Kinome profiling of Non-Hodgkin Lymphoma identifies Tyro3 as a therapeutic target in PEL
Non-Hodgkin lymphomas (NHL) make up the majority of lymphoma diagnoses and represent a very diverse set of malignancies. We sought to identify kinases uniquely upregulated in different NHL subtypes. Using Multiplexed Inhibitor Bead-mass spectrometry (MIB/MS), we found Tyro3 was uniquely upregulated and important for cell survival in primary effusion lymphoma (PEL), which is a viral lymphoma infected with Kaposi’s sarcoma-associated herpesvirus (KSHV).
Sample Processing Protocol
Suspension cells were pelleted and washed three times in cold PBS. Pellets were lysed on ice in MIB lysis buffer containing protease and phosphatase inhibitors. Cell lysates were sonicated, clarified, and the supernatant was syringe-filtered through a 0.2 µm SFCA membrane. Protein concentration was determined by Bradford assay and around 2.5 mg were used for each sample. The filtered lysate was brought to 1 M NaCl and passed through a column of multiplexed kinase inhibitor-conjugated beads (MIBs) consisting of Sepharose-conjugated Purvalanol B, PP58, CTx-0294885, VI-16832, and two custom-synthesized pan-kinase inhibitor compounds, UNC-8088A and UNC-2142A. The MIBs were washed with 5 mL of high-salt buffer and 5 mL of low-salt buffer. The columns were washed a final time with 1 mL 0.1% SDS before elution in 1 mL of 0.5% SDS. Eluted kinases were reduced (dithiothreitol) and alkylated (iodoacetamide) prior to being concentrated with Amicon Ultra centrifugal filters and detergent was removed from the concentrated eluate by chloroform/methanol extraction. Protein pellets were resuspended in 50 mM HEPES (pH 8.0) and were digested overnight with sequencing grade modified trypsin. Peptides were dried down in a speed-vac and cleaned with C-18 spin columns according to the manufacturer instructions. Peptides were resuspended in 5% acetonitrile and 0.1% formic acid, and 25-50% injected onto a Thermo Easy-Spray 75uM x 25cm C-18 column via an Easy nanoLC-1000. Peptides were separated as a single fraction on a 300 minute gradient (5-40% acetonitrile) and identified by a Q-Exactive mass spectrometer. Parameters: 3e6 AGC MS1, 80ms MS1 max inject time, 1e5 AGC MS2, 100ms MS2 max inject time, 20 loop count, 1.8 m/z isolation window, 45s dynamic exclusion.
Data Processing Protocol
Spectra were identified using MaxQuant software and the Uniprot/Swiss-Prot database. Peptide abundance was calculated using label-free quantification (LFQ). Mass spectrometry runs were normalized by natural log-transformed and median-centering LFQ values on a common-captured set of 151 kinases. Heat maps were generated using the Euclidian hierarchical clustering program from GENE-E.
Jason Wong, University of North Carolina at Chapel Hill
Blossom Andrea Damania, Department of Microbiology and Immunology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, USA. ( lab head )
Corresponding dataset(s) in other omics resources
GEO: GSE114791; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE114791