Oncogenic ETS fusions promote DNA damage and proinflammatory responses via pericentromeric RNAs in extracellular vesicles
Peter Ruzanov, … , Lincoln D. Stein, Poul H. Sorensen
Peter Ruzanov, … , Lincoln D. Stein, Poul H. Sorensen
Published March 26, 2024
Citation Information: J Clin Invest. 2024;134(9):e169470. https://doi.org/10.1172/JCI169470.
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Oncogenic ETS fusions promote DNA damage and proinflammatory responses via pericentromeric RNAs in extracellular vesicles

Abstract

Aberrant expression of the E26 transformation-specific (ETS) transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS:FLI1 and EWS:ERG fusions in Ewing sarcoma (EwS) and TMPRSS2:ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS:FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV, and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs), and are highly elevated in plasma of patients with EwS with metastatic disease. High human satellite 2 and 3 (HSAT2,3) levels in EWS:FLI1- or ERG-expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle, and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA, and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs that transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics.

Authors

Peter Ruzanov, Valentina Evdokimova, Manideep C. Pachva, Alon Minkovich, Zhenbo Zhang, Sofya Langman, Hendrik Gassmann, Uwe Thiel, Marija Orlic-Milacic, Syed H. Zaidi, Vanya Peltekova, Lawrence E. Heisler, Manju Sharma, Michael E. Cox, Trevor D. McKee, Mark Zaidi, Eve Lapouble, John D. McPherson, Olivier Delattre, Laszlo Radvanyi, Stefan E.G. Burdach, Lincoln D. Stein, Poul H. Sorensen

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Figure 5

ERG binds to pericentromeric regions and activates HSAT2,3 expression and DNA damage pathways in PCa cells.

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ERG binds to pericentromeric regions and activates HSAT2,3 expression an...
(A) Chromosomal distribution of the HSAT contigs assembled from VCaP cell-derived ERG ChIP-Seq reads. ERG-high (18 hour DHT) versus ERG-low (0 hour DHT), P ≤ 0.001, 2-way ANOVA. (B) Highest-scoring consensus motif in ERG ChIP-Seq contigs. (C) Experimental outline (top) and immunoblotting detection of the endogenous ERG protein in androgen-deprived (–) or R1881-stimulated VCaP cells pretreated with ERG or scrambled control (ctrl) siRNAs (bottom). (D) Representation of satellite RNAs in androgen-deprived (ERG-low) and R1881-stimulated (ERG-high) and ERG KD VCaP cells. (E) Chromosomal distribution of HSAT2,3 RNA-Seq reads; Y-axis, square-root transformed. Each plot represents mean RPM ± SEM from 2 biological replicates. Comparisons made ERG-high versus -low cells; *P < 0.05, unpaired 2-tailed t test. (F) RT-qPCR of indicated RNAs in VCaP EVs after 48 hour treatment with R1881. Values normalized to the exogenously added spike-in MS2 RNA ± SD (n = 3). Fold change relative to untreated control; *P < 0.001, unpaired 2-tailed t test. (G) Immunoblotting of benign prostate cells expressing ERG or vector control (top) and RT-qPCR of HSAT2,3 expression in these cells (bottom). Values normalized to GAPDH RNA ± SEM (n = 2), representative of 1 of 2 independent experiments. Fold change relative to vector control; *P < 0.05, **P < 0.01, ***P < 0.001, unpaired 2-tailed t test. (H) RT-ddPCR of indicated RNAs in EVs from cells in G. Data are mean ± SD. Comparisons to EVs from vector control cells, ***P < 0.001, ****P < 0.0001, unpaired 2-tailed t test. (I and J) GSEA of hallmark pathways upregulated (I) or downregulated (J) in HSAT2,3-high PCa tumors (n = 98). Circle size indicates gene set size, and circle color indicates the FDR adjusted q value. Note that none of downregulated pathways reached statistical significance. Common pathways detected in PCa and EwS tumors are depicted in green.