Selective histone methyltransferase G9a inhibition reduces metastatic development of Ewing sarcoma through the epigenetic regulation of NEU1

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

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

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor of children and young adults in which the principal driver is a fusion gene, EWSR1-FLI1. Although the essential role of EWSR1-FLI1 protein in the regulation of oncogenesis, survival, and tumor progression processes has been described in-depth, little is known about the regulation of chimeric fusion-gene expression. Here, we demonstrate that the active nuclear HDAC6 in EWS modulates the acetylation status of specificity protein 1 (SP1), consequently regulating the SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of the 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. High expression of HDAC6 in primary EWS tumor samples from patients correlates with a poor prognosis in two independent series accounting 279 patients. Notably, a combination treatment of a selective HDAC6 and doxorubicin (a DNA damage agent used as a standard therapy of EWS patients) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for patients with EWS.Research in the E.D.A. lab is supported by Asociación Española Contra el Cáncer (AECC), the Ministry of Science of Spain-FEDER (CIBERONC, PI1700464, PI2000003, RD06/0020/0059)S. D.G.D. and L.H.P. are supported by CIBERONC (CB16/12/00361). D.G.D., M.J.R. and L.H.P. are PhD researchers funded by the Consejería de Salud, Junta de Andalucía (PI-0197-2016, ECAI F2-0012-2018 and PI-0013-2018, respectively).Peer reviewe

Polycomb and KDM6A Roles in the Epigenetic Dynamics of Ewing Sarcoma Tumorigenesis

[eng] Ewing sarcoma (ES) is a highly aggressive tumour of the bone and soft tissue that typically affects children, adolescents, and young adults. The fusion protein EWSR1‐FLI1 is the main genetic alteration found that acts as a pioneer factor during early ES tumorigenesis. EWSR1– FLI1 binds GGAA microsatellites causing remodelling of enhancers and genome reprogramming. Among the proteins cooperating with EWSR1‐FLI1 at enhancers, our group has described that the Polycomb subunit RING1B co‐localizes genome‐wide and promotes oncogene recruitment and transcriptional activation of key enhancer genes. In this thesis, we show that RING1B is a critical factor for ES tumour growth and, together with other E3 ubiquitin ligases, is deregulated by the neddylation pathway inhibitor MLN4924 (pevonedistat). Treatment of ES cell lines with the inhibitor promotes RING1B loss from EWSR1‐FLI1–activated targets as well as eviction of EWSR1‐FLI1, thereby deregulating gene expression. Lack of neddylation caused by the drug appears to be the mechanism behind the in vitro and in vivo degradation of RING1B. We also study how introduction of EWSR1‐FLI1 to a putative cell‐of‐origin deregulates distribution of the histone repressive mark H3K27me3. Although the global levels of this histone modification are maintained, we observe a perturbed distribution. Specifically, gain or loss of H3K27me3 occurs in some EWSR1‐FLI1–repressed or –activated regions, respectively, which coincide with enrichment of its writer, EZH2, and its eraser, KDM6A, in a transformed ES cell line. Finally, we demonstrate that targeting the tumorigenic distribution of H3K27me3 by combined inhibition of EZH2 (with GSK126) and KDM6A (with GSKJ4) causes a synergic cytotoxic response in ES cell lines. Altogether, our data provide further insight into the epigenetic mechanisms underlying EWSR1‐FLI1–mediated transformation and reveal new targets for future clinical trials

Polycomb and KDM6A Roles in the Epigenetic Dynamics of Ewing Sarcoma Tumorigenesis

Ewing sarcoma (ES) is a highly aggressive tumour of the bone and soft tissue that typically affects children, adolescents, and young adults. The fusion protein EWSR1‐FLI1 is the main genetic alteration found that acts as a pioneer factor during early ES tumorigenesis. EWSR1– FLI1 binds GGAA microsatellites causing remodelling of enhancers and genome reprogramming. Among the proteins cooperating with EWSR1‐FLI1 at enhancers, our group has described that the Polycomb subunit RING1B co‐localizes genome‐wide and promotes oncogene recruitment and transcriptional activation of key enhancer genes. In this thesis, we show that RING1B is a critical factor for ES tumour growth and, together with other E3 ubiquitin ligases, is deregulated by the neddylation pathway inhibitor MLN4924 (pevonedistat). Treatment of ES cell lines with the inhibitor promotes RING1B loss from EWSR1‐FLI1–activated targets as well as eviction of EWSR1‐FLI1, thereby deregulating gene expression. Lack of neddylation caused by the drug appears to be the mechanism behind the in vitro and in vivo degradation of RING1B. We also study how introduction of EWSR1‐FLI1 to a putative cell‐of‐origin deregulates distribution of the histone repressive mark H3K27me3. Although the global levels of this histone modification are maintained, we observe a perturbed distribution. Specifically, gain or loss of H3K27me3 occurs in some EWSR1‐FLI1–repressed or –activated regions, respectively, which coincide with enrichment of its writer, EZH2, and its eraser, KDM6A, in a transformed ES cell line. Finally, we demonstrate that targeting the tumorigenic distribution of H3K27me3 by combined inhibition of EZH2 (with GSK126) and KDM6A (with GSKJ4) causes a synergic cytotoxic response in ES cell lines. Altogether, our data provide further insight into the epigenetic mechanisms underlying EWSR1‐FLI1–mediated transformation and reveal new targets for future clinical trials

Polycomb and KDM6A Roles in the Epigenetic Dynamics of Ewing Sarcoma Tumorigenesis

Ewing sarcoma (ES) is a highly aggressive tumour of the bone and soft tissue that typically affects children, adolescents, and young adults. The fusion protein EWSR1‐FLI1 is the main genetic alteration found that acts as a pioneer factor during early ES tumorigenesis. EWSR1– FLI1 binds GGAA microsatellites causing remodelling of enhancers and genome reprogramming. Among the proteins cooperating with EWSR1‐FLI1 at enhancers, our group has described that the Polycomb subunit RING1B co‐localizes genome‐wide and promotes oncogene recruitment and transcriptional activation of key enhancer genes. In this thesis, we show that RING1B is a critical factor for ES tumour growth and, together with other E3 ubiquitin ligases, is deregulated by the neddylation pathway inhibitor MLN4924 (pevonedistat). Treatment of ES cell lines with the inhibitor promotes RING1B loss from EWSR1‐FLI1–activated targets as well as eviction of EWSR1‐FLI1, thereby deregulating gene expression. Lack of neddylation caused by the drug appears to be the mechanism behind the in vitro and in vivo degradation of RING1B. We also study how introduction of EWSR1‐FLI1 to a putative cell‐of‐origin deregulates distribution of the histone repressive mark H3K27me3. Although the global levels of this histone modification are maintained, we observe a perturbed distribution. Specifically, gain or loss of H3K27me3 occurs in some EWSR1‐FLI1–repressed or –activated regions, respectively, which coincide with enrichment of its writer, EZH2, and its eraser, KDM6A, in a transformed ES cell line. Finally, we demonstrate that targeting the tumorigenic distribution of H3K27me3 by combined inhibition of EZH2 (with GSK126) and KDM6A (with GSKJ4) causes a synergic cytotoxic response in ES cell lines. Altogether, our data provide further insight into the epigenetic mechanisms underlying EWSR1‐FLI1–mediated transformation and reveal new targets for future clinical trials

RING1B recruits EWSR1-FLI1 and cooperates in the remodeling of chromatin necessary for Ewing sarcoma tumorigenesis

Ewing sarcoma (EwS) is an aggressive tumor that affects adolescents and young adults. EwS is defined by a chromosomal translocation, EWSR1-FLI1 being the most common, that causes genome reprogramming through remodeling of enhancers. Here, we describe an unexpected function of RING1B, which is highly expressed in EwS. While retaining its repressive activity at Polycomb developmental regulated genes, RING1B colocalizes with EWSR1-FLI1 at active enhancers. We demonstrate that RING1B is necessary for the expression of key EWSR1-FLI1 targets by facilitating oncogene recruitment to their enhancers. Knockdown of RING1B impairs growth of tumor xenografts and expression of genes regulated by EWSR1-FLI1 bound enhancers. Pharmacological inhibition of AURKB with AZD1152 increases H2Aub levels causing down-regulation of RING1B/EWSR1-FLI1 common targets. Our findings demonstrate that RING1B is a critical modulator of EWSR1-FLI1–induced chromatin remodeling, and its inhibition is a potential therapeutic strategy for the treatment of these tumors.Asociación Española Contra el Cáncer (AECC) GCB13131578Asociación Pablo Ugarte (APU)Instituto de Salud Carlos III PI16/00245Ministerio de Economía, Industria y Competitividad (MEIC) BFU2016-75008-PFundación Vencer el Cáncer (VEC

Ewing Sarcoma Meets Epigenetics, Immunology and Nanomedicine: Moving Forward into Novel Therapeutic Strategies.

Ewing Sarcoma (EWS) is an aggressive bone and soft tissue tumor that mainly affects children, adolescents, and young adults. The standard therapy, including chemotherapy, surgery, and radiotherapy, has substantially improved the survival of EWS patients with localized disease. Unfortunately, this multimodal treatment remains elusive in clinics for those patients with recurrent or metastatic disease who have an unfavorable prognosis. Consistently, there is an urgent need to find new strategies for patients that fail to respond to standard therapies. In this regard, in the last decade, treatments targeting epigenetic dependencies in tumor cells and the immune system have emerged into the clinical scenario. Additionally, recent advances in nanomedicine provide novel delivery drug systems, which may address challenges such as side effects and toxicity. Therefore, therapeutic strategies stemming from epigenetics, immunology, and nanomedicine yield promising alternatives for treating these patients. In this review, we highlight the most relevant EWS preclinical and clinical studies in epigenetics, immunotherapy, and nanotherapy conducted in the last five years

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

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor of children and young adults in which the principal driver is a fusion gene, EWSR1-FLI1. Although the essential role of EWSR1-FLI1 protein in the regulation of oncogenesis, survival, and tumor progression processes has been described in-depth, little is known about the regulation of chimeric fusion-gene expression. Here, we demonstrate that the active nuclear HDAC6 in EWS modulates the acetylation status of specificity protein 1 (SP1), consequently regulating the SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of the 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. High expression of HDAC6 in primary EWS tumor samples from patients correlates with a poor prognosis in two independent series accounting 279 patients. Notably, a combination treatment of a selective HDAC6 and doxorubicin (a DNA damage agent used as a standard therapy of EWS patients) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for patients with EWS.Ministerio de Ciencia de España y fondos FEDER. PI1700464, PI2000003 y RD06/0020/0059Consejería de Salud, Junta de Andalucía. PI-0197-2016, ECAI F2-0012-2018 y PI-0013-2018