Cód. SSPA: IBiS-C-11
Ewing Sarcoma (ES) is a malignant bone and soft-tissue tumor mainly affecting children and young adults. Advances in terms of multimodal therapies in patients presenting localized disease, lead to a surprising overall survival of 70% at 5-year diagnostic. Despite this, patients presenting multifocal/metastatic or refractory disease present disappointing overall survival rates of around 20%.
From a molecular point of view, ES is characterized by the presence of reciprocal chromosomal translocations, given rise to fusion genes which in turn are translated into chimerical proteins (mostly involving EWSR1-ETS members). These functional chimerical proteins represent the major oncogenic event in ES. They typically involve the EWSR1 gene and a member of the ETS family, mostly FLI1, in about 85% of the cases. This aberrant transcription factor deregulates the transcription of target genes such as IGF1/IGF1R related genes, among many others.
Despite the evident role of the ES fusion protein, recent evidence has demonstrated that other secondary alterations also play an enormous part in terms of tumor development, progression and even clinical prognostic.
The main research line of our group is based on the definition and characterization of these secondary alterations and how they may affect ES biology. Our goal is to establish new therapeutic targets as well as biomarkers of response to treatment based on preclinical studies focused on alternative/novel therapies.
Detailed Objectives
Discovery and validation of molecular mechanisms of tumor initiation and progression in sarcomas, namely in Ewing Sarcoma, our model of study.
Translate these molecular mechanisms to the clinic in the form of biomarkers either of response to treatment or therapeutic monitoring.
Establish stable collaborations with academic/ pharmaceutical groups interested in our research.
Research Lines:
1. Discovery and validation of molecular mechanisms of tumor initiation and progression in Ewing Sarcoma.
The mechanisms of tumor initiation of Ewing Sarcoma remain unclear until this day. Despite this, the mesenchymal stem cell has been postulated as the most probable cell of origin of this tumor entity. Our group, developed an Ewing sarcoma cell line where the EWSR1-FLI1 stable interference led to a decrease of the intrinsic tumor characteristics followed by an increase of mesenchymal stem cells characteristics, namely differentiation capacity and phenotype. This study revealed a new target of the chimerical protein, TOPK1. Also, the in vivo implantation of the interfered tumor cells resulted in the production of smaller tumors in comparison to the parental cell line. (Herrero-Martín D et al, Br J Cancer. 2009 Jul 7;101(1):80-90.)
More recently, in collaboration with other European groups and within the Europoan Project EuroBONET, we have studied the phenotype of Mesenchymal stem cells derived from Ewing Sarcoma patients in comparison to Ewing Sarcoma cells and Ewing Sarcoma samples. We observed that Ewing Sarcoma patients are similar to other mesenchymal stem cells and show no differential phenotype. However, Ewing sarcoma cells present some phenotypic features of mesenchymal stem cells.
Also, the detailed study of CD99 expression in a large set of mesenchymal stem cells revealed that the inhibition of this receptor showed no impairment on this cell type survival. (Amaral AT et al, PLoS One, 2014, )
2. Discovery, functional validation and further clinical translation of new therapeutic targets from genomic, integrative proteomic studies in Ewing Sarcoma.
Pre-clinical in vitro and in vivo studies described the protein HSP90 (Heat Shock Protein 90) as a biomarker of response to treatment with anti-IGF1R (Insulin-like growth factor receptor 1) inhibitors (namely ADW). Integrative proteomic analysis revealed that ES cell lines resistant to treatment with ADW, expressed higher levels of HSP90. This fact was later validated by RNA interference and in vivo studies in xenograft models. Also, Histopathological analysis of Ewing sarcoma samples demonstrated that in fact, in patients, the differential expression of HSP90 was a marker of worst response to treatment. (Martins AS et al, Cancer Res. 2008 Aug 1;68(15):6260-70.)
More recently, in a multicentre retrospective study, we demonstrated through CGH arrays the prognostic value of a particular secondary alteration. The gain of chromosome long arm (1qG) was present in approximately 30% of a total of 67 tumor samples. Furthermore, we performed a transcriptomic study with 38 tumor samples and finally established the 1qG fingerprint. This fingerprint was characterized by the differential expression of 74 genes located on 1q. Cases presenting this 1qG fingerprint were correlated with cell cycle deregulation, relapse and worst survival. Some genes with described over expression resulting from this 1qG in these cases were CDT2 and PARP1. (Mackintosh C et al, Oncogene. 2012 Mar 8;31(10):1287-98)
Design and development of diagnostic tools which will allow the selection of patients for specific treatments, with particular emphasis to FISH and molecular signatures related with the over expression of 1q located genes.
Retrospective Studies showed the clinical relevance of secondary alterations in Ewing Sarcoma, namely, alterations of copy number (CNAs), 1q and 8q Gains, 16q losses, among others. The next objective is to establish in prospective studies the prognostic value of CNAs in Ewing Sarcoma patients, through the development of FISH probes (for the evaluation of 1qG and 16q losses) and genes such as CDT2 and PARP1.
Preclinical evaluation of experimental/commercial drugs on Ewing Sarcoma cell lines (previously validated in the European Project EuroBonet) and in xenograft models.
a) Taking together that CDT2 belongs to a subtype of complexes with ubiquitin ligase activity (known as Cullin-RING ligases, CRL) and that these complexes con be inhibited with a potent specific new drug (MLN4924) we decided to study the effectiveness of MLN4924 in Ewing Sarcoma cell lines. Our results showed a high sensitivity of Ewing Sarcoma cell lines, with a dose dependent mechanism of response. At concentrations lower than the IC50 of proliferation (concentration needed to achieve the death of 50% of the cell population), we observed moderate apoptosis, with a delay in the G2-phase mostly due to the accumulation of WEE1. On the other hand, at concentrations higher than the IC50 of proliferation we observed a delay in the S-phase and high apoptotic index. More importantly, an efficient antitumoral effect was observed in our in vivo model. (Mackintosh C et al, Oncogene. 2013 Mar 14; 32(11):1441-51.)
b) PARP proteins are crucial for cell survival and are involved in several mechanisms such as DNA damage repair, cell cycle, apoptosis induction, angiogenesis and tumor formation and tumor progression. This fact evidently renders PARP proteins as interesting therapeutic targets. Although studies in other solid tumors suggested that PARP inhibitors were mostly active in tumors with deficiencies/mutations in DNA repair mechanisms, and that in Ewing Sarcoma these have not been described, the use of PARP inhibitors in combination with other chemotherapeutic agents (Temozolamide and radiotherapy) has shown great results in recent in vitro / in vivo studies. In our lab, we are focused in studying possible drug combinations of PARP inhibitors and other DNA-related agents in in vitro and in vivo models in Ewing Sarcoma.
c) Another key mechanism related to malignancy is the over expression of the IGF1-IGF1R axis in Ewing Sarcoma. Initially, we studied the effects of small molecules (ADW), which by competition of IGF1 exert an antagonistic action, blocking IGF1R activation. This tyrosine kynase inhibitor is an important regulator of cell proliferation in Ewing Sarcoma. ADW demonstrated an efficient anti-tumoral activity in comparison to traditional chemotherapeutic agents such as Vincristine or Doxorubicin. Interestingly, resistance to anti-IGF1R therapies has been related to the IGF2/IR dependency as well as with HSP90 over expression. So, more recently, and within the European project EuroSARC, we have studied the effects of a dual inhibitor of IGF1R/IR in Ewing Sarcoma cell lines, Linsitinb. In fact, we combined Linsitinib with DNA damage inducing agent, Trabectedin, and observed high levels of synergism.
Development of epigenetic studies based in the activity of the chimerical protein EWSR1/FLI1 in models of inducible ectopic expression. These studies include chromatin remodeling, non-coding RNAs and pre-clinical in vitro and in vivo studies with epigenetic-based drugs.
Secondary genetic alterations and epigenetic alterations might help to explain the most aggressive forms of Ewing Sarcoma and reveal new promising therapeutic targets. In this study we propose the stratification of Ewing sarcoma patients according to their epigenetic profile. We are currently developing and validating diagnostic tools which will allow us to molecularly characterize Ewing Sarcoma patients through techniques which can be easily translated to the clinical reality. Finally, we plan to further study the targets found through in vivo and in vitro studies and according to their effectiveness applied them to future clinical trials in Ewing Sarcoma patients.
