Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition.
Targeted therapeutics that block signal transduction through the RAS-RAF-MEK and PI3K-AKT-mTOR pathways offer significant promise for the treatment of human malignancies. Dual inhibition of MAP/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) with the potent and selective small-molecule inhibitors GDC-0973 and GDC-0941 has been shown to trigger tumor cell death in preclinical models. Here we have used phosphomotif antibodies and mass spectrometry (MS) to investigate the effects of MEK/PI3K dual inhibition during the period immediately preceding cell death. Upon treatment, melanoma cell lines responded by dramatically increasing phosphorylation on proteins containing a canonical DNA damage-response (DDR) motif, as defined by a phosphorylated serine or threonine residue adjacent to glutamine, [s/t]Q. In total, >2,000 [s/t]Q phosphorylation sites on >850 proteins were identified by LC-MS/MS, including an extensive network of DDR proteins. Linear mixed-effects modeling revealed 101 proteins in which [s/t]Q phosphorylation was altered significantly in response to GDC-0973/GDC-0941. Among the most dramatic changes, we observed rapid and sustained phosphorylation of sites within the ABCDE cluster of DNA-dependent protein kinase. Preincubation of cells with the inhibitors of the DDR kinases DNA-dependent protein kinase or ataxia-telangiectasia mutated enhanced GDC-0973/GDC-0941-mediated cell death. Network analysis revealed specific enrichment of proteins involved in RNA metabolism along with canonical DDR proteins and suggested a prominent role for this pathway in the response to MEK/PI3K dual inhibition.
Departments of Protein Chemistry, Translational Oncology, Nonclinical Biostatistics, and Bioinformatics and Computational Biology, Genentech, Inc., South San Francisco, CA 94080.
Donald S
DS
Kirkpatrick
Daisy J
DJ
Bustos
Taner
T
Dogan
Jocelyn
J
Chan
Lilian
L
Phu
Amy
A
Young
Lori S
LS
Friedman
Marcia
M
Belvin
Qinghua
Q
Song
Corey E
CE
Bakalarski
Klaus P
KP
Hoeflich
D001384:Azetidines / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D004249:DNA Damage / Q000502:physiology*; D006801:Humans; D007191:Indazoles / Q000494:pharmacology; D016014:Linear Models; D008545:Melanoma / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010880:Piperidines / Q000494:pharmacology; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D053719:Tandem Mass Spectrometry / Q000379:methods
C532162:2-(1H-indazol-4-yl)-6-(4-methanesulfonylpiperazin-1-ylmethyl)-4-morpholin-4-ylthieno(3,2-d)pyrimidine; D001384:Azetidines; D007191:Indazoles; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D010880:Piperidines; D013449:Sulfonamides; D020929:Mitogen-Activated Protein Kinase Kinases; C574276:cobimetinib
PI3 kinase; PRKDC; phosphoproteomics