Aminetou Mohamed

Background: AZA is the standard of care for patients with either HRMDS or AML with 20-30% blasts (Oligoblastic AML). The response rate is ~50% with a median response duration of 12-15 months. The mechanisms underlying primary and secondary resistance are poorly understood and it remains difficult to accurately predict which patients will respond and in responders, to predict secondary resistance. The main factors demonstrated or suggested to interfere with response to AZA and/or survival include marrow blast percentage, performance status, IPSS cytogenetic risk, presence of peripheral blasts, transfusion dependency, IPSS-R risk, mutations of TET2, P53, IDH1/2, and DNMT3A, elevated expression of BCL2L10, Fas, or PI-PLCbeta1. Deregulation of microRNAs is a hallmark of MDS, yet its consequences on AZA efficacy have not been specifically addressed in HRMDS. Methods: We measured the expression of 754 miRNAs in SKM1 MDS cells resistant or sensitive to AZA. miRNAs of interest were ectopically expressed or specifically inhibited in HEK293T cells which were assayed for AZA sensitivity (MTT). Seven miRNAs were quantified in bone marrow mononuclear cells deriving from 75 patients with HRMDS (n= 50) or oligoblastic AML treated with AZA [median age 74, 24 females, median cycle number 6, (1-39)]. Results: Seven miRNAs (miR-125a-5p, miR-99b-5p, miR-126, miR-126*, hsa-let-7c, miR-34b-3p, and miR-10b*) that include 6 tumor-suppressor miRNAs, were found differentially expressed between AZA-sensitive versus -resistant cells; all being transcriptionally repressed in resistant cells. In silico, 5 of these miRNAs were found to target the…

DNMT1 protein expression in SKM1 AZA-sensitive (-S) versus AZA-resistant clones (-R). The Figure corresponds to the Figure 1B with signal quantification performed with ImageJ software. The data are expressed as mean {plus minus} SD of three independent experiments, with each performed in triplicate (Mann-Whitney test, p<0.05).

Pathway enrichment analysis. For ontology analysis, gene lists were analyzed using DAVID software (KEGG pathways). The complete set of genes featured in the microarrays was used as the reference background. The three numbers on the right represent the number of deregulated mRNAs, the overall number of genes within the pathway and the p value, respectively. Data are presented for genes targeted by at least one of the 7 miRNAs and that were found repressed in SKM1 AZA resistant cells.

Purpose: Azacitidine inhibits DNA methyltransferases, including DNMT1, and is currently the standard of care for patients with higher-risk myelodysplastic syndrome (HRMDS) or low blast count acute myeloid leukemia (AML).Experimental Design: The expression of 754 miRNAs was compared in azacitidine-resistant and azacitidine-sensitive myelodysplastic syndrome cells. We investigated the role of differentially expressed miRNAs on DNMT1 expression and azacitidine resistance in vitro. We next evaluated anti-DNMT1 miRNA expression in pretreatment bone marrow samples derived from 75 patients treated with azacitidine for HRMDS or AML.Results: Seven miRNAs, including 5 that in silico targeted the DNMT1 3′ UTR, were repressed in azacitidine-resistant cells in which DNMT1 protein levels were significantly higher. Ectopic anti-DNMT1 miRNA expression decreased DNMT1 expression and increased azacitidine sensitivity, whereas specific inhibition of endogenous anti-DNMT1 miRNAs increased DNMT1 express...

Approximately one-third of expressed genes are misspliced in AML, opening the possibility that additional factors than splicing factor mutations might cause RNA missplicing in these diseases. AML cells harbor a constellation of epigenetic modifications and regularly express large amounts of WT1 transcripts. Histone acetylation/methylation and DNA CpG methylation favor either exon skipping or inclusion, mainly through interfering with RNA Pol II-mediated elongation. This can result either from the binding of various factors on Pol II or alternatively from the recruitment of DNA binding factors that create roadblocks to Pol II-induced elongation. WT1 exhibits pleiotropic effects on mRNA splicing, which mainly result from the binding properties of WT1 via its zinc fingers domains to DNA, RNA, and proteins. Through the repression of the kinase SRPK1, WT1 modifies the splicing of VEGF, which plays important roles in hematopoiesis and angiogenesis. At the protein level, WT1 interacts with...

The present study shows that the XmnI polymorphism (rs7482144) at -158 C → T of Gγ gene is well associated with increased expression of HbF among sickle cell homozygotes in Mauritania. The prevalence of homozygous and heterozygous XmnI polymorphism is respectively of 36% and 34%, whereas negative individuals for this polymorphism are of 30% in the Mauritania population. This XmnI polymorphism induces increased HbF synthesis. The most found haplotype in Mauritania is the Senegalese type(77.7%) which was described as a relative protector by the presence of HbF followed by the beninese haplotype (8.8%) , the ArabIndian (5.5%), the Bantu (4.4%) and the two atypical haplotypes 1 and 2 (2.2 and 1.1%) reflecting the multiethnic character of Mauritania.

Parmi les nombreuses perturbations participant a l'heterogeneite bioclinique des leucemies aigues myeloides (LAM), les anomalies d'epissage ont recemment pu etre identifiees grâce au developpement des techniques de microarray et du sequencage a haut debit. Apres une revue bibliographique abordant les possible mecanismes et consequences des perturbation de l'epissage dans les LAM, nous avons dans un deuxieme article, analyse les patrons d'epissage associes a l'expression d'oncogenes connus pour interferer avec le spliceosome, l'ARN et la chromatine, ainsi qu'a la resistance aux principales drogues utilisees dans la prise en charge des LAM. Nos resultats identifient des signatures specifiques et originales, de nombreux evenements concernant des genes non deregules au plan transcriptionnel. Parmi ces perturbation nous avons, dans une troisieme etude, evalue l'impact clinique d'une perturbation d'epissage de l'ARN messager de TET2. Les res...

Background. The ATP binding cassette transporter 3 (ABCA3) has been recently found to induce a significant reduction in cytotoxicity following exposure to anthracyclines, mitoxantrone, etoposide, Ara-C, vincristine, and rituximab. ABCA3 acts through the modulation of multivesicular bodies (MVB) and contributes to drug sequestration in late endosomal organelles, i.e. MVB and lysosomes. Studies having investigated the prognostic impact of ABCA3 expression in AML have yielded conflicting results as ABCA3 expression has both been reported to exert unfavorable or neutral effects on patient outcomes. In addition, the small sample size of these studies precluded the use of multivariate analyses. Methods. Our goal was to investigate the prognostic impact of ABCA3 expression in adult patients with AML treated with IC with or without gemtuzumab ozogamicin (GO). To this end we investigated the relationship between ABCA3 expression and EFS in a representative series of 221 AML homogeneously tre...

Background. We have recently investigated transcriptional and posttranscriptional changes in AML cells (Oncotarget, 2016, 7, 2889-909) and identified ABCA3 expression, TET2 expression and TET2 exon 2 skipping (TET2E2S) as prognostic factors in pediatric AML (See companion abstracts from Ceraulo et al.). The present study was conducted in order to test whether the combination of these prognostic factors might help stratifying patients according to their relapse risk. Methods. Samples derived from 120 patients with available high-quality RNA and enrolled in the French ELAM2 protocol. Patients were classified according to their standardized cytogenetic and molecular (NPM1 mutations, FLT3-ITD, CEBPA double mutations) risk subgroups. Treatment consisted of 1 induction course (AraC and mitoxantrone) and 3 consolidation courses (course 1 and 3 with high dose AraC); all children with either intermediate or high-risk disease were candidates for hematopoietic stem cell transplant in complete ...

Background. In AML, the frequency of TET2 mutations increases with age ranging from 1-5% in children to 6-27% in adults. These mutations have both been reported to exert unfavorable or neutral effects on patient outcomes. Besides mutations, fluctuation in TET2 expression has been evidenced in AML while we recently found that TET2 exon 2 skipping (TET2E2S) is associated with favorable outcome in adult AML (Mint-Mohamed et al., ASH 2014 and submitted). Here we investigated the effect TET2 expression and TET2E2S in homogeneously treated adult and pediatric AML patients with intensive chemotherapy (IC). Methods. Samples derived from 341 patients with available high-quality RNA and enrolled in the ELAM2 protocol (n=120) and the ALFA-0701 protocol (n=221).qRTPCR amplification of 2 conserved TET2 mRNA sequences was performed with GUS and ABL as reference genes. TET2 exon 2 skipping was quantified throughexon(E)-specific qRTPCR (qESPCR) amplification of E1E3 (spliced) and E2E3 (unspliced) T...

Background. Despite progress in the molecular and genetic classification of pediatric acute myeloid leukemia (AML), the prognosis remains heterogeneous. The ATP-binding cassette transporter A3 (ABCA3) seems specifically involved in the resistance of pediatric AML to intensive chemotherapy. However, studies having investigated the prognostic impact of ABCA3 expression have yielded conflicting results with respect to patient outcomes while the small sample size of these studies precluded the use of multivariate analysis. Here we investigated the prognostic impact of ABCA3 expression in a representative series of homogeneously treated pediatric AML. Methods. Samples derived from 233 patients with available high-quality RNA and enrolled in the ELAM2 protocol (NCT00149162). qRTPCR amplification of 2 conserved ABCA3 mRNA sequences was performed with GUS and ABL as reference genes. Primer sets were complementary to exons 6-7 and exons 19-20 junctions. Patients were classified according to ...

2392 In humans, the majority of all protein-coding transcripts contain introns that are removed by mRNA splicing carried out by spliceosomes. Mutations in the spliceosome machinery have recently been identified using whole-exome/genome technologies in myelodysplastic syndromes (MDS) and in acute myeloid leukemia (AML). In MDS the frequency of somatic spliceosomal mutations (SSM) range from 1–3% for U2AF1 in RARS/RCMD-RS to more than 70% for SF3B1 in ARSI. These values are significantly lower in AML whereas AML cells cumulate numerous splicing defects. Beside SSMs, one can propose that alternative splicing (AS) might be disturbed by other processes such as abnormal protein-protein interactions. DEK and WT1 are 2 oncogenes overexpressed in most patients with AML. They physiologically influence AS through physical interactions with the heterodimer U2AF1/U2AF2 involved in the recognition of splice acceptor site by the splicing machinery. It is therefore possible that the leukemogenic ov...

Background: AZA is the standard of care for patients with either HRMDS or AML with 20-30% blasts (Oligoblastic AML). The response rate is ~50% with a median response duration of 12-15 months. The mechanisms underlying primary and secondary resistance are poorly understood and it remains difficult to accurately predict which patients will respond and in responders, to predict secondary resistance. The main factors demonstrated or suggested to interfere with response to AZA and/or survival include marrow blast percentage, performance status, IPSS cytogenetic risk, presence of peripheral blasts, transfusion dependency, IPSS-R risk, mutations of TET2, P53, IDH1/2, and DNMT3A, elevated expression of BCL2L10, Fas, or PI-PLCbeta1. Deregulation of microRNAs is a hallmark of MDS, yet its consequences on AZA efficacy have not been specifically addressed in HRMDS. Methods: We measured the expression of 754 miRNAs in SKM1 MDS cells resistant or sensitive to AZA. miRNAs of interest were ectopica...

The nucleoside analogue cytarabine (AraC) has served as the backbone of acute myeloid leukemia (AML) treatment for nearly forty years. About one-third of expressed genes are abnormally spliced in AML yet alternative exon usage (AEU) plays a role in the plasticity of tumor cells and may influence the response to treatment. Here the exon expression profiles of the erythroleukemia K562 cell line were compared to that of its AraC-resistant variant K562/AraC through Affymetrix HTA2 exon arrays. 5140 exon events harbored by 2583 genes distinguished the 2 cell lines. Among these, the skipping of TET2 exon 2 was identified in K562 cells sensitive to AraC whereas TET2 gene expression remained unchanged at the whole transcript level. The results were confirmed by exon-specific RTPCR (ESPCR). Microarray analysis did not evidenced any significant change in mRNA splicing for the 10 remaining exons of the TET2 gene. TET2 is a dioxygenase that catalyzes the conversion of 5-methylcytosine (5-mC) to...

ATP binding cassette (ABC) transporters are a superfamily of highly conserved membrane proteins that transport a wide variety of substrates across cell membranes and confer drug resistance against a wide range of chemotherapeutic agents. We recently found that WT1, which is regularly overexpressed in AML and interact with the splicing machinery, modifies the splicing of ABC transporters A2, A3, A5, and C2. For ABCA3, WT1 knock-down in three AML cell line coupled with Affymetrix HTA2 exon arrays analysis confirmed by exon-specific PCR revealed that WT1 influences the skipping of exon 19. ABCA3 belongs in the ABC subclass and induces a significant reduction in cytotoxicity observed following exposure to DNR, mitoxantrone, etoposide, Ara-C and vincristine. The ABCA3 domain encoded by exon 19 (amino acid 805-847) is localized at the junction of the first nucleotide-binding domain and the second transmembrane domain, and is involved in ATP hydrolysis. In silico, skipping of exon 19 delet...

Purpose: Azacitidine inhibits DNA methyltransferases, including DNMT1, and is currently the standard of care for patients with higher-risk myelodysplastic syndrome (HRMDS) or low blast count acute myeloid leukemia (AML).Experimental Design: The expression of 754 miRNAs was compared in azacitidine-resistant and azacitidine-sensitive myelodysplastic syndrome cells. We investigated the role of differentially expressed miRNAs on DNMT1 expression and azacitidine resistance in vitro We next evaluated anti-DNMT1 miRNA expression in pretreatment bone marrow samples derived from 75 patients treated with azacitidine for HRMDS or AML.Results: Seven miRNAs, including 5 that in silico targeted the DNMT1 3' UTR, were repressed in azacitidine-resistant cells in which DNMT1 protein levels were significantly higher. Ectopic anti-DNMT1 miRNA expression decreased DNMT1 expression and increased azacitidine sensitivity, whereas specific inhibition of endogenous anti-DNMT1 miRNAs increased DNMT1 expr...

In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2S(high) patients were found to be younger than TET2(low) patients without a difference in the rate of complete remission. However, TET2E2S(high) patients exhibited a significantly lower CIR (p<10(-4)). TET2E2S and FLT3-ITD, but not age or NPM1 mutation status were independent prognostic factors for DFS and event-free survival (EFS), while TET2E2S was the sole prognostic factor that we identified for overall survival (OS). In both ...

In addition to spliceosome gene mutations, oncogene expression and drug resistance in AML might influence exon expression. We performed exon-array analysis and exon-specific PCR (ESPCR) to identify specific landscapes of exon expression that are associated with DEK and WT1 oncogene expression and the resistance of AML cells to AraC, doxorubicin or azacitidine. Data were obtained for these five conditions through exon-array analysis of 17 cell lines and 24 patient samples and were extended through qESPCR of samples from 152 additional AML cases. More than 70% of AEUs identified by exon-array were technically validated through ESPCR. In vitro, 1,130 to 5,868 exon events distinguished the 5 conditions from their respective controls while in vivo 6,560 and 9,378 events distinguished chemosensitive and chemoresistant AML, respectively, from normal bone marrow. Whatever the cause of this effect, 30 to 80% of mis-spliced mRNAs involved genes unmodified at the whole transcriptional level. T...

Approximately one-third of expressed genes are misspliced in AML, opening the possibility that additional factors than splicing factor mutations might cause RNA missplicing in these diseases. AML cells harbor a constellation of epigenetic modifications and regularly express large amounts of WT1 transcripts. Histone acetylation/methylation and DNA CpG methylation favor either exon skipping or inclusion, mainly through interfering with RNA Pol II-mediated elongation. This can result either from the binding of various factors on Pol II or alternatively from the recruitment of DNA binding factors that create roadblocks to Pol II-induced elongation. WT1 exhibits pleiotropic effects on mRNA splicing, which mainly result from the binding properties of WT1 via its zinc fingers domains to DNA, RNA, and proteins. Through the repression of the kinase SRPK1, WT1 modifies the splicing of VEGF, which plays important roles in hematopoiesis and angiogenesis. At the protein level, WT1 interacts with...

Background: The use of primaquine (PQ) for radical treatment of Plasmodium vivax in carriers of G6PD deficiency (G6PDd) constitutes the main factor associated with severe haemolysis in G6PDd. The current study aimed to estimate the incremental cost-effectiveness ratio of using a rapid diagnostic test (RDT) to detect G6PDd in male patients with P. vivax malaria in the Brazilian Amazon, in comparison with the routine indicated by the Programme for Malaria Control, which does not include this evaluation. Methods: A cost-effectiveness analysis of estimated RDT use was carried out for the Brazilian Amazon for the year 2013, considering the perspective of the Brazilian Public Health System. Using decision trees, estimates were compared for two different RDT strategies for G6PDd in male individuals infected with P. vivax before being prescribed PQ, with the routine indicated in Brazil, which does not include prior diagnosis of G6PDd. The first strategy considered the combined use of RDT BinaxNOW ® G6PD (BX-G6PD) in municipalities with more than 100,000 inhabitants and the routine programme (RP) for the other municipalities. Operational limitations related to the required temperature control and venous blood collection currently restrict the use of RDT BX-G6PD in small municipalities. The second strategy considered the use of the RDT CareStart ™ G6PD (CS-G6PD) in 100 % of the municipalities. The analysis was carried out for the outcomes: " adequately diagnosed case " and " hospitalization avoided ". Results: For the outcome " adequately diagnosed case " , comparing the RDT strategies based on RDT with the routine control programme (RP), the CS-G6PD strategy was the most cost-effective, with BX-G6PD extendedly dominating (the ICER of BX-G6PD compared with RP was higher than the ICER of CS-G6PD compared with RP). CS-G6PD dominated the other strategies for the " hospitalization avoided " outcome. Conclusion: The CS-G6PD strategy is cost-effective for adequately diagnosing cases and avoiding hospitalization. This information can help in decision-making, both in incorporating prior diagnosis in the use of PQ and to promote greater safety among G6PD deficient individuals in the Brazilian Amazon P. vivax endemic areas.