Ewings Sarcoma Preclinical Collaboration

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Ewings Sarcoma Preclinical Collaboration

• Melinda S. Merchant, MD, PhD Dept of Pediatrics
• Marc Ladanyi, MD Dept of Pathology
• Hakim Djaballah, PhD High Throughput Core
• Neal Shukla, MD Pediatric Oncology Fellow

Memorial Sloan-Kettering Cancer Center New York,
New York
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Background An aggressive tumor in need of new
therapeutics

• Ewings Sarcoma Bone/soft tissue tumor of
  childhood and adolescence
• Poor prognosis in metastatic disease despite
  multi-modality therapy
• Chemo resistance frequent with recurrence
• No Ewings sarcoma samples included in NCI
  screening of compounds for anti-tumor activity
• Need new therapeutics specifically tested
  against Ewings Sarcoma

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Goals

• To identify active agents against Ewings sarcoma
• Cytotoxic compounds which kill or inhibit growth
  of Ewings tumor cells
• Agents which decrease expression of translocation
  specific to this disease
• To increase number of new therapeutics in the
  pipeline for clinical application

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Project Design

• Scale up
• Two representative Ewings sarcoma cell lines
• Determine appropriate growth conditions
• Transfection
• Develop GFP assay for readout of impact on
  translocation
• Pilot screening
• determine assay reproducibility using fully
  automated screening robotic platform, mimicking a
  real HTS run
• A validation library of 2,880 compounds will be
  used for this validation step
• High-Throughput screen
• 200,000 compounds
• Validate hits from screening in panel of cell
  lines

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Molecular Strategy
EWS-FLI1 translocation
? Ewings cell
EWS-FLI1 fusion protein
downstream gene
Promotor region

• ?
• Reporter construct is transfected into cells so
  that binding of promotor region by fusion protein
  will drive GFP expression
• Thus, Cells have green fluorescence when EWS-FLI1
  is active

Green Fluorescent Protein
Promotor region
? A compound that causes decreased EWS-FLI1
activity will result in less green detected
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Strategy in context
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High Throughput Screening Core

• Both synthetic chemicals and natural products
• obtained from sources such as plants,
  micro-organisms, fungi, and deep sea algae
• Strategic collaborations with industry for the
  supply of chemicals and to provide expertise in
  medicinal chemistry optimization
• ChemBridge Research Laboratories (USA), Specs
  (Holland), AnalytiCon Discovery (Germany),
  MicroSource (USA), and Prestwick Chemical
  (France).
• These compounds are available without major
  intellectual property issues.