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WEE1 accumulation and deregulation of S-phase proteins mediate MLN4924 potent inhibitory effect on Ewing sarcoma cells

Abstract

Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor of children and young adults in which finding effective new targeted therapies is imperative. Here, we report an in-depth preclinical study of the investigational cullin-RING ubiquitin ligase (CRL) inhibitor MLN4924 in ES, as we have recently demonstrated the implication of a CRL component in the ES pathogenesis. First, our results support a high sensitivity of ES cells to MLN4924 growth inhibition both in vitro (14 ES cell lines tested, median IC50=81 nM) and in tumor xenografts (tumor regression achieved with 60 mg/kg BID, subcutaneously, n=9). Second, we report a dual mechanism of action of MLN4924 in ES cells: while a wide range of MLN4924 concentrations (30–300 nM) trigger a G2 arrest that can only be rescued by WEE1 kinase inhibition or depletion, saturating doses of the drug (>300 nM) cause a delay in S-phase progression concomitant with unbalanced CDK2-Cyclin E and CDK2-Cyclin A relative levels (accumulation of the first and depletion of the latter). The aberrant presence of CDC6 in the nucleus at late S-phase cell cycle stage confirmed the loss of CDK2-Cyclin A-specific functions. Remarkably, other mechanisms explored (P27 accumulation and DNA damage signaling pathways) were found unable to explain MLN4924 effects, strengthening the specificity of our findings and suggesting the absence of functionality of some CRL substrates accumulated in response to MLN4924. This study renders a rationale for clinical trials and contributes molecular mechanisms for a better understanding of this promising antitumoral agent.

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Acknowledgements

We thank Cristina Teodosio and Martín Pérez de Andrés for their kind help in flow cytometry studies. Daniel J García-Domínguez is supported by a grant from the María García Estrada Foundation. Research in Enrique de Alava’s lab is also supported by the Ministry of Economy and Competitiveness of Spain-FEDER (PI081828, RD06/0020/0059, PI1100018, ISCIII_postdoc grant CD06/00001) and the European Commission (FP7-HEALTH-2011-two-stage, Project ID 278742 EUROSARC).

Author contributions: CM conceived the study and its design, analyzed the data and wrote the manuscript. The experimental work was accomplished by CM and DJGD. DJGD and JLO carried out the work with animals. AGP, PGS, MPS and EDA participated in conceptualization of the study and in critical discussion of data. All authors revised and edited the manuscript.

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Correspondence to C Mackintosh or E de Álava.

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Peter G Smith was an employee of Millennium Pharmaceuticals, Inc. at the time of this study. There are no other competing financial interests.

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Supplementary Information accompanies the paper on the Oncogene website

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Mackintosh, C., García-Domínguez, D., Ordóñez, J. et al. WEE1 accumulation and deregulation of S-phase proteins mediate MLN4924 potent inhibitory effect on Ewing sarcoma cells. Oncogene 32, 1441–1451 (2013). https://doi.org/10.1038/onc.2012.153

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