Live-cell microscopy reveals small molecule inhibitor effects on MAPK pathway dynamics

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.

Figure 1. Automated Ras-mediated Raf redistrbution analysis.

(A) Graphical representation of raw data from Raf-redistribution assay analysis (left, mCherry-KRas in red, Venus-CRaf in green) nuclear and PM masking (center) and radial intensity ratio scanning (right) show image analysis approach. Scale bar is 20 µm and applies to all panels. (B) Schematic of bicistronic vector used to generate stable cell lines. (C) Images of bicsitronic Flp-In stable cell lines expressing Venus-CRaf with eCFP-KRasWT and eCFP-KRasG12D. Cells were stained with Hoechst to demonstrate the percentage of cells that express the fluorescent-fusion proteins (>99% for both cell lines). Scale bars are 20 µm and apply to corresponding magnifications in all panels. (D) CRaf PM/cytoplasmic ratio measurement of KRasWT and KRasG12D stable cell line, p<0.001. (E) eCFP-KRasG12D and Venus-CRaf expressing cells were treated with a serial dilution of MCP110 between 100 µM and 24 nM for 2.5 hrs, example images shown. Scale bar is 20 µm and applies to all panels. (F) CRaf PM/cytoplasmic intensity ratios were measured for MCP110 serial dilution in (E).

Figure 2. CRaf RBCRD detects endogenous RasGTP.

(A) HEK 293T cells were transfected with Venus-RBDCRD, eCFP-CAAX PM reporter (PMem), and mCherry-H2B to label nuclei. Scale bar is 20 µm and applies to all panels. (B) KRas protein levels were reduced in NCI-H727 cells using 2 different siRNA oligos against KRas or control oligo and then transfected with Venus-RBDCRD. Scale bar is 20 µm and applies to all panels. (C) Quantification of RBDCRD PM targeting in NCI-H727 with KRas siRNA. (D) Hec1A isogenic cell ines (Parental, Ras^WT/- and Ras^G12D/-) were transfected with Venus-RBDCRD and imaged live. RBDCRD PM targeting was then measured for each cell line. (E) Serum starved HEK 293T cells were stimulated with 100 ng/ml EGF, and imaged every 15 sec, PM targeting of RBDCRD was then measured.

Figure 3. Raf priming and MAPK negative feedback inhibition act to recruit CRaf to the PM.

(A) The Raf inhibitors AZD628, GDC0879 and PLX4720 along with Mek-i A were dosed in eCFP-KRasG12D and Venus-CRaf expressing HEK 293 cells, example images shown. Scale bar is 20 µm and applies to all panels. (B) PM/cytoplasmic CRaf intensity for images such as those in (A) was measured 4 hours after compound addition. (C) Mek-i A was compared to Mek-i B and Erk-i in CRaf redistribution assay. (D) Chart shows EC₅₀ values for both the CRaf PM targeting assay and biochemical kinase activity. (E) Cytoplasmic pERK was measured in cells dosed wih Raf, Mek and Erk inhibitors by immuno-fluorescence. (F) Kinetics of Raf inhibitor priming and feedback were compared by conducting time lapse analysis of cells treated with GDC0879 and Mek-i A, frames acquired every 2 min.

Figure 4. Combination of Raf priming with Mek/Erk inhibition has an additive effect on MAPK signaling.

(A) KRasG12D/CRaf HEK 293 cells were dosed with varying concentration of Mek-i A and constant dose of 12 nM AZD628, 2 µM PLX4720 or 300 nM GDC0879, as indicated, and CRaf PM targeting was measured after 4 hrs. (B) CRaf PM targeting was measured for the combination of Mek-i B with 12 nM AZD628, 2 µM PLX4720 or 300 nM GDC0879. (C) CRaf PM targeting was measured for the combination of Erk-i with12 nM AZD628, 2 µM PLX4720 or 300 nM GDC0879. (D) Top: Immunoblot of Braf:Craf heterodimers in cells treated with GDC-0879 (0.03, 0.1, 0.3, 1 µM) for 4 hours. Heterodimer formation observed with GDC-0879 alone is further increased upon co-administration of 1 µM MEK inhibitor Mek-i A. Bottom: CRaf IP kinase activity assays from lysates of cells treated as above. CRaf was immunoprecipitated from treated cells and kinase activity towards recombinant MEK was tested in vitro. Co-administration of Mek-i A with GDC0879 resulted in a dose-dependent increase in maximal CRaf kinase activity across all GDC0879 doses. For the CRaf kinase activity assays, relative phospho-MEK levels measured with MSD pMEK ELISA assay are shown. (E) Four-day proliferation curves of Ras^WT/Raf^WT cells (SW48 colon carcinoma) upon combination treatment of GDC-0879 with increasing concentrations of Mek-i A show induction of viable cell counts compared to MEK inhibitor treatment alone in a dose dependent manner with a maximal effect at co-administration of 1 µM of GDC0879. (F) Model describing the localization effect of Raf, Mek and Erk inhibitors on Raf cellular localization. Ras activation causes CRaf recruitment to the PM, priming of CRaf or inhibition of negative downstream feedback increase CRaf targeting to the PM. Combination of priming and feedback inhibition additively promote CRaf PM targeting.

Figure 5. RBDCRD response to MAP-kinase inhibitors.

(A) RBDCRD PM targeting was measured in Venus-RBDCRD stable cell line after 4 hr treatment with AZD628, GDC0879, PLX4720, Mek-i A, Mek-i B, or Erk-i. (B) phospho-ERK intensity was measured by immuno-fluorescence in cells treated as in (A). (C) RBDCRD PM targeting was measured in cells treated with a constant dose of 250 nM GDC0879 with a dose curve of the other small molecule inhibitor, showing the dominant effect of Mek and Erk inhibitor-induced targeting. (D) Time-lapse experiments show the kinetics of RBDCRD PM targeting and displacement upon treatment with 250 nM GDC0879, 20 nM Mek-i A or the combination of both. (E) Time-lapse experiment as in (D) where GDC0879 and Mek-i A were added sequentially with a 28.5 min. time gap between compound additions. (F) Schematic model describing RBDCRD cellular behavior in the presence of MAPK inhibitors.

Figure 6. Visualization of the labeled MAPK pathway.

(A) Example images show induced parental KRasG12D/CRaf cell line, induced and uninduced 4-color cell line where TagBFP-Mek1 and mCherry-Erk2 are constitutively expressed. Scale bar is 20 µm and applies to all panels. (B) Nuclear localization of TagBFP-Mek1 and mCherry-Erk2 was measured in MAPK cell line where eCFP-KRasG12D and Venus-CRaf were either induced or uninduced. (C) MAPK cell line was dosed with Raf, Mek and Erk inhbitors, CRaf PM localization was measured. (D) The nuclear/cytoplasmic ratio of mCherry-Erk1 was measured in MAPK cell line where KRasG12D and CRaf expression were induced. (E) The nuclear/cytoplasmic ratio of mCherry-Erk1 was measured in the MAPK cell line where KRasG12D and CRaf were not induced. (F) Nuclear pERK was measured in the HEK 293 KRasG12D/CRaf/Mek1/Erk2 cell line by immuno-fluorescence 5hrs after inhibitor treatment. (G) Nuclear pERK was measured as in (F) in the HEK 293 cell line expressing TagBFP-Mek1 and mCherry-Erk2 where expression of KRasG12D and CRaf is not induced.