Cancer is a complex disease characterized by genetic alterations affecting oncogenes and tumor suppressors. High proliferation rates in cancer cells stress the endoplasmic reticulum (ER), leading to the unfolded protein response (UPR) and the integrated stress response (ISR). ISR, involving kinases like PERK, HRI, GCN2, and PKR, phosphorylates eIF2α (p-eIF2α), impacting global translation and regulating stress response genes. While ISR is upregulated in many cancers, its role in lung adenocarcinoma (LUAD) is not fully understood. LUAD, the most common type of lung cancer, has a poor prognosis, and approximately 30% of cases are associated with KRAS mutations, which often prove resistant to therapy. This study investigates the role of ISR, specifically the PERK/p-eIF2α axis, in KRAS-driven LUAD development and explores its potential as a therapeutic target.

Existing literature highlights the connection between endoplasmic reticulum stress, unfolded protein response (UPR), and tumorigenesis. The integrated stress response (ISR), a crucial stress-response pathway, has been implicated in cancer development, but its specific role in LUAD and KRAS-driven cancers remains largely unclear. Studies have shown that the phosphorylation of eIF2α (p-eIF2α), a key component of ISR, influences translation and gene expression. However, a detailed understanding of how this affects LUAD development and specifically its interaction with KRAS mutations is lacking. This research builds upon previous work demonstrating the involvement of the ISR in various cancers and explores its specific contribution to the pathogenesis of KRAS-mutant LUAD. The study also examines the potential of targeting ISR components as a novel treatment strategy for this particularly aggressive form of lung cancer.

The study involved multiple approaches: 1. **Analysis of human LUAD samples:** Tissue microarrays (TMAs) from 928 primary human LUADs were analyzed for p-eIF2α expression to determine its correlation with patient survival and tumor characteristics. Immunohistochemistry (IHC) was used to assess p-eIF2α levels, and statistical analysis was performed to evaluate its prognostic significance. 2. **Mouse models of KRAS-driven lung cancer:** KRAS^{+/LSL-KRAS G12D} mice were crossed with mice with either a conditional homozygous S51A mutation of the eIF2S1 allele (eIF2αA/A) or wild-type eIF2S1 (eIF2αS/S) to investigate the role of p-eIF2α in lung tumor formation. Lentiviruses expressing CRE recombinase and TP53 shRNA were used to induce lung tumorigenesis. Ultrasound imaging, H&E staining, and IHC were used to monitor tumor growth and characterize tumor types. RNA sequencing (RNA-seq) was conducted on KRAS G12D eIF2αS/S and eIF2αA/A tumor cells to identify differentially expressed genes. Upstream regulator analysis was performed using Ingenuity Pathway Analysis (IPA). 3. **In vitro studies:** Human LUAD cell lines with KRAS mutations were used to investigate the relationship between mutant KRAS, PERK/p-eIF2α signaling, and p-ERK. The effects of PERK inhibition using GSK2606414 and ISR inhibition using ISRIB were evaluated on cell survival and colony formation. siRNAs targeting DUSP6 and PERK were employed to study their roles. 4. **In vivo therapeutic studies:** Xenograft and orthotopic transplantation models were used to assess the therapeutic effects of PERK and ISR inhibitors (GSK2606414 and ISRIB) on tumor growth in mice. Ultrasound imaging and IHC were used to monitor tumor response to treatment. Specific techniques included polysome profiling to study mRNA translation, real-time qPCR, and multiplex fluorescent IHC for single-cell analyses. Statistical analyses were performed using appropriate methods, such as Kaplan-Meier survival analysis, Cox proportional hazards models, and t-tests.

The study revealed a strong association between increased p-eIF2α levels and poor prognosis in 928 human LUAD patients. Higher p-eIF2α levels correlated with invasive growth patterns and increased tumor cell proliferation (Ki67 positivity). In mouse models of KRAS-driven lung cancer, genetic inactivation of eIF2α (eIF2αA/A) significantly reduced tumor growth and prolonged survival compared to wild-type mice (eIF2αS/S). Mechanistically, p-eIF2α was shown to suppress DUSP6 expression translationally, leading to increased p-ERK levels and enhanced tumor cell survival. Upstream regulator analysis identified PERK activation and the upregulation of pro-tumorigenic pathways (CTNNB1, LHX1, HIF1A, IL6) and downregulation of tumor suppressors (HOXA10, ESRRA, ASXL1, MFN2). Mutant KRAS upregulated the PERK/p-eIF2α arm in human LUAD cells, rendering them more sensitive to PERK inhibition. Both PERK inhibition (using GSK2606414) and ISR inhibition (using ISRIB) significantly reduced tumor growth in multiple mouse models, including orthotopic transplantation of LLC cells and syngeneic KRAS G12D models, leading to prolonged survival. ISRIB, a memory-enhancing drug with limited toxicity, showed particularly promising anti-tumor effects. Notably, the relationship between p-eIF2α and p-ERK was confirmed at the single-cell level in human LUAD specimens, supporting the mechanistic link established in mouse models.

The findings demonstrate the crucial role of the PERK/p-eIF2α axis of ISR in KRAS-driven LUAD tumorigenesis. The translational repression of DUSP6 by p-eIF2α, resulting in increased p-ERK, is a key mechanism driving tumor growth. The study identified both the clinical significance of p-eIF2α as a prognostic marker in human LUAD and the preclinical efficacy of ISR inhibitors as potential therapeutic agents. ISRIB, a relatively safe drug, showed potent anti-tumor activity, offering a promising avenue for treating this often-resistant cancer type. The upregulation of pro-tumorigenic pathways and downregulation of tumor suppressors by p-eIF2α further underlines the importance of targeting this pathway. The study highlights the potential of utilizing ISR inhibitors for a broader range of KRAS mutations beyond G12C, addressing a significant unmet clinical need.

This research establishes the integrated stress response (ISR), specifically the PERK/p-eIF2α arm, as a critical driver of KRAS-mutant lung adenocarcinoma. The translational repression of DUSP6 by p-eIF2α resulting in increased p-ERK signaling was identified as a key mechanism. The significant therapeutic potential of ISR inhibitors, particularly ISRIB, was demonstrated in preclinical models. These findings support further investigation of ISR-targeted therapies for treating this aggressive and often treatment-resistant cancer, potentially extending to other KRAS-mutant cancers. Future studies should focus on clinical trials to assess the efficacy and safety of ISR inhibitors in patients with KRAS-driven LUAD.

While the study used multiple model systems (human LUAD samples, mouse models, and in vitro studies), the results may not fully translate to all clinical scenarios. The mouse models, though useful, may not perfectly replicate the complexity of human LUAD development. The study focused primarily on the PERK/p-eIF2α arm of the ISR, and further investigation is needed to explore the roles of other ISR branches. Larger-scale clinical trials are needed to definitively establish the clinical benefit of ISR inhibitors in LUAD patients.