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 8

R-loop accumulation is a feature of murine CLL with Sf3b1 mutation and Atm deletion.

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R-loop accumulation is a feature of murine CLL with Sf3b1 mutation and A...
(A) Quantification of centromeric p-RPA signal in Nalm-6 Cas9 SF3B1-WT and -MT cells with and without ATM knockdown. Two-tailed paired t test, Nalm-6 WT vs. SF3B1 MT, or vs. ATM MT, or vs. DM, P < 0.0001; ATM MT vs. DM, P < 0.0001; SF3B1 MT vs. DM, P < 0.0001. The number of chromosomes quantified ranges from 56 to 113. (B and C) Quantification of 2-dimensional cross-sectional area of the entire body of chromosomes (B) and spindle length and width (C) in metaphases of cells described in A. Box plots show the median and 25th and 75th percentiles, with whiskers extending to minimum and maximum values. Dots represent biological replicates. Two-tailed unpaired t test followed by Bonferroni’s post hoc test. (D) Left: Representative images of R-loops detected by IF with S9.6 antibody (red) in WT, Sf3b1-MT, Atm-deleted (MT), and Sf3b1-MT and Atm-deleted (DM) murine splenic B cells. Scale bars: 5 μm. Right: Quantification of S9.6 nuclear fluorescence intensity. Number of mice used for each genotype is indicated. The number of cells quantified ranges from 2135 to 3690. Center lines show the medians. Two-tailed unpaired t test followed by Bonferroni’s post hoc test. (E) Top: Dotblot assay using splenic B cells derived from DM mice without and with CLL. Bottom: Relative S9.6 signal quantification normalized over ssDNA signal. Each bar represents 1 biological replicate. (F) DRIP-qPCR analysis of R-loop enrichment over negative (Snrpn) and positive (c-Myc, Snord116) loci for R-loop accumulation, over representative genes (Prkce, Drosha, Ddx17, Parp8, Pouf5l, and Akt) and centromeric regions (minor satellites), in normal and CLL B cells derived from DM mice. RNH1 treatment is included as control. Data are presented as mean ± SEM (n = 3, technical replicates). One-way ANOVA Tukey’s test. Untreated vs. RNH1-treated is significant for all samples tested (P < 0.0001).