Funded Research:
CD99 Engagement and Apoptosis in Ewing's Sarcoma

An ESUN Announcement

April 15, 2011: The Liddy Shriver Sarcoma Initiative is pleased to announce the funding of a $50,000 research grant, "CD99 engagement and apoptosis in Ewing’s sarcoma: role of mitochondria and mitochondrial DNA mutations." The study is being conducted by Michela Rugolo, PhD and Katia Scotlandi, PhD. Dr. Rugolo is with the Department of Evolutionary and Experimental Biology, University of Bologna in Bologna, Italy and Dr. Scotlandi is with the Laboratory of Experimental Oncology at the Rizzoli Institute in Bologna, Italy.

This grant is made possible by very generous donations made in memory of Michael Lio (Strike out Sarcoma) and in memory of Peter Skelton (Peter Skelton Sarcoma Research Foundation) and by the friends and families of Ryan Glenny, Paul Roth, Mike Homan, Craig Goris, Richard Pollak, and Joe Rivas, who lost their lives to this disease; and donations by friends and families to honor Jeremy Weingrod and Alex Franke, who are still fighting it.

In their grant application, Drs. Rugolo and Scotlandi noted that:

"The therapy of sarcomas is still firmly entrenched in conventional cytotoxic drugs and patients are still paying the price of severe toxicity in best cases or suffering inadequate treatments when they have metastases at diagnosis. In addition, although sarcomas have a devastating impact on young patients, they are too rare to attract the interest of big-pharma. This project handles the problem of the therapeutic impasse in Ewing’s sarcoma by focusing on two aspects:

1. The definition of the molecular mechanisms of apoptosis induced by CD99 engagement, with emphasis on mitochondrial involvement. CD99 has been identified as a very interesting therapeutic target for EWS. However, being a rare target, clinical development of specific drugs is unlikely in the next period of time. The identification of the molecular mechanisms underlying CD99-driven apoptosis will define more common targets, for which drugs may already be developed and available. To achieve our purpose we will measure the mitochondrial membrane potential with the cationic dye TMRM by using fluorescence microscopy, following the experimental protocol previously described, in EWS cells treated with anti-CD99 antibodies that were already described of therapeutic interest. In addition, we will investigate the role of the mitochondrial apoptogenic factors AIF and Omi/HtrA2, which are known to induce caspase-independent cell death, by measuring their release from mitochondria into cytosol. As an alternative mechanism, we will analyze whether permeabilization of the lysosomal membranes, resulting in the release of lysosomal proteases into the cytosol, can play a role in CD99-driven apoptosis.

2. The comprehensive analysis of mitochondrial DNA mutations in clinical samples and cell lines. Considering that CD99-induced apoptosis seems a very promising therapeutic target for EWS, identification of other cellular regulators of apoptosis may help to disclose  novel mechanisms potentially useful for therapy. Mitochondrial DNA mutations have been associated with tumorigenesis and alterations of apoptotic abilities of cells. However no data have been reported in sarcomas so far. To this purpose, mitochondrial genome sequencing, heteroplasmy evaluation and assessment of pathogenic potential of mutations will be performed in clinical samples and a panel of cell lines, as previously described (Gasparre et al. 2007). In particular, retrospective material from Rizzoli Institute tissue bank archival and samples collected during the study will be used. In addition we will analyse a panel of 22 EWS cell lines, including cells commonly used in the different labs and cell lines established at the Rizzoli Institute. Identification of the same mutations in EWS tumors and cell lines will allow us to perform functional studies, to establish a direct link between mDNA mutations, ability of EWS cells to respond to CD99- and drug-induced apoptosis, and tumorigenesis, with the aim to obtain a better definition of combined therapies with targeted drugs and conventional chemotherapy. Furthermore, exploring mDNA mutations may provide novel findings to be used also as predictors of prognosis and help stratifying patients at diagnosis based on risks."

They summarize the focus of this study as follows:

  1. Elucidation of key events occurring downstream CD99-driven apoptosis that can be targeted for drug design and therapeutic intervention; 
  2. Identification of new diagnostic and prognostic markers that by categorizing Ewing’s sarcomas according to mutations in mtDNA can be used to develop modifications in risk-based therapy and minimize toxicity to the "good risk" patients.


Read more about this study in the article, "CD99 Engagement and Apoptosis in Ewing’s Sarcoma: Role of Mitochondria and Mitochondrial DNA Mutations," which appears in the April 2011 issue of ESUN.

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