Discovery of a novel B-Raf fusion protein related to c-Met drug resistance.

ABSTRACT

In recent years, there have been notable advances with the development of anticancer drugs including those targeting protein tyrosine kinases such as the c-Met receptor, which has been implicated in the development and progression of several cancers. However, despite such progress, drug resistance continues to be the single most important cause of cancer treatment failure, and understanding the mechanisms of drug resistance remains a major hurdle in treating patients with recurrent disease. PF-04217903 is a small-molecule c-Met kinase inhibitor that potently inhibits c-Met-driven processes such as cell growth (proliferation and survival), motility, invasion, and morphology of a variety of tumor cells. Resistance to PF-04217903 was observed in GTL-16, a gastric carcinoma cell line with a constitutively activated c-Met receptor. In this report, mass spectrometry (MS) based quantitative phosphoproteomic analysis was used to determine changes in signaling pathways in the parental cells in response to c-Met inhibition and to investigate the changes in protein levels and related canonical pathways in both parental and PF-04217903 resistant (R3) clones in response to c-Met inhibition. The quantitative MS workflow included phosphoprotein enrichment of cell lysates from six treatment conditions in-solution digestion, chemical labeling of peptides with a set of 6-plex isobaric tandem mass tags (TMT), HILIC fractionation, phosphopeptide enrichment, and nano LC-MS/MS on a LTQ-Orbitrap mass spectrometer. An investigation of these quantitative datasets using Ingenuity Pathways Analysis (IPA) revealed pathway changes in the various treatments that were consistent with previously observed transcriptomic and phenotypic changes. Proteomic analysis also revealed an increase in B-Raf expression in R3 clones. Expression profiling confirmed that B-Raf gene copy number was up-regulated and also indicated the presence of a mutated form of B-Raf. Using a bottom-up MS approach, SND-1 was identified as the B-Raf fusion partner. The discovery of this novel B-Raf fusion protein presents a novel target with potential clinical implications in the treatment of patients resistant to c-Met inhibitors.