Abstract 5198: Identification of microRNAs involved in DNA damage response in malignant B cells and their biological and clinical relevance
Authors: Cerna Katerina , Oppelt Jan , Radova Lenka , Musilova Katerina , Tom Nikola , Pardy Filip , Malcikova Jitka , Plevova Karla , Tichy Boris , Brychtova Yvona , Doubek Michael , Trbusek Martin , Mayer Jiri , Koca Jaroslav , Calogero Raffaele , Pospisilova Sarka , Mraz Marek 
Published: May 2015

Abstract
We and others have shown that expression of miRNAs influences the biology of B cell malignancies (Calin et al., 2005; Mraz et al., 2009, 2012, 2013). The aim of this study was to identify miRNAs involved in the apoptotic response of malignant B cells. Purified primary B cells of chronic lymphocytic leukemia (CLL) patients (pts.) were treated in vitro with fludarabine (F) (LC50 dose of 3.5 μg/ml; 48 h). Five paired (n=10) samples (with and without F) were analyzed using 2 NGS platforms-SOLiD (ABI) and HiSeq (Illumina). The obtained reads were mapped to miRBase using 3 tools (CLC Genomic Workbench, SHRiMP2, miRanalyzer) and data analyzed by a pair-wise comparison with edgeR and baySeq packages (Bioconductor 2.13). The overlap of these 3+2 bioinformatic approaches identified 6 miRNAs significantly changed with DNA damage. RNA-Seq validation on 5 additional paired samples (n=10) confirmed the changed expression of all 6 previously identified miRNAs (3 down-, 3 up-regulated). The most constantly up-regulated was miR-34a, which was previously shown to be regulated by p53 (He et al., 2005; Mraz et al., 2009). To test the importance of miR-34a in vivo, we collected samples from CLL pts. (n=51) treated with F, cyclophosphamide and rituximab (FCR) regimen. miR-34a was induced in all samples after F administration (day 2, p&lt;0.001). Surprisingly, the lower basal and induced levels of miR-34a correlated with significantly (p&lt;0.05) shorter time to treatment failure, suggesting its strong prognostic potential. We further determined the expression of miR-34a in a large cohort of CLL pts. (n=158) using an in-house designed assay for its copy-number quantification. We defined a cut-point (number of miR-34a copies) that segregates pts. with extremely unfavorable prognosis (overall survival [OS] 1.37 yrs. vs. not reached; p=0.0001; HR=3.89; CI=2.05-7.39) and this was independent of routinely used prognostic markers (FISH, IgHV, age, sex) in a multivariate analysis. We have previously described that low levels of miR-34a associate with TP53 abnormalities, so we limited the analysis to wt-TP53 samples (n=116). In this multivariate analysis miR-34a was the strongest predictor of OS (1.29 yrs. vs. not reached; p=0.002; HR=9.82; CI=2.30-42.05). The molecular pathways affected by miR-34a levels in B cells are largely unknown. However, miR-34a shares 51 predicted target mRNAs (evolutionary conserved) with at least 1 other F induced miRNA, suggesting that they might cooperate in the regulation of these genes. The pathways regulated by these miRNAs are currently being investigated using integrated analysis of miRNA and transcriptome profiling of CLL samples (n=100).
Supported by: NGS-PTL (FP7-HEALTH-2012-INNOVATION-1, no. 306242); EHA Research Fellowship award; Grant Agency of Czech Rep.; IGA MZ CR NT11218-6/2010; CZ.1.07/2.4.00/17.0042; MUNI/A/0723/2012; CZ.1.07/2.3.00/30.0009, co-financed from EU and Czech Rep.
Citation Format: Katerina Cerna, Jan Oppelt, Lenka Radova, Katerina Musilova, Nikola Tom, Filip Pardy, Jitka Malcikova, Karla Plevova, Boris Tichy, Yvona Brychtova, Michael Doubek, Martin Trbusek, Jiri Mayer, Jaroslav Koca, Raffaele Calogero, Sarka Pospisilova, Marek Mraz. Identification of microRNAs involved in DNA damage response in malignant B cells and their biological and clinical relevance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5198. doi:10.1158/1538-7445.AM2014-5198