Splicing targeting drugs highlight intron retention as an actionable vulnerability in advanced prostate cancer.

BACKGROUND: Advanced prostate cancer (PC) is characterized by insensitivity to androgen deprivation therapy and chemotherapy, resulting in poor outcome for most patients. Thus, advanced PC urgently needs novel therapeutic strategies. Mounting evidence points to splicing dysregulation as a hallmark of advanced PC. Moreover, pharmacologic inhibition of the splicing process is emerging as a promising option for this disease. METHOD: By using a representative androgen-insensitive PC cell line (22Rv1), we have investigated the genome-wide transcriptomic effects underlying the cytotoxic effects exerted by three splicing-targeting drugs: Pladienolide B, indisulam and THZ531. Bioinformatic analyses were performed to uncover the gene structural features underlying sensitivity to transcriptional and splicing regulation by these treatments. Biological pathways altered by these treatments were annotated by gene ontology analyses and validated by functional experiments in cell models. RESULTS: Although eliciting similar cytotoxic effects on advanced PC cells, Pladienolide B, indisulam and THZ531 modulate specific transcriptional and splicing signatures. Drug sensitivity is associated with distinct gene structural features, expression levels and cis-acting sequence elements in the regulated exons and introns. Importantly, we identified PC-relevant genes (i.e. EZH2, MDM4) whose drug-induced splicing alteration exerts an impact on cell survival. Moreover, computational analyses uncovered a widespread impact of splicing-targeting drugs on intron retention, with enrichment in genes implicated in pre-mRNA 3'-end processing (i.e. CSTF3, PCF11). Coherently, advanced PC cells displayed high sensitivity to a specific inhibitor of the cleavage and polyadenylation complex, which enhances the effects of chemotherapeutic drugs that are already in use for this cancer. CONCLUSIONS: Our study uncovers intron retention as an actionable vulnerability for advanced PC, which may be exploited to improve therapeutic management of this currently incurable disease.

The DNA/RNA helicase DHX9 orchestrates the KDM2B-mediated transcriptional regulation of YAP1 in Ewing sarcoma.

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.

RNA Editing in Cancer Progression.

Coding and noncoding RNA molecules play their roles in ensuring cell function and tissue homeostasis in an ordered and systematic fashion. RNA chemical modifications can occur both at bases and ribose sugar, and, similarly to DNA and histone modifications, can be written, erased, and recognized by the corresponding enzymes, thus modulating RNA activities and fine-tuning gene expression programs. RNA editing is one of the most prevalent and abundant forms of post-transcriptional RNA modification in normal physiological processes. By altering the sequences of mRNAs, it makes them different from the corresponding genomic template. Hence, edited mRNAs can produce protein isoforms that are functionally different from the corresponding genome-encoded variants. Abnormalities in regulatory enzymes and changes in RNA-modification patterns are closely associated with the occurrence and development of various human diseases, including cancer. To date, the roles played by RNA modifications in cancer are gathering increasing interest. In this review, we focus on the role of RNA editing in cancer transformation and provide a new perspective on its impact on tumorigenesis, by regulating cell proliferation, differentiation, invasion, migration, stemness, metabolism, and drug resistance.

The RNA-binding protein FUS/TLS interacts with SPO11 and PRDM9 and localize at meiotic recombination hotspots.

In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11ß and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.

Inhibition of the PI3K/AKT/mTOR signaling promotes an M1 macrophage switch by repressing the ATF3-CXCL8 axis in Ewing sarcoma.

Ewing sarcomas are aggressive pediatric tumors of bone and soft tissues driven by in frame chromosomal translocations that yield fusion proteins guiding the oncogenic program. Promising alternative strategies to ameliorate current treatments involve inhibition of the PI3K/AKT/mTOR pathway. In this study, we identified the activating transcription factor 3 (ATF3) as an important mediator of the PI3K/AKT/mTOR pathway in Ewing sarcoma cells. ATF3 exerted its pro-tumoral activity through modulation of several chemokine-encoding genes, including CXCL8. The product of CXCL8, IL-8, acts as a pro-inflammatory chemokine critical for cancer progression and metastasis. We found that ATF3/IL-8 axis impacts macrophages populating the surrounding tumor microenvironment by promoting the M2 phenotype. Our study reveals valuable information on the PI3K/AKT/mTOR derived chemokine signaling in Ewing sarcoma cells: by promoting ATF3 and CXCL8 downregulation, inhibition of the PI3K/AKT/mTOR signaling promotes a proinflammatory response leading to upregulation of the protective anti-tumoral M1 macrophages.

Oncogenic Dysregulation of Circulating Noncoding RNAs: Novel Challenges and Opportunities in Sarcoma Diagnosis and Treatment.

Sarcomas comprise a heterogeneous group of rare mesenchymal malignancies. Sarcomas can be grouped into two categories characterized by different prognosis and treatment approaches: soft tissue sarcoma and primary bone sarcoma. In the last years, research on novel diagnostic, prognostic or predictive biomarkers in sarcoma management has been focused on circulating tumor-derived molecules as valuable tools. Liquid biopsies that measure various tumor components, including circulating cell-free DNA and RNA, circulating tumor cells, tumor extracellular vesicles and exosomes, are gaining attention as methods for molecular screening and early diagnosis. Compared with traditional tissue biopsies, liquid biopsies are minimally invasive and blood samples can be collected serially over time to monitor cancer progression. This review will focus on circulating noncoding RNA molecules from liquid biopsies that are dysregulated in sarcoma malignancies and discuss advantages and current limitations of their employment as biomarkers in the management of sarcomas. It will also explore their utility in the evaluation of the clinical response to treatments and of disease relapse. Moreover, it will explore state-of-the-art techniques that allow for the early detection of these circulating biomarkers. Despite the huge potential, current reports highlight poor sensitivity, specificity, and survival benefit of these methods, that are therefore still insufficient for routine screening purposes.

Functional Interaction Between the Oncogenic Kinase NEK2 and Sam68 Promotes a Splicing Program Involved in Migration and Invasion in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents the most aggressive breast cancer subtype. Poor prognosis in TNBC is partly due to lack of efficacious targeted therapy and high propensity to metastasize. Dysregulation of alternative splicing has recently emerged as a trait of TNBC, suggesting that unveiling the molecular mechanisms underlying its regulation could uncover new druggable cancer vulnerabilities. The oncogenic kinase NEK2 is significantly upregulated in TNBC and contributes to shaping their unique splicing profile. Herein, we found that NEK2 interacts with the RNA binding protein Sam68 in TNBC cells and that NEK2-mediated phosphorylation of Sam68 enhances its splicing activity. Genome-wide transcriptome analyses identified the splicing targets of Sam68 in TNBC cells and revealed a common set of exons that are co-regulated by NEK2. Functional annotation of splicing-regulated genes highlighted cell migration and spreading as biological processes regulated by Sam68. Accordingly, Sam68 depletion reduces TNBC cell migration and invasion, and these effects are potentiated by the concomitant inhibition of NEK2 activity. Our findings indicate that Sam68 and NEK2 functionally cooperate in the regulation of a splicing program that sustains the pro-metastatic features of TNBC cells.

The DNA/RNA helicase DHX9 contributes to the transcriptional program of the androgen receptor in prostate cancer.

BACKGROUND: Prostate cancer (PC) is the most commonly diagnosed male malignancy and an important cause of mortality. Androgen deprivation therapy is the first line treatment but, unfortunately, a large part of patients evolves to a castration-resistant stage, for which no effective cure is currently available. The DNA/RNA helicase DHX9 is emerging as an important regulator of cellular processes that are often deregulated in cancer. METHODS: To investigate whether DHX9 modulates PC cell transcriptome we performed RNA-sequencing analyses upon DHX9 silencing in the androgen-responsive cell line LNCaP. Bioinformatics and functional analyses were carried out to elucidate the mechanism of gene expression regulation by DHX9. Data from The Cancer Genome Atlas were mined to evaluate the potential role of DHX9 in PC. RESULTS: We found that up-regulation of DHX9 correlates with advanced stage and is associated with poor prognosis of PC patients. High-throughput RNA-sequencing analysis revealed that depletion of DHX9 in androgen-sensitive LNCaP cells affects expression of hundreds of genes, which significantly overlap with known targets of the Androgen Receptor (AR). Notably, AR binds to the DHX9 promoter and induces its expression, while Enzalutamide-mediated inhibition of AR activity represses DHX9 expression. Moreover, DHX9 interacts with AR in LNCaP cells and its depletion significantly reduced the recruitment of AR to the promoter region of target genes and the ability of AR to promote their expression in response to 5α-dihydrotestosterone. Consistently, silencing of DXH9 negatively affected androgen-induced PC cell proliferation and migration. CONCLUSIONS: Collectively, our data uncover a new role of DHX9 in the control of the AR transcriptional program and establish the existence of an oncogenic DHX9/AR axis, which may represent a new druggable target to counteract PC progression.

Somatic Mutations in Core Spliceosome Components Promote Tumorigenesis and Generate an Exploitable Vulnerability in Human Cancer.

Alternative pre-mRNA processing enables the production of distinct mRNA and protein isoforms from a single gene, thus greatly expanding the coding potential of eukaryotic genomes and fine-tuning gene expression programs. Splicing is carried out by the spliceosome, a complex molecular machinery which assembles step-wise on mRNA precursors in the nucleus of eukaryotic cells. In the last decade, exome sequencing technologies have allowed the identification of point mutations in genes encoding splicing factors as a recurrent hallmark of human cancers, with higher incidence in hematological malignancies. These mutations lead to production of splicing factors that reduce the fidelity of the splicing process and yield splicing variants that are often advantageous for cancer cells. However, at the same time, these mutations increase the sensitivity of transformed cells to splicing inhibitors, thus offering a therapeutic opportunity for novel targeted strategies. Herein, we review the recent literature documenting cancer-associated mutations in components of the early spliceosome complex and discuss novel therapeutic strategies based on small-molecule spliceosome inhibitors that exhibit strong anti-tumor effects, particularly against cancer cells harboring mutations in spliceosomal components.

EWS splicing regulation contributes to balancing Foxp1 isoforms required for neuronal differentiation.

Alternative splicing is a key regulatory process underlying the amplification of genomic information and the expansion of proteomic diversity, particularly in brain. Here, we identify the Ewing sarcoma protein (EWS) as a new player of alternative splicing regulation during neuronal differentiation. Knockdown of EWS in neuronal progenitor cells leads to premature differentiation. Transcriptome profiling of EWS-depleted cells revealed global changes in splicing regulation. Bioinformatic analyses and biochemical experiments demonstrated that EWS regulates alternative exons in a position-dependent fashion. Notably, several EWS-regulated splicing events are physiologically modulated during neuronal differentiation and EWS depletion in neuronal precursors anticipates the splicing-pattern of mature neurons. Among other targets, we found that EWS controls the alternative splicing of the forkhead family transcription factor FOXP1, a pivotal transcriptional regulator of neuronal differentiation, possibly contributing to the switch of gene expression underlying the neuronal differentiation program.

pncCCND1_B Engages an Inhibitory Protein Network to Downregulate CCND1 Expression upon DNA Damage.

Promoter-associated noncoding RNAs (pancRNAs) represent a class of noncoding transcripts driven from the promoter region of protein-coding or non-coding genes that operate as cis-acting elements to regulate the expression of the host gene. PancRNAs act by altering the chromatin structure and recruiting transcription regulators. PncCCND1_B is driven by the promoter region of CCND1 and regulates CCND1 expression in Ewing sarcoma through recruitment of a multi-molecular complex composed of the RNA binding protein Sam68 and the DNA/RNA helicase DHX9. In this study, we investigated the regulation of CCND1 expression in Ewing sarcoma cells upon exposure to chemotherapeutic drugs. Pan-inhibitor screening indicated that etoposide, a drug used for Ewing sarcoma treatment, promotes transcription of pncCCND1_B and repression of CCND1 expression. RNA immunoprecipitation experiments showed increased binding of Sam68 to the pncCCND1_B after treatment, despite the significant reduction in DHX9 protein. This effect was associated with the formation of DNA:RNA duplexes at the CCND1 promoter. Furthermore, Sam68 interacted with HDAC1 in etoposide treated cells, thus contributing to chromatin remodeling and epigenetic changes. Interestingly, inhibition of the ATM signaling pathway by KU 55,933 treatment was sufficient to inhibit etoposide-induced Sam68-HDAC1 interaction without rescuing DHX9 expression. In these conditions, the DNA:RNA hybrids persist, thus contributing to the local chromatin inactivation at the CCND1 promoter region. Altogether, our results show an active role of Sam68 in DNA damage signaling and chromatin remodeling on the CCND1 gene by fine-tuning transitions of epigenetic complexes on the CCND1 promoter.

RNA Targeting in Inherited Neuromuscular Disorders: Novel Therapeutic Strategies to Counteract Mis-Splicing.

Neuromuscular disorders represent multifaceted abnormal conditions, with little or no cure, leading to patient deaths from complete muscle wasting and atrophy. Despite strong efforts in the past decades, development of effective treatments is still urgently needed. Advent of next-generation sequencing technologies has allowed identification of novel genes and mutations associated with neuromuscular pathologies, highlighting splicing defects as essential players. Deciphering the significance and relative contributions of defective RNA metabolism will be instrumental to address and counteract these malignancies. We review here recent progress on the role played by alternative splicing in ensuring functional neuromuscular junctions (NMJs), and its involvement in the pathogenesis of NMJ-related neuromuscular disorders, with particular emphasis on congenital myasthenic syndromes and muscular dystrophies. We will also discuss novel strategies based on oligonucleotides designed to bind their cognate sequences in the RNA or targeting intermediary of mRNA metabolism. These efforts resulted in several chemical classes of RNA molecules that have recently proven to be clinically effective, more potent and better tolerated than previous strategies.

A ligand-insensitive UNC5B splicing isoform regulates angiogenesis by promoting apoptosis.

The Netrin-1 receptor UNC5B is an axon guidance regulator that is also expressed in endothelial cells (ECs), where it finely controls developmental and tumor angiogenesis. In the absence of Netrin-1, UNC5B induces apoptosis that is blocked upon Netrin-1 binding. Here, we identify an UNC5B splicing isoform (called UNC5B-Δ8) expressed exclusively by ECs and generated through exon skipping by NOVA2, an alternative splicing factor regulating vascular development. We show that UNC5B-Δ8 is a constitutively pro-apoptotic splicing isoform insensitive to Netrin-1 and required for specific blood vessel development in an apoptosis-dependent manner. Like NOVA2, UNC5B-Δ8 is aberrantly expressed in colon cancer vasculature where its expression correlates with tumor angiogenesis and poor patient outcome. Collectively, our data identify a mechanism controlling UNC5B's necessary apoptotic function in ECs and suggest that the NOVA2/UNC5B circuit represents a post-transcriptional pathway regulating angiogenesis.

Alternative Splicing: Recent Insights into Mechanisms and Functional Roles.

Alternative splicing generates multiple protein isoforms from one primary transcript and represents one of the major drivers of proteomic diversity in human cells [...].

Exercise-mediated downregulation of MALAT1 expression and implications in primary and secondary cancer prevention.

Long non-coding RNAs (lncRNAs) play critical roles in various biological functions and disease processes including cancer. The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was initially identified as a lncRNA with elevated expression in primary human non-small cell lung tumors with high propensity to metastasize, and subsequently shown to be highly expressed in numerous other human cancers including breast, ovarian, prostate, cervical, endometrial, gastric, pancreatic, sarcoma, colorectal, bladder, brain, multiple myeloma, and lymphoma. MALAT1 is deeply involved in several physiological processes, including alternative splicing, epigenetic modification of gene expression, cellular senescence, healthy aging, and redox homeostasis. The aim of this work was to investigate the modulation exerted by a single bout of endurance exercise on the level of MALAT1 expression in peripheral blood mononuclear cells (PBMCs) from healthy male donors displaying different training status and redox homeostasis features. Our findings show that MALAT1 is downregulated after acute endurance exercise in subjects whose fitness level guarantee a high expression of SOD1 and SOD2 antioxidant genes and low levels of endogenous oxidative damage. In vitro protocols in Jurkat lymphoblastoid cells exposed to pro-oxidant environment confirmed the link between MALAT1 expression and antioxidant gene modulation, documenting p53 phosphorylation and its recruitment to MALAT1 promoter. Remarkably, analyses of Microarray-Based Gene Expression Profiling revealed high MALAT1 expression in leukemia patients in comparison to healthy control and a significant negative correlation between MALAT1 and SOD1 expression. Collectively our results highlight the beneficial effect of a physically active lifestyle in counteracting aberrant cancer-related gene expression programs by improving the redox buffering capacity.

Emerging Contribution of PancRNAs in Cancer.

"Cancer" includes a heterogeneous group of diseases characterized by abnormal growth beyond natural boundaries. Neoplastic transformation of cells is orchestrated by multiple molecular players, including oncogenic transcription factors, epigenetic modifiers, RNA binding proteins, and coding and noncoding transcripts. The use of computational methods for global and quantitative analysis of RNA processing regulation provides new insights into the genomic and epigenomic features of the cancer transcriptome. In particular, noncoding RNAs are emerging as key molecular players in oncogenesis. Among them, the promoter-associated noncoding RNAs (pancRNAs) are noncoding transcripts acting in cis to regulate their host genes, including tumor suppressors and oncogenes. In this review, we will illustrate the role played by pancRNAs in cancer biology and will discuss the latest findings that connect pancRNAs with cancer risk and progression. The molecular mechanisms involved in the function of pancRNAs may open the path to novel therapeutic opportunities, thus expanding the repertoire of targets to be tested as anticancer agents in the near future.

Dissecting the transcriptional regulatory networks of promoter-associated noncoding RNAs in development and cancer.

In-depth analysis of global RNA sequencing has enabled a comprehensive overview of cellular transcriptomes and revealed the pervasive transcription of divergent RNAs from promoter regions across eukaryotic genomes. These studies disclosed that genomes encode a vast repertoire of RNAs beyond the well-known protein-coding messenger RNAs. Furthermore, they have provided novel insights into the regulation of eukaryotic epigenomes, and transcriptomes, including the identification of novel classes of noncoding transcripts, such as the promoter-associated noncoding RNAs (pancRNAs).PancRNAs are defined as transcripts transcribed within few hundred bases from the transcription start sites (TSSs) of protein-coding or non-coding genes. Unlike the long trans-acting ncRNAs that regulate expression of target genes located in different chromosomal domains and displaying their function both in the nucleus and in the cytoplasm, the pancRNAs operate as cis-acting elements in the transcriptional regulation of neighboring genes. PancRNAs are very recently emerging as key players in the epigenetic regulation of gene expression programs in development and diseases.Herein, we review the complex epigenetic network driven by pancRNAs in eukaryotic cells, their impact on physiological and pathological states, which render them promising targets for novel therapeutic strategies.

Poison-Exon Inclusion in DHX9 Reduces Its Expression and Sensitizes Ewing Sarcoma Cells to Chemotherapeutic Treatment.

Alternative splicing is a combinatorial mechanism by which exons are joined to produce multiple mRNA variants, thus expanding the coding potential and plasticity of eukaryotic genomes. Defects in alternative splicing regulation are associated with several human diseases, including cancer. Ewing sarcoma is an aggressive tumor of bone and soft tissue, mainly affecting adolescents and young adults. DHX9 is a key player in Ewing sarcoma malignancy, and its expression correlates with worse prognosis in patients. In this study, by screening a library of siRNAs, we have identified splicing factors that regulate the alternative inclusion of a poison exon in DHX9 mRNA, leading to its downregulation. In particular, we found that hnRNPM and SRSF3 bind in vivo to this poison exon and suppress its inclusion. Notably, DHX9 expression correlates with that of SRSF3 and hnRNPM in Ewing sarcoma patients. Furthermore, downregulation of SRSF3 or hnRNPM inhibited DHX9 expression and Ewing sarcoma cell proliferation, while sensitizing cells to chemotherapeutic treatment. Hence, our study suggests that inhibition of hnRNPM and SRSF3 expression or activity could be exploited as a therapeutic tool to enhance the efficacy of chemotherapy in Ewing sarcoma.

Small molecule inhibition of Ewing sarcoma cell growth via targeting the long non coding RNA HULC.

Ewing sarcomas (ES) are aggressive pediatric cancers of bone and soft tissues characterized by in frame chromosomal translocations giving rise to chimeric transcription factors, such as EWS-FLI1. An emerging strategy to block EWS-FLI1 activity is represented by the small molecule YK-4-279, which binds to EWS-FLI1 and alters its transcriptional activity. The specific effectors of the anti-oncogenic activity of YK-4-279 are still largely unknown. Herein, by performing a high-throughput screening we identify the lncRNA HULC (Highly Upregulated in Liver Cancer) as a prominent target of YK-4-279 activity in ES cells. High levels of HULC correlate with ES aggressiveness, whereas HULC depletion reduces ES cell growth. Mechanistically, we find that HULC promotes the expression of TWIST1 oncogene by sponging miR-186. Downregulation of HULC upon treatment with YK-4-279 reduces the expression of TWIST1 by unleashing miR-186 and favoring its binding to TWIST1 transcripts. Notably, high levels of miR-186 and low levels of TWIST1 correlate with better prognosis in ES patients. Our results disclose a novel oncogenic regulatory circuit mediated by HULC lncRNA that is disrupted by the small molecule YK-4-279, with promising therapeutic implications for ES treatment.

The Promoter-Associated Noncoding 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 patients with Ewing sarcoma compared with other sarcomas and that the pncCCND1_B RNA, a previously uncharacterized CCND1 promoter-associated noncoding (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 IGF1 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. SIGNIFICANCE: A pncRNA-based mechanism represses expression of CCND1 through the formation of a protein-RNA complex and provides new therapeutic opportunities for patients with Ewing sarcoma.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/14/3570/F1.large.jpg.