Protein expression profiling

Workpackage 6

Objectives

Comparisons by kinome analysis the different subsets of MSC with normal counterparts attempting to assess cell signaling pathways in MSC, but also to elucidate cell signaling pathways that are more, or less, active in MSC cells relative to their normal cell counterparts.

Description of work

The experiments will start with BM aspirates from newly diagnosed MM patients or healthy donors for purification of MM cells and plasma cells, respectively. MM and plasma cells will be purified by high speed cell sorting The yield of MM cells (or plasma cells) will vary from patient to patient (or donor to donor), but we anticipate that we will in most cases be able to obtain 0.3X106-1X106 MM or plasma cells from each aspirate. In addition to sorting MM or plasma cells we will simultaneously sort 0.3X106-1X106 CD19+CD138- cells for each marrow aspirate for comparison with the kinome of MM or plasma cells. Furthermore, other subsets of MSC will be obtained from workpackage 2 in similar sorts. After we obtain PepChip data for each cell sample we will then determine their respective kinomes using the data and statistical analysis tools available in the following software packages: ArrayVision Evaluation 8.0 (Molecular Dynamics, USA), Microsoft Excel and SPSS 11.0. This will involve initial image acquisition and quantification using ArrayVision software where saturated spots and spots with signal lower than or equal to background are identified and excluded from the analysis.

To determine the shared kinome for all ten cell samples analyzed, we will calculate the average level of phosphorylation for each arrayed kinase substrate in each individual MM, plasma cell or precursor cell sample and then determine whether the mean for this individual sample is significantly different from the mean level of phosphorylation for the other 9 cell samples analyzed using statistical analysis toole (e.g. Wilcoxon test) in the SPSS 11.o software package. If the phosphorylation level for a given substrate in a specific patient/donor sample is found to be significantly different from that found for other cell samples then that kinase substrate will be excluded from the shared kinome for that cell type. By asking SPS 11.0 to query the mean level of phosphorylation of each spot on the array for all samples analyzed, we will systematically identify those substrate phosphorylation levels that are shared amongst all ten cell samples analyzed. This will establish the "shared kinome" for each subset of MM cells, MSC and normal cells. However, to identify the portion of the MM or MSC shared kinome that differs from that for the shared kinome of normal plasma cells, we will use an optimized Pearson correlation coefficient dissimilarity measurement in order to extract phosphorylation levels that are significantly (P less than 0.05) up- or down-regulated.

Deliverables

• Kinome analysis on 10 MM plasma cell and 10 normal plasma cell samples.
• Kinome analysis on 10 CD20+/CD138- from the same patients as in 1.
• Kinome analysis for each group of defined MSC and their normal counterparts.
• Confirmation of obtained signaling pathways.

Milestones

• Validate that emerging PepChip technology to be applied to determine a kinome signature specific to B subsets in the myeloma hierarchy.
• Pursuing "private kinome" signatures for MM - those that are specific to a given patient, or a subset of patients. These "private kinomes" could serve as predictors of disease progression, response to therapy and survival.
• Data will be posted to a publicly accessible website.