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 6

SF3B1 mutation modulates R-loop metabolism through SERBP1 alternative splicing.

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SF3B1 mutation modulates R-loop metabolism through SERBP1 alternative s...
(A) IGV of RNA-Seq reads covering the cryptic 3′ splice site of the SERBP1 gene in SF3B1-WT and -MT Nalm-6 cells. (B) Immunoblots of HEK293T SF3B1-WT and -MT cells overexpressing either FLAG empty vector (EV) or SERBP1 FLAG-tagged isoforms. (C) Quantification of centromeric p-RPA immunofluorescence signal normalized to background near centromeres from cells described in B. SF3B1-MT EV cell line results are reported in 2 different graphs for better visualization. Wilcoxon’s paired test. HEK293T WT EV vs. SF3B1-MT EV, P < 0.0001; SF3B1-MT EV vs. SF3B1-MT SERBP1 normal isoform, P < 0.0001; SF3B1-MT EV vs. SF3B1-MT SERBP1 alternative isoform, P = NS. The number of chromosomes quantified ranges from 39 to 50. (D) Representative R-loops (left) and relative quantification (right) from dotblot assay in cells from B. Bars represent mean; dots represent biological replicates. One-way ANOVA comparison test. (E) Alkaline comet assay in cells as in B. Box plots show the median and 25th and 75th percentiles, with whiskers extending to minimum and maximum values. One-way ANOVA Dunnett’s multiple test. Comets quantified range from 783 to 995. (F) Left: Representative immunoblot of HEK293T cells as in B, treated for the indicated times with cycloheximide (CHX). Right: FLAG and SERBP1 immunoblot quantification normalized over GAPDH. (G) eCLIP-qPCR performed with HEK293T cells transfected as in B. SF3B2, ATP5F1B, MTR, PKM, ZFR, and DYNLL1 were selected based on SERBP1 predicted mRNA target and R-loop–forming genes associated with SF3B1 mutation. SNRPN, NOP10, and UQRCB were selected as negative controls.