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Supplementary Table S4. The complete list of identified phosphopeptides after (pY) enrichment and TMT-based quantitative phosphoproteomics analysis in KRASG12C mutant lung cancer cell lines (n=8) with mean-centered log abundance values.

Supplementary Table S1. Relative quantification of peptides representing total KRAS, WT KRAS, free KRAS G12C protein and drug bound KRAS G12C Protein Levels.

Supplementary Table S9. The correlation analysis between ERBB2/ERBB3 and EMT score in SPORE 442 KRASG12C lung cancer mutants.

Supplementary Table S2. The complete list of identified phosphopeptides after (pY) enrichment in ARS-1620 treated cells with fold change quantisation and p-value calculation.

Supplementary Table S5. The complete list of identified proteins after TMT-based quantitative proteomic analysis in KRASG12C mutant lung cancer cell lines (n=8) with mean-centered log abundance values.

Supplementary Fig. S1. Differential response (2D and 3D culture) of KRASG12C specific covalent inhibitors (ARS-1620 and AMG510) in KRASG12C lung cancer cell lines and signaling rebound following KRASG12C inhibition. Supplementary Fig. S2. MetaCore literature network after ARS-1620 treatment in H358 cells. Supplementary Fig. S3. TMT design for expression and pY comparison in 8 KRASG12C lung cancer cell lines and two group comparison of phosphosites Supplementary Fig. S4. Analysis of CCLE data set for EMT markers and ERBB3 gene expression; and PCA representing 105 probeset TGFβ-EMT signatures (Gordian et al. 2019) translated into proteins expression to describe EMT activity in KRASG12C lung cancer cell lines. Supplementary Fig. S5. Impact of SHP2 inhibitors (SHP-099 and RMC-4550) on signaling in H358 cells and cell viability analysis following treatment with SHP2 inhibitors or in the combination with ARS-1620 in H358 and H1792 cells. Supplementary Fig. S6. Literature-based signaling network after KRASG12C inhibition in H1792 cells and cell viability analysis following treatment with ARS-1620 (1µM), AZD-4547 (1µM) and their combination in NSCLCG12C mutant cell lines. Supplementary Fig. S7. Effect of TGFβ stimulation on cell viability in response to KRASG12C inhibition and different combination strategies. Supplementary Fig. S8. Heatmap showing mean-centered log abundance expression values for indicated phosphosites/protein expression in KRASG12C mutant cell lines and E vs M 2-group comparison performed for FGFR1 gene expression in CCLE data set. Supplementary Fig. S9. Literature-based signaling network after KRASG12C inhibition in Calu1 cells and impact on signaling following treatment with ARS-1620 plus drugs known to target AXL Receptor.

Detailed Supplementary Methods

Legends to Supplementary Figures and Tables

Supplementary Table S3. The complete list of identified phosphopeptides after global (pS/T/Y) phosphopeptides enrichment in ARS-1620 treated cells with fold change quantisation and p-value calculation.

Supplementary Table S7. The mean-centered CCLE normalized log2 RNA-seq counts for indicated genes and TGFβ-EMT analysis of 15 KRASG12C NSCLC cell lines.

Purpose:Covalent inhibitors of KRASG12C specifically target tumors driven by this form of mutant KRAS, yet early studies show that bypass signaling drives adaptive resistance. Although several combination strategies have been shown to improve efficacy of KRASG12C inhibitors (KRASi), underlying mechanisms and predictive strategies for patient enrichment are less clear.Experimental Design:We performed mass spectrometry–based phosphoproteomics analysis in KRASG12C cell lines after short-term treatment with ARS-1620. To understand signaling diversity and cell type–specific markers, we compared proteome and phosphoproteomes of KRASG12C cells. Gene expression patterns of KRASG12C cell lines and lung tumor tissues were examined.Results:Our analysis suggests cell type–specific perturbation to ERBB2/3 signaling compensates for repressed ERK and AKT signaling following ARS-1620 treatment in epithelial cell type, and this subtype was also more responsive to coinhibition of SHP2 and SOS1. Conversely, both high basal and feedback activation of FGFR or AXL signaling were identified in mesenchymal cells. Inhibition of FGFR signaling suppressed feedback activation of ERK and mTOR, while AXL inhibition suppressed PI3K pathway. In both cell lines and human lung cancer tissues with KRASG12C, we observed high basal ERBB2/3 associated with epithelial gene signatures, while higher basal FGFR1 and AXL were observed in cells/tumors with mesenchymal gene signatures.Conclusions:Our phosphoproteomic study identified cell type–adaptive responses to KRASi. Markers and targets associated with ERBB2/3 signaling in epithelial subtype and with FGFR1/AXL signaling in mesenchymal subtype should be considered in patient enrichment schemes with KRASi.