Biology subjects

de Alava, E.

Publications and source records attributed to de Alava, E..

9 recordsLinked to original sources

bioRxiv2026

EXO1 Facilitates MiDAS and Prevents Genome Instability and Cell Death in Ewing Sarcoma

Ewing sarcoma (EwS) is an aggressive malignancy driven by EWSR1::ETS fusions, predominantly EWSR1::FLI1. Previous efforts using both direct and indirect approaches to target these chimeric oncoproteins have yielded limited clinical benefit. Although EWSR1::FLI1 is a well-known source of replication stress and genome instability, targeting DNA damage response (DDR) factors that mitigate these effects remain poorly understood. Here, we identified a marked dependency of EwS cells on exonuclease 1 (EXO1). We demonstrate that EXO1 is essential for EwS cell survival and tumor growth, highlighting its potential as a novel therapeutic target. Intriguingly, we unveil that EXO1 loss impairs mitotic DNA synthesis (MiDAS), promoting EWSR1::FLI1-associated genome instability and cell death. Collectively, our results support the idea that targeting DDR factors, which counteract replication stress and/or DNA damage induced by fusion oncoproteins, represents a promising therapeutic option for EwS.

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↗

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↗

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↗

bioRxiv2023

EWS::FLI1-DHX9 interaction promotes Ewing sarcoma sensitivity to DNA topoisomerase 1 poisons by altering R-loop metabolism

Drug resistance is one of the major factors associated with poor outcome of cancer patients. Treatment of Ewing sarcoma (EwS), an aggressive neoplasm mainly affecting children, adolescents and young adults, is associated with therapy failure and tumor relapse in 30-80% of the cases. Thus, it supports the need to explore the mechanisms modulating drug activity. Here, we describe a novel mechanism of drug sensitivity based on the role of EWS::FLI1 in R-loop metabolism. Our results demonstrate that EWS::FLI1 promotes R-loop formation favoring the interaction between DHX9 and elongating RNA polymerase II. In addition, we discovered that EWS::FLI1 kidnaps DHX9 preventing the resolution of TOP1 poisoning-associated R-loops. Our findings indicate that R-loops accumulation promotes replicative stress, genome instability and cell sensitivity to SN-38. Collectively, these results uncover a novel mechanism behind EwS sensitivity to genotoxic agents, with relevant implications for EwS treatment.

cancer biology↗

bioRxiv2021

HDAC6 regulates expression of the oncogenic driver EWSR1-FLI1 through the EWSR1 promoter in Ewing sarcoma

Ewing sarcoma (EWS) is an aggressive developmental sarcoma driven by a fusion gene, EWSR1-FLI1. However, little is known about the regulation of EWSR1-FLI1 chimeric fusion gene expression. Here, we demonstrate that active nuclear HDAC6 in EWS modulates acetylation status of specificity protein 1 (SP1), consequently regulating SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. Overexpression of HDAC6 in primary EWS tumor clinical samples correlates with a poor prognosis. Notably, a combination treatment of a selective HDAC6 inhibitor and doxorubicin (standard of care in EWS) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for EWS patients.

cancer biology↗

bioRxiv2020

NOVEL NUCLEAR ROLE OF HDAC6 IN PROGNOSIS AND THERAPEUTIC TARGET FOR COLORECTAL CANCER

Histone deacetylase 6 (HDAC6) inhibition is a potential treatment of a wide range of cancer types via the acetylation of diverse proteins in the cytoplasm. However, the regulation of histone acetylation and the maintenance of higher-order chromatin structure remains unidentified. Here, we investigated the effect of selective inhibition of HDAC6 by histone acetylation, chromatin relaxation assays, co-immunoprecipitation, acetylome peptide array and in vivo RNA microarray. Our data shows that nuclear HDAC6 physically interacts with the Histone 4 lysine 12 residue, and that HDAC6 inhibition increases acetylation specifically at this residue in several cancer types. Inhibition induces major chromatin structure modulation, but has no equivalent effect on knockout HDAC6-/- MEF cells. We identified several novel HDAC6-deacetylated substrates and high expression of HDAC6 in colorectal cancer (CRC) tissue association with reduced levels of H4K12ac and independent of the key CRC driver mutations, but positively associated with EGFR expression. Furthermore, in vivo HDAC6 inhibition induces significant tumor regression in a CRC xenograft mice model with significant changes in the expression of functional nuclear genes. We also demonstrated that a DNA damaging agent in combination with selective HDAC6 inhibition is effective and acts synergistically, inducing chromatin relaxation and increased cell death in CRC cells. CRC tissues (Normal versus tumor; n=58 matched pairs) together with TCGA data analysis of 467 CRC patients showed that high HDAC6 expression is associated with metastasis, overall and disease-free survival, and is an independent risk factor of CRC stage progression. Our findings designate a new role for nuclear HDAC6 both in cancer prognosis and as a new therapeutic target for CRC and other types of cancer. HighlightHistone deacetylases 6 activity; Chromatin relaxation; Histone modifications; Gene array; DOX: doxorubicin; OXA: oxaliplatin; 5-FU: fluorouracil; Ac: acetylation; MNase: Micrococal nuclease.

cancer biology↗

bioRxiv2019

Hippo pathway effectors YAP1/TAZ induce a EWS-FLI1-opposing gene signature and associate with disease progression in Ewing Sarcoma

YAP1 and TAZ (WWTR1) oncoproteins are the final transducers of Hippo tumor suppressor pathway. Deregulation of the pathway leads to YAP1/TAZ activation fostering tumorigenesis in multiple malignant tumor types, including sarcoma. However, oncogenic mutations within the core components of the Hippo pathway are uncommon. Ewing Sarcoma (EwS), a pediatric cancer with low mutation rate, is characterized by a canonical fusion involving EWSR1 gene, and FLI1 as the most common partner. The fusion protein is a potent driver of oncogenesis but secondary alterations are scarce, and little is known about other biological factors that determine the risk of relapse or progression. We have observed YAP1/TAZ expression and transcriptional activity in EwS cell lines. Analyses of 55 primary human EwS samples revealed that high YAP1/TAZ expression was associated with progression of the disease and predicted poorer outcome.\n\nWe did not observe recurrent SNV or copy number gains/losses in Hippo pathway-related loci. However, differential CpG methylation of RASSF1 locus -a regulator of Hippo pathway- was observed in EwS cell lines compared with mesenchymal stem cells, the putative cell of origin of EwS. Hypermethylation of RASSF1 correlated with the transcriptional silencing of the tumor suppressor isoform RASFF1A, and transcriptional activation of the protumorigenic isoform RASSF1C promoting YAP1/TAZ activation. Knockdown of YAP1/TAZ decreased proliferation and invasion abilities of EwS cells, and revealed that YAP1/TAZ transcription activity is inversely correlated with the EWS-FLI1 transcriptional signature. This transcriptional antagonism could be partly explained by EWS-FLI1-mediated transcriptional repression of TAZ. Thus, YAP1/TAZ may override the transcriptional program induced by the fusion protein, contributing to the phenotypic plasticity determined by dynamic fluctuation of the fusion protein, a recently proposed model for disease dissemination in EwS.

cancer biology↗

bioRxiv2017

Robust diagnosis of Ewing sarcoma by immunohistochemical detection of super-enhancer-driven EWSR1-ETS targets

Ewing sarcoma is an undifferentiated bone-associated cancer. Although molecular detection of pathognomonic EWSR1-ETS fusions such as EWSR1-FLI1 enables definitive diagnosis, substantial confusion can arise if molecular diagnostics are unavailable. Diagnosis based solely on the conventional immunohistochemical marker CD99 is unreliable due to its abundant expression in morphological mimics. This study aimed to identify novel diagnostic immunohistochemical markers for Ewing sarcoma.\n\nWe analyzed 768 expression microarrays representing 21 tumor entities including Ewing-like sarcomas to nominate candidate biomarkers. These candidates were validated by immunohistochemistry (IHC) in a tissue microarray (TMA) comprising 174 samples. Microarray, chromatin immunoprecipitation and sequencing (ChIP-Seq) data, and reporter assays were employed to analyze their EWSR1-FLI1-dependency.\n\nOur comparative expression analyses revealed that ATP1A1, BCL11B, and GLG1 constitute specific markers for Ewing sarcoma. Analysis of ChIP-Seq and microarray datasets showed that their expression is EWSR1-FLI1-dependent. This outcome corresponded to EWSR1-FLI1-binding to proximal super-enhancers, which showed high activity in reporter assays. Consistently, high ATP1A1, BCL11B, and GLG1 expressions were detected by IHC. Automated cut-off-finding and combination-testing in the TMA demonstrated that detection of high BCL11B and/or GLG1 expression is sufficient to reach 96% specificity for Ewing sarcoma. While 88% of tested Ewing-like sarcomas displayed strong CD99-immunoreactivity, none displayed combined high expression of BCL11B and GLG1.\n\nCollectively, we provide evidence that ATP1A1, BCL11B, and GLG1 are EWSR1-FLI1 targets, of which BCL11B and GLG1 offer a fast, simple and cost-efficient way to diagnose Ewing sarcoma by IHC. We anticipate that these markers will significantly reduce the number of misdiagnosed patients, and thus improve patient care.

pathology↗