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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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 1

SF3B1 mutation triggers cen-R-loop accumulation.

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SF3B1 mutation triggers cen-R-loop accumulation. (A) R-loop level quant...
(A) R-loop level quantified by dotblot assay with S9.6 antibody in K562, HEK293T, and Nalm-6 SF3B1-WT and -MT cells. Serial DNA dilutions starting from 4 mg (K562 and Nalm-6) or 2 mg (HEK293T). Single-strand DNA (ssDNA) blotting was used as loading control. Top: Representative image. Bottom: S9.6 signal quantification over ssDNA signal. Bar graphs represent mean; dots represent biological replicates. Two-way paired ANOVA test. (B) Representative images (top) of neutral comet assay for double-strand breaks in Nalm-6 SF3B1-WT and -MT cells with overexpression of either empty vector (EV) or RNaseH1 (RNH1) and relative comet tail moment (bottom) in 3 biological replicates. Scale bars: 100 μm. Total comets quantified range from 435 to 742 cells. Box plots show the median and 25th and 75th percentiles, with whiskers extending to minimum and maximum values. Two-tailed unpaired t test followed by Bonferroni’s post hoc test. (C) Cen-R-loops are recognized and coated by phospho–RPA S33 (p-RPA). RNAPII, RNA polymerase II. Created with BioRender (biorender.com). (D) Top: RNH1 WT and mutant vectors. NLS, nuclear localization signal; LR, linker region. Bottom: Detection of overexpression of RNH1 with V5 tag by immunoblot in Nalm-6 SF3B1-WT and -MT cells overexpressing either WT or WKKD RNH1 protein. GAPDH was used as loading control. (E) Representative images of cen-R-loops detected by p-RPA (red) and ACA (green) immunofluorescence. Scale bars: 2 μm. (F) Quantified centromeric p-RPA signal normalized to background signal near centromeres (see Methods). Graphs represent mean ± SEM. The number of chromosomes quantified ranges from 46 to 67. SF3B1 MT overexpressing WKKD vs. WT RNH1, P = 0.0001, Wilcoxon’s paired test.