A novel KMT2A::AFF1-derived fusion circRNA regulates mitochondrial metabolism in t(4;11) B-cell acute lymphoblastic leukemia.

The t(4;11) translocation, which drives KMT2A::AFF1 fusion gene expression, is associated with poor prognosis in pediatric and adult B-cell Acute Lymphoblastic Leukemia (B-ALL). KMT2A::AFF1 fusion circular RNAs (f-circRNAs) have been identified in B-ALL, though their contribution to leukemia has not yet been outlined. We identified a novel recurrent KMT2A::AFF1-derived back-splicing junction joining AFF1 exon 7 to KMT2A exon 3 (AK_7_3) in SEM, RS4;11, and ALL-PO B-ALL cell lines, all of which carry the t(4;11)(q24;q23)/KMT2A::AFF1 translocation. This sequence was observed in RT-PCR products encompassing two splicing variants of KMT2A exon 4, which are also present in the linear KMT2A::AFF1 chimera. AK_7_3 was also found in 12/18 pediatric and 17/24 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 patients. Whole-transcriptome analysis of AK_7_3-knockdown SEM cells identified upregulated genes involved in oxidative stress response and regulation of apoptosis. Cell death analysis confirmed the pro-apoptotic impact of AK_7_3 silencing. Confocal analyses, Seahorse bioenergetic profiling, and electron microscopy revealed an overproduction of reactive oxygen species, increased mitochondrial membrane potential, mitochondrial respiration, and dysmorphic mitochondria after AK_7_3 knockdown in KMT2A::AFF1 cell lines. In summary, we identified AK_7_3 as a novel f-circRNA back-splicing junction recurrent in t(4;11)-positive B-ALL patients at both diagnosis and relapse. Our data provide evidence for a functional role of this molecule as an anti-apoptotic agent that affects mitochondrial function, suggesting its potential involvement in KMT2A::AFF1-driven B-ALL leukemogenesis.