WT1-associated missplicing in acute myeloid leukemia

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1 WT1-associated missplicing in acute myeloid leukemia
Aminetou Mint Mohamed, Morgan Thenoz, Delphine Maucort-Boulch, Meyling Cheok, Hussein Mortada, Didier Auboeuf, Mohamed El-Hamri, Catherine Koering, Isabelle Tigaud, Charles Dumontet, Emeline Cros, Christiane Pinatel, Olivier Nibourel, Claude Preudhomme, Xavier Thomas, Franck Nicolini, Françoise Solly, Eric Wattel, and Franck Mortreux CNRS, ENS, Lyon University Hospital, Lyon, France Background and Aim METHODS About one third of expressed genes are abnormally spliced in AML (1) Causes, mechanisms, and consequences of missplicing remain incompletely understood WT1 overexpressed in the majority of AML Possesses pleiotropic effect on mRNA splicing: represses the splicing-factor kinase SRPK1 (2) physically interacts with the splicing factors WTAP (3), RBM4 (4), and U2AF1/U2AF2 (5) (Figure 1). Aim: To determine whether and how WT1 modifies alternative exon usage (AEU) in AML and to test if these changes have a clinical impact Three AML cell lines: Kazumi, MOLM13, and KG1 were knocked down or not for WT1 through shRNA (Figure 2) Total RNA was analyzed using the Exon microarray platform GeneChip HTA2 (Affymetrix). Microarray data were cross-compared between cell lines and only statistically significant modifications (p<0.05) shared by at least 2 cell lines (MOLM13, Kasumi-1 and KG1) were selected (Figure 3) WT1-specific alternative exon usage (AEU) 4 ATP-binding cassette transporter mRNA (ABC-A2, -A3, -A5, and -C3) Quantified through quantitative exon-specific PCR (qESPCR) In cell lines In 132 AML MOLM13 KG1 KASUMI WT1 Vinculin PLKO shWT1 55KDa 110 KDa PLKO* shWT1 WT1 expression (arbitrary units) Figure 2. MOLM13, Kasumi-1, and KG1 AML cells were infected with lentiviral vectors encoding shWT1 or empty control vectors (PLKO). Protein lysates were prepared and western blot analysis was performed using antibodies to for WT1 (left). RNA was prepared and qRTPCR was performed (right) .   Figure 1. WT1 interact with the splicing machinery RESULTS Distribution of alternative exon usages in AML cell lines after WT1 expression For the two cell categories (WT1- high versus WT1- low), the venn diagrams (Figure 4) show the distribution of genes modified in whole gene expression level (white), alternative exon usage (dark grey), or both (light grey) For gene ontology analysis, gene lists were analyzed using DAVID software (KEGG pathways). The complete set of genes featured in microarrays was used as reference background. Top pathways are presented (p<0.05). Exon array data were confirmed by exon-specific PCR (75%, Figure 5) WT1-dependent missplicing of ABC transporter mRNAs in AML cells in vivo Typical results are representred for ABC A3 (Figure 6). The Figure represents the AEUs identified with FATERDB (top, 1), the position of oligonucleotides used for qESPCR (2), results of qESPCR for the 3 cell lines (bottom, left), and fresh AML samples (bottom, right), and the correlation between WT1 expression and ABC transporter expression in the 132 fresh AML samples and in the 37 donors (bottom, right) Similar results were obtained for ABC-A3, -A2, -C3, and-A5 A statistically significant correlation linked WT1 expression and the distribution of the 4 ABC transporter isoforms in the 132 AML diagnostic samples (Figure 6, p<0,01 for each, Spearman Rank Correlation) No correlation was observed with control samples. As an additional control experiment, qESPCR was carried out with the 132 AML for 2 AEUs of the TET2 gene mRNA that were not found to be linked with WT1 expression ex vivo. In contrast to ABC transporters, no correlation could be evidenced between WT1 expression and the distribution of the 4 TET2 isoforms in fresh AML samples. ABC transporter mRNA missplicing at diagnosis influences AML outcome in patients treated with intensive chemotherapy. Univariate analysis (Figure 7, Table 1) ABC-A3 exons expression, i.e. exon skipping of exon 19, significantly affected both OS and DFS; higher the level of isoform expression, poorer the outcome. Alternatively, an elevated expression level of the alternative ABC-A isoform was associated with statistically significant better OS and DFS. Multivariate analysis (Table 2), Age, cytogenetic, and ABC A3 exon 19 skipping were identified to be independent prognostic factors for OS and DFS Age and ABC-A3 exon exclusion were identified to be independent prognostic factor for OS in the 49 patients with normal karyotype. Figure 4. Distribution of quantitative and qualitative gene modifications in WT1 high versus WT1 low cells. Figure 5. Validation of microarray-predicted exon events through exon-specific RT-PCR Figure 6. WT1-dependent missplicing of ABC transporter mRNAs in AML cells in vivo. Figure 7. ABC transporter mRNA missplicing at diagnosis influences AML outcome in patients treated with intensive chemotherapy. CONCLUSIONS References In AML, WT1 affects AEUs of numerous key genes involved in hematologic differentiation, leukemogenesis, and resistance to chemotherapy 4/4 tested AEUs displayed the same correlation with WT1 expression in vivo and ex vivo. This correlation was specific because it was not observed in control samples or in AML with 2 TET2 AEUs found WT1-independent ex vivo. These results help explain The deregulated pattern of AEUs observed in AML cells where spliceosome gene mutations represent a rare event. The pathogenic effect of WT1 expression in AML ABC-A3 missplicing possesses a strong prognostic impact Rather than whole gene transcription analysis, qESPCR represents a promising tool for assessing AML aggressiveness at the time of diagnostic in patients with normal or abnormal karyotype. Adamia, S., et al. A Genome-wide aberrant RNA splicing in patients with acute myeloid leukemia identifies novel potential disease markers and therapeutic targets. Clinical cancer research : an official journal of the American Association for Cancer Research (2013). Amin, E.M., et al. WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing. Cancer cell 20, (2011). Little, N.A., et al. Identification of WTAP, a novel Wilms' tumour 1-associating protein. Human molecular genetics 9, (2000). Markus, M.A., et al. WT1 interacts with the splicing protein RBM4 and regulates its ability to modulate alternative splicing in vivo. Experimental cell research 312, (2006). Davies, R.C., et al. WT1 interacts with the splicing factor U2AF65 in an isoform-dependent manner and can be incorporated into spliceosomes. Genes & development 12, (1998).


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