Quantitative Analysis of Protein Expression Changes Induced by Geldanamycin in NPM-ALK Positive Lymphoma Cells by Functional Proteomics.

A subset of anaplastic large cell lymphomas are characterized by the t(2;5)(p23;q35) chromosomal translocation. Fusion of the nucleophosmin (NPM) gene on chromosome 5q35 with the anaplastic lymphoma kinase (ALK) gene on chromosome 2p23 results in the constitutive expression of the NPM-ALK oncoprotein. Heat shock protein 90 (HSP90) is a molecular chaperone involved in the folding of a number of substrate proteins implicated in cancer. The list of known HSP90 client proteins is expanding and includes oncogenes, cell cycle-associated proteins, and kinases including NPM-ALK. The ansamycin-based anti-cancer drug, geldanamycin (GA) has been shown to inhibit HSP90 by interfering with its function as a molecular chaperone, resulting in ubiquitin-mediated proteasomal degradation of its client proteins. To date, the cellular effects of inhibition of HSP90 by GA remain largely uncharacterized. Here, we describe a functional proteomics approach to identify the differentially expressed proteins in NPM-ALK positive lymphoma cells exposed to GA. GA induced a dose- and time-dependent decrease in cell viability with an IC₅₀ of 20μM, which was associated with a G2/M cell cycle arrest followed by caspase-3 mediated apoptosis. Equal amounts of total cell lysates from DMSO-control and GA-treated SUDHL-1 cells were labeled with either the light or heavy cleavable ICAT™ reagent. Samples were combined, digested with trypsin, and separated by strong cation exchange chromatography and avidin affinity chromatography. Peptides were subjected to liquid chromatography (LC), electrospray ionization (ESI) and tandem mass spectrometry (MS/MS). The resulting peptides were searched against the NCBI and Swiss-Prot databases using SEQUEST™ for protein identification and relative quantitation determined by XPRESS™. A total of 117 proteins were differentially expressed by greater than 1.5-fold of which 48 were overexpressed and 69 were underexpressed in GA-treated cells relative to control. The 48 overexpressed proteins included those that are involved in apoptosis, protein degradation, cell communication, and cell cycle regulation. Of the 69 underexpressed proteins, many included proteins involved in ubiquitin-proteasome function (proteasome beta 2 subunit, HSP90-organizing protein, CBL-C), cytokines (IL-14 precursor), and DNA repair. Notably, a number of previously reported targets of HSP90/70 (male germ cell-associated kinase and IL-26 precursor) were identified. Our results provide insights into the role of HSP90 in the pathogenesis of NPM-ALK positive lymphomas and demonstrate the diverse cellular functions that are deregulated as a consequence of HSP90 inhibition.