Biology subjects

Prada, E.

Publications and source records attributed to Prada, E..

5 recordsLinked to original sources

bioRxiv2026

RERE and the Mediator complex cooperate with EWSR1::FLI1 in the reprogramming of Translation and Alternative Splicing, the latter being a therapeutically targetable vulnerability in Ewing sarcoma

Ewing Sarcoma (ES) is an aggressive neoplasm arising in bones and soft tissues driven by the oncogenic fusion EWSR1::FLI1. Through epigenetic deregulation, EWSR1::FLI1 generates de novo super-enhancers that control the expression of key genes for tumor cell maintenance. By an integrative in silico analysis, we identified the subunit of the Mediator complex MED13L and RERE, a member of the atrophin family of arginine-glutamic acid dipeptide repeat-containing proteins, as genes regulated by EWSR1::FLI1-bound super-enhancers. We confirmed that EWSR1::FLI1 regulates MED13L and RERE expression in ES cell lines and showed that these proteins are highly expressed in Ewing primary tumors. Besides the well-established role of the Mediator complex in transcriptional regulation given its association with the RNA polymerase II, in ES cells the DNA binding sites of MED13L overlap with those of RERE and EWSR1::FLI1 in genes that control protein translation and alternative splicing (AS). Accordingly, the expression of various spliceosome components is co-regulated by MED13L, RERE and the oncogene, leading to AS in ES cells. We identified RBM39, a splicing factor downregulated after MED13L and RERE depletion, as a direct transcriptional target of EWSR1::FLI1. Consistently, in vitro viability experiments using indisulam, which induces selective DCAF15-dependent proteosome degradation of RBM39, demonstrate ES cells highly and specifically sensitive to RBM39 inhibition. In vivo experiments with mice xenografted with ES cells show complete tumor regression with indisulam, highlighting the potential of this approach as a novel and promising therapeutic strategy for Ewing sarcoma. STATEMENT OF SIGNIFICANCEEwing sarcoma (ES) is characterized by FET::ETS oncoproteins that act as pioneer transcription factors. Here, we identified two genes controlled by EWSR1::FLI1-bound super-enhancers, MED13L and RERE, and characterized the mechanism by which these proteins cooperate with the oncogene to regulate RNA metabolism and ribosomal processes in ES cells. These findings have led to the identification of the splicing factor RBM39 as a vulnerability in ES, as supported by the extraordinary sensitivity of these tumors to monotherapy with RBM39 degrader indisulam.

cancer biology↗

bioRxiv2026

USP7 sustains PAX3::FOXO1 enhancer reprogramming and represents a therapeutic vulnerability in Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and is often associated with dismal outcomes, underscoring the urgent need for new therapeutic strategies. RMS arises from embryonic skeletal muscle precursor cells that fail to complete the myogenic differentiation program. Fusion-positive rhabdomyosarcoma (FP-RMS), defined by the presence of recurrent gene fusions such as PAX3::FOXO1 or PAX7::FOXO1, is associated with the poorest overall survival. The encoded fusion oncoprotein cause epigenetic reprogramming that defines the biology and behavior of FP-RMS. Polycomb repressive complex 1 (PRC1)-mediated chromatin regulation contributes to the control of developmental gene programs. Here, we investigate the dependency of RMS on epigenetic remodeling mediated by the PRC1.1 subunits ubiquitin specific protease 7 (USP7) and really interesting new gene 1B (RING1B). We found that USP7 is overexpressed in RMS samples, and that high expression correlates with poor patient prognosis. USP7 and RING1B bind to H3K27ac-enriched regions and colocalize with PAX3::FOXO1 at active enhancers controlling key tumorigenic genes in FP-RMS. Moreover, both shRNA-mediated depletion and pharmacological inhibition of USP7 downregulate PAX3::FOXO1 enhancer-driven genes, induce skeletal muscle differentiation and significantly inhibit FP-RMS tumor growth in vivo. Altogether, our findings identify USP7 as a critical regulator of PAX3::FOXO1-bound enhancers and highlight a novel therapeutic opportunity in RMS based on epigenetic dependencies.

cancer biology↗

bioRxiv2025

EWS-FLI1 Expression in Human Embryonic MSCs Leads to Transcriptional Reprograming, Defective DNA Damage Repair and Ewing Sarcoma

Ewing sarcoma (ES) is an aggressive bone and soft tissue neoplasm characterized by EWSR1/ETS rearrangements and whose cellular origin remains unclear. EWS-FLI1 expression in human pediatric mesenchymal stem cells (hpMSCs) induces a quantitatively and qualitatively different transcriptional response than its expression in human adult MSCs (haMSCs), but fails to form tumors in vivo. ES cells have early developmental lineage signatures distinct from postnatal MSCs. Here, we have generated MSCs from experimental teratomas out of human embryonic stem cells (heSCs). Transduction of these human embryonic mesenchymal stem cells (heMSCs) with EWS-FLI1 results in the acquisition of an ES transcriptome, although the oncogene does not preferentially bind to promoters, but to intronic and intergenic microsatellites with >10 CA dinucleotides and GGAA repeats, respectively. In heMSCs, EWS-FLI1 directly regulates BRCA1 expression, although EWS-FLI1-expressing cells show defects in DNA damage repair. Xenografting of EWS-FLI1-transduced heMSCs resulted in the formation of tumors expressing characteristic ES markers. In summary, EWS-FLI1 enforces an aberrant transcriptome and endows in vivo transforming capacity when expressed in an undifferentiated early heMSC. Our approach represents an innovative experimental method for understanding critical aspects of the biology of developmental tumors, from leukemia to sarcomas, in which few (even single) genetic alterations are able to transform a fetal stem cell.

cancer biology↗

bioRxiv2023

KDM6 demethylases mediate EWSR1-FLI1-driven oncogenic transformation in Ewing Sarcoma

Ewing Sarcoma (EwS) is an aggressive bone and soft tissue tumor driven by the fusion oncoprotein EWSR1-FLI1. This aberrant transcription factor binds to GGAA microsatellites, causing epigenetic reprogramming through the formation of active neo-enhancers in a permissive cellular context. Inhibition of the oncogene remains challenging and current efforts instead seek to exploit emergent epigenetic treatments targeting EWSR1-FLI1 cofactors. Here, stemming from the genome-wide redistribution of H3K27me3 upon expression of EWSR1-FLI1 in pediatric hMSC, we unravel the contribution of the H3K27me3 demethylases KDM6A and KDM6B in transcriptional activation at EWSR1-FLI1 enhancers. We found that KDM6A has a demethylase-independent role in recruiting the SWI/SNF member BRG1 at EWSR1-FLI1-primed enhancers containing single GGAA motif, which is critical for EwS tumor growth. Conversely, KDM6B demethylates H3K27me3 at EWSR1-FLI1-active enhancers containing multimeric GGAA repeats and its deletion synergizes with EZH2 inhibitors. Our results highlight KDM6 demethylases as EWSR1-FLI1 cofactors with potential for future targeted therapies.

cancer biology↗

bioRxiv2020

Sequential combinations of chemotherapeutic agents with BH3 mimetics to treat rhabdomyosarcoma and avoid resistance.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood and adolescence. Refractory/relapsed RMS patients present a bad prognosis that combined with the lack of specific biomarkers difficult the development of new therapies. We here utilize dynamic BH3 Profiling (DBP), a functional predictive biomarker that measures net changes in mitochondrial apoptotic signaling, to identify anti-apoptotic adaptations upon treatment. We use this information to guide the use of BH3 mimetics to specifically inhibit BCL-2 pro-survival proteins, defeat resistance and avoid relapse to therapy. Indeed, we found that BH3 mimetics that selectively target BCL-xL and MCL-1 synergistically enhance the effect of the clinically used chemotherapeutic agents vincristine and doxorubicin in RMS cells. We validated this strategy in vivo using a RMS patient-derived xenograft (PDX) model and observed a reduction on tumor growth with a tendency to its stabilization with the sequential combination of vincristine and the MCL-1 inhibitor S63845. Finally, we identified the molecular mechanism by which RMS cells acquire resistance to vincristine: through the anti-apoptotic protein MCL-1 for which we observed an enhanced binding between MCL-1 and BID after drug exposure, which is suppressed by the sequential addition of S63845. In conclusion, our findings validate the use of DBP as a functional assay to predict treatment effectiveness in RMS and provide a rationale for BH3 mimetic combination with chemotherapeutic agents to avoid tumor resistance, improve treatment efficiency and decrease undesired secondary effects.

cancer biology↗