Epigenetic changes in human model KMT2A leukemias highlight early events during leukemogenesis.

Chromosomal translocations involving KMT2A gene are one of the most common genetic alterations found in pediatric acute myeloid leukemias (AML) although the molecular mechanisms that initiate the disease remain incompletely defined. To elucidate these initiating events we have used a human model system of AML driven by the KMT2A-MLLT3 (KM3) fusion. More specifically, we investigated changes in DNA methylation, histone modifications, and chromatin accessibility at each stage of our model system and correlated these with expression changes. We observe the development of a profound hypomethylation phenotype in the early stages of leukemic transformation after KM3 addition along with loss of expression of stem cell associated genes along with skewed expression in other genes such as S100A8/9 implicated in leukemogenesis. In addition, early increases in the expression of the lysine demethylase KDM4B was functionally linked to these expression changes as well as other key transcription factors. Remarkably, our ATAC-seq data showed that there were relatively few leukemiaspecific changes and the vast majority corresponded to open chromatin regions and transcription factor clusters previously observed in other cell types. Integration of the gene expression and epigenetic changes revealed the adenylate cyclase gene ADCY9 as an essential gene in KM3-AML, and suggest the potential for autocrine signalling through the chemokine receptor CCR1 and CCL23 ligand. Together, our results suggest that KM3 induces subtle changes in the epigenome while co-opting the normal transcriptional machinery to drive leukemogenesis.

High-throughput Cos-Seq screen with intracellular Leishmania infantum for the discovery of novel drug-resistance mechanisms.

Increasing drug resistance towards first line antimony-derived compounds has forced the introduction of novel therapies in leishmaniasis endemic areas including amphotericin B and miltefosine. However, their use is threatened by the emergence and spread of drug-resistant strains. In order to discover stage-dependent resistance genes, we have adapted the Cos-Seq approach through the introduction of macrophage infections in the pipeline. A L. infantum intracellular amastigote population complemented with a L. infantum cosmid library was submitted to increasing concentrations of miltefosine, amphotericin B and pentavalent antimonials in experimental infections of THP-1 cells. For each step of selection, amastigotes were extracted and cosmids were isolated and submitted to next-generation sequencing, followed by subsequent gene-enrichment analyses. Cos-Seq screen in amastigotes revealed four highly enriched loci for antimony, five for miltefosine and one for amphotericin B. Of these, a total of seven cosmids were recovered and tested for resistance in both promastigotes and amastigotes. Candidate genes within the pinpointed genomic regions were validated using single gene overexpression in wild-type parasites and/or gene disruption by means of a CRISPR-Cas9-based approach. This led to the identification and validation of a stage-independent antimony-resistance gene (LinJ.06.1010) coding for a putative leucine rich repeat protein and a novel amastigote-specific miltefosine-resistance gene (LinJ.32.0050) coding for a member of the SEC13 family of WD-repeat proteins. This study further reinforces the power of Cos-Seq approach to discover novel drug-resistance genes, some of which are life-stages specific.

The Prostaglandin F Synthase Activity of the Human Aldose Reductase AKR1B1 Brings New Lenses to Look at Pathologic Conditions.

Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2α are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2/EP2 and PGF2α/FP may constitute a functional dyad with physiological relevance comparable to the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2α production in response to IL-1ß in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231) also known as ALDR1 or ALR2 is a functional PGF2α synthase in different models of living cells and tissues. Using human endometrial cells, prostate, and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1ß is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2α production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1ß particularly around the multiple stress response region containing two putative antioxidant response elements adjacent to TonE and AP1. We also show that AKR1B1 is able to regulate PGE2 production through PGF2α acting on its FP receptor and that aldose reductase inhibitors like alrestatin, Statil (ponalrestat), and EBPC exhibit distinct and characteristic inhibition of PGF2α production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies.

The human aldose reductase AKR1B1 qualifies as the primary prostaglandin F synthase in the endometrium.

CONTEXT: Prostaglandins (PGs) E2 and PGF2α are produced in the endometrium and are important for menstruation and fertility. Dysmenorrhea is associated with increased production of PGF2α relative to PGE2, and the opposite is true for menorrhagia. The pathways leading to PGE2 biosynthesis are well described, but little is known for PGF2α. Aldoketoreductase (AKR)-1C3, the only PGF synthase identified in the human, cannot explain the production of PGF2α by endometrial cells. AKR1B1 appears to be an alternate candidate with promising therapeutic value. OBJECTIVE: The objective of the study was to address whether AKR1B1 (gene ID 231) is a functional PGF2α synthase in the human endometrium and a valid therapeutic target for menstrual pain. DESIGN: The design of the study was basic laboratory analyses to identify gene expression and protein levels associated with PGF2α production in endometrial tissues and endometrial cells from cycling women aged between 23 and 52 yr undergoing biopsies or hysterectomy for diverse gynecological disorders. RESULTS: AKR1B1 is expressed at a high level during the menstrual cycle during the secretory phase and in both epithelial and stromal cells, whereas AKR1C3 was found only in epithelial cells. Purified recombinant AKR1B1 protein, gene silencing, and transient transfection experiments all concur to demonstrate that this enzyme is a functional PGF synthase. Ponalrestat, a specific inhibitor developed to block AKR1B1 activity, reduced PGF2α production in response to IL-1ß in both cultured endometrial cells and endometrial explants. CONCLUSIONS: The human aldose reductase AKR1B1 currently associated with diabetes complications is also a highly functional PGF synthase responsible for PGF2α production in the human endometrium and a potential target for treatment of menstrual disorders.

Gene expression profile of androgen modulated genes in the murine fetal developing lung.

BACKGROUND: Accumulating evidences suggest that sex affects lung development. Indeed, a higher incidence of respiratory distress syndrome is observed in male compared to female preterm neonates at comparable developmental stage and experimental studies demonstrated an androgen-related delay in male lung maturation. However, the precise mechanisms underlying these deleterious effects of androgens in lung maturation are only partially understood. METHODS: To build up a better understanding of the effect of androgens on lung development, we analyzed by microarrays the expression of genes showing a sexual difference and those modulated by androgens. Lungs of murine fetuses resulting from a timely mating window of 1 hour were studied at gestational day 17 (GD17) and GD18, corresponding to the period of surge of surfactant production. Using injections of the antiandrogen flutamide to pregnant mice, we hunted for genes in fetal lungs which are transcriptionally modulated by androgens. RESULTS: Results revealed that 1844 genes were expressed with a sexual difference at GD17 and 833 at GD18. Many genes were significantly modulated by flutamide: 1597 at GD17 and 1775 at GD18. Datasets were analyzed by using in silico tools for reconstruction of cellular pathways. Between GD17 and GD18, male lungs showed an intensive transcriptional activity of proliferative pathways along with the onset of lung differentiation. Among the genes showing a sex difference or an antiandrogen modulation of their expression, we specifically identified androgen receptor interacting genes, surfactant related genes in particularly those involved in the pathway leading to phospholipid synthesis, and several genes of lung development regulator pathways. Among these latter, some genes related to Shh, FGF, TGF-beta, BMP, and Wnt signaling are modulated by sex and/or antiandrogen treatment. CONCLUSION: Our results show clearly that there is a real delay in lung maturation between male and female in this period, the latter pursuing already lung maturation while the proper is not yet fully engaged in the differentiation processes at GD17. In addition, this study provides a list of genes which are under the control of androgens within the lung at the moment of surge of surfactant production in murine fetal lung.