Natural killer cell lymphoma (NKL) is a highly aggressive form of hematolymphoid malignancy that arises from natural killer cells, a critical component of the innate immune system. Early diagnosis of the tumor is critical however there are no biomarkers which are useful currently for the diagnosis and monitoring of patients with NKLs. We hypothesized that comprehensive analysis of differentially expressed proteins by NKL and normal NK cells would be a rational strategy for the discovery of NKL biomarkers and lead to better understanding of pathogenetic mechanisms of NKL. We used total cell lysates obtained from highly enriched peripheral blood NK cells (CD3-, CD56dim) and an Epstein-Barr virus negative, IL-2 dependent cell line (NK-92MI) for quantitative proteomic analysis. Isotopecoded affinity tags (ICATTM)-labeling followed by liquid chromatography-tandem mass spectrometry analyses (LC-MS/MS) identified 114 differentially expressed proteins in NKL compared to the normal NK cells. Out of these, 80 were upregulated 1.5-fold or greater and 34 were downregulated 1.5-fold or greater in NKL. Proteins within diverse cellular functional categories including cell signaling, transcriptional regulation, DNA metabolism, and immune response were differentially expression. Furthermore, known proteins relevant to the function of NK cells such as perforin, granzyme and defensins were downregulated in the NKL cell line. We selected eight proteins to validate the changes identified by ICAT-LC-MS/MS using Western blot analysis. HSP90, PCNA, alpha-enolase, GSTP1, galectin-1, and PARK7 were upregulated, and lactoferrin and annexin A1 were downregulated by western blot analysis demonstrating high concordance with the results of ICAT-LC-MS/MS analysis. We validated the functional significance of HSP90 as a potential therapeutic target in NKL by investigating the effect of inhibitors of HSP90 (geldanamycin) on the survival of an NKL cell line. Inhibition of HSP90 led to significant decrease in cell viability and apoptotic cell death as confirmed by western blotting demonstration of PARP-1 induction and detection of its cleavage products. This study represents the first global quantitative proteomic cataloguing of proteins expressed by NKL and normal NK cells, and reveals potential candidate proteins that may be useful for the diagnosis and function as therapeutic targets in patients with NKL.

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