Live-Cell Microscopy Reveals Small Molecule Inhibitor Effects on MAPK Pathway Dynamics

Daniel J Anderson,Jenni K Durieux,Mary J C Ludlam,Ryan Alvarado,Peter K Jackson,Georgia Hatzivassiliou,Kyung Song,Sudha Agarwal

doi:10.1371/journal.pone.0022607

Abstract

Oncogenic mutations in the mitogen activated protein kinase (MAPK) pathway are prevalent in human tumors, making this pathway a target of drug development efforts. Recently, ATP-competitive Raf inhibitors were shown to cause MAPK pathway activation via Raf kinase priming in wild-type BRaf cells and tumors, highlighting the need for a thorough understanding of signaling in the context of small molecule kinase inhibitors. Here, we present critical improvements in cell-line engineering and image analysis coupled with automated image acquisition that allow for the simultaneous identification of cellular localization of multiple MAPK pathway components (KRas, CRaf, Mek1 and Erk2). We use these assays in a systematic study of the effect of small molecule inhibitors across the MAPK cascade either as single agents or in combination. Both Raf inhibitor priming as well as the release from negative feedback induced by Mek and Erk inhibitors cause translocation of CRaf to the plasma membrane via mechanisms that are additive in pathway activation. Analysis of Erk activation and sub-cellular localization upon inhibitor treatments reveals differential inhibition and activation with the Raf inhibitors AZD628 and GDC0879 respectively. Since both single agent and combination studies of Raf and Mek inhibitors are currently in the clinic, our assays provide valuable insight into their effects on MAPK signaling in live cells.

Highlights

The mitogen activated protein kinase (MAPK) pathway containing Raf, Mek and Erk is a central downstream axis of Ras signaling involved in Ras-driven transformation [1,2]
We achieved good spatial resolution covering the equatorial plasma membrane (PM) and the adjacent cytoplasm, a critical approach for subsequent automated analysis (Figure S1a). Both RBD and fulllength CRaf showed little targeting to the PM when cotransfected with wild type (WT) KRas, but higher membrane localization when these constructs were cotransfected with oncogenic KRasG12D (Figure S1b)
When cells transiently co-transfected with eCFP-KRas and Venus-CRaf were subjected to this pixel ratio measurement approach, oncogenic KRasG12D caused an increase in both RBD and FL-CRaf targeting to the PM (Figure S1b)

Summary

Introduction

The mitogen activated protein kinase (MAPK) pathway containing Raf, Mek and Erk is a central downstream axis of Ras signaling involved in Ras-driven transformation [1,2]. Ras and Raf harbor activating mutations in 30% and 8% of human tumors, respectively [3,4,5], making these oncoproteins critical targets for oncology drug development Inhibitors of both Mek and Raf are currently in clinical trials. Three recent studies have investigated such ATP-mimetic Raf inhibitors in BRaf-WT cells, showing that these inhibitors have the ability to activate MAPK signaling in cells with WT BRaf [9,10,11]. This activation is attributed to inhibitorinduced priming of the Raf kinase as indicated by Raf dimerization, targeting of Raf to plasma membrane (PM)-localized Ras and subsequent downstream MAPK pathway activation. There is significant promise in targeting the MAPK pathway as a therapeutic strategy, the effects of small molecule kinase inhibitors on normal and tumor cells must be well understood to ensure success in the clinic

Methods

Results

Conclusion