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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, TET2E2Shigh patients were found to be younger than TET2low patients without a difference in the rate of complete remission. However, TET2E2Shigh patients exhibited a significantly lower CIR (p

// Aminetou Mint Mohamed 1 , Marie Balsat 1 , Morgan Thenoz 1 , Catherine Koering 1 , Lea Payen-Gay 2 , Meyling Cheok 3 , Hussein Mortada 4 , Didier Auboeuf 4 , Christiane Pinatel 5 , Mohamed El-Hamri 6 , Charles Dumontet 7 , Emeline Cros 7 , Pascale Flandrin-Gresta 1, 8 , Olivier Nibourel 4 , Claude Preudhomme 4 , Mauricette Michallet 1, 6 , Xavier Thomas 6 , Franck Nicolini 6 , Francoise Solly 1, 8 , Denis Guyotat 1, 9 , Lydia Campos 1, 8 , Eric Wattel 1, 6, * , Franck Mortreux 1, * 1 Universite Lyon 1, CNRS UMR5239, Oncovirologie et Biotherapies, Faculte de Medecine Lyon Sud, ENS – HCL, Pierre Benite, France 2 INSERM, UMR-S1052, Centre de Recherche en Cancerologie de Lyon, Lyon, France 3 Jean-Pierre Aubert Center, INSERM U837, Facteurs de persistance des cellules leucemiques, Institute for Cancer Research in Lille, Lille cedex, France 4 Centre de Recherche sur le Cancer de Lyon, Inserm, Epissage alternatif et progression tumorale, Lyon, France 5 Centre de Recherche sur le Cancer de Lyon, Inserm, Echappement aux systemes de sauvegarde et plasticite cellulaire, Lyon, France 6 Universite Lyon I, Service d’Hematologie, Pavillon Marcel Berard, Centre Hospitalier Lyon-Sud, Pierre Benite, France 7 Centre de Recherche sur le Cancer de Lyon, Inserm, Anticorps anticancer, Lyon, France 8 Universite de Saint Etienne, Laboratoire d’Hematologie, CHU de Saint-Etienne, Saint-Etienne, France 9 Institut de Cancerologie de la Loire, CHU de Saint-Etienne, Saint Priest en Jarez, France * These authors have contributed equally to this work Correspondence to: Eric Wattel, e-mail: eric.wattel@chu-lyon.fr Franck Mortreux, e-mail: franck.mortreux@ens-lyon.fr Keywords: acute myeloid leukemia, alternative splicing, WT1, DEK, multidrug resistance Received: March 16, 2015 Accepted: April 28, 2015 Published: May 12, 2015 ABSTRACT 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. These AEUs unmasked new functional pathways that are distinct from those generated by transcriptional deregulation. These results also identified new putative pathways that could help increase the understanding of the effects mediated by DEK or WT1, which may allow the targeting of these pathways to prevent resistance of AML cells to chemotherapeutic agents.

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 expression and triggered azacitidine resistance. In patients treated with azacitidine, decreased expression of anti-DNMT1 miRNAs was associated with poor outcome. miR-126* had the strongest prognostic impact. Patients with miR-126*low myelodysplastic syndrome had significantly lower response rates (P = 0.04) and higher relapse rates (P = 0.03), as well as shorter progression-free (PFS; P = 0.004) and overall survival (OS; P = 0.004). Multivariate analysis showed that age, miR-126* expression, and revised International Prognostic Scoring System risk independently predicted PFS and OS. In 15 patient samples collected over time, decreased miRNA expression levels were associated with secondary resistance. Conclusions: A decreased expression of anti-DNMT1 miRNAs might account for azacitidine resistance in HRMDS and AML, and measuring miRNA expression before and during treatment might help predict primary or secondary azacitidine resistance. Clin Cancer Res; 23(12); 3025–34. ©2016 AACR.

International audience; Replicative senescence is a hallmark of chronic liver diseases including chronic hepatitis B virus (HBV) infection, whereas HBV-encoded oncoproteins HBx and preS2 have been found to overcome senescence. HBx possesses a C-terminal truncation mainly in hepatocellular carcinomas but also in noncancerous liver tissues. Here, by cell counting, BrdU incorporation, MTT proliferation assay, cell cycle analysis, SA-betagal staining and Western blotting in primary and malignant cells, we investigated the effect of HBx C-terminal mutants on cellular senescence. HBx C-terminal mutants were found to trigger cellular senescence in primary MRC5 cells, and malignant liver cells Huh7, and SK-Hep1. In contrast, these mutants promoted the proliferation of HepG2 malignant liver cells. The pro-senescent effect of HBx relied on an increased p16(INK4a) and p21(Waf1/Cip1) expression, and a decreased phosphorylation of Rb. Together, these results suggest that the two main variants of HBx present in HBV-infected liver possess opposite effects on cellular senescence that depend on the phenotype of infected cells