Determining the ERK-regulated phosphoproteome driving KRAS-mutant cancer

Abstract

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.

Fig. 1.. ERK signaling is critical for KRAS-regulated PDAC growth.

(A) Crystal structure of ERK2 bound to the ATP analogue ANP; activating ERK2 mutations (and analogous ERK1 mutations) and the TEY activation loop are shown (PDB: 4NIF). (B) Immunoblots showing mutant ERK expression and rescue of downstream signaling in Pa16C, MIA PaCa-2, and UM53 cells with doxycycline (Dox)-inducible activated ERK1^SD (R84S/S170D) or ERK2^SD (R67S/S153D) treated with the KRAS^G12D (MRTX1133, 20 nM) or KRAS^G12C (MRTX1257, 20 nM) inhibitor for 24 hours. Dox (1 μg/mL) was added 24 hours prior to drug treatments. Blots are representative of three or more independent experiments. Quantification of pRSK and MYC are shown in fig. S1C. (C) Cell viability assays of Pa16C, MIA PaCa-2, UM53 cells with Dox-inducible expression of activated ERK1^SD (R84S/S170D) or ERK2^SD (R67S/S153D) in response to KRAS^G12C/D (MRTX1257, MRTX1133) or ERK1/2 inhibitor (SCH772984). Dox treatment (1 μg/mL) was added 24 hours prior to drug treatments. Graphs show the mean percent growth of three replicates +/− standard error of the mean. (D) RPPA Spearman correlation coefficients comparing log₂(FC) of signals from 147 antibodies for the individual six cell lines and all cell lines considered together. Cells were treated with siRNAs targeting NS (non-specific control), KRAS, MAPK3 (ERK1) and/or MAPK1 (ERK2), for 72 hours as in (fig. S1E). Statistical analysis was performed from four biological replicates. The individual treatments and full protein list can be found in fig. S1G and data S1. (E) RNA transcript expression changes induced by 24-hour KRAS^G12D inhibition (MRTX1133, 100 nM) versus DMSO control in Pa16C cells expressing activated ERK1^SD (cyan), ERK2^SD (pink), or GFP control (black). Genes are ranked by expression changes in GFP expressing cells. Dox (1 μg/mL) was added 24 hours prior to drug treatments. Statistical analysis was performed from two biological replicates. Genes shown for ERK1^SD and ERK2^SD (points) are those with significant DE induced by KRAS^G12D inhibition in GFP control cells (adj. p-val. < 0.01). (F) Venn diagrams representing the overlap between genes upregulated (G12Di UP) or downregulated (G12Di DN) by KRAS^G12D inhibition in GFP expressing cells (green; |log2FC| > 0.25 and adj. p-val. < 0.01) and genes rescued by ERK1^SD (cyan) and ERK2^SD (pink). The center overlap (purple) depicts genes rescued by both ERK1^SD and ERK2^SD, the genes in only the green area are not rescued by either, in the cyan area rescued by only ERK1^SD, and in the purple area by only ERK2^SD. The term “rescue” indicates an expression change (log2FC) less extreme than the GFP control but no p-values were used to represent the degree of those expression changes.

Fig. 2.. Phosphoproteomic determination of the ERK regulated phosphoproteome in KRAS mutant PDAC.

(A) Flow diagram of the MIB/MS experiment was created with BioRender.com. (B) MIB/MS analyses of six KRAS mutant cell lines treated for 1 or 24 hours with ERKi (SCH772984, 1 μM). (C) Experimental workflow for proteomics analyses, created with BioRender.com. (D) Differentially expressed phosphosites following 1 and 24 hours ERK inhibition in the same six PDAC cell lines as in (A and B). Phosphosites previously reported in the ERK Compendium are highlighted, and top significantly dysregulated sites identified within the ERK Compendium as direct substrates of ERK are labeled. (E) Overlap between ERK-dependent phosphosites (p < 0.05) or phosphoproteins in PDAC and phosphosites/phosphoproteins reported in the CPTAC PDAC dataset (fdr < 0.05), ERK Compendium, PhosphoSitePlus (PSP) regulatory sites, and Post-Translational Signature Database (PTMsigDB). (F) Differentially expressed phosphosites following 24-hour KRAS^G12C inhibition (MRTX1257, 20 nM, G12Ci) or RAS(ON) multi-selective tri-complex inhibition (RMC-7977, 10 nM, RASi). ERK-dependent phosphosites (p<0.05, 24 h) are annotated by blue (downregulated) or red (upregulated) points. ERK-regulated phosphosites at 1 and 24 hours are annotated as bars below the volcano plot.

Fig. 3.. The ERK phosphoproteome regulates diverse functional pathways comprised of proteins enriched in ERK binding motifs.

(A) Representative images of pERK (red), total ERK (green), and nuclear DAPI (grey) immunofluorescence in Pa16C cells treated with ERKi (SCH772984, 1 μM) or DMSO for 24 hours (scale bar = 75 μm), quantified in (B) Dashed lines represent median, and statistics represent a Mann-Whitney test on 113–655 individual cells from 5–10 fields of at least two replicates. (C) Representative pseudo-colored FRET intensity of Pa16C cells stably expressing untargeted EKAR4 (EKAR4) or EKAR4 tagged with either a nuclear localization signal (nuc-EKAR4) or nuclear export signal (cyto-EKAR4) treated with ERKi (SCH772984, 1 μM) or DMSO for 4 or 24 hours (scale bar = 20 μm), quantified in (D). Statistical analysis performed as in (B). (E) Subcellular association of phosphoproteins with differentially regulated phosphosites and enrichment (red/blue, p<0.05) at 1 hour and 24 hours ERKi. Statistical analysis of enrichment determined by one-tailed hypergeometric testing and p values corrected by Benjamini-Hochberg. (F) Proportion of phosphosites containing the p[S/T]-P phospho motif together with an ERK-binding D or DEF motif within the phosphoprotein. Proportions were determined within the background of all detected phosphosites, and sites differentially downregulated at 1 hour and 24 hours ERKi (p < 0.01, log₂(FC)<0). Statistical analysis of enrichment determined as in (E). (G) Subcellular association of phosphoproteins with ERK binding and phosphorylation motifs as in (F). Point color and size represent relative enrichment and significance of enrichment, respectively. Points are highlighted if statistically enriched (red, adj. p < 0.05). Enrichment was determined using one-tailed hypergeometric enrichment using all detected phosphosites as background. (H) Membership of phosphoproteins within top enriched gene sets for cytoskeleton-associated phosphoproteins with downregulated phosphosites at 1 hour ERKi. Phosphoproteins are colored by the presence of ERK-binding D or DEF motifs and outlined in green if a differentially regulated phosphosite was detected with a p[S/T]-P motif. Size of phosphoproteins is proportional to the number of downregulated phosphosites, and pathways are sized by the number of phosphoprotein members. Differentially regulated phosphosites are also shown and likewise colored green if the phosphosite contains a p[S/T]-P motif. (I) Immunoblots showing mutant ERK expression and downstream signaling in Pa16C expressing dox-inducible ERK1^SD, ERK1^SDN (R84S/S170D/D338N), and ERK1^SDA (R84S/S170D/Y280A) treated with the KRAS^G12D inhibitor (MRTX1133, 20 nM) for 24 hours. Dox (1 μg/mL) was added 24 hours prior to drug treatments. Blots are representative of three or more independent experiments and quantification of pRSK and MYC are in fig. S3Q. (J) Viability of Dox-inducible ERK1^SD, ERK1^SDN, and ERK1^SDA cell lines treated with a dose escalation of G12Di (MRTX1133). Dox (1 μg/mL) was added 24 hours prior to drug treatments. Graphs show the mean percent growth of three replicates +/− standard error of the mean.

Fig. 4.. ERK regulates a dynamic phosphoproteome by engaging downstream kinases.

(A) Epigenetic regulator, transcription factor, kinase, phosphatase, and E3 ubiquitin ligase phosphoproteins with downregulated phosphosites and their ERK binding DEF and/or D motifs. The number of phosphoproteins containing a downregulated known regulatory phosphorylation is annotated with a red triangle. (B) Relative kinase activities calculated for 303 S/T kinases at 1 hour and 24 hours ERKi using kinase motif specificities (37). Line color represents the direction of change from 1 to 24 hours ERKi, and line transparency represents the magnitude of change. (C) Kinase-substrate networks at 1 and 24 hours ERKi. Networks were constructed using kinase motif specificities (37), kinase-substrate specificities from PhosphoSitePlus (25), kinase-substrate interactions from PhosphoNetworks (38) and BioGRID (39). Nodes are sized and colored by their strength in network, and shape represents protein group. Edges are colored by direction of association with up- or downregulated phosphosites. Edge width represents the magnitude of interaction score. The top quantile of kinases is labeled in each network; text size is scaled with node strength. (D) Comparison of kinase-motif enrichment (log₂(freq. factor)) derived from ERK-dependent phosphoproteome versus RAS-dependent phosphoproteomes as shown in Fig 2F. Fill color represents the significance of enrichment in the ERK-dependent phosphoproteome and size represents the significance in the KRAS^G12C- or RAS-dependent phosphoproteome.

Fig. 5.. ERK signaling is highly integrated with RHO GTPase signaling.

(A) Overlap of KEGG, Gene Ontology (GO), and Reactome gene sets significantly enriched in (p < 0.01, left) or containing any gene associated with (right) phosphoproteins reported in the ERK Compendium (21) or the ERK-dependent PDAC phosphoproteome. Gene set enrichment was determined using one-tailed hypergeometric testing using the entire proteome as background for both phosphoprotein lists; p values corrected by Benjamini-Hochberg. (B) Terms of top Reactome gene sets significantly enriched in the ERK Compendium (left), in the ERK dependent PDAC phosphoproteome (middle), or in the ERK dependent PDAC phosphoproteome but not the ERK Compendium (right). Terms are sized and colored by significance of enrichment. (C) Signed significance from gene enrichment analysis of up- or downregulated phosphoproteins (log₂(FC) < 0 or > 0; adj. p < 0.05) at 1 and 24 hours ERKi. Up- and downregulated phosphoprotein enrichment is represented by fill color (red and blue respectively) with number of phosphoproteins annotated as size by foreground matches. (D) Isolated network for edges connecting to proteins within the Reactome “RHO GTPase cycle” and “RHO GTPase effectors” gene sets with interaction scores greater than one standard deviation. Edges are colored by time point (1 or 24 hours) and phosphosite change (up- or downregulated) and edge-node associations are outlined (Down at 1 hour, Down at 24 hours, Up at 1 hour, Up at 24 hours). Top kinases by strength are labeled and node sized by strength. (E) Representative images of phalloidin (green) and nuclear DAPI (blue) immunofluorescence in Pa14C and Pa16C cells treated with ERKi (1 μM, SCH772984) or DMSO for 24 hours or 72 hours (scale bar = 50 μm). (F) Quantification of relative integrative intensity of F-actin from (E). Solid line represents median, dashed line represents upper and lower quartiles, and statistics represent a Mann-Whitney test on 113–655 individual cells from 5–10 fields of at least two replicates.

Fig. 6.. Genetic dependencies of the ERK dependent PDAC phosphoproteome.

(A) Annotated Chronos dependency rank plot, ranked by DepMap dependency scores for ERK-regulated phosphoproteins. Median Chronos dependency across 43 KRAS MT PDAC cell lines for each phosphoprotein is represented as a point on the rank plot. The line for each point represents upper and lower quartiles and is colored by the median dependency. Annotations indicate membership in the subcellular compartment or Reactome gene set. Enrichment significance was determined by one-tailed hypergeometric testing of phosphoproteins with median Chronos scores < −0.5 (dotted line); p values corrected by Benjamini-Hochberg. (B) Selectivity of genetic dependencies for ERK regulated phosphoproteins towards KRAS MT or KRAS WT cancers. Cell lines from cancers of the pancreas, large intestine, biliary tract, lung, stomach, endometrium, cervix, or ovary were characterized as either KRAS mutant, including G12A/C/D/R/S/V, G13D, or Q61K/H/L/R mutations, or KRAS WT. The mean difference in Chronos dependency (y-axis) in KRAS MT and KRAS WT cell lines is shown for each ERK-regulated phosphoprotein with respect to the mean Chronos dependency in KRAS MT PDAC (x-axis). Genes are colored and sized by adjusted p values calculated by two-way ANOVA and Tukey’s HSD post hoc test between KRAS MT and KRAS WT cell lines, with cancer type as an independent variable. (C) Density histogram of mean genetic dependencies of the 23 genes with significantly selective dependencies in KRAS MT cancers as shown in (B), separated by KRAS MT PDAC versus other cancers from (B) defined as KRAS MT or KRAS WT. Statistical differences determined by Games-Howell. (D) Kinase-substrate network from 1 and 24 hours ERKi treatments with node size and color representing median genetic dependency of the 43 KRAS MT PDAC cell lines. Edges represent interaction scores as in Fig. 4C.