SF3B1 mutation and ATM deletion co-drive leukemogenesis via centromeric R-loop dysregulation

RNA splicing factor SF3B1 is recurrently mutated in various cancers, particularly in hematological malig-nancies. We previously reported that co-expression of Sf3b1 mutation and Atm deletion in B cells, but not either lesion alone, leads to the onset of chronic lymphocytic leukemia (CLL) with CLL cells harboring chromosome amplification. However, the exact role of Sf3b1 mutation and Atm deletion in chromosomal instability (CIN) remains unclear. Here, we demonstrate that SF3B1 mutation promotes centromeric R-loop (cen-R-loop) accumulation, leading to increased chromosome oscillation, impaired chromosome segrega-tion, altered spindle architecture and aneuploidy, which can be alleviated by removal of cen-R-loop and exaggerated by deletion of ATM. Aberrant splicing of key genes involved in R-loop processing underlies augmentation of cen-R-loop as overexpression of the normal isoform, but not the altered form, mitigates mitotic stress in SF3B1 mutant cells. Our study underscores the critical role of novel splice variants in link-ing RNA splicing dysregulation and CIN, and highlights cen-R-loop augmentation as a key mechanism for leukemogenesis. DNA:RNA hybrids (R-loop) were profiled in Nalm-6 SF3B1-K700K and SF3B1-K700E by high troughput sequencing. DNA:RNA hybrids immunoprecipitation (DRIP) were performed with S9.6 antibody extracted from hybridoma cell line. RNA analysis were performed in SF3B1-K700K and SF3B1-K700E HEK293T cells.