Abstract 1858: Development of a multiplex panel of biomarkers to assess energy metabolism in cancer.

Background: Cancer cells have an altered glucose metabolism, which not only permits them to survive under hypoxic conditions but confers a distinct growth advantage by sustaining higher proliferation rates, invasiveness, and subsequent distant metastasis. This unique difference in glucose metabolism from healthy cells opens up a selective mechanism by which to kill cancer cells, bypassing normal healthy cells. Several enzymes in glucose metabolism are attractive targets for development of first-in-class therapeutic agents. We have undertaken development of a multiplex panel involving biomarkers involved in glucose metabolism to support drug development and to interrogate glycolytic pathway in cancer patients. Methods: The panel includes quantitative determinations of protein levels of hexokinase-2 (HK2), pyruvate kinase isoform-2 (PKM2), lactate dehydrogenase alpha (LDHa), and phosphorylated pyruvate dehydrogenase E1-alpha (phospho-PDHE1a). The sandwich immunoassays were built on the Luminex® xMAP technology platform using MagPlex magnetic beads and cloned proteins are employed as calibrators. The panel is designed to measure protein concentrations in fractionated cell extracts from flash frozen tumor biopsies. Results: We have completed development of three sandwich immunoassays for HK2, PK-M2, and LDHa on Luminex platform. Suitable capture antibodies as well as phycoerythrin labeled detection antibodies have been identified and tested for sandwich formation and multiplexing using recombinant calibrators and cell lysates. The multiplex is currently standardized for optimal assay performance. Known pharmacological modulators (3-bromopyruvate, bortezumib, oxamate, dichloroacetate) of HK2, PKM2, LDHa, and PDHE1a were used in vitro to demonstrate expected directional changes in biomarkers in the panel. Conclusions: We have developed a multiplex panel to measure protein levels of three key molecules involved in energy metabolism in cancer cells. The multiplex panel provides a critical tool, in the clinical setting, to support ongoing therapeutic strategies aimed at targeting the energy provision pathway in cancer patients. Importantly, the panel can be used to survey metabolic phenotype of different tumors to identify type and stage of cancer most susceptible to inhibition of glycolysis. Funded by NCI Contract No HHSN261200800001E. Citation Format: Meenakshi P. Balakrishnan, Ralph E. Parchment, Melinda G. Hollingshead, Joseph E. Tomaszewski, Robert J. Kinders, James H. Doroshow, Apurva K. Srivastava. Development of a multiplex panel of biomarkers to assess energy metabolism in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1858. doi:10.1158/1538-7445.AM2013-1858