The promoter associated non-coding RNA pncCCND1_B assembles a protein-RNA complex to regulate cyclin D1 transcription in Ewing sarcoma

Most Ewing sarcomas are characterized by the in frame chromosomal translocation t(11;22), generating the EWS-FLI1 oncogene. EWS-FLI1 protein interacts with the RNA helicase DHX9 and affects transcription and processing of genes involved in neoplastic transformation, including CCND1 (the cyclin D1 gene), which contributes to cell cycle dysregulation in cancer. In this study, we found that CCND1 expression is significantly higher in Ewing sarcoma patients compared to other sarcomas and that the pncCCND1_B RNA, a previously uncharacterized CCND1 promoter-associated non-coding (pnc) transcript, is expressed in Ewing sarcoma cells. PncCCND1_B interacted with the RNA-binding protein Sam68 and repressed CCND1 expression. Notably, knockdown of Sam68 affected pncCCND1_B subcellular localization and cyclin D1 expression. Pharmacologic impairment of DHX9/EWS-FLI1 interaction promoted RNA-dependent association of Sam68 with DHX9 and recruitment of Sam68 to the CCND1 promoter, thus repressing it. Conversely, mitogenic stimulation of Ewing sarcoma cells with IGF-1 impaired Sam68/DHX9 interaction and positively regulated CCND1 expression. These studies uncover a fine-tuned modulation of the proto-oncogene CCND1 in Ewing sarcoma cells via alternative complexes formed by DHX9 with either EWS-FLI1 or pncCCND1_B-Sam68.