Abstract 5112: Proteomic investigation of melphalan resistance in multiple myeloma to support selection of combination therapy
Authors: Xiang Yun , Wood Elizabeth R. , Gancar Jessica , Oguz Umut , Cubitt Christopher L. , Dalton William S. , Shain Kenneth H. , Koomen John M. 
Published: June 2012

Abstract
Introduction: Proteomic analyses using iTRAQ and reaction monitoring mass spectrometry are used to examine the mechanism of melphalan resistance and build an assessment platform to enable future selection of combination therapy in patients.
Experimental Procedures: Drug IC50 values and interactions are assessed using cell viability measurements. Protein expression analysis using isobaric tags for relative and absolute quantification (iTRAQ) was used to compare melphalan resistant cell lines (8226/LR5 and U266/LR6) to their isogenic, naïve counterparts (RPMI-8226, U266). In this study, iTRAQ was used to identify proteins that have changed in expression level during the development of melphalan resistance in multiple myeloma (MM). In addition to manual selection of differentially expressed proteins, the resulting quantitative data were coalesced into pathway models using Metacore, GeneGO for further analysis. For targeted protein detection and quantification, liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM) assays are developed and implemented.
Data Summary: Melphalan resistant cells are more susceptible to other chemotherapy agents, including steroids, proteasome inhibitors, and geldanamycin derivatives, than their isogenic, naïve counterparts. To investigate the differences, iTRAQ showed numerous changes in protein expression, including redox stress components, mitochondrial proteins, and heat shock proteins (HSPs). LC-MRM assays were developed for proteins detected in the iTRAQ experiments that are current chemotherapy targets, e.g. HSPs and proteasome components. Furthermore, assays have also been developed for additional protein targets of other chemotherapy agents, e.g. topoisomerases and the glucocorticoid receptor, that were not detected in the iTRAQ results. LC-MRM was then used to examine the expression of these proteins in the four model cell lines listed above and in the naïve cells following the onset of drug treatment. Endogenous proteins were quantified in less than 10,000 cells. These assays can be combined to develop an assessment tool to help elucidate the mechanisms of drug resistance and enable the assessment of myeloma cells from patients’ bone marrow aspirates.
Conclusions: Quantification of the proteomes of myeloma cell lines using iTRAQ provided insights into the mechanism of melphalan resistance in multiple myeloma. Investigation of the expression of these proteins and other drug targets in myeloma cell lines has produced an LC-MRM platform that can be translated for patient assessment to assist in selection of chemotherapy combinations.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5112. doi:10.1158/1538-7445.AM2011-5112