U2AF1 Splicing Factor Mutation and Cajal Bodies in Myeloid Malignancies

Splicing factor (SF) mutations are a hallmark of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), occurring in over 50% of patients. Among these, mutations in U2AF1 are recurrent and associated with poor clinical outcomes in MDS. SF mutations are known to cause widespread RNA splicing alterations that drive ineffective hematopoiesis, and recent findings demonstrated that these alterations are associated to changes in the biology of membraneless organelles. Cajal bodies (CBs) are dynamic, membraneless nuclear organelles that serve as key hubs for genome organization and RNA splicing kinetics. Beyond their role in RNA processing, CBs are also critically involved in telomere biology. They serve as the assembly point of TERC telomerase RNA and TERT telomerase enzyme into the nascent telomerase ribonucleoprotein complex, facilitating telomerase trafficking and activity to telomeres. To investigate the link between U2AF1 mutations and CB biology and function in the context of myeloid malignancies, we first engineered human erythroleukemia (HEL) cell lines to express exogenous FLAG-tagged U2AF1 wild-type and S34F mutant under doxycycline induction. After cell validation, we employed immunofluorescence (IF) staining followed by confocal microscopy to quantitatively assess CB number and nuclear distribution in S34F mutant cells compared to wild-type controls. Our findings reveal that S34F mutant HEL cells exhibit a significant upregulation of CBs compared to wild-type cells, as evidenced by increased mean CB number and intensity. This increased in CBs could be associated to an increase telomerase activity, thereby explaining the clonal advantage of S34F mutant cells in the bone marrow microenvironment.