CO01 | A novel highly recurrent fusion circRNA from the kmt2a::aff1 fusion gene impacting mitochondrial metabolism in B-ALL with the t(4;11)(q21.3-q22.1;q23.3) translocation

Abstract

Introduction: Fusion circRNAs (f-circRNAs) may be generated by the back-splicing of linear fusion transcripts derived from genomic rearrangements. Recently, f-circRNAs from the KMT2A::AFF1 fusion gene, originated by a balanced t(4;11)(q21.3-q22.1;q23.3), were detected by bioinformatics approaches in the B-cell Acute Lymphoblastic Leukemia (B-ALL) RS4;11 cell line, as well as in patients, though their contribution to leukemia has not yet been studied.
In this framework, we identified a novel KMT2A::AFF1 f-circRNA back-splicing junction (BSJ) in B-ALL cell lines, investigated its oncogenic role in vitro, and determined its possible recurrence in B-ALL patients harboring the t(4;11) rearrangement.
Methods: KMT2A::AFF1 BSJs were investigated by PCR approaches and Sanger sequencing in RNAse R-treated and untreated samples of SEM, ALL-PO, and RS4;11 B-ALL cell lines with the t(4;11) translocation, as well as in pediatric and adult B-ALL patients, both with and without the t(4;11) translocation. The f-circRNA functional role was explored using total RNA-seq profiling of Dicer substrate small-interfering RNA (DsiRNA) BSJ knockdown cells, combined with cell death assays, confocal laser scanning microscopy (CLSM), Seahorse analysis, and transmission electron microscopy (TEM).
Results: RT-PCR/Sanger sequencing revealed a BSJ fusing AFF1 exon 8 to KMT2A exon 2 (AK_8_2), enriched in RNAse R-treated versus untreated samples, in three B-ALL cell lines, all carrying the t(4;11)(q24;q23)/KMT2A::AFF1 translocation. This sequence was also found in 8/10 pediatric and 7/10 adult t(4;11)-positive B-ALL patients, including three paired diagnosis-relapse samples, whereas it was absent in 23 t(4;11)-negative B-ALL samples. Total RNA-seq profiling of AK_8_2 knocked-down SEM cells identified upregulated genes involved in oxidative stress response and apoptosis regulation. Cell death analysis confirmed the pro-apoptotic impact of AK_8_2 silencing. Confocal analyses and Seahorse bioenergetic profiling demonstrated overproduction of reactive oxygen species, increased mitochondrial membrane potential, and enhanced mitochondrial function, accompanied by dysmorphic mitochondria, as observed by TEM, after AK_8_2 knockdown.
Conclusions: We identified AK_8_2 as a novel f-circRNA BSJ recurrent in t(4;11)-positive B-ALL patients at both diagnosis and relapse. Our data provide evidence of the functional role of this RNA molecule in apoptosis and mitochondrial metabolism, suggesting its potential involvement in B-ALL leukemogenesis.