Doron Tolomeo, Michela Bardini, Santina Venuto, Grazia Visci, Angelo Lonoce, Lucrezia Calamo, Ingrid Cifola, Marco Severgnini, Maria Mastrodonato, Daniela Semeraro, Luigia Valsecchi, Mario Mauri, Roberta La Starza, Martina Lucarelli, Amalia Azzariti, Simona Serratì, Anna Ferrari, Giorgia Simonetti, Giovanni Martinelli, Nicola Santoro, Monica Fumagalli, Gaia Di Timoteo, Ilaria Saltarella, Martina Milani, Orietta Spinelli, Nicoletta Bertorello, Giovanni Cazzaniga, Grazia Fazio, Clelia Tiziana Storlazzi
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.