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. 2021 Jun 1;81(11):3051-3066.
doi: 10.1158/0008-5472.CAN-20-2435. Epub 2021 Mar 16.

Alterations in the Global Proteome and Phosphoproteome in Third Generation EGFR TKI Resistance Reveal Drug Targets to Circumvent Resistance

Xu Zhang #  1 , Tapan K Maity #  2 , Karen E Ross  3 , Yue Qi  2 , Constance M Cultraro  2 , Meriam Bahta  2 , Stephanie Pitts  2 , Meghana Keswani  2 , Shaojian Gao  2 , Khoa Dang P Nguyen  2 , Julie Cowart  4 , Fatos Kirkali  2 , Cathy Wu  4 , Udayan Guha  1
Affiliations

Alterations in the Global Proteome and Phosphoproteome in Third Generation EGFR TKI Resistance Reveal Drug Targets to Circumvent Resistance

Xu Zhang et al. Cancer Res. .

Abstract

Lung cancer is the leading cause of cancer mortality worldwide. The treatment of patients with lung cancer harboring mutant EGFR with orally administered EGFR tyrosine kinase inhibitors (TKI) has been a paradigm shift. Osimertinib and rociletinib are third-generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard of care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here we characterized the proteome and phosphoproteome of a series of isogenic EGFR-mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs, comprising the most comprehensive proteomic dataset resource to date to investigate third generation EGFR TKI resistance in lung adenocarcinoma. Unbiased global quantitative mass spectrometry uncovered alterations in signaling pathways, revealed a proteomic signature of epithelial-mesenchymal transition, and identified kinases and phosphatases with altered expression and phosphorylation in TKI-resistant cells. Decreased tyrosine phosphorylation of key sites in the phosphatase SHP2 suggests its inhibition, resulting in subsequent inhibition of RAS/MAPK and activation of PI3K/AKT pathways. Anticorrelation analyses of this phosphoproteomic dataset with published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures program predicted drugs with the potential to overcome EGFR TKI resistance. The PI3K/MTOR inhibitor dactolisib in combination with osimertinib overcame resistance both in vitro and in vivo. Taken together, this study reveals global proteomic alterations upon third generation EGFR TKI resistance and highlights potential novel approaches to overcome resistance. SIGNIFICANCE: Global quantitative proteomics reveals changes in the proteome and phosphoproteome in lung cancer cells resistant to third generation EGFR TKIs, identifying the PI3K/mTOR inhibitor dactolisib as a potential approach to overcome resistance.

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Figures

Fig. 1.
Fig. 1.
Summary of SILAC-based quantitative proteome and phosphoproteome analyses of isogenic 3rd generation EGFR TKI-sensitive and resistant lung adenocarcinoma cells. (A) Experimental workflow showing treatment of SILAC-labelled cells, enrichment of phosphopeptides, and detection by tandem mass spectrometry. TKI-sensitive H1975, osimertinib-resistant AZR3/AZR4 cells, and rociletinib-resistant COR1/COR10 cells were treated with DMSO or the corresponding TKI (Left panel). Summary table showing the number of proteins and phosphosites identified in each experiment (Right panel). (B) Venn diagrams of proteins (left panel) and phosphosites (right panel) identified in osimertinib and rociletinib experiments. (C) Hierarchical clustering of proteins (left panel) and phosphosites (right panel) based on SILAC ratios. Columns represent different cell lines treated as indicated. Rows represent quantified proteins or phosphopeptides identified in all experimental conditions.
Fig. 2.
Fig. 2.
GO classification analyses for localization and function of proteins with altered abundance, and heatmaps of protein SILAC ratios (TKI-resistant/sensitive) of translation regulators and EMT proteins and ratio of transcripts of EMT genes (TKI-resistant/sensitive). (A) Percentage of differentially expressed proteins in TKI-resistant cells with specific subcellular localization (up) and function (bottom). (B) Heatmap of SILAC ratios of protein abundance (TKI-resistant/sensitive) of selected translation regulators across all experiments demonstrate significantly more alterations in rociletinib-resistant cells. (C) Hierarchical clustering of quantified EMT proteins in three state SILAC experiments based on SILAC ratios of protein abundance in presence and absence of corresponding TKI. (D) Hierarchical clustering of transcript ratios of EMT genes quantified by qPCR.
Fig. 3.
Fig. 3.
Comparison of protein abundance differences between osimertinib-resistant and rociletinib-resistant cells treated with respective TKI. (A) Differentially expressed proteins in TKI-resistant cells compared to the sensitive cells. 60 differentially expressed proteins were common to all four resistant cell lines. (B) Heatmap of SILAC ratios of protein abundance (TKI-resistant/sensitive) and hierarchical clustering of the 60 differentially expressed proteins common to all TKI-resistant cell lines. (C) Protein network of the 60 common differentially expressed proteins together with EGFR by STRING analysis. Many of these proteins are direct and indirect downstream targets of EGFR. (D) Heatmap of SILAC ratios of phosphorylation abundance (TKI-resistant/sensitive) and hierarchical clustering of the 49 differentially phosphorylated phosphosites common to all TKI-resistant cell lines.
Fig. 4.
Fig. 4.
Validation of differentially phosphorylated phosphosites and differentially expressed proteins in osimertinib- or rociletinib-resistant cells. (A-B) Western blots showing changes in phosphorylation and total protein expression without TKI treatment and upon 1 hour of rociletinib (100 nM) or osimertinib (50 nM) treatment in H1975, COR1, COR10, AZR3, and AZR4 cells. Bar graphs show the relative quantification of phosphorylated proteins normalized to total protein expression in each cell line.
Fig. 5.
Fig. 5.
Phosphorylation alteration of different phosphosites of the phosphatases PTPN11. (A) Multiple phosphorylation sites of PTPN11 identified and quantified in both osimertinib and rociletinib resistant cells. The SILAC ratios indicate the relative abundance between the resistant and H1975 sensitive cells (upper panel). Annotated MS/MS spectra (left) and MS spectra of their parent ions (middle and right) of the phosphopeptides VpYENVGLMQQQK (Y584) (middle panel) and IQNTGDpYYDLYGGEK (Y62) (lower panel) of PTPN11 showing the relative abundance changes between the sensitive and resistant cells in osimertinib and rociletinib experiment. (B) Western blots showing changes in phosphorylation and total protein expression of PTPN11 and EGFR without TKI treatment and upon 1 hour of rociletinib (100 nM) or osimertinib (50 nM) treatment in H1975, COR1, COR10, AZR3, and AZR4 cells. (C) Schematic showing the role of SHP2 in the activation of RAS/MAPK and inhibition of PI3K/AKT signaling pathways downstream from EGFR.
Fig. 6.
Fig. 6.
Networks generated by upstream kinase analysis of significantly altered phosphopeptide substrates in TKI-resistant cells using iPTMNet. (A) AZR3/H1975 in the presence of osimertinib; (B) AZR4/H1975 in the presence of osimertinib; (C) COR1/H1975 in the presence of rociletinib; (D) COR10/H1975 in the presence of rociletinib. The scale bars show the log2 SILAC ratio of phosphorylation (resistant/sensitive). Phosphosites in red are hyperphosphorylated and blue are hypophosphorylated in the resistant cells.
Fig. 7.
Fig. 7.
In vitro and in vivo sensitivity of osimertinib and rociletinib resistant cells to dactolisib, a PI3K/mTOR inhibitor, and in vitro sensitivity of osimertinib resistant cells to VX-970, an ATR inhibitor. (A) Cell viability curves of H1975, H1975-AZR 3 and 4 cells showing the effect of osimertinib, dactolisib, and dactolisib in presence of 2 μM of osimertinib treatment. The EC50 values and fold resistant of the AZR3 and AZR4 cells compared to H1975 sensitive cells are shown. (B) Cell viability curves of H1975, H1975-COR 1 and 10 cells showing the effect of rociletinib, dactolisib, and dactolisib in presence of 2 μM of rociletinib treatment. The EC50 values and fold resistance of COR1 and COR10 cells compared to H1975 sensitive cells are shown. (C-D) Cell viability curves of PC9, PC9-OsiR-NCI1 (C) and HCC827, HCC827-OsiR-NCI1 (D) cell lines showing the effect of osimertinib, dactolisib, and dactolisib in presence of 2 μM of osimertinib treatment. The corresponding EC50 of each drug is shown in the right panels. (E) Cell viability curves of H1975, H1975-AZR 3 and 4 cell lines showing the effect of VX-970, and VX-970 in presence of 2 μM of osimertinib treatment. The EC50 of VX970 alone or in presence of 2μM osimertinib are shown in the right panel. (F) Western blots showing changes in phosphorylation and total protein expression without TKI treatment and upon 1 hour of dactosilib (50nM), osimertinib (50nM) or a combination of dactolisib (50nM) and osimertinib (50nM) treatment in H1975, H1975-AZR3, and H1975-AZR4 cells (G) Western blots showing changes in phosphorylation and total protein expression without TKI treatment and upon 1 hour of dactosilib (50nM) or osimertinib (50nM) or a combination of dactolisib (50nM) and osimertinib (50nM) treatment treatment in PC9, PC9-OsiR-NCI1, HCC827, and HCC827-OsiR-NCI1 cells. (H-J) Dactolisib in combination with osimertinib inhibits tumor growth of H1975-AZR3 xenografts in vivo. The in vivo growth of xenograft tumors is shown for 20 days of treatment (H) and the percent tumor growth at Day 20 (I) and Day 36 (J) of treatment is shown.
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