Abstract 668: Phosphoproteomic characterization of selumetinib action in KRAS mutant lung cancer
Authors: Kim Jae-Young , Welsh Eric A. , Fang Bin , Kinose Fumi , Koomen John M. , Haura Eric B. 
Published: August 2015

In the field of lung cancer therapeutics, KRAS-mutant lung cancer is a significant problem due to limited therapeutic options and poor prognosis. Pathway inhibition of the RAS-driven RAF-MEK-ERK pathway using small-molecule kinase inhibitors has been a key focus for treating cancers driven by oncogenic RAS; however, significant clinical responses are lacking. Our initial experiments assessed the efficacy of the clinical MEK inhibitor AZD6244 (selumetinib) in a panel of non-small cell lung cancer cell lines harboring oncogenic KRAS mutations. None of the cell lines showed pronounced apoptosis nor did we observe significant reduction in cell viability in nano-molar range (&lt;1 μM). Given that cancer cells can rapidly rewire signaling cascades to adapt to loss of survival signaling, we hypothesized that a phosphoproteomics approach would provide further insight into adaptive signaling alteration associated with AZD6244. To this aim, we assessed global phosphoproteome change after AZD6244 treatment (1 μM, 24hr) in A427 and A549 lung adenocarcinoma cell lines harboring oncogenic KRAS mutations. We identified 7,730 unique phosphopeptides (corresponding to 3,137 unique phosphoproteins) after removing low-confidence phosphopeptides. We defined phosphopeptide significantly altered by AZD6244 as one that has had quantity changed by two-fold, corresponding to one standard deviation of fold change within the data. This resulted in 882 increased and 831 decreased phosphopeptides after MEK inhibition. As expected, AZD6244 decreased ERK phosphorylation and induced feedback increase of MEK and B- and C-RAF phosphorylation. In addition, we observed that AZD6244 increased tyrosine phosphorylation (Y1172) and decreased serine phosphorylation (S1045) of EGFR, which are involved in kinase activation and receptor trafficking, respectively. We also found reduced inhibitory serine phosphorylation of MET (S985), suggesting MEK inhibition could lead to MET activation by relieving inhibitory serine phosphorylation. These observations raise the possibility that MEK inhibition leads to feedback activation of RTKs, as well as its downstream activation (e.g., wild-type RAS). Notably, AZD6244 increased phosphorylation of KSR-1, a scaffolding protein required for assembly of MAPK signaling complex as well as phosphorylation of GEF-H1, a recently reportedly novel regulator of KSR-1 required for proper activation of RAS-driven MAPK activation. Collectively, our phosphoproteomics results indicate altered phosphorylation of a series of signaling proteins in MAPK signaling cascade, presumably contributing feedback activation of RAF, MEK and ERK reactivation following MEK inhibition. We are currently characterizing the importance of GEF-H1 and KSR-1 in lung cancer cell growth and survival and their role in adaptive signaling change associated with MEK inhibition. Updated work will be presented.

Citation Format: Jae-Young Kim, Eric A. Welsh, Bin Fang, Fumi Kinose, John M. Koomen, Eric B. Haura. Phosphoproteomic characterization of selumetinib action in KRAS mutant lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 668. doi:10.1158/1538-7445.AM2015-668