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. Overall design: 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.

RNA剪接因子SF3B1在多种癌症中频发突变，尤其在血液系统恶性肿瘤（hematological malignancies）中。本团队此前研究显示，在B细胞中同时存在Sf3b1突变与Atm缺失（而非单一的这两种损伤之一），会诱发伴有染色体扩增的慢性淋巴细胞白血病（chronic lymphocytic leukemia, CLL）。然而，Sf3b1突变与Atm缺失在染色体不稳定性（chromosomal instability, CIN）中的确切作用仍不明确。本研究证实，SF3B1突变会促进着丝粒R环（centromeric R-loop, cen-R-loop）的积累，进而引发染色体振荡加剧、染色体分离受损、纺锤体结构异常以及非整倍体；该表型可通过清除cen-R-loop得到缓解，而ATM缺失则会使其加重。参与R环（R-loop）加工的关键基因的异常剪接是cen-R-loop积累增强的基础：过表达正常剪接异构体（而非异常剪接异构体），可缓解SF3B1突变细胞中的有丝分裂应激。本研究强调了新型剪接变体在连接RNA剪接失调与染色体不稳定性（CIN）中的关键作用，并揭示cen-R-loop积累增强是白血病发生的核心机制之一。整体实验设计：通过高通量测序对Nalm-6细胞中SF3B1-K700K与SF3B1-K700E模型的DNA:RNA杂交链（R-loop）进行了检测分析；采用从杂交瘤细胞系中提取的S9.6抗体完成DNA:RNA杂交链免疫沉淀（DNA:RNA hybrids immunoprecipitation, DRIP）实验；同时对SF3B1-K700K与SF3B1-K700E的HEK293T细胞开展了RNA表达分析。