RNF4 sustains Myc-driven tumorigenesis by facilitating DNA replication
Joonyoung Her, … , Haiyan Zheng, Samuel F. Bunting
Joonyoung Her, … , Haiyan Zheng, Samuel F. Bunting
Published March 26, 2024
Citation Information: J Clin Invest. 2024;134(10):e167419. https://doi.org/10.1172/JCI167419.
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Research Article Genetics Oncology

RNF4 sustains Myc-driven tumorigenesis by facilitating DNA replication

Abstract

The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4–conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.

Authors

Joonyoung Her, Haiyan Zheng, Samuel F. Bunting

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Figure 2

Rnf4Δ/Δ B cells show defects in DNA replication.

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Rnf4Δ/Δ B cells show defects in DNA replication. (A) Analysis of chromo...
(A) Analysis of chromosome aberrations in B lymphocytes treated overnight with or without 2 μM olaparib or 1 μM cisplatin. Statistical differences between the means of the treated and nontreated samples are shown. (B) Flow cytometry analysis of CFSE dilution to measure B cell growth over 72 hours. Chart shows quantification of cell doublings based on CFSE fluorescence. (C) Western blot analysis of caspase-3 cleavage and γ-H2AX after no treatment (NT) or after overnight recovery from treatment with 2 Gy of ionizing radiation (IR), 0.4 μM aphidicolin (APH), 2.5 μM cisplatin (CDDP), or 100 μM MMS. (D) Sample flow cytometry data quantifying cell viability after treatment with gemcitabine (GEM). Gated population shows the viable, propidium iodide–negative population. Graph shows proportion of cells that became nonviable 24 hours after treatment with either hydroxyurea (HU) (4 mM, 3 hours), APH (40 μM, 2 hours), or GEM (250 nM, 2 hours). (E) Immunofluorescent detection of 53BP1 G1 nuclear bodies (green) in B cells after in vitro culture. Cyclin A staining (red) reveals S/G2-phase cells. Scale bars: 10 μm. (F) EdU uptake measured by flow cytometry. (G) Analysis of nascent DNA tract length in WT and Rnf4Δ/Δ splenic B cells by DNA combing. Mean ± SD of n = 3 experiments shown. (H) Stability of nascent DNA measured by fiber analysis after 4 mM HU treatment. Mean ± SD of n = 3 experiments shown. (I) Western blot showing induction of p-CHK1 in WT and Rnf4Δ/Δ cells after IR (30 Gy, 2 hours recovery), APH (0.4 μM, overnight), GEM (100 nM, 2 hours), or MMS (200 μM, 3 hours). Error bars in A, B, and D–F show SD of the mean, with P values calculated by unpaired 2-tailed t test. P values in G and H were calculated by paired, 2-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.