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Targeting EWS/FLI1 activity in ewing sarcoma


Boro, Aleksandar. Targeting EWS/FLI1 activity in ewing sarcoma. 2012, University of Zurich, Faculty of Science.

Abstract

Ewing sarcoma (ES) is the second most frequent bone cancer in childhood. Clinically, ES appears as very aggressive osteolytic tumor with early tendency for development of metastasis. It belongs to the group of small-round-blue-cell tumors and is comprised of largely undifferentiated cells. The unique feature of this tumor is presence of the balanced t(11;22)(q24;q12) translocation in more than 85% of cases. This gene rearrangement results in the expression of a chimeric fusion protein where RNA binding domain of EWS is exchanged by the DNA binding domain of the ets transcription factor FLI1, thus generating a dysregulated transcription factor EWS/FLI1. More than 18 less represented alternative translocations involving EWS and other ets protein family members have been described since. Extensive evidence supports the fact that EWS/FLI1 is an essential oncogenic component of ES development. Its oncogenic activity is thought to be mediated through inappropriate regulation of target genes that are crucial for the fully malignant phenotype. Apart from having transforming and tumorigenic potential, even more important is that EWS/FLI1 appears to be necessary for tumor cell maintenance. For these reasons EWS/FLI1 represents an attractive target. However, it is widely accepted that transcription factors are ‘’undruggable’’, and EWS/FLI1 as intrinsically disordered protein is not prone to direct inhibition by a small molecule in the classical sense. No targeted agents have been routinely introduced into therapy of ES. Even though in the last few decades there has been considerable progress in both diagnosis as well as treatment of the localised disease, only 15% of patients with metastatic disease survive. Current treatment regimens of ES are not fully exploiting the fact that specific inhibition of signalling pathways is now possible. More specifically, it is not known whether specific pathway could affect the activity of EWS/FLI1. In this thesis, we characterized PHLDA1 as a novel direct target gene whose expression is repressed by EWS/FLI1. Moreover, the role of PHLDA1 in ES was addressed in this study. Using this gene and additional specific well-characterized target genes such as NROB1, NKX2.2, and CAV1, all activated by EWS/FLI1, as a read-out system, we established a screening system that is capable of detecting compounds targeting activity and/or expression of EWS/FLI1 either 6 directly or indirectly. This system is an important simplification of the previously applied gene expression based high-throughput screening, represents a more targeted approach than screenings based on survival or proliferation only, and is more robust than a screening approach based on a single-gene reporter assay. We screened a small-molecule compound library enriched for FDA-approved drugs for substances that modulated the expression of EWS/FLI1 target genes. Among a hit-list of 9 well known drugs such as camptothecin, fenretinide, etoposide and doxorubicin, we also identified the kinase inhibitor midostaurin (PKC412). Subsequent experiments demonstrated that midostaurin is able to induce apoptosis in a panel of 6 ES cell lines in vitro and can significantly suppress xenograft tumor growth in vivo. These results suggest that midostaurin might be a novel drug that is active against ES cells which might act by modulating the expression of EWS/FLI1 target genes. In the second part of this thesis, in order to identify molecular pathway(s) that may contribute to the transcriptional activity and oncogenic properties of EWS/FLI1, we used our well established screening approach and performed screening of a small library of 153 targeted inhibitors covering all major signaling pathways. We discovered PI3K inhibitors as potent modulators of EWS/FLI1 expression. This finding was confirmed in several ES cell lines and off target effects of the PI3K inhibitors was excluded by performing genetic loss of function experiments. Analysis of the EWS/FLI1 promoter region using various deletion constructs in reporter gene assays determined two 12bp minimal elements where transcription factor(s) under PI3K control is binding. Elucidating the direct link between PI3K and EWS/FLI1 is of importance, and identity of the responsible transcription factor(s) might provide novel therapeutic opportunities. Results presented in this thesis clearly demonstrate that screening approach here established can be used for both screening for compounds effective against ES as well as for screening targeted inhibitors leading to better understanding of the biology of ES. Together it should lead to development of novel therapeutic approaches for the treatment of ES.

Abstract

Ewing sarcoma (ES) is the second most frequent bone cancer in childhood. Clinically, ES appears as very aggressive osteolytic tumor with early tendency for development of metastasis. It belongs to the group of small-round-blue-cell tumors and is comprised of largely undifferentiated cells. The unique feature of this tumor is presence of the balanced t(11;22)(q24;q12) translocation in more than 85% of cases. This gene rearrangement results in the expression of a chimeric fusion protein where RNA binding domain of EWS is exchanged by the DNA binding domain of the ets transcription factor FLI1, thus generating a dysregulated transcription factor EWS/FLI1. More than 18 less represented alternative translocations involving EWS and other ets protein family members have been described since. Extensive evidence supports the fact that EWS/FLI1 is an essential oncogenic component of ES development. Its oncogenic activity is thought to be mediated through inappropriate regulation of target genes that are crucial for the fully malignant phenotype. Apart from having transforming and tumorigenic potential, even more important is that EWS/FLI1 appears to be necessary for tumor cell maintenance. For these reasons EWS/FLI1 represents an attractive target. However, it is widely accepted that transcription factors are ‘’undruggable’’, and EWS/FLI1 as intrinsically disordered protein is not prone to direct inhibition by a small molecule in the classical sense. No targeted agents have been routinely introduced into therapy of ES. Even though in the last few decades there has been considerable progress in both diagnosis as well as treatment of the localised disease, only 15% of patients with metastatic disease survive. Current treatment regimens of ES are not fully exploiting the fact that specific inhibition of signalling pathways is now possible. More specifically, it is not known whether specific pathway could affect the activity of EWS/FLI1. In this thesis, we characterized PHLDA1 as a novel direct target gene whose expression is repressed by EWS/FLI1. Moreover, the role of PHLDA1 in ES was addressed in this study. Using this gene and additional specific well-characterized target genes such as NROB1, NKX2.2, and CAV1, all activated by EWS/FLI1, as a read-out system, we established a screening system that is capable of detecting compounds targeting activity and/or expression of EWS/FLI1 either 6 directly or indirectly. This system is an important simplification of the previously applied gene expression based high-throughput screening, represents a more targeted approach than screenings based on survival or proliferation only, and is more robust than a screening approach based on a single-gene reporter assay. We screened a small-molecule compound library enriched for FDA-approved drugs for substances that modulated the expression of EWS/FLI1 target genes. Among a hit-list of 9 well known drugs such as camptothecin, fenretinide, etoposide and doxorubicin, we also identified the kinase inhibitor midostaurin (PKC412). Subsequent experiments demonstrated that midostaurin is able to induce apoptosis in a panel of 6 ES cell lines in vitro and can significantly suppress xenograft tumor growth in vivo. These results suggest that midostaurin might be a novel drug that is active against ES cells which might act by modulating the expression of EWS/FLI1 target genes. In the second part of this thesis, in order to identify molecular pathway(s) that may contribute to the transcriptional activity and oncogenic properties of EWS/FLI1, we used our well established screening approach and performed screening of a small library of 153 targeted inhibitors covering all major signaling pathways. We discovered PI3K inhibitors as potent modulators of EWS/FLI1 expression. This finding was confirmed in several ES cell lines and off target effects of the PI3K inhibitors was excluded by performing genetic loss of function experiments. Analysis of the EWS/FLI1 promoter region using various deletion constructs in reporter gene assays determined two 12bp minimal elements where transcription factor(s) under PI3K control is binding. Elucidating the direct link between PI3K and EWS/FLI1 is of importance, and identity of the responsible transcription factor(s) might provide novel therapeutic opportunities. Results presented in this thesis clearly demonstrate that screening approach here established can be used for both screening for compounds effective against ES as well as for screening targeted inhibitors leading to better understanding of the biology of ES. Together it should lead to development of novel therapeutic approaches for the treatment of ES.

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Additional indexing

Item Type:Dissertation
Referees:Wenger R H, Schäfer B, Pruschy M
Communities & Collections:04 Faculty of Medicine > University Children's Hospital Zurich > Medical Clinic
04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2012
Deposited On:17 Jan 2013 09:07
Last Modified:07 Dec 2017 18:27

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