# Unveiling unique protein and phosphorylation signatures in lung adenocarcinomas with and without ALK, EGFR, and KRAS genetic alterations

## TL;DR

This study identifies unique protein and phosphorylation patterns in lung cancer tumors with specific genetic mutations, which could help improve diagnosis and treatment strategies.

## Contribution

The study reveals distinct proteomic and phosphoproteomic signatures associated with EGFR, KRAS, and EML4–ALK mutations in lung adenocarcinoma.

## Key findings

- EGFR-mutated tumors showed unique protein expression patterns and downregulated pathways related to membrane and vesicle transport.
- Altered activities of 10 kinases, including CDK2, were identified in EML4–ALK samples.
- Proteomic differences included proteins like cathepsin L and STING, and phosphoproteins linked to RNA splicing like SRSF1.

## Abstract

Genetic alterations in key oncogenes have been frequently identified in lung adenocarcinoma (LUAD), including genes encoding epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), and anaplastic lymphoma kinase (ALK). In this pilot study, we aimed to characterize the differences in enriched biological pathways and phosphorylation events between LUAD tumors harboring EGFR, KRAS, or echinoderm microtubule‐associated protein‐like 4 (EML4)–ALK oncogenic alterations and triple wild‐type LUAD tumors (WT, without EML4–ALK, KRAS, or EGFR alterations) by mass spectrometry (MS)‐based quantitative proteomics and phosphoproteomics. We analyzed tumor regions of 82 formalin‐fixed paraffin‐embedded (FFPE) tissue sections with 6, 23, 31, and 22 samples from the EML4–ALK, EGFR, KRAS, and WT sample groups, respectively. A total of 1377 to 2189 proteins and 73 to 1781 phosphosites were quantified in these analyses. Based on the results, the samples clustered according to their genetic alteration type, and EGFR‐mutated samples showed unique protein expression patterns. Membrane organization, vesicle organization, and vesicle‐mediated transport Gene Ontology Biological Process (GOBP) terms were significantly downregulated in EGFR‐mutated samples compared to the other sample groups. Changes in 36 proteins and 52 phosphosites were also identified as potentially specific to a given genetic alteration. Many of these proteins have previously been linked to EGFR or KRAS mutations [e.g., cathepsin L, stimulator of interferon genes protein (STING)], whereas several phosphoproteins are associated with RNA splicing [e.g., serine/arginine‐rich splicing factor 1 (SRSF1), SRSF2, and SRSF7 proteins]. Kinase–substrate enrichment analysis indicated altered activities of 10 kinases, including mitogen‐activated protein kinases (MAPKs) and cyclin‐dependent kinases (CDKs). For example, CDK2 activity was elevated in EML4–ALK samples compared to the other sample groups. Our results could provide significant insights into further studies that could contribute to developing improved diagnostic and therapeutic strategies for LUAD.

Proteomic and phosphoproteomic analyses were performed on lung adenocarcinoma (LUAD) tumors with EGFR, KRAS, or EML4–ALK alterations and wild‐type cases. Distinct protein expression and phosphorylation patterns were identified, especially in EGFR‐mutated tumors. Key altered pathways included vesicle transport and RNA splicing. The findings highlight molecular differences that could support the refinement of LUAD classification and guide future therapeutic development.