Proteomics as a Functional Tool in Evaluating Bortezomib Treatment and Drug Resistance Mechanism.

Multiple Myeloma (MM) is an incurable plasma cell disorder. Bortezomib (PS-341, Velcade™) is a novel proteosome inhibitor with significant activity in multiple myeloma, although subsets of patients remain refractory to the activity of the drug. Hence, better characterization of the interactions of this drug with classical resistance mechanisms may identify improved treatment applications. Materials and methods: We examined resistance mechanism after bortezomib treatment by using paired sensitive and resistant cell lines (MM1.S and OPM-2) in MM. As the cell lines used were not isogenic, we wanted to ensure that the changes seen were related to drug treatment only by using a corresponding solid tumor counterpart; thyroid carcinoma cell lines (KAT-18 and WRO). By using paired cell lines from different tumor types and applying a stringent exclusion criteria, any proteins that are related to cell line or cancer type phenomenon would be excluded and any protein changes seen would be related to ‘real’ changes that were attributed to bortezomib treatment. Samples were analysed using 2D-DIGE, a technique based on pre-electrophoretic labelling of samples with one of three spectrally resolvable fluorescent CyDyes (Cy2, Cy3, and Cy5) allowing multiplexing of samples into the same gel. All 2D-DIGE images were scanned and collected on a Typhoon Fluorescent Imager. Pooled samples were used as an internal standard to quantify expression changes with statistical significance. Statistics and quantitation of protein expression were carried out initially using DeCyder Biological Variation Analysis (BVA) software before performing subsequent Extended Data Analysis (EDA). Proteins of interest were picked from preparative gels and full scan mass spectra were recorded in profile mode and tandem mass spectra in centroid mode. The peptides were identified using the information in the tandem mass spectra by searching against the SWISS PROT database using SEQUEST.

18 proteins have been identified to be differentially expressed in the sensitive cell line compared to the resistant cell lines. 6 proteins were up-regulated and 12 down-regulated in the sensitive cell lines compared to the resistant cell lines (t-test 0.02) and all proteins were >1.3-fold differentially expressed. Of the proteins that are significantly changed, caspase 3 is down-regulated in sensitive cell lines after bortezomib treatment and heat shock protein 70 (Hsp70) is up-regulated. Caspase 3 is a key apoptosis executioner and is known to be responsible for the proteolytic cleavage of many proteins when cells are treated with bortezomib. Hsp70 is a chaperone protein for both proapoptotic and antiapoptotic proteins; it has been demonstrated to be upregulated with bortezomib treatment. These two proteins correlate with what is known about how bortezomib affects cells and validates our results. Other proteins that were significantly changed are involved in transcriptional regulation. These proteins provide an interesting insight into how bortezomib treatment affects cellular processes and provide clues into resistance mechanisms.

As the field is now aware, bortezomib is more than a proteosome inhibitor and it affects other pathways in cellular function which would be difficult to evaluate using conventional analytical methods. Proteomics is a novel way to evaluate bortezomib's effect in cellular pathways and also drug resistance mechanisms. We are in the process of validating the other novel proteins identified in our screen using small molecule inhibitors to these proteins and/or knockdown studies to validate these proteins even further.

Richardson:Millenium: Membership on an entity's Board of Directors or advisory committees, Research Funding; MLNM: speakers bureau up to 7/1/09; Celgene: Membership on an entity's Board of Directors or advisory committees, speakers bureau up to 7/1/09. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding. Anderson:Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.