SF3B1 mutation and ATM deletion codrive leukemogenesis via centromeric R-loop dysregulation
Martina Cusan, … , Ren-Jang Lin, Lili Wang
Martina Cusan, … , Ren-Jang Lin, Lili Wang
Published July 18, 2023
Citation Information: J Clin Invest. 2023;133(17):e163325. https://doi.org/10.1172/JCI163325.
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Research Article Oncology

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

Abstract

RNA splicing factor SF3B1 is recurrently mutated in various cancers, particularly in hematologic malignancies. We previously reported that coexpression 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 demonstrated that SF3B1 mutation promotes centromeric R-loop (cen-R-loop) accumulation, leading to increased chromosome oscillation, impaired chromosome segregation, altered spindle architecture, and aneuploidy, which could be alleviated by removal of cen-R-loop and exaggerated by deletion of ATM. Aberrant splicing of key genes involved in R-loop processing underlay augmentation of cen-R-loop, as overexpression of the normal isoform, but not the altered form, mitigated mitotic stress in SF3B1-mutant cells. Our study identifies a critical role of splice variants in linking RNA splicing dysregulation and CIN and highlights cen-R-loop augmentation as a key mechanism for leukemogenesis.

Authors

Martina Cusan, Haifeng Shen, Bo Zhang, Aijun Liao, Lu Yang, Meiling Jin, Mike Fernandez, Prajish Iyer, Yiming Wu, Kevyn Hart, Catherine Gutierrez, Sara Nik, Shondra M. Pruett-Miller, Jeremy Stark, Esther A. Obeng, Teresa V. Bowman, Catherine J. Wu, Ren-Jang Lin, Lili Wang

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Figure 4

SF3B1 mutation–associated R-loops have minimal overlapping with splice variants.

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SF3B1 mutation–associated R-loops have minimal overlapping with splice ...
(A) Quantification of genome-wide bona fide R-loops in Nalm-6 WT and SF3B1 MT detected by DRIP-seq. (B) Volcano plot of differential bona fide R-loops between SF3B1-MT and -WT cells. Significant differential peaks cutoff as FDR < 0.05 and fold change > 1.5. (C) Genomic distribution of differential upregulated bona fide R-loops associated with SF3B1 mutation with UpSet and PieChart plots. Intersection size indicates the number of R-loops. The black dots connected with lines represent overlapped R-loops. (D) Integrative Genomics Viewer (IGV) of R-loops profiled by DRIP-seq over indicated upregulated and downregulated genes. (E) Validation of differential R-loop peaks in D by DRIP-qPCR assay. RNH1 treatment is included as background control. Graphs represent qPCR results of biological duplicates; fold change over paired RNH1 treatment is presented as mean ± SEM; 2-tailed unpaired t test. (F) Quantification of centromeric bona fide R-loops detected by DRIP-seq. (G) SF3B1 mutation–associated cen-R-loops validated using DRIP-qPCR in mitotic cells with and without SF3B1 mutation. Chromosome 1 (Chr1) centromere, pericentromere, and telomeric 1q TERRA regions tested for R-loop accumulation. SNRPN was used as negative control. RNH1-treated samples were used as an R-loop background control. Graphs represent qPCR results expressed as percentage of input mean ± SEM. Dots represent technical replicates of 2 biological replicates. (H) Venn diagram demonstrates overlap between Nalm-6 SF3B1 MT–associated alternative splice variants and bona fide R-loop peaks in WT (blue) and MT (red) SF3B1.