Next Generation RNA Sequencing of Acute Promyelocytic Leukemia (APL) Identifies Novel Long Non Coding RNAs Including New Variants of MIR181A1HG That Are Differentially Expressed during Myeloid Differentiation

Introduction: Recently we identified a recurrent acquired genomic deletion on chromosome 1q as a potential new marker in approximately 14% of APL patients predicting a significantly increased risk of relapse (Nowak D et al., Genes Chromosomes and Cancer 2012). The deleted region contains the coding sequences for the microRNAs hsa-mir-181a1 and hsa-mir-181b1, which have been implicated as prognostic factors in Acute Myeloid Leukemia (AML) and a corresponding host gene (MIR181A1HG). To elucidate biologic mechanisms associated with the described genomic deletion we performed targeted sequencing of the affected region and RNA sequencing of APL samples carrying the deletion versus samples not carrying the deletion with subsequent validation of novel variants of MIR181A1HG.

Methods: Explorative sequencing of genomic DNA in the chromosomal subband 1q31.3, pos. 197073900-197196158 (hg18) was performed using the amplicon sequencing workflow of the Roche 454 platform sequencing 5000 bp fragments tiling a region of approximately 120 kb on n=3 APL samples. Corresponding patient samples from molecular remission were used as germline controls. Whole transcriptome sequencing of poly-A enriched RNA was performed on n=6 samples of bone marrow blasts of APL patients either carrying a deletion of the mir181a1/b1 coding region (n=3) or not carrying a deletion (n=3). RNA Sequencing was performed using the HiSeq2000 platform. Data analysis was carried out using Bowtie vers. 2.2.30, TopHat vers. 2.0.12 for alignment and mapping and the Cufflinks package vers. 2.2.1 for transcriptome assembly and expression analysis all using default settings and hg19 as reference genome. Validation of newly identified variants and differential expression of MIR181A1HG was carried out by RACE PCR and qRT-PCR on cDNA from primary leukemic blasts of APL patients (n=45), CD34+ cells from healthy donors (n=29). In vitro differentiation assays with concomitant gene expression analysis of MIR181A1HG variants were performed with CD34+ cells from healthy donors.

Results: Genomic sequencing of the recurrently deleted region revealed no somatically acquired mutations in the analyzed APL samples. Differential gene expression analysis using FPKM values (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) inferred from RNA sequencing data of APL samples carrying a genomic deletion of 1q31.3 versus non-deleted samples identified n=58 genes significantly downregulated in deleted samples and n=31 upregulated genes. Interestingly, among the differentially regulated genes, BAALC, a factor recently shown to be prognostically relevant in APL was significantly upregulated 13 fold in the unfavourable group of samples with 1q31.3 deletions. Furthermore, RNA sequencing revealed numerous new isoforms of known transcripts as well as novel long non-conding RNA (lncRNA) sequences. Among these were a total of 6 new transcript variants of the MIR181A1HG gene in the recurrently deleted region on chromosome 1q31.3. One novel 5600bp lncRNA covering the coding regions for the hsa-mir-181a1/b1 was 24 fold overexpressed in samples carrying the recurrent 1q31.3 deletions. Expression analysis of MIR181A1HG in blasts of APL patients, CD34+ cells, unselected bone marrow cells and granulocytes of healthy donors revealed significantly elevated levels of MIR181A1HG in APL cells as compared to healthy CD34+ cells and almost absent expression in unselected bone marrow and granulocytes. This indicated a possible role for MIR181A1HG in APL blasts and hematopoietic stem cells. Subsequent in vitro differentiation experiments of primary healthy CD34+ cells showed that MIR181A1HG is downregulated 7 fold within 14 days of cytokine induced myeloid differentiation. Furthermore, MIR181A1HG was downregulated 5 fold during ATRA induced differentiation of NB4 cells.

Conclusion: RNA sequencing of APL cells demonstrated numerous novel uncharacterized lncRNAs whose expression is associated with clinical risk and which merit further investigation. Identification of novel isoforms of MIR181A1HG, which are highly expressed in APL blasts and purified CD34+ cells suggest a potential role for this lncRNA in hematopoietic stem cells and response to ATRA induced differentiation of APL cells.