Evaluating melanoma drug response and therapeutic escape with quantitative proteomics.
The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.
From the ‡Departments of Molecular Oncology.
Vito W
VW
Rebecca
From the ‡Departments of Molecular Oncology.
Elizabeth
E
Wood
From the ‡Departments of Molecular Oncology.
Inna V
IV
Fedorenko
From the ‡Departments of Molecular Oncology.
Kim H T
KH
Paraiso
From the ‡Departments of Molecular Oncology.
H Eirik
HE
Haarberg
From the ‡Departments of Molecular Oncology.
Yi
Y
Chen
From the ‡Departments of Molecular Oncology.
Yun
Y
Xiang
¶Cutaneous Oncology.
Amod
A
Sarnaik
¶Cutaneous Oncology.
Geoffrey T
GT
Gibney
¶Cutaneous Oncology.
Vernon K
VK
Sondak
From the ‡Departments of Molecular Oncology, ‖Chemical Biology and Molecular Medicine, Moffitt Cancer Center and Research Institute, Tampa, Florida 33612 john.koomen@moffitt.org keiran.smalley@moffitt.org.
John M
JM
Koomen
From the ‡Departments of Molecular Oncology, ¶Cutaneous Oncology, john.koomen@moffitt.org keiran.smalley@moffitt.org.
Keiran S M
KS
Smalley
D000818:Animals; D053961:Azabicyclo Compounds / Q000494:pharmacology; D001562:Benzimidazoles / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D019008:Drug Resistance, Neoplasm; D020558:GTP Phosphohydrolases / Q000235:genetics; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D013058:Mass Spectrometry; D008545:Melanoma / Q000188:drug therapy* / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D016328:NF-kappa B / Q000378:metabolism; D009368:Neoplasm Transplantation; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010795:Phthalic Acids / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D040901:Proteomics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors; D034622:RNA Interference; D034741:RNA, Small Interfering; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D014183:Transplantation, Heterologous; D000077484:Vemurafenib; D060449:Wnt Signaling Pathway / Q000235:genetics; D051176:beta Catenin / Q000235:genetics
C517975:AZD 6244; D053961:Azabicyclo Compounds; D001562:Benzimidazoles; D014408:Biomarkers, Tumor; D018841:HSP90 Heat-Shock Proteins; D007211:Indoles; D008565:Membrane Proteins; D016328:NF-kappa B; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010795:Phthalic Acids; D010880:Piperidines; D034741:RNA, Small Interfering; D013449:Sulfonamides; C559121:XL 888; D051176:beta Catenin; D000077484:Vemurafenib; D020797:Receptor, Platelet-Derived Growth Factor beta; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D051057:Proto-Oncogene Proteins c-akt; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human; C577197:crenolanib