Abstract
Ewing sarcomas (ES) are aggressive paediatric tumours of bone and soft tissues. Resistance to chemotherapy and high propensity to metastasize remain the main causes of treatment failure. Thus, identifying novel targets for alternative therapeutic approaches is urgently needed. DNA/RNA helicases are emerging as crucial regulators of many cellular processes often deregulated in cancer. Among them, DHX9 is up-regulated in ES and collaborates with EWS-FLI1 in ES transformation. We report that DHX9 silencing profoundly impacts on the oncogenic properties of ES cells. Transcriptome profiling combined to bioinformatic analyses disclosed a gene signature commonly regulated by DHX9 and the Lysine Demethylase KDM2B, with the Hippo pathway regulator YAP1 as a prominent target. Mechanistically, we found that DHX9 enhances H3K9 chromatin demethylation by KDM2B and favours RNA Polymerase II recruitment, thus promoting YAP1 expression. Conversely, EWS-FLI1 binding to the promoter represses YAP1 expression. These findings identify the DHX9/KDM2B complex as a new druggable target to counteract ES malignancy.
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Data availability
The data generated in this study are publicly available in Gene Expression Omnibus (GEO) at GSE214257.
References
Grünewald TG, Alonso M, Avnet S, Banito A, Burdach S, Cidre-Aranaz F, et al. Sarcoma treatment in the era of molecular medicine. EMBO Mol Med. 2020;12:e11131.
Grünewald TGP, Cidre-Aranaz F, Surdez D, Tomazou EM, de Álava E, Kovar H, et al. Ewing sarcoma. Nat Rev Dis Prim. 2018;4:5.
Balamuth NJ, Womer RB. Ewing’s sarcoma. Lancet Oncol. 2010;11:184–92.
Riggi N, Suvà ML, Stamenkovic I. Ewing’s Sarcoma. N. Engl J Med. 2021;384:154–64.
Erkizan HV, Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg JS, Yuan L, et al. A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing’s sarcoma. Nat Med. 2009;15:750–6.
Riggi N, Knoechel B, Gillespie SM, Rheinbay E, Boulay G, Suvà ML, et al. EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma. Cancer Cell. 2014;26:668–81.
Kovar H. Dr. Jekyll and Mr. Hyde: the two faces of the FUS/EWS/TAF15 protein family. Sarcoma. 2011;2011:837474.
Cidre-Aranaz F, Alonso J. EWS/FLI1 target genes and therapeutic opportunities in Ewing sarcoma. Front Oncol. 2015;5:162.
Fidaleo M, De Paola E, Paronetto MP. The RNA helicase A in malignant transformation. Oncotarget. 2016;7:28711–23.
Cristini A, Groh M, Kristiansen MS, Gromak N. RNA/DNA hybrid interactome identifies DXH9 as a molecular player in transcriptional termination and R-loop-associated DNA damage. Cell Rep. 2018;23:1891–905.
Lee T, Paquet M, Larsson O, Pelletier J. Tumor cell survival dependence on the DHX9 DExH-box helicase. Oncogene. 2016;35:5093–105.
Chakraborty P, Huang JTJ, Hiom K. DHX9 helicase promotes R-loop formation in cells with impaired RNA splicing. Nat Commun. 2018;9:4346.
Gulliver C, Hoffmann R, Baillie GS. The enigmatic helicase DHX9 and its association with the hallmarks of cancer. Future Sci OA. 2020;7:FSO650.
Fidaleo M, Svetoni F, Volpe E, Miñana B, Caporossi D, Paronetto MP. Genotoxic stress inhibits Ewing sarcoma cell growth by modulating alternative pre-mRNA processing of the RNA helicase DHX9. Oncotarget. 2015;6:31740–57.
Palombo R, Paronetto MP. pncCCND1_B Engages an Inhibitory Protein Network to Downregulate CCND1 Expression upon DNA Damage. Cancers (Basel) 2022; 14.
Palombo R, Verdile V, Paronetto MP. Poison-exon inclusion in DHX9 reduces its expression and sensitizes ewing sarcoma cells to chemotherapeutic Treatment. Cells. 2020;9:328.
Chellini L, Pieraccioli M, Sette C, Paronetto MP. The DNA/RNA helicase DHX9 contributes to the transcriptional program of the androgen receptor in prostate cancer. J Exp Clin Cancer Res. 2022;41:178.
Liu S, He L, Wu J, Wu X, Xie L, Dai W, et al. DHX9 contributes to the malignant phenotypes of colorectal cancer via activating NF-κB signaling pathway. Cell Mol Life Sci. 2021;78:8261–81.
Cao S, Sun R, Wang W, Meng X, Zhang Y, Zhang N, et al. RNA helicase DHX9 may be a therapeutic target in lung cancer and inhibited by enoxacin. Am J Transl Res. 2017;9:674–82.
Erkizan HV, Schneider JA, Sajwan K, Graham GT, Griffin B, Chasovskikh S et al. RNA helicase A activity is inhibited by oncogenic transcription factor EWS-FLI1. Nucleic Acids Res. 2015.
Palombo R, Frisone P, Fidaleo M, Mercatelli N, Sette C, Paronetto MP. The promoter-associated noncoding RNA. Cancer Res. 2019;79:3570–82.
Kollareddy M, Sherrard A, Park JH, Szemes M, Gallacher K, Melegh Z, et al. The small molecule inhibitor YK-4-279 disrupts mitotic progression of neuroblastoma cells, overcomes drug resistance and synergizes with inhibitors of mitosis. Cancer Lett. 2017;403:74–85.
Spriano F, Chung EYL, Gaudio E, Tarantelli C, Cascione L, Napoli S, et al. The ETS inhibitors YK-4-279 and TK-216 are novel antilymphoma agents. Clin Cancer Res. 2019;25:5167–76.
Xue J, Li S, Shi P, Chen M, Yu S, Hong S, et al. The ETS inhibitor YK-4-279 suppresses thyroid cancer progression independently. Front Oncol. 2021;11:649323.
Rahim S, Minas T, Hong SH, Justvig S, Çelik H, Kont YS, et al. A small molecule inhibitor of ETV1, YK-4-279, prevents prostate cancer growth and metastasis in a mouse xenograft model. PLoS ONE. 2014;9:e114260.
Povedano JM, Li V, Lake KE, Bai X, Rallabandi R, Kim J, et al. TK216 targets microtubules in Ewing sarcoma cells. Cell Chem Biol. 2022;29:1325–32.e1324
Ludwig JA, Meyers PA, Dirksen U. Ewing’s sarcoma. N. Engl J Med. 2021;384:1476.
Yan M, Yang X, Wang H, Shao Q. The critical role of histone lysine demethylase KDM2B in cancer. Am J Transl Res. 2018;10:2222–33.
Vacík T, Lađinović D, Raška I. KDM2A/B lysine demethylases and their alternative isoforms in development and disease. Nucleus. 2018;9:431–41.
He S, Huang Q, Hu J, Li L, Xiao Y, Yu H, et al. EWS-FLI1-mediated tenascin-C expression promotes tumour progression by targeting MALAT1 through integrin α5β1-mediated YAP activation in Ewing sarcoma. Br J Cancer. 2019;121:922–33.
Bierbaumer L, Katschnig AM, Radic-Sarikas B, Kauer MO, Petro JA, Högler S, et al. YAP/TAZ inhibition reduces metastatic potential of Ewing sarcoma cells. Oncogenesis. 2021;10:2.
Rodríguez-Núñez P, Romero-Pérez L, Amaral AT, Puerto-Camacho P, Jordán C, Marcilla D, et al. Hippo pathway effectors YAP1/TAZ induce an EWS-FLI1-opposing gene signature and associate with disease progression in Ewing sarcoma. J Pathol. 2020;250:374–86.
Katschnig AM, Kauer MO, Schwentner R, Tomazou EM, Mutz CN, Linder M, et al. EWS-FLI1 perturbs MRTFB/YAP-1/TEAD target gene regulation inhibiting cytoskeletal autoregulatory feedback in Ewing sarcoma. Oncogene. 2017;36:5995–6005.
Truong DD, Lamhamedi-Cherradi SE, Ludwig JA. Targeting the IGF/PI3K/mTOR pathway and AXL/YAP1/TAZ pathways in primary bone cancer. J Bone Oncol. 2022;33:100419.
Padeken J, Methot SP, Gasser SM. Establishment of H3K9-methylated heterochromatin and its functions in tissue differentiation and maintenance. Nat Rev Mol Cell Biol. 2022;23:623–40.
Toretsky JA, Erkizan V, Levenson A, Abaan OD, Parvin JD, Cripe TP, et al. Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res. 2006;66:5574–81.
Filion C, Labelle Y. The oncogenic fusion protein EWS/NOR-1 induces transformation of CFK2 chondrogenic cells. Exp Cell Res. 2004;297:585–92.
Wei GH, Badis G, Berger MF, Kivioja T, Palin K, Enge M, et al. Genome-wide analysis of ETS-family DNA-binding in vitro and in vivo. EMBO J. 2010;29:2147–60.
Liu M, Roth A, Yu M, Morris R, Bersani F, Rivera MN, et al. The IGF2 intronic miR-483 selectively enhances transcription from IGF2 fetal promoters and enhances tumorigenesis. Genes Dev. 2013;27:2543–8.
Myöhänen S, Baylin SB. Sequence-specific DNA binding activity of RNA helicase A to the p16INK4a promoter. J Biol Chem. 2001;276:1634–42.
Nakajima T, Uchida C, Anderson SF, Lee CG, Hurwitz J, Parvin JD, et al. RNA helicase A mediates association of CBP with RNA polymerase II. Cell. 1997;90:1107–12.
Chakraborty P, Hiom K. DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination. Nat Commun. 2021;12:4126.
Palombo R, Paronetto MP. Engages an inhibitory protein network to downregulate. Cancers (Basel) 2022; 14.
Chakraborty P, Grosse F. Human DHX9 helicase preferentially unwinds RNA-containing displacement loops (R-loops) and G-quadruplexes. DNA Repair (Amst). 2011;10:654–65.
He J, Shen L, Wan M, Taranova O, Wu H, Zhang Y. Kdm2b maintains murine embryonic stem cell status by recruiting PRC1 complex to CpG islands of developmental genes. Nat Cell Biol. 2013;15:373–84.
He J, Kallin EM, Tsukada Y, Zhang Y. The H3K36 demethylase Jhdm1b/Kdm2b regulates cell proliferation and senescence through p15(Ink4b). Nat Struct Mol Biol. 2008;15:1169–75.
Jiang Y, Li C, Wu Q, An P, Huang L, Wang J, et al. Iron-dependent histone 3 lysine 9 demethylation controls B cell proliferation and humoral immune responses. Nat Commun. 2019;10:2935.
Meng Z, Moroishi T, Guan KL. Mechanisms of Hippo pathway regulation. Genes Dev. 2016;30:1–17.
Becker JS, Nicetto D, Zaret KS. H3K9me3-dependent heterochromatin: barrier to cell fate changes. Trends Genet. 2016;32:29–41.
Chung YR, Schatoff E, Abdel-Wahab O. Epigenetic alterations in hematopoietic malignancies. Int J Hematol. 2012;96:413–27.
Wiencke JK, Zheng S, Morrison Z, Yeh RF. Differentially expressed genes are marked by histone 3 lysine 9 trimethylation in human cancer cells. Oncogene. 2008;27:2412–21.
Sankar S, Theisen ER, Bearss J, Mulvihill T, Hoffman LM, Sorna V, et al. Reversible LSD1 inhibition interferes with global EWS/ETS transcriptional activity and impedes Ewing sarcoma tumor growth. Clin Cancer Res. 2014;20:4584–97.
Biedler JL, Helson L, Spengler BA. Morphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture. Cancer Res. 1973;33:2643–52.
Seeger RC, Rayner SA, Banerjee A, Chung H, Laug WE, Neustein HB, et al. Morphology, growth, chromosomal pattern and fibrinolytic activity of two new human neuroblastoma cell lines. Cancer Res. 1977;37:1364–71.
Whang-Peng J, Triche TJ, Knutsen T, Miser J, Kao-Shan S, Tsai S, et al. Cytogenetic characterization of selected small round cell tumors of childhood. Cancer Genet Cytogenet. 1986;21:185–208.
Wang Y, Einhorn P, Triche TJ, Seeger RC, Reynolds CP. Expression of protein gene product 9.5 and tyrosine hydroxylase in childhood small round cell tumors. Clin Cancer Res. 2000;6:551–8.
Acknowledgements
This work was supported by grants from the Associazione Italiana Ricerca sul Cancro (AIRC) (IG21877) to MPP, and from the Italian Ministry of Health (SG-2019-12371596) to LC.
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Conceptualization, LC and MPP; methodology, LC; formal analysis, LC; investigation, LC and MPP; IHC, DB; mouse xenografts, LC and MS; data curation, LC and MPP; writing-original draft preparation, LC and MPP; writing-review and editing, CS and MPP; supervision, MPP; project administration, MPP; funding acquisition, LC and MPP. The authors have read and agreed to the published version of the manuscript.
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Chellini, L., Scarfò, M., Bonvissuto, D. et al. The DNA/RNA helicase DHX9 orchestrates the KDM2B-mediated transcriptional regulation of YAP1 in Ewing sarcoma. Oncogene 43, 225–234 (2024). https://doi.org/10.1038/s41388-023-02894-1
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DOI: https://doi.org/10.1038/s41388-023-02894-1
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