Biology subjects

Cidre-Aranaz, F.

Publications and source records attributed to Cidre-Aranaz, F..

At least 19 recordsLinked to original sources

bioRxiv2026

Functional spatial transcriptomics uncover LMO7 as a fusion-regulated and clinically relevant driver of metastasis in Ewing sarcoma

Metastatic dissemination represents the major determinant of poor clinical outcome across cancer entities. Yet, how driver oncogenes shape transcriptional programs facilitating metastasis is poorly understood. In Ewing sarcoma (EwS) - a highly aggressive pediatric bone and soft-tissue sarcoma driven by chimeric FET::ETS transcription factors - low activity of the fusion oncoproteins is thought to promote metastasis, but the underlying molecular mechanisms remain largely elusive. Here, using spatially resolved functional transcriptomics in EwS patient tumors, we identify a distinct transcriptional state at the invasive tumor front, that in contrast to the tumor core, is characterized by lower FET::ETS activity and induction of the multifunctional shuttle LIM domain only protein 7 (LMO7). Integrating these data with clinical information reveals that high LMO7 expression is associated with poor outcomes. Gene network analysis of patient tumors and integrated proteomic and transcriptomic profiling of EwS cell lines following inducible LMO7 silencing highlight LMO7 as a central regulatory hub orchestrating epithelial-mesenchymal transition (EMT) and cytoskeletal remodeling in EwS. Functional experiments demonstrate that LMO7 silencing decreases clonogenicity and migratory capacity in vitro and suppresses primary tumor growth and metastatic dissemination in vivo. Collectively, these findings identify LMO7 as a clinically relevant effector of FET::ETS fusions in EwS, and illustrate how integrating functional, spatial and clinical data can uncover oncogene-driven effectors of metastasis.

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

Targeting CXADR-mediated AKT signaling suppresses tumorigenesis and enhances chemotherapy efficacy in Ewing sarcoma

Distant metastasis is the leading cause of mortality in Ewing sarcoma (EwS) - a malignant bone or soft-tissue cancer mainly affecting children, adolescents, and young adults. Despite continuous efforts in understanding its pathogenesis, the molecular mechanisms driving EwS metastasis remain poorly understood, thus limiting the potential for therapeutic progress. Here, we identify the tight junction component Coxsackievirus and Adenovirus receptor (CXADR) as a critical regulator of cancer progression and metastasis in EwS. Differential gene expression analysis of patient tumors from two independent cohorts revealed that elevated CXADR levels are associated with metastatic disease and poor overall survival. In functional experiments, conditional CXADR knockdown reduced the growth of EwS cell line models in vitro, and suppressed local tumorigenesis. Notably, CXADR knockdown completely abrogated metastasis formation in vivo. Integration of transcriptome profiling and mechanistic studies uncovered that CXADR promotes the activation of AKT signaling, likely through complex formation with PTEN. Consequently, pharmacological targeting of AKT using the FDA-approved pan-AKT inhibitor Capivasertib showed CXADR-dependent cytotoxicity, with enhanced efficacy if combined with the EwS standard-of-care chemotherapeutic agent Vincristine. Collectively, our findings establish CXADR as a prognostic and predictive biomarker in EwS, highlighting AKT inhibition combined with chemotherapy as a promising strategy for patients with high CXADR expression. Together, these findings support a precision medicine approach combining molecular stratification and targeted therapies to improve patient outcomes in metastatic EwS.

cancer biology↗

bioRxiv2025

Exploration of oncogenic cooperation between germline variation and somatic mutation in prostate cancer progression

Prostate carcinoma (PCa) is the most common cancer of men, associated with a still unresolved issue of accurate risk-stratification. While recent advances in omics technologies have provided clues as to how molecular changes shape the onset and progression of PCa, it remains largely unclear whether germline variants and somatic mutations cooperate to contribute to PCa progression and outcome. Thus, we explored whether oncogenic cooperation between regulatory germline variants and somatic driver mutations can help explain why some PCa patients develop a more aggressive phenotype, which may have implications for risk-adapted medical treatment. Here, by employing an integrative functional genomics approach, we identified receptor-type protein-tyrosine phosphatase kappa (PTPRK) as a TMPRSS2::ERG (TE)-modulated gene associated with PCa progression whose expression is controlled by cooperation of the TE-fusion with a regulatory single nucleotide polymorphism (SNP). Analysis of available clinically annotated patient cohorts demonstrated that PTPRK is overexpressed in TE-positive PCa tumors and associated with higher Gleason scores and metastatic disease. TE knockdown in PCa cell lines reduced PTPRK expression, while ectopic overexpression of the fusion in TE-negative PCa cell lines and prostatic epithelium cells induced its expression. Functionally, PTPRK silencing inhibited cellular proliferation, cell cycle progression, and clonogenic growth of PCa cells, which was mirrored by dysregulation of corresponding gene and protein signatures in global transcriptomic and phospho-proteomic analyses after PTPRK knockdown. Analysis of TE ChIP-Seq and Hi-C data from PCa cells highlighted a proximal TE-bound DNA element whose TE-dependent enhancer activity was validated in reporter assays and which could be abrogated by a regulatory SNP. Collectively, our results provide evidence of how exploration of oncogenic cooperation may help to identify novel biomarkers and potentially druggable pathways and highlight the role of the regulatory genome in PCa progression.

cancer biology↗

bioRxiv2025

cIAP1 inhibitor of apoptosis is a tumor suppressor in Ewing sarcoma

Ewing sarcoma (EwS) is a highly aggressive pediatric malignancy driven by EWSR1::ETS fusion oncoproteins -primarily EWSR1::FLI1- which deregulate genes essential for differentiation, proliferation, and cell survival. To uncover key downstream targets of this fusion involved in cell differentiation, we combined transcriptomic profiling of EwS cell lines following EWSR1::FLI1 inhibition with gene ontology analysis, a clinically annotated gene expression dataset derived from EwS patient material and network analyses. This integrative approach identified inhibitor of apoptosis protein 1 (cIAP1, alias BIRC2) as an EWSR1::FLI1-suppresed gene. Despite its known oncogenic role in many cancers, cIAP1 showed minimal expression in EwS. Using inducible cIAP1 re-expression models in EwS cells, we demonstrated that cIAP1 re-expression suppresses proliferation, clonogenic growth, and 3D spheroid formation in vitro. Transcriptomic and proteomic analyses revealed that low cIAP1 expression enhances proliferation-related gene signatures, which are inhibited upon cIAP1 re-expression. In vivo xenograft models revealed that cIAP1 re-expression significantly reduces tumor growth, mitotic activity, and Ki-67 positivity, while increasing tumor necrosis and apoptosis. These findings highlight an unexpected tumor-suppressive role for cIAP1 in fusion-driven sarcomas, contrasting with its pro-survival function in other cancers. Collectively, our results identify cIAP1 as a prognostically relevant, EWSR1::FLI1-regulated hub whose re-expression disrupts tumor progression, offering a potential therapeutic strategy to restore tumor-suppressive pathways in EwS.

cancer biology↗

bioRxiv2025

EWSR1::ETS-low cells promote metabolic reprogramming of the tryptophan-kynurenine-AHR axis, immunosuppression, and poor outcome in Ewing sarcoma

The extent to which dynamic changes in oncogene activity shape cancer cell metabolism and drive disease progression remains poorly understood. Ewing sarcoma (EwS), driven by EWSR1::ETS fusion transcription factors, constitutes an ideal model to interrogate this question, as fluctuations in fusion activity direct divergent transcriptional programs. While EWSR1::ETS-high cells display a rather sessile but proliferative phenotype, EWSR1::ETS-low cells are more invasive. Yet, the mechanisms underlying these different phenotypes remain poorly characterized. Here, by employing an integrative functional metabolomics approach, we link reduced EWSR1::ETS activity in primary EwS tumors to adverse clinical outcome and pronounced activation of the aryl hydrocarbon receptor (AHR) pathway. Low EWSR1::ETS states foster tryptophan catabolism and accumulation of the AHR agonist kynurenine, which in turn promotes an immunosuppressive tumor microenvironment characterized by impaired natural killer (NK) cell cytotoxicity and enrichment of immunoregulatory infiltrates. Functionally, AHR silencing restores NK cell-mediated tumor recognition, while also directly suppressing EwS cell proliferation, clonogenicity, and spheroid growth in plasma-like media. Genetic inhibition of AHR reduces tumor burden and metastatic competence in xenograft models. These findings reveal a mechanistic link between oncogene fluctuation, amino acid metabolism, and immune evasion, positioning AHR as a central mediator of EwS progression and a tractable therapeutic vulnerability.

cancer biology↗

bioRxiv2025

Dynamic modelling of EWS::FLI1 fluctuations reveals molecular determinants of phenotypic tumor plasticity and prognosis in Ewing sarcoma

The mechanisms underlying tumor cell plasticity driving drug resistance and disease progression remain poorly understood. In Ewing sarcoma (EwS), variations in EWS::FLI1 (EF) activity have been associated with epithelial-mesenchymal plasticity (EMP). Using degron technology, we titrated endogenous EF in an EwS cell line and linked phenotypic states to distinct EF thresholds. Strikingly, modest EF depletion promoted a pro-metastatic phenotype, that diminished upon near-complete EF loss. Nascent RNA sequencing revealed distinct gene clusters with heterogenous response patterns to varying EF dosage. Target genes most sensitive to subtle EF depletion contained GGAA microsatellites in EF-bound enhancers. Furthermore, we identified Kruppel-like zinc-finger transcription factors associated with EF-repressed EMP genes. Transient EF depletion followed by rapid restoration to simulate oncoprotein fluctuations identified persistently dysregulated genes associated with poor prognosis. This study underscores the therapeutic challenge of insufficient EF inhibition and provides a foundation for exploiting oncoprotein dynamics to uncover therapeutic vulnerabilities in fusion-driven cancers. Beyond EwS, our results underscore the broader impact of oncoprotein dosage dynamics in cancers with otherwise quiet genomes. SIGNIFICANCEWe report EwS as a paradigm for the importance of oncogene fluctuations in tumor cell plasticity and disease progression. Effective therapeutic strategies must ensure complete EF depletion to prevent inadvertent metastasis.

cancer biology↗

bioRxiv2025

High Tau Expression Correlates with Reduced Invasion and Prolonged Survival in Ewing Sarcoma

The microtubule-associated protein Tau (encoded by the MAPT gene) is linked to a family of neurodegenerative disorders defined as tauopathies, which are characterized by its brain accumulation in neurofibrillary tangles and neuropil threads. Newly described Tau functions comprise DNA protection, chromatin remodeling, p53 regulation and cell fate modulation, suggesting a role of Tau in oncogenesis. Bioinformatic-supported characterization of Tau in cancer reveals robust expression in bone cancer cells, in particular Ewing sarcoma (EwS) cell lines. EwS is an aggressive cancer caused by a fusion of members of the FET and ETS gene families, primarily EWSR1::FLI1. Here we found that MAPT is a EWSR1::ETS target gene and that higher Tau expression in EwS cells inhibited their migratory and invasive behavior, consistent with a more immobile and proliferative phenotype observed in EwS. Indeed, we report that high Tau expression is associated with improved overall survival of EwS patients. We also show that the sessile but proliferative phenotype of EWSR1::ETS-high cells may result from a modulatory role of Tau on focal adhesion to extracellular matrix proteins. Our data highlight the utility of determining Tau expression as a prognostic factor in EwS as well as the opportunity to target Tau expression as an innovative EwS therapy.

cancer biology↗

bioRxiv2024

Loss of SMARCB1 evokes targetable epigenetic vulnerabilities in Epithelioid Sarcoma

Dysfunction of epigenetic modulators, such as the SWI/SNF complex, is a wide-spread but relatively ill-defined feature of a broad spectrum of cancer entities. Among SWI/SNF-mutant entities, SMARCB1-deficient cancers, such as the highly aggressive Epithelioid Sarcoma (EpS), are characterized by this genetic event in an otherwise rather silent mutational landscape. This renders EpS an ideal model to study how epigenetic reprogramming by a single mutation can contribute to tumorigenesis. Hence, to characterize and compare the function of the SMARCB1-deficient, residual and the physiological SWI/SNF complex in cancer, we generated a panel of SMARCB1 re-expressing EpS cell lines and employed a functional multi-omics approach. Here, we show that SWI/SNF holds canonical characteristics of both tumor-suppressors and proto-oncogenes due to its multi-faceted role in the regulation of the epigenome. Our data indicates that the loss of SMARCB1 causes an overall loss of SWI/SNF chromatin affinity at cis-regulatory enhancer elements, inducing a preference for uncontrolled proliferation and cell cycle progression as opposed to development and differentiation. We further demonstrate that EpS cell lines depend on residual SWI/SNF action to maintain clonogenicity and proliferation. Consequently, EpS cell lines exhibit markedly increased sensitivity to pharmacological inhibition of the residual SWI/SNF when compared with SWI/SNF-proficient cancer entities. Collectively, our results from the EpS model shed new light on how a single mutation can rewire the pleiotropic effects of an epigenetic master regulator and provide inroads for therapeutic intervention.

cancer biology↗

bioRxiv2024

Super-enhancer-driven CACNA2D2 is an EWSR1::WT1 signature gene encoding a diagnostic marker for desmoplastic small round cell tumor (DSRCT)

Desmoplastic small round cell tumor (DSRCT) is a highly aggressive cancer predominantly occurring in male adolescents and young adults. The lack of a comprehensive understanding on the biology of the disease is paralleled by its dismal survival rates (5-20%). To overcome this challenge, we first identified and prioritized urgently needed resources for clinicians and researchers. Thus, we established genome-wide single-cell RNA-sequencing and bulk proteomic data of in vitro and in vivo-generated knockdown models of the pathognomonic DSRCT fusion oncoprotein (EWSR1::WT1) and combined them with an original systems-biology-based pipeline including patient data and the largest histology collection of DSRCTs and morphological mimics available to date. These novel tools were enriched with curated public datasets including patient- and cell line-derived ChIP-seq, bulk and single-cell RNA-seq studies resulting in a multi-model and multi-omic toolbox for discovery analyses. As a proof of concept, our approach revealed the alpha-2/delta subunit of the voltage-dependent calcium channel complex, CACNA2D2, as a highly overexpressed, super-enhancer driven, direct target of EWSR1::WT1. Single-cell and bulk-level analyses of patient samples and xenografted cell lines highlighted CACNA2D2 as a critical component of our newly established EWSR1::WT1 oncogenic signature, that can be employed to robustly identify DSRCT in reference sets. Finally, we show that CACNA2D2 is a highly sensitive and specific single biomarker for fast, simple, and cost-efficient diagnosis of DSRCT. Collectively, we establish a large-scale multi-omics dataset for this devastating disease and provide a blueprint of how such toolbox can be used to identify new and clinically relevant diagnostic markers, which may significantly reduce misdiagnoses, and thus improve patient care.

cancer biology↗

bioRxiv2024

Physiologically refined cell culture conditions uncover oncogene-dependent metabolic signatures in Ewing sarcoma spheroids

Ewing sarcoma (EwS) cell line culture largely relies on standard techniques, which do not recapitulate physiological conditions. Here, we report on a physiologically improved, feasible, and cost-efficient EwS cell culture technique employing an advanced medium composition, reduced fetal calf serum, and spheroidal growth. Functional in vitro assays and transcriptome profiling demonstrated that these refined conditions better recapitulate proliferation rates of patient tumors as well as hypoxic conditions relevant for EwS pathophysiology. Moreover, transcriptional signatures associated with the oncogenic activity of the EwS-specific FET::ETS fusion transcription factors in the refined culture conditions were shifted from proliferative towards metabolic gene signatures. The herein presented optimized physiological EwS cell culture technique provides a broadly applicable approach for enhanced in vitro modeling relevant to advancing EwS research and the validity of experimental results. MOTIVATIONCell culture remains the main platform to model EwS for research purposes. Yet, concerns exist about the limitations of standard in vitro techniques to adequately reflect physiological conditions. In this study, we refined EwS cell culture methods to increase modeling capacity while ensuring a practical and cost-effective handling, thereby broadening their applicability within the scientific community.

cancer biology↗

bioRxiv2024

Glutaredoxin 3 (GLRX3) confers a fusion oncogene-dependent vulnerability to Ewing sarcoma

Ewing sarcoma (EwS) is a highly aggressive bone and soft-tissue associated cancer for which there are no effective targeted therapeutics available. Genetically, EwS is driven by aberrantly active EWSR1::ETS fusion transcription factors, most commonly EWSR1::FLI1. Despite their unique expression in EwS, all attempts to effectively target these fusion oncoproteins clinically were not yet successful, wherefore alternative targets are required. Here, we functionally characterize the evolutionarily conserved oxidative stress regulator glutaredoxin 3 (GLRX3) as a EwS-specific and EWSR1::FLI1-dependent vulnerability. Through integration of transcriptome-profiling, conditional drug screens in 3D cultures, and functional experiments, we discover that GLRX3 promotes EwS growth in vitro and in vivo, and that it has a key role in mitigation of oxidative stress and maintenance of iron homeostasis. These GLRX3 functions can be exploited in both GLRX3-high and -low expressing EwS cells by targeted therapeutics including CDK4/6 inhibitors and inducers of apoptotic and ferroptotic cell death. Collectively, our results exemplify how the interplay of an evolutionarily conserved oxidative stress regulator with a dominant oncogene can promote malignancy but provide opportunities for predictive diagnostics and personalized therapy.

cancer biology↗

bioRxiv2024

Somatic gene delivery for flexible in vivo modeling of high-risk sarcoma

A particular challenge hampering therapeutic advancements for high-risk sarcoma patients is the broad spectrum of molecularly distinct sarcoma entities and the corresponding lack of suitable model systems to recapitulate and study these diseases. To overcome this predicament, we developed a novel genetically-controlled, yet versatile mouse modeling platform allowing delivery of different genetic lesions by electroporation (EPO) of the thigh muscle wildtype mice. This optimized sarcoma EPO-GEMM (EPO-based genetically engineered mouse model) platform allowed the generation of ten biologically distinct sarcoma entities, including Synovial Sarcoma (SS), fusion-positive and fusion-negative Rhabdomyosarcoma (RMS), Alveolar Soft Part Sarcoma (ASPS), Undifferentiated Pleomorphic Sarcoma (UPS) and Infantile Fibrosarcoma (IFS). Comprehensive molecular profiling and cross-species analyses confirmed faithful recapitulation of the human disease, including the expression of relevant immunotherapy targets. Syngeneic allografting enabled reliable preservation and scalability of Sarcoma-EPO-GEMMs for treatment trials, such as B7-H3-directed CAR-T cell therapy in an immunocompetent background.

cancer biology↗

bioRxiv2023

Genomic and phenotypic stability of fusion-driven pediatric Ewing sarcoma cell lines

Human cancer cell lines are the mainstay of cancer research. Recent reports showed that highly mutated adult carcinoma cell lines (mainly HeLa and MCF-7) present striking diversity across laboratories and that long-term continuous culturing results in genomic/transcriptomic heterogeneity with strong phenotypical implications. This highlighted how despite human cell line models being powerful tools for cancer research, the findings derived from their use may present limitations in terms of reproducibility. However, to what extent these conclusions can be generalized to the majority of cancer cell lines remained unexplored. Here, we hypothesized that oligomutated pediatric sarcoma cell lines driven by a chimeric oncogenic transcription factor (COTF), such as Ewing sarcoma (EwS), were genetically and phenotypically more stable than the previously investigated (adult) carcinoma cell lines. A comprehensive molecular and phenotypic characterization of multiple EwS cell line strains in direct comparison to the HeLa and MCF-7 cell lines, together with a simultaneous analysis during 12 months of continuous cell culture showed that COTF-driven pediatric sarcoma cell line strains are genomically more stable than adult carcinoma strains, display remarkably stable and homogenous transcriptomes, and exhibit uniform and stable drug response. The analysis of multiple EwS cell lines subjected to long-term continuous culture conditions revealed that variable degrees of genomic/transcriptomic/phenotypic may be observed among COTF-driven cell lines, further exemplifying that the potential for reproducibility of in vitro scientific results may be rather understood as a spectrum, even within the same tumor entity.

cell biology↗

bioRxiv2022

Chromosome 8 gain drives poor patient outcome via expression of 4E-BP1 in Ewing sarcoma

Chromosome 8 (chr8) gains are common in cancer. However, their potential contribution to tumor heterogeneity is largely unexplored. Ewing sarcoma (EwS) is characterized by pathognomonic FET::ETS fusions but a general paucity of other recurrent somatic mutations that could explain the observed clinical diversity. In EwS, chr8 gains are the second most common genetic alteration rendering EwS an ideal model to investigate the relevance of chr8 gains in an otherwise silent genomic context. Here, we report that chr8 gain-driven gene expression patterns correlate with poor overall survival of EwS patients. This effect is predominantly mediated by increased expression of the translation initiation factor binding protein 4E-BP1 encoded by EIF4EBP1 on chr8. High EIF4EBP1 expression showed the strongest association with poor patient survival among all chr8-encoded genes and correlated with chr8 gains in EwS tumors. Similar findings were made in numerous entities of The Cancer Genome Atlas (TCGA). Integrated multi-omics profiling uncovered that 4E-BP1 orchestrates a pro-proliferative proteomic network. Consistently, silencing of 4E-BP1 in the EwS model reduced cell proliferation, clonogenicity, spheroidal growth in vitro, and tumorigenesis in vivo. Drug screens and functional assays revealed that high 4E-BP1 expression sensitizes for pharmacological CDK4/6 inhibition in preclinical models. Collectively, we establish chr8 gains and high 4E-BP1 expression as prognostic biomarkers in EwS and demonstrate that their association with patient outcome is primarily mediated by 4E-BP1 orchestrating a pro-proliferative proteomic network sensitizing EwS for CDK4/6 inhibitors. Our data suggest that testing for chr8 gains may improve risk-stratification and therapeutic management in EwS and other cancers.

cancer biology↗

bioRxiv2022

A palmitate-rich metastatic niche enables metastasis growth via p65 acetylation

Cancer cells outgrowing in distant organs of metastasis rewire their metabolism to fuel on the available nutrients. While this is often considered an adaptive pressure limiting metastasis formation, some nutrients available at the metastatic site naturally or through changes in organ physiology may inherently promote metastatic growth. We find that the lung, a frequent site of metastasis, is a lipid-rich environment. Moreover, we observe that pathological conditions such as pre-metastatic niche formation and obesity further increase the availability of the fatty acid palmitate in the lung. We find that targeting palmitate processing inhibits spheroid growth in vitro and metastasis formation in lean and obese mice. Mechanistically, we discover that breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a (CPT1a)-dependent manner. Lysine acetyltransferase 2a (KAT2a), whose expression is promoted by palmitate availability, relies on the available acetyl-CoA to acetylate the NF-{kappa}B subunit p65. This favors nuclear location of p65 and activates a pro-metastatic transcriptional program. Accordingly, deletion of KAT2a phenocopies CPT1a silencing in vitro as well as in vivo and patients with breast cancer show co-expression of both proteins in metastases across palmitate-rich metastatic sites. In conclusion, we find that palmitate-rich environments foster metastasis growth by increasing p65 acetylation resulting in elevated NF-{kappa}B signaling.

cancer biology↗

bioRxiv2022

Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer types including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity. Here, we show in the EwS model that - capitalizing on neomorphic DNA-binding preferences - the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes. We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to the otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor types typically considered to be immunologically cold. Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.

cancer biology↗

bioRxiv2021

Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation

Cell lines have been essential for major discoveries in cancer including Ewing sarcoma (EwS). EwS is a highly aggressive pediatric bone or soft-tissue cancer characterized by oncogenic EWSR1-ETS fusion transcription factors converting polymorphic GGAA-microsatellites (mSats) into neo-enhancers. However, further detailed mechanistic evaluation of gene regulation in EwS have been hindered by the limited number of well-characterized cell line models. Here, we present the Ewing Sarcoma Cell Line Atlas (ESCLA) comprising 18 EwS cell lines with inducible EWSR1-ETS knockdown that were profiled by whole-genome-sequencing, DNA methylation arrays, gene expression and splicing arrays, mass spectrometry-based proteomics, and ChIP-seq for EWSR1-ETS and histone marks. Systematic analysis of these multi-dimensional data illuminated hundreds of new potential EWSR1-ETS target genes, the nature of EWSR1-ETS-preferred GGAA-mSats, and potential indirect modes of EWSR1-ETS-mediated gene regulation. Moreover, we identified putative co-regulatory transcription factors and heterogeneously regulated EWSR1-ETS target genes that may have implications for the clinical heterogeneity of EwS. Collectively, our freely available ESCLA constitutes an extremely rich resource for EwS research and highlights the power of leveraging multidimensional and comprehensive datasets to unravel principles of heterogeneous gene regulation by dominant fusion oncogenes.

genomics↗