A METABOLOMIC APPROACH TO TARGETING ASS1 DEFICIENT SARCOMAS
A better understanding of the biology and metabolism of sarcoma growth is necessary to develop novel therapies. Argininosuccinate synthase 1 (ASS1) is the rate-limiting enzyme in the conversion of citrulline to arginine. When ASS1 is not expressed, arginine becomes an essential amino acid to a cancer cell that must be delivered from the diet. Immunohistochemical analysis of 45 subtypes of sarcoma demonstrates that ASS1 is not expressed in over 85% of 619 out of 701 patient samples, thus making this a common enzymatic defect that can be exploited therapeutically. We have demonstrated sarcomas that lack ASS1 die when treated with a combination of pegylated-arginine-deiminase (ADI) and chloroquine. As the natural escape mechanism is re-expression of ASS1 in a tumor, a better understanding of the metabolomic changes that occur with the treatment of ADI is needed for better therapeutic exploitation. Preliminary data from leiomyosarcoma cell line metabolic mass spectrometry have demonstrated that ADI down-regulates pyruvate kinase M2 (PKM2), the enzyme responsible for the shuttling of glucose metabolites to the pentose phosphate shunt – the Warburg effect. In addition, there is up-regulation in glutaminase which converts glutamine to glutamic acid for ATP generation through the tricarboxylic acid cycle. The natural consequence of this is depletion of glutathione, the free radical scavenger. In vitro experiments have demonstrated that cell lines treated with ADI have increased cell death with glutamine withdrawal. Uniquely, we have discovered that synovial sarcoma cell lines are naturally sensitive to glutamine withdrawal. Finally, we have found a lack of expression of PKM2 in osteosarcoma and Ewing’s sarcoma. We hypothesize that: metabolomic characterization of synovial sarcoma, osteosarcoma and Ewing’s sarcoma will identify additional targets for synthetic lethality with ADI in ASS1- deficient sarcomas. We propose two aims: Specific Aim 1: To determine the metabolic changes induced by ADI using mass spectroscopy metabolomics of synovial sarcoma cell lines. Unlike other sarcomas, synovial sarcomas are uniquely sensitive to glutamine withdrawal. Using metabolomic mass spectroscopy we will determine not only the metabolic consequences of ADI on the treatment of synovial sarcoma, but also the pathways associated with this unique glutamine sensitivity. The identified defects will be validated with functional assays and western blotting. Specific Aim 2: To determine the metabolic changes induced by ADI using mass spectroscopy metabolomics of Ewing’s sarcoma and Osteosarcoma. As bone sarcoma cell lines do not express PKM2, the metabolic changes associated with ADI may be different than those associated with soft tissue sarcoma. Using metabolic mass spectroscopy we will determine the metabolic consequences of ADI on Ewing’s and Osteosarcoma. The identified defects will be validated with functional assays and western blotting.