FUSION CIRCRNAS FROM THE KMT2A:AFF1 CHIMERIC GENE IN PEDIATRIC B-ALL PATIENTS AND CELL LINES HARBORING THE T(4;11)(Q21.3-Q22.1;Q23.3) TRANSLOCATION

Introduction: Circular RNAs (circRNAs) are endogenous transcripts originated as back-splicing products. Their circular structure confers higher stability than linear transcripts, resulting in their involvement, when deregulated, in several human diseases, including cancer. Increasing literature documents a predominant non-coding role as micro RNA sponges or transcriptional regulators, affecting the expression of genes involved in cell proliferation, invasion, apoptosis, and angiogenesis. circRNAs are also generated by the back-splicing of linear fusion transcripts derived from genomic rearrangements, giving rise to fusion circRNAs (f-circRNAs), as demonstrated in hematological and solid tumors harboring recurrent chromosomal rearrangements. Methods: By analyzing six bone marrow (BM) samples from pediatric patients at diagnosis of B-cell Acute Lymphoblastic Leukemia (BALL) and two cell lines (SEM and MV4-11), we investigated if the linear KMT2A:AFF1 fusion transcript, originated by the recurrent balanced t(4;11)(q21.3-q22.1;q23.3) translocation, might generate f-circRNAs. We performed RT-PCR and Sanger sequencing experiments with divergent primers, designed on each linear fusion transcript sequence, on RNAse R-digested RNA samples, using a pool of normal BM samples and the pre-B cell line NALM-6 as negative controls. RT-qPCR also evaluated the KMT2A:AFF1 f-circRNA expression in positive and negative samples, including B-ALL patients with the t(12;21) translocation or a normal karyotype. Results: The RT-PCR and Sanger sequencing results in SEM and MV4-11 cell lines indicated the generation of three f-circRNAs showing the back-splicing of AFF1 exon 8 with either KMT2A exon 2 or exon 1 and AFF1 exon 11 with KMT2A exon 2. The obtained products were not digested by RNAse R treatment, confirming their circular structure. Notably, the first and the third isoforms were also observed in pediatric B-ALL patients with the t(4;11) translocation, but not in patients without this rearrangement, indicating specificity for t(4;11) positive patients. Furthermore, one patient, tested in samples at onset and relapse, displayed the persistence of both f-circRNAs isoforms. Conclusions: Identifying specific f-circRNAs from the KMT2A:AFF1 fusion transcript is of relevance, as this evidence could help better understand the role of the t(4;11) rearrangement in the biology of pediatric B-ALL. The oncogenic role of such transcripts is under evaluation by in vitro studies. Furthermore, circRNAs and f-circRNAs are considered novel “liquid biopsy” biomarkers for early and non-invasive diagnosis of tumors, as well as therapeutic targets in human cancer. Therefore, such molecules may be crucial for future diagnostic/followup approaches and personalized therap