Phosphoproteomics Reveals MAPK Inhibitors Enhance MET- and EGFR-Driven AKT Signaling in KRAS-Mutant Lung Cancer

Abstract

Pathway inhibition of the RAS-driven MAPK pathway using small-molecule kinase inhibitors has been a key focus for treating cancers driven by oncogenic RAS, yet significant clinical responses are lacking. Feedback reactivation of ERK driven by drug-induced RAF activity has been suggested as one of the major drug resistance mechanisms, especially in the context of oncogenic RAS. To determine if additional adaptive resistance mechanisms may co-exist, we characterized global phosphoproteomic changes after MEK inhibitor selumetinib (AZD6244) treatment in KRAS-mutant A427 and A549 lung adenocarcinoma cell lines employing mass spectrometry-based phosphoproteomics. We identified 9,075 quantifiable unique phosphosites (corresponding to 3,346 unique phosphoproteins), of which 567 phosphosites were more abundant and 512 phosphosites were less abundant after MEK inhibition. Selumetinib increased phosphorylation of KSR-1, a scaffolding protein required for assembly of MAPK signaling complex, as well as altered phosphorylation of GEF-H1, a novel regulator of KSR-1 and implicated in RAS-driven MAPK activation. Moreover, selumetinib reduced inhibitory serine phosphorylation of MET at Ser985 and potentiated HGF- and EGF-induced AKT phosphorylation. These results were recapitulated by pan-RAF (LY3009120), MEK (GDC0623), and ERK (SCH772984) inhibitors, which are currently under early-phase clinical development against RAS-mutant cancers. Our results highlight the unique adaptive changes in MAPK scaffolding proteins (KSR-1, GEF-H1) and in RTK signaling, leading to enhanced PI3K-AKT signaling when the MAPK pathway is inhibited.

Dual inhibiting OCT4 and AKT potently suppresses the propagation of human cancer cells

“…However, when used as single agents, inhibitors targeting the above epigenetic modulators and their cell surface receptors did not yield satisfactory clinical outcomes3940. In most cases, inhibiting the targeted components results in compensatory activation of their upstream regulators or parallel signaling pathways414243. Thus, combinations of targeted treatments have been employed to circumvent such problems in order to yield clinical benefits4344.…”

HDAC8 Regulates a Stress Response Pathway in Melanoma to Mediate Escape from BRAF Inhibitor Therapy

“…Each of the fractions was enriched for phosphopeptides using a Phos-SELECT Iron Affinity Gel (Millipore Sigma; ref. 16). Mass spectrometry data were acquired on a QExactive mass spectrometer coupled to a U3000 RSLCnano system (ThermoFisher) as described previously (16).…”

“…16). Mass spectrometry data were acquired on a QExactive mass spectrometer coupled to a U3000 RSLCnano system (ThermoFisher) as described previously (16). Two technical replicates were performed for the immuneenriched phosphotyrosine samples as well as each of the immobilized metal affinity chromatography (IMAC)-enriched fractions.…”

Targeted phosphoproteomics of the Ras signaling network reveal regulatory mechanisms mediated by oncogenic KRAS

“…Several prior phosphoproteomic studies examined oncogenic KRAS-driven signaling in lung cancer. The most notable of these used stable isotope labeling techniques (SILAC and iTRAQ) for proteome-scale analysis of phosphorylation in oncogenic KRAS-driven lung adenocarcinoma cell lines [59] and patientderived tumors [60]. These studies have identified candidate phosphorylation sites that might be involved in oncogenic KRAS signaling, some of which are also profiled in the current work.…”