Topoisomerase IIIβ protects from tumorigenesis and immune dysregulation

Abstract

Topoisomerase III-beta (Top3b) reduces nucleic acid torsional stress and intertwining generated during RNA and DNA metabolism while protecting the genome from pathological R-loops, which otherwise result in DNA breakage and genome instability. By studying Top3b knockout mice (Top3b-KO), we find that the loss of Top3b accelerates the development of spontaneous lymphoid tumors arising in spleens and lymph nodes, the organs with prominent Top3b expression. Aging Top3b-KO mice also display splenomegaly and systemic immune alterations including neutrophilia and lymphopenia suggestive of chronic inflammation. At the molecular level, Top3b deficiency causes genome-wide R-loop accumulation in splenocytes as measured by CUT&Tag sequencing. Increased R-loops is associated with genomic DNA breaks and activation of immune signaling pathways including the IL-6 signaling, interleukin-7 signaling and cGAS-STING. Moreover, knocking-out Top3b promotes the rapid development of syngeneic EL4 T-cell lymphomas. In conclusion, our work implies that, in addition to its role in preserving the nervous system, Top3b protects from tumorigenesis and immune dysregulations.

Keywords: Cell Biology; DNA breaks; Immune dysregulation; Lymphoma; R-loop; Topoisomerase.

Fig. 1.. Loss of Top 3b predisposes to spontaneous lymphomagenesis.

(A) Elevated incidence of lymphomas in Top3b-KO mice. Data are mean ± SEM. *p < 0.05 calculated using two-tailed Student’s t-test. (B) Representative splenic structural alterations at 15 months of age. Paraffin-embedded spleen sections stained with hematoxylin and eosin (H&E) and B220, CD3 and F4/80 antibodies show diffuse white pulp expansion in Top3b-KO mice. Scale bar, 1 mm. (C) Representative H&E staining of spleens from 15-month-old WT and Top3b-KO female mice. Note dark staining of lymphocytes within the follicular compartment with distinct borders in WT spleen as compared to the pale immature lymphocytes with high rate of mitosis (white arrow) invading into and expanding follicular compartment in Top3b-KO. Higher magnifications of white box areas are shown on the right, Scale bars, 50 μm. (D) Representative low magnification images of lymphomas (upper panel) in the spleen and lymph node from 21-month-old Top3b-KO female mouse. Note that neoplastic cells occupy the entire spleen and lymph node. Five-μm paraffin-embedded sections stained with H&E. Scale bar, 1 mm. Microscopic features (lower panel) of lymphoid neoplasms outlined by white boxes in upper panel (D) show diffuse expansion of uniform neoplastic cells with distinct border, round nuclei, and scant cytoplasm morphologically resembling lymphoblasts. H&E staining. Scale bars, 100 μm. (E) Immunohistochemical staining (IHC) of splenic lymphoma (inset) with anti-B220 and anti-CD3 antibodies. Most neoplastic cells are strongly positive for B220, with a smaller population of CD3⁺ T cells. Scale bar, 100 μm. (F) Representative IHC images with lymphocyte-specific (B220, CD3) and proliferation (Ki67) markers in Payer’s patch lymphoma from a 22-month-old Top3b-KO male mouse. Scale bars, 500 μm. Nuclei were co-stained with hematoxylin.

Fig. 2.. Splenomegaly and immune dysregulation in Top3b-KO mice.

(A) Spleen-to-body weight ratio in female and male Top3b-KO mice. Mean ± SEM, left to right: n = 15/5/22/4/25/8/33/17 mice. (B) Increased splenocytes and Flow cytometry analysis (FACS) of splenic B220⁺, CD4⁺ T and CD8⁺ T cells; Left to right: n = 6/6/5–6/3–5 mice. Data are mean ± SEM. (C) Blood analysis showing increase in neutrophils, decrease in lymphocytes and elevation of neutrophil-to-leucocyte ratio in Top3b-KO female mice. Data are mean ± SEM, n =3–5 mice per group. (D) Serum interleukin-6 (IL6) levels in 11–15 months female mice: WT (n = 8), Top3b-KO (n = 9). (E) Frequency of immune subsets in liver tissues of WT (n=4) and Top3b-KO (n=8) mice at 15–19 months of age. Data are mean ± SEM. p values were determined using unpaired two tailed Welch’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: non-significant.

Fig. 3.. Loss of Top3b results in genome-wide accumulation of R-loops.

(A) Experimental design. (B) Representative slot blot showing increased accumulation of R-loops in Top3b-KO mouse splenocytes compared to WT. Genomic DNA was probed with S9.6 antibody. (C) Quantitation of 3 independent slot-blot experiments. Data are plotted as means, n = 3 per genotype. Statistical significance was calculated using two-tailed Student’s t-test, *p < 0.05. (D) R-loop CUT&Tag-seq signals for WT and KO splenocytes in regions ± 2 Kb from the R-loop centers. Heatmap color intensity represents R-loop CUT&Tag-seq signals level. (E) Coverage-plot of normalized R-loop peak signals in 31,922 genes showing R-loops enrichment in 23,553 genes (upper left, read) and depletion in 8,369 genes (lower right, blue) in Top3b-KO mice. FPKM, Fragments Per Kilobase Million. (F) Distribution of R-loop peak intensity expressed across different genomic features. (G) Average R-loop signal intensity is positively correlated with gene length. 31,922 genes were divided into four different quartiles: Very short (< 0.1 kb), Short (≥ 0.1 kb and < 1 kb), Medium (≥ 1 kb and < 10 kb) and Long (≥ 10 kb), and distribution of average R-loop signal intensity in each quartile is plotted. p values were determined using the Mann Whitney test. ****p < 0.0001.

Fig. 4.. Top3b loss leads to dysregulation of the immunotransciptome.

(A) Heatmap showing distribution of RNA signal across differentially down-regulated (862) and up-regulated (509) genes in three independent RNA-seq experiments in Top3b-KO splenocytes. (B) Volcano plots showing some of the differentially expressed genes (DEGs) in Top3b-KO splenocytes. DEGs were analyzed using LIMMA. (C) Functional annotation of the significantly altered immune pathways of DEGs (1,371) determined by Ingenuity Pathway Analysis.

Fig. 5.. Increased DNA breaks and cGas-Sting protein levels in Top3b-KO splenocytes.

(A) Lack of correlation between R-loop levels and RNA-expression. (B) Examples of R-loop signals (IVG tracing) in representative genomic regions determined by CUT&Tag-seq in WT (green) and KO (red) splenocytes. R-loop signals were group auto-scaled with the Top3b; scales are indicated at right. (C) Representative image of immunofluorescence staining of 3-month-old female mouse splenocytes with anti-gamma-H2AX (γH2AX) antibody. n = 3 mice per genotype. Nuclei were co-stained with DAPI and outlined with white lines. Scale bar, 10 μm. (D) Quantification of γH2AX foci number and foci intensity shown in (C). 200 nuclei were analyzed by Random-Forest ML pixel-classifier per each mouse. Data are means ± SEM; ****p < 0.0001, unpaired, two-tailed Student’s t-test. (E-F) Detection and quantification of γH2AX (E), cGas (F) and Sting (G) by Western blotting. GAPDH was used as loading control. n = 4 mice per genotype. Data are means ± SEM; **p < 0.01, ***p < 0.001 unpaired, two-tailed Student’s t-test.

Fig. 6.. Top3b-deficiency promotes syngeneic growth of EL4 lymphoma.

(A) Experimental scheme. 10,000 EL4 tumor cells stably transfected with a luciferase reporter gene were injected subcutaneously (s.c.) into the right flank of 3-month-old female mice (n = 5 for each genotype). Data are representative of two biological independent experiments with similar results. (B) Kinetics of tumor growth. Tumor size was determined using a digital caliper twice a week. Data were analyzed by unpaired, two-tailed Student’s t-test. *p < 0.05, ns, not significant. (C) Tumor weights 21 days after EL4 transplantation. Photographs on the right show differences in tumor size at the end of experiments. Data are means ± SEM; **p < 0.01, two-tailed Student’s t-test, ns, not significant. (D) Representative bioluminescence images 21 days after transplantation of EL4 cells. (E) Quantification of bioluminescence imaging as total flux of photons per second. Data are means ± SEM; *p < 0.05, two-tailed Student’s t-test, ns, not significant.