Oscar Martã­nEz Tirado

Metastatic Ewing sarcoma (EwS) remains a major clinical challenge, highlighting the need to identify molecular drivers of disease dissemination. Through integrated transcriptomic, functional, and mechanistic analyses, we identify PARP14 as a key regulator of EwS metastatic progression. PARP14 is consistently upregulated in metastatic lesions across spontaneous and xenograft mouse models as well as in patient samples. Genetic and pharmacologic depletion of PARP14 (via siRNA knockdown, CRISPR-Cas9 knockout, or PROTAC-mediated degradation) significantly impaired EwS cell migration and invasion in vitro, without affecting proliferation or clonogenic capacity. In vivo, PARP14 loss delayed tumor growth and reduced metastatic burden, supporting a functional role in disease progression. Conversely, IFNγ-induced PARP14 upregulation and stable overexpression enhanced EwS cell migration and increased lung metastasis formation. Transcriptomic profiling of PARP14-deficient cells revealed downregulation of invasive signatures and enrichment of inflammatory and p53-related pathways, together with gene sets associated with chromatin remodeling. Subcellular fractionation and interactome analyses showed that PARP14 localizes to cytoplasmic, nuclear, and chromatin-associated compartments, and interacts with chromatin, cytoskeletal and ECM regulators. ChIP analyses further demonstrated PARP14 binding to promoters enriched for NRF1 motifs, suggesting a role as a transcriptional co-regulator influencing gene networks relevant to EwS progression. Together, our results establish PARP14 as a contributor to EwS dissemination and uncover a previously unappreciated chromatin-associated role for this protein, supporting its potential as both a biomarker and therapeutic target in metastatic EwS.

Ewing sarcoma (EWS) is the second most common bone tumor affecting children and young adults, with dismal outcomes for patients with metastasis at diagnosis. Mechanisms leading to metastasis remain poorly understood. To deepen our knowledge on EWS progression, we have profiled tumors and metastases from a spontaneous metastasis mouse model using a multi‐omics approach. Combining transcriptomics, proteomics, and methylomics analyses, we identified signaling cascades and candidate genes enriched in metastases that could be modulating aggressiveness in EWS. Phenotypical validation of two of these candidates, cyclic AMP‐responsive element‐binding protein 1 (CREB1) and lipoxygenase homology domain‐containing protein 1 (LOXHD1), showed an association with migration and clonogenic abilities. Moreover, previously described CREB1 downstream targets were present amongst the metastatic‐enriched results. Combining the different omics datasets, we identified FYVE, RhoGEF, and PH domain‐containing protein 4 (FGD4) as a CREB1 target interconnecting the different EWS biological layers (RNA, protein and methylation status) and whose high expression is associated with worse clinical outcome. Further studies will provide insight into EWS metastasis mechanisms and ultimately improve survival rates for EWS patients.

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor with high susceptibility to metastasize. The underlying molecular mechanisms leading to EWS metastases remain poorly understood. Epigenetic changes have been implicated in EWS tumor growth and progression. Linking epigenetics and metastases may provide insight into novel molecular targets in EWS and improve its treatment. Here, we evaluated the effects of a selective G9a histone methyltransferase inhibitor (BIX01294) on EWS metastatic process. Our results showed that overexpression of G9a in tumors from EWS patients correlates with poor prognosis. Moreover, we observe a significantly higher expression of G9a in metastatic EWS tumor as compared to either primary or recurrent tumor. Using functional assays, we demonstrate that pharmacological G9a inhibition using BIX01294 disrupts several metastatic steps in vitro, such as migration, invasion, adhesion, colony formation and vasculogenic mimicry. Moreover, BIX01294 reduces tumor growth and metastases in two spontaneous metastases mouse models. We further identified the sialidase NEU1 as a direct target and effector of G9a in the metastatic process in EWS. NEU1 overexpression impairs migration, invasion and clonogenic capacity of EWS cell lines. Overall, G9a inhibition impairs metastases in vitro and in vivo through the overexpression of NEU1. G9a has strong potential as a prognostic marker and may be a promising therapeutic target for EWS patients.

Diamond nanoparticles (nanodiamonds) can transport active drugs in cultured cells as well as in vivo. However, in the latter case, methods allowing the determination of their bioavailability accurately are still lacking. A nanodiamond can be made fluorescent with a perfectly stable emission and a lifetime ten times longer than that of tissue autofluorescence. Taking advantage of these properties, we present an automated quantification method of fluorescent nanodiamonds (FND) in histological sections of mouse organs and tumors, after systemic injection. We use a home-made time-delayed fluorescence microscope comprising a custom pulsed laser source synchronized on the master clock of a gated intensified array detector. This setup allows ultra-high-resolution images (120 Mpixels in size) of whole mouse organ sections to be obtained, with subcellular resolution and single-particle sensitivity. As a proof-of-principle experiment, we quantified the biodistribution and aggregation state of new cationic FNDs capable of transporting small interfering RNA inhibiting the oncogene responsible for Ewing sarcoma. Image analysis showed a low yield of nanodiamonds in the tumor after intravenous injection. Thus, for the in vivo efficacy assay, we injected the nanomedicine into the tumor. We achieved a 28-fold inhibition of the oncogene. This method can readily be applied to other nanoemitters with ≈100 ns lifetime.

RING1B is necessary for the expression of key EWSR1-FLI1 targets by promoting oncogene recruitment to enhancers.

Bone sarcomas are rare cancers which often present with metastatic disease and are still associated with poor survival rates. Studies in the last decade have identified that exosomes, a type of extracellular vesicle released by cells, play an important role in tumour progression and dissemination. Through the transfer of their cargo (RNAs, proteins, and lipids) across cells, they are involved in cellular cross-talk and can induce changes in cellular behaviour. Exosomes have been shown to be important in metastasis organotropism, induction of angiogenesis and vascular permeability, the education of cells towards a pro-metastatic phenotype or the interaction between stromal and tumour cells. Due to the importance exosomes have in disease progression and the high incidence of metastasis in bone sarcomas, recent studies have evaluated the implications of these extracellular vesicles in bone sarcomas. In this review, we discuss the studies that evaluate the role of exosomes in osteosarcoma, Ewing sarcoma, and preliminary data on chondrosarcoma.

One largely unknown question in cell biology is the discrimination between inconsequential and functional transcriptional events with relevant regulatory functions. Here, we find that the oncofetalHMGA2gene is aberrantly reexpressed in many tumor types together with its antisense transcribed pseudogeneRPSAP52.RPSAP52is abundantly present in the cytoplasm, where it interacts with the RNA binding protein IGF2BP2/IMP2, facilitating its binding to mRNA targets, promoting their translation by mediating their recruitment on polysomes and enhancing proliferative and self-renewal pathways. Notably, downregulation ofRPSAP52impairs the balance between the oncogeneLIN28Band the tumor suppressorlet-7family of miRNAs, inhibits cellular proliferation and migration in vitro and slows down tumor growth in vivo. In addition, high levels ofRPSAP52in patient samples associate with a worse prognosis in sarcomas. Overall, we reveal the roles of a transcribed pseudogene that may display properties of an oncofetal master regulator in human cancers.

(2019). Targeting fusion proteins: a double edge sword?. Expert Opinion on Therapeutic Targets. Ahead of Print.