P415: TARGETED 3RD-GENERATION (NANOPORE) SEQUENCING REVEALS MANY NOVEL CIRCULAR RNAS OF THE APOPTOSIS-RELATED BCL2L12 GENE, EXPRESSED IN HUMAN CELLS FROM ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES

Abstract

Background: Circular RNAs (circRNAs) are non-coding RNAs generated via back-splicing. They are indirect – yet pivotal – regulators of gene expression, as many of them bind microRNAs (miRNAs) and render them unavailable to exert their direct regulatory functions. They also interact with chromatin, altering its structure and affecting transcription of several genes. Lastly, though considered as non-coding, few of them have also been shown to encode short, functional polypeptides. Due to their multifaceted role and the deregulation of their expression in cancer, circRNAs are related to the development and progression of malignancies, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). BCL2L12 is a prominent member of the apoptosis-related BCL2 family, with an already established role in AML. Moreover, BCL2L12 mRNA expression has been associated with chemoresistance of malignant bone marrow blasts. However, the identity and role of the circRNAs produced by alternative back-splicing of BCL2L12 primary transcripts in MDS and AML has not been investigated, so far. Aims: We sought to determine the identity of BCL2L12 circRNAs expressed in MDS and AML cell lines and to explore their putative interactions with miRNAs having an established role in these disease entities. Methods: MDS-L cells [MDS with del(5q) and complex karyotype], NB-4 and HL-60 cells (acute promyelocytic leukemia with and without PML-RARA, respectively), U-937 and THP-1 cells (acute monocytic leukemia), KASUMI-1 cells (AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1) and MOLM-13 cells (AML with t(9;11)(p21.3;q23.3); KMT2A-MLLT3) were propagated. Total RNA was extracted from these seven cell lines; 2μg of each RNA extract were reverse-transcribed using random hexamers. Next, nested PCR with divergent primers was performed, starting from each exon of the BCL2L12 gene; thus, only cDNAs from BCL2L12 circRNAs were amplified. After having mixed and purified all nested-PCR products of each cell line cDNA, nanopore sequencing libraries were built. Following nanopore sequencing and basecalling in a MinION Mk1C 3rd-generation sequencer, sequencing reads were aligned and corrected using Minimap2 and TranscriptClean, respectively. circRNA identification was performed using an in-house–built, PERL-based algorithm; miRDB was used for prediction of their interactions with miRNAs. Results: We discovered 83 novel BCL2L12 circRNAs, 9 of which were common between MDS-L cells and at least one AML cell line. The exon structure of these novel circRNAs showed a remarkable diversity, comprising exons also encountered in messenger RNAs (mRNAs) of BCL2L12 as intronic regions. This phenomenon was mostly observed in MDS-L cells. Several BCL2L12 circRNAs in NB-4 and HL-60 cells exhibited complete intron retention; additionally, a novel exon was present at a remarkable frequency in NB-4 cells. Moreover, several BCL2L12 circRNAs are predicted to sponge miRNAs with a key role in AML (miR-7-5p, miR-125b-5p and miR-182-5p) and/or MDS (miR-150-5p). Summary/Conclusion: Numerous circRNAs are produced through alternative back-splicing of the primary BCL2L12 transcripts. The circRNA expression profile of this gene differs among the MDS and AML cell lines, as well as among the six AML cell lines. Intronic regions of this apoptosis-related gene are included in several circRNAs, augmenting the repertoire of miRNAs predicted to be bound by BCL2L12 transcripts. This phenomenon was more common in the MDS cell line, followed by both acute promyelocytic leukemia cell lines. Our data suggest an important regulatory role for this gene in MDS and AML subtypes.