR-ABL1 fusion transcripts. We investigated the precise features of the molecular rearrangements and the minimal residual disease follow-up for these five patients. Three resulted from new rearrangements between the BCR and ABL1 sequences (the breakpoints being located within BCR exon 13 in two cases and within BCR exon 18 in one case). The other two cases revealed a complex e8-[ins]-a2 fusion transcript involving a third partner gene, PRDM12 and SPECC1L, respectively. Moreover, single nucleotide polymorphism-array analysis performed in the latter two cases showed copy number alterations shared by the two patients, thus identifying genes that were deleted during rearrangement and suggesting their potential role in CML pathogenesis. Interestingly, we highlight that the prognosis of alterations, such as the presence of an e8a2 transcript or the deletion of various genes, which have been controversial, may be definitively erased by the introduction of tyrosine kinase inhibitors (TKIs).

XY Sox9 embryonic loss-of-function mouse mutants show complete sex reversal and produce partially fertile XY oocytes.

Gonadal differentiation is the first step of mammalian sex determination. The expression of the Y chromosomal testis determining factor Sry leads to up-regulation of the transcription factor Sox9 which promotes testis differentiation. Previous studies showed that Sox9 deficiency induces expression of ovarian markers in XY mutant fetal gonads before they die. To better understand the genome-wide transcriptional profile underlying this process we compared samples from XY Sf1:Cre(Tg/+); Sox9(flox/flox) mutant gonads in which Sox9 is ablated in Sertoli-precursor cells during early stages of gonad development to XX Sox9(flox/flox) ovaries and XY Sox9(flox/flox) testes at E13.5. We found a complex mRNA signature that indicates wide-spread transcriptional de-regulation and revealed for XY mutants at E13.5 an intermediate transcript profile between male and female gonads. However, XY Sf1:Cre(Tg/+); Sox9(flox/flox) mutant gonads develop as ovaries containing XY developing follicles at P0 but less frequently so than in XX control ovaries. Furthermore, we studied the extent to which developing XY mutant ovaries are able to mediate adult fertility and observed that XY oocytes from XY mutant ovaries are competent for fertilization; however, two thirds of them fail to develop beyond two-cell stage embryos. Taken together, we found that XY Sf1:Cre(Tg/+); Sox9(flox/flox) females are capable of producing viable offspring albeit at a reduced level.

Distinct epithelial gene expression phenotypes in childhood respiratory allergy.

Epithelial cell contribution to the natural history of childhood allergic respiratory disease remains poorly understood. Our aims were to identify epithelial pathways that are dysregulated in different phenotypes of respiratory allergy. We established gene expression signatures of nasal brushings from children with dust mite-allergic rhinitis, associated or not associated with controlled or uncontrolled asthma. Supervised learning and unsupervised clustering were used to predict the different subgroups of patients and define altered signalling pathways. These profiles were compared with those of primary cultures of human nasal epithelial cells stimulated with either interleukin (IL)-4, IL-13, interferon (IFN)-α, IFN-ß or IFN-γ, or during in vitro differentiation. A supervised method discriminated children with allergic rhinitis from healthy controls (prediction accuracy 91%), based on 61 transcripts, including 21 T-helper cell (Th) type 2-responsive genes. This method was then applied to predict children with controlled or uncontrolled asthma (prediction accuracy 75%), based on 41 transcripts: nine of them, which were down-regulated in uncontrolled asthma, are directly linked to IFN. This group also included GSDML, which is genetically associated with asthma. Our data revealed a Th2-driven epithelial phenotype common to all children with dust mite allergic rhinitis. It highlights the influence of epithelially expressed molecules on the control of asthma, in association with atopy and impaired viral response.

Non-del(5q) myelodysplastic syndromes-associated loci detected by SNP-array genome-wide association meta-analysis.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell malignancies. Known predisposing factors to adult MDS include rare germline mutations, cytotoxic therapy, age-related clonal hematopoiesis, and autoimmune or chronic inflammatory disorders. To date, no published studies characterizing MDS-associated germline susceptibility polymorphisms exist. We performed a genome-wide association study of 2 sample sets (555 MDS cases vs 2964 control subjects; 352 MDS cases vs 2640 control subjects) in non-del(5q) MDS cases of European genomic ancestry. Meta-analysis identified 8 MDS-associated loci at 1q31.1 (PLA2G4A), 3p14.1 (FAM19A4), 5q21.3 (EFNA5), 6p21.33, 10q23.1 (GRID1), 12q24.32, 15q26.1, and 20q13.12 (EYA2) that approached genome-wide significance. Gene expression for 5 loci that mapped within or near genes was significantly upregulated in MDS bone marrow cells compared with those of control subjects (P < .01). Higher PLA2G4A expression and lower EYA2 expression were associated with poorer overall survival (P = .039 and P = .037, respectively). Higher PLA2G4A expression is associated with mutations in NRAS (P < .001), RUNX1 (P = .012), ASXL1 (P = .007), and EZH2 (P = .038), all of which are known to contribute to MDS development. EYA2 expression was an independently favorable risk factor irrespective of age, sex, and Revised International Scoring System score (relative risk, 0.67; P = .048). Notably, these genes have regulatory roles in innate immunity, a critical driver of MDS pathogenesis. EYA2 overexpression induced innate immune activation, whereas EYA2 inhibition restored colony-forming potential in primary MDS cells indicative of hematopoietic restoration and possible clinical relevance. In conclusion, among 8 suggestive MDS-associated loci, 5 map to genes upregulated in MDS with functional roles in innate immunity and potential biological relevance to MDS.

An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes.

Two collections of oligonucleotides have been designed for preparing pangenomic human and mouse microarrays. A total of 148,993 and 121,703 oligonucleotides were designed against human and mouse transcripts. Quality scores were created in order to select 25,342 human and 24,109 mouse oligonucleotides. They correspond to: (i) a BLAST-specificity score; (ii) the number of expressed sequence tags matching each probe; (iii) the distance to the 3' end of the target mRNA. Scores were also used to compare in silico the two microarrays with commercial microarrays. The sets described here, called RNG/MRC collections, appear at least as specific and sensitive as those from the commercial platforms. The RNG/MRC collections have now been used by an Anglo-French consortium to distribute more than 3500 microarrays to the academic community. Ad hoc identification of tissue-specific transcripts and a approximately 80% correlation with hybridizations performed on Affymetrix GeneChiptrade mark suggest that the RNG/MRC microarrays perform well. This work provides a comprehensive open resource for investigators working on human and mouse transcriptomes, as well as a generic method to generate new microarray collections in other organisms. All information related to these probes, as well as additional information about commercial microarrays have been stored in a freely-accessible database called MEDIANTE.

Live Staphylococcus aureus and bacterial soluble factors induce different transcriptional responses in human airway cells.

To characterize the response of respiratory epithelium to infection by Staphylococcus aureus (S. aureus), human airway cells were incubated for 1 to 24 h with a supernatant of a S. aureus culture (bacterial supernatant), then profiled with a pangenomic DNA microarray. Because an upregulation of many genes was noticed around 3 h, three independent approaches were then used to characterize the host response to a 3-h contact either with bacterial supernatant or with live bacteria: 1) a DNA microarray containing 4,200 sequence-verified probes, 2) a semiquantitative RT-PCR with a set of 537 pairs of validated primers, or 3) ELISA assay of IL-8, IL-6, TNFalpha, and PGE(2). Among others, Fos, Jun, and EGR-1 were upregulated by the bacterial supernatant and by live bacteria. Increased expression of bhlhb2 and Mig-6, promoter regions which harbor HIF responding elements, was explained by an increased expression of the HIF-1alpha protein. Activation of the inducible form of cyclooxygenase, COX-2, and of the interleukins IL-1, IL-6, and IL-8, as well as of the NF-kappaB pathway, was observed preferentially in cells in contact with bacterial supernatant. Early infection was characterized by an upregulation of anti-apoptotic genes and a downregulation of pro-apoptotic genes. This correlated with a necrotic, rather than apoptotic cell death. Overall, this first global description of an airway epithelial infection by S. aureus demonstrates a larger global response to bacterial supernatant (in term of altered genes and variation factors) than to exponentially growing live bacteria.

Genetic differences between Asian and Caucasian chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a common hematological malignancy in Western countries. However, this disease is very rare in Asian countries. It is not clear whether the mechanisms of development of CLL in Caucasians and Asians are the same. We compared genetic abnormalities in Asian and Caucasian CLL using 250k GeneChip arrays. Both Asian and Caucasian CLL had four common genetic abnormalities: deletion of 13q14.3, trisomy 12, abnormalities of ATM (11q) and abnormalities of 17p. Interestingly, trisomy 12 and deletion of 13q14.3 were mutually exclusive in both groups. We also found that deletions of miR 34b/34c (11q), caspase 1/4/5 (11q), Rb1 (13q) and DLC1 (8p) are common in both ethnic groups. Asian CLL more frequently had gain of 3q and 18q. These suggest that classic genomic changes in the Asian and Caucasina CLL are same. Further, we found amplification of IRF4 and deletion of the SP140/SP100 genes; these genes have been reported as CLL-associated genes by previous genome-wide-association study. We have found classic genomic abnormalities in Asian CLL as well as novel genomic alteration in CLL.

Two alternatively spliced 5'BCR/3'JAK2 fusion transcripts in a myeloproliferative neoplasm with a three-way t(9;18;22)(p23;p11.3;q11.2) translocation.

Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs) are known to harbor alterations of the tyrosine kinase JAK2 (9p24), resulting in the constitutive autoactivation of the encoded protein. Here, we report an unclassifiable MPN case, BCR/ABL1-negative, showing a three-way t(9;18;22)(p23;p11.3;q11.2) translocation, which generates a 5'BCR/3'JAK2 gene by fusing BCR at intron 1 to JAK2 at intron 14 on the derivative chromosome 22. The fusion gene produced two alternatively spliced 5'BCR/3'JAK2 transcripts, fusing in-frame BCR exon 1 to JAK2 exon 15 and exon 17. This is the first report of the simultaneous occurrence of two BCR/JAK2 fusion transcripts in the same sample and of the longer transcript isoform (BCR exon 1 fused to JAK2 exon 15). Notably, both BCR/JAK2 encoded fusion proteins are predicted to juxtapose the coiled-coil dimerization domain of BCR to the catalytically inactive pseudokinase domain (JH2), entirely or partially deprived of the inhibitory region 1 (IR1). Interestingly, IR1 is involved in the auto-inhibitory interaction with the JAK2 kinase domain (JH1), which may result in deregulation of JAK2 activity.

Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway.

Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. However, the mechanisms governing motile cilia biosynthesis remain poorly elucidated. We identify miR-449 microRNAs as evolutionarily conserved key regulators of vertebrate multiciliogenesis. In human airway epithelium and Xenopus laevis embryonic epidermis, miR-449 microRNAs strongly accumulated in multiciliated cells. In both models, we show that miR-449 microRNAs promote centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. We established Notch1 and its ligand Delta-like 1(DLL1) as miR-449 bona fide targets. Human DLL1 and NOTCH1 protein levels were lower in multiciliated cells than in surrounding cells, decreased after miR-449 overexpression and increased after miR-449 inhibition. In frog, miR-449 silencing led to increased Dll1 expression. Consistently, overexpression of Dll1 mRNA lacking miR-449 target sites repressed multiciliogenesis, whereas both Dll1 and Notch1 knockdown rescued multiciliogenesis in miR-449-deficient cells. Antisense-mediated protection of miR-449-binding sites of endogenous human Notch1 or frog Dll1 strongly repressed multiciliogenesis. Our results unravel a conserved mechanism whereby Notch signalling must undergo miR-449-mediated inhibition to permit differentiation of ciliated cell progenitors.

MiR-129-5p is required for histone deacetylase inhibitor-induced cell death in thyroid cancer cells.

The molecular mechanism responsible for the antitumor activity of histone deacetylase inhibitors (HDACi) remains elusive. As HDACi have been described to alter miRNA expression, the aim of this study was to characterize HDACi-induced miRNAs and to determine their functional importance in the induction of cell death alone or in combination with other cancer drugs. Two HDACi, trichostatin A and vorinostat, induced miR-129-5p overexpression, histone acetylation and cell death in BCPAP, TPC-1, 8505C, and CAL62 cell lines and in primary cultures of papillary thyroid cancer (PTC) cells. In addition, miR-129-5p alone was sufficient to induce cell death and knockdown experiments showed that expression of this miRNA was required for HDACi-induced cell death. Moreover, miR-129-5p accentuated the anti-proliferative effects of other cancer drugs such as etoposide or human α-lactalbumin made lethal for tumor cells (HAMLET). Taken together, our data show that miR-129-5p is involved in the antitumor activity of HDACi and highlight a miRNA-driven cell death mechanism.

Gene expression profiling in human gastric mucosa infected with Helicobacter pylori.

Pathogenic mechanisms associated with Helicobacter pylori infection enhance susceptibility of the gastric epithelium to carcinogenic conversion. We have characterized the gene expression profiles of gastric biopsies from 69 French Caucasian patients, of which 43 (62%) were infected with H. pylori. The bacterium was detected in 27 of the 42 antral biopsies examined and in 16 of the 27 fundic biopsies. Infected biopsies were selected for the presence of chronic active gastritis, in absence of metaplasia and dysplasia of the gastric mucosa. Infected antral and fundic biopsies exhibited distinct transcriptional responses. Altered responses were linked with: (1) the extent of polymorphonuclear leukocyte infiltration, (2) bacterial density, and (3) the presence of the virulence factors vacA, babA2, and cagA. Robust modulation of transcripts associated with Toll-like receptors, signal transduction, the immune response, apoptosis, and the cell cycle was consistent with expected responses to Gram-negative bacterial infection. Altered expression of interferon-regulated genes (IFITM1, IRF4, STAT6), indicative of major histocompatibility complex (MHC) II-mediated and Th1-specific responses, as well as altered expression of GATA6, have previously been described in precancerous states. Upregulation of genes abundantly expressed in cancer tissues (UBD, CXCL13, LY96, MAPK8, MMP7, RANKL, CCL18) or in stem cells (IFITM1 and WFDC2) may reveal a molecular switch towards a premalignant state in infected tissues. Tissue microarray analysis of a large number of biopsies, which were either positive or negative for the cag-A virulence factor, when compared to each other and to noninfected controls, confirmed observed gene alterations at the protein level, for eight key transcripts. This study provides 'proof-of-principle' data for identifying molecular mechanisms driving H. pylori-associated carcinogenesis before morphological evidence of changes along the neoplastic progression pathway.

Transcriptional signature of epidermal keratinocytes subjected to in vitro scratch wounding reveals selective roles for ERK1/2, p38, and phosphatidylinositol 3-kinase signaling pathways.

Covering denuded dermal surfaces after injury requires migration, proliferation, and differentiation of skin keratinocytes. To clarify the major traits controlling these intermingled biological events, we surveyed the genomic modifications occurring during the course of a scratch wound closure of cultured human keratinocytes. Using a DNA microarray approach, we report the identification of 161 new markers of epidermal repair. Expression data, combined with functional analysis performed with specific inhibitors of ERK, p38(MAPK) and phosphatidylinositol 3-kinase (PI3K), demonstrate that kinase pathways exert very selective functions by precisely controlling the expression of specific genes. Inhibition of the ERK pathway totally blocks the wound closure and inactivates many early transcription factors and EGF-type growth factors. p38(MAPK) inhibition only delays "healing," probably in line with the control of genes involved in the propagation of injury-initiated signaling. In contrast, PI3K inhibition accelerates the scratch closure and potentiates the scratch-dependent stimulation of three genes related to epithelial cell transformation, namely HAS3, HBEGF, and ETS1. Our results define in vitro human keratinocyte wound closure as a repair process resulting from a fine balance between positive signals controlled by ERK and p38(MAPK) and negative ones triggered by PI3K. The perturbation of any of these pathways might lead to dysfunction in the healing process, similar to those observed in pathological wounding phenotypes, such as hypertrophic scars or keloids.

Early gene expression in wounded human keratinocytes revealed by DNA microarray analysis.

Wound healing involves several steps: spreading of the cells, migration and proliferation. We have profiled gene expression during the early events of wound healing in normal human keratinocytes with a home-made DNA microarray containing about 1000 relevant human probes. An original wounding machine was used, that allows the wounding of up to 40% of the surface of a confluent monolayer of cultured cells grown on a Petri dish (compared with 5% with a classical 'scratch' method). The two aims of the present study were: (a) to validate a limited number of genes by comparing the expression levels obtained with this technique with those found in the literature; (b) to combine the use of the wounding machine with DNA microarray analysis for large-scale detection of the molecular events triggered during the early stages of the wound-healing process. The time-courses of RNA expression observed at 0.5, 1.5, 3, 6 and 15 h after wounding for genes such as c-Fos, c-Jun, Egr1, the plasminogen activator PLAU (uPA) and the signal transducer and transcription activator STAT3, were consistent with previously published data. This suggests that our methodologies are able to perform quantitative measurement of gene expression. Transcripts encoding two zinc finger proteins, ZFP36 and ZNF161, and the tumour necrosis factor alpha-induced protein TNFAIP3, were also overexpressed after wounding. The role of the p38 mitogen-activated protein kinase (p38MAPK) in wound healing was shown after the inhibition of p38 by SB203580, but our results also suggest the existence of surrogate activating pathways.

Escherichia coli cytotoxic necrotizing factor 1 inhibits intestinal epithelial wound healing in vitro after mechanical injury.

Cytotoxic necrotizing factor type 1 (CNF1) from Escherichia coli activates the small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42) by catalyzing their deamidation at a specific glutamine residue. Since RhoA, Rac, and Cdc42 play a pivotal role in cell migration during the early phase of wound repair, we investigated whether CNF1 was able to interfere with wound healing in intestinal epithelial monolayers (T84 cells). After mechanical injury, we found that CNF1 blocks epithelial wound repair within 48 h. This effect was characterized by cell elongation and filopodium formation on the leading edge, in association with permanent phosphorylation of the focal adhesion kinase (FAK) via Rho activation. Moreover, inhibition of Rho kinase with Y-27632 decreased CNF1-mediated permanent FAK phosphorylation, leading to complete restitution of wound repair within 24 h. In addition, we found that CNF1 induced upregulation of mitogen-activated protein kinases (MAPK) activation. Moreover, activation of Rac and MAPK by CNF1 increased matrix metalloproteinase 9 expression in wounded T84 monolayers. Taken together, these results provide evidence that CNF1 strongly impairs intestinal epithelial wound healing.

Coordinated regulation of genes for secretion in tobacco at late developmental stages: association with resistance against oomycetes.

Besides the systemic acquired resistance (SAR) induced in response to microbial stimulation, host plants may also acquire resistance to pathogens in response to endogenous stimuli associated with their own development. In tobacco (Nicotiana tabacum), the vegetative-to-flowering transition comes along with a susceptibility-to-resistance transition to the causal agent of black shank disease, the oomycete Phytophthora parasitica. This resistance affects infection effectiveness and hyphal expansion and is associated with extracellular accumulation of a cytotoxic activity that provokes in vitro cell death of P. parasitica zoospores. As a strategy to determine the extracellular events important for restriction of pathogen growth, we screened the tobacco genome for genes encoding secreted or membrane-bound proteins expressed in leaves of flowering plants. Using a signal sequence trap approach in yeast (Saccharomyces cerevisiae), 298 clones were selected that appear to encode for apoplastic, cell wall, or membrane-bound proteins involved in stress response, in plant defense, or in cell wall modifications. Microarray and northern-blot analyses revealed that, at late developmental stages, leaves were characterized by the coordinate up-regulation of genes involved in SAR and in peroxidative cross-linking of structural proteins to cell wall. This suggests the potential involvement of these genes in extracellular events that govern the expression of developmental resistance. The analysis of the influence of salicylic acid on mRNA accumulation also indicates a more complex network for regulation of gene expression at a later stage of tobacco development than during SAR. Further characterization of these genes will permit the formulation of hypotheses to explain resistance and to establish the connection with development.