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

Introduction: Fusion circRNAs (f-circRNAs) may be generat ed 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 con tribution to leukemia has not yet been studied. In this frame work, we identified a novel KMT2A::AFF1 f-circRNA back-s plicing junction (BSJ) in B-ALL cell lines, investigated its oncogenic role in vitro, and determined its possible recur rence in B-ALL patients harboring the t(4;11) rearrange ment. Methods: KMT2A::AFF1 BSJs were investigated by PCR ap proaches and Sanger sequencing in RNAse R-treated and un treated 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 us ing total RNA-seq profiling of Dicer substrate small-interfer ing 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 translo cation. 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 reac tive oxygen species, increased mitochondrial membrane po tential, 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.