Phosphoproteomic study of the effect of ALK inhibition alongside DUSP6 gene inactivation in KELLY neuroblastoma cells
Description
The hypothesis behind this research was that the DUSP6 phosphatase regulates signaling from the ALK tyrosine kinase in neuroblastoma cells, and loss of DUSP6 would enhance the effectiveness of ALK inhibitors in these cancer cells. The phosphoproteomics study was carried out to understand which phosphopeptides change in neuroblastoma cells when DUSP6 is lost, and how these are affected by co-treatment with lorlatinib. KELLY tumour cells were previously engineered to express Cas9 and a DUSP6-targeting guide RNA. KELLY parental cells and derived cells lacking DUSP6 expression were then treated with 30nM lorlatinib for 1 hr and cell extracts processed for phosphopeptide analysis using mass spec. Samples were made as quadruplicate biological replicates. The data provide an indication of novel protein effectors of DUSP6 (not determined if direct or indirect substrates) in these tumour cells and how some of these may be co-regulated by ALK. It is found that DUSP6 does not significantly impinge on ERK signaling as it is does in other cell types, but may be a co-regulator of pathways involving N-Myc and EE2FK. The data can be mined for further information concerning how DUSP6 functions in KELLY tumour cells, what the signaling effect is of lorlatinib in these cells, and how the two pathway interact. The work is part of the PhD thesis of Elliott Thompson, to be found in UCL Discovery (https://discovery.ucl.ac.uk/id/eprint/10148906/).
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Phosphoproteomics experiments were performed using mass spectrometry as reported (1, 2). In brief, frozen cell pallets were lysed in 8M urea buffer and supplemented with phosphatase inhibitors (10 mM Na3VO4, 100 mM β-glycerol phosphate and 25 mM Na2H2P2O7 (Sigma)). Proteins were digested into peptides using trypsin as previously described (3, 4). Phosphopeptides were enriched from total peptides by TiO2 chromatography essentially as reported previously (5). Dried phosphopeptides were dissolved in 0.1% TFA and analysed by nanoflow ultimate 3000 RSL nano instrument was coupled on-line to a Q Exactive plus mass spectrometer (Thermo Fisher Scientific). Gradient elution was from 3% to 28% solvent B in 90 min at a flow rate 250 nL/min with solvent A being used to balance the mobile phase (buffer A was 0.1% formic acid in water and B was 0.1% formic acid in acetonitrile) . The spray voltage was 1.95 kV and the capillary temperature was set to 255 ºC. The Q-Exactive plus was operated in data dependent mode with one survey MS scan followed by 15 MS/MS scans. The full scans were acquired in the mass analyser at 375- 1500m/z with the resolution of 70 000, and the MS/MS scans were obtained with a resolution of 17 500. MS raw files were converted into Mascot Generic Format using Mascot Distiller (version 2.6.1) and searched against the SwissProt database (release Sep 2018) restricted to human entries using the Mascot search daemon (version 2.6.0). Allowed mass windows were 10 ppm and 25 mmu for parent and fragment mass to charge values, respectively. Variable modifications included in searches were oxidation of methionine, pyro-glu (N-term) and phosphorylation of serine, threonine and tyrosine. Phosphopeptide quantification was performed using in-house software Pescal as described before (3). The resulting quantitative data was parsed into excel files for further normalisation and statistical analysis. 1. Hijazi, M. et al. Reconstructing kinase network topologies from phosphoproteomics data reveals cancer-associated rewiring. Nature Biotechnology,2020. 38(4): p. 493-502 2. Casado, P. et al. Kinase-substrate enrichment analysis provides insights into the heterogeneity of signaling pathway activation in leukemia cells. Sci. Signal. 6, rs6 (2013) 3. Alcolea, M.P., et al., Phosphoproteomic analysis of leukemia cells under basal and drug-treated conditions identifies markers of kinase pathway activation and mechanisms of resistance. Mol Cell Proteomics, 2012. 11(8): p. 453-66. 4. Montoya, A., et al., Characterization of a TiO(2) enrichment method for label-free quantitative phosphoproteomics. Methods, 2011. 54(4): p. 370-8. 5. Larsen, M.R., et al., Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics, 2005. 4(7): p. 873-86
Institutions
- University College LondonEngland, London