title: Monitoring a nuclear factor-κB signature of drug resistance in multiple myeloma.
abstract: The emergence of acquired drug resistance results from multiple compensatory mechanisms acting to prevent cell death. Simultaneous monitoring of proteins involved in drug resistance is a major challenge for both elucidation of the underlying biology and development of candidate biomarkers for assessment of personalized cancer therapy. Here, we have utilized an integrated analytical platform based on SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring mass spectrometry, a versatile and powerful tool for targeted quantification of proteins in complex matrices, to evaluate a well-characterized model system of melphalan resistance in multiple myeloma (MM). Quantitative assays were developed to measure protein expression related to signaling events and biological processes relevant to melphalan resistance in multiple myeloma, specifically: nuclear factor-κB subunits, members of the Bcl-2 family of apoptosis-regulating proteins, and Fanconi Anemia DNA repair components. SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring methods were developed for quantification of these selected target proteins in amounts of material compatible with direct translation to clinical specimens (i.e. less than 50,000 cells). As proof of principle, both relative and absolute quantification were performed on cell line models of MM to compare protein expression before and after drug treatment in naïve cells and in drug resistant cells; these liquid chromatography-multiple reaction monitoring results are compared with existing literature and Western blots. The initial stage of a systems biology platform for examining drug resistance in MM has been implemented in cell line models and has been translated to MM cells isolated from a patient. The ultimate application of this platform could assist in clinical decision-making for individualized patient treatment. Although these specific assays have been developed to monitor MM, these techniques are expected to have broad applicability in cancer and other types of disease.
authors.affiliation: Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute University of South Florida, Tampa, FL 33612, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Xiang
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Remily-Wood
authors.forename: Vasco
authors.initials: V
authors.lastname: Oliveira
authors.forename: Danielle
authors.initials: D
authors.lastname: Yarde
authors.forename: Lili
authors.initials: L
authors.lastname: He
authors.forename: Jin Q
authors.initials: JQ
authors.lastname: Cheng
authors.forename: Linda
authors.initials: L
authors.lastname: Mathews
authors.forename: Kelly
authors.initials: K
authors.lastname: Boucher
authors.forename: Christopher
authors.initials: C
authors.lastname: Cubitt
authors.forename: Lia
authors.initials: L
authors.lastname: Perez
authors.forename: Ted J
authors.initials: TJ
authors.lastname: Gauthier
authors.forename: Steven A
authors.initials: SA
authors.lastname: Eschrich
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Shain
authors.forename: William S
authors.initials: WS
authors.lastname: Dalton
authors.forename: Lori
authors.initials: L
authors.lastname: Hazlehurst
authors.forename: John M
authors.initials: JM
authors.lastname: Koomen
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis; D001854:Bone Marrow Cells / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D019008:Drug Resistance, Neoplasm; D004591:Electrophoresis, Polyacrylamide Gel; D051856:Fanconi Anemia Complementation Group Proteins / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008558:Melphalan / Q000494:pharmacology* / Q000627:therapeutic use; D009101:Multiple Myeloma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D016328:NF-kappa B / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization; D053668:Syndecan-1 / Q000378:metabolism; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism; D051841:Transcription Factor RelB / Q000235:genetics / Q000378:metabolism
chemical_list: D018906:Antineoplastic Agents, Alkylating; D051856:Fanconi Anemia Complementation Group Proteins; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D020033:Protein Isoforms; C498143:RELA protein, human; C497593:RELB protein, human; C508241:SDC1 protein, human; D053668:Syndecan-1; D051996:Transcription Factor RelA; D051841:Transcription Factor RelB; D008558:Melphalan