KRAS-mutant Lung Adenocarcinoma Research Articles

USP13 facilitates a ferroptosis-to-autophagy switch by activation of the NFE2L2/NRF2-SQSTM1/p62-KEAP1 axis dependent on the KRAS signaling pathway

ABSTRACT Macroautophagy/autophagyis a lysosomal-regulated degradation process that participates incellular stress and then promotes cell survival or triggers celldeath. Ferroptosis was initially described as anautophagy-independent, iron-regulated, nonapoptotic cell death.However, recent studies have revealed that autophagy is positivelyassociated with sensitivity to ferroptosis. Nonetheless, themolecular mechanisms by which these two types of regulated cell death(RCD) modulate each other remain largely unclear. Here, we screened85 deubiquitinating enzymes (DUBs) and found that overexpression ofUSP13 (ubiquitin specific peptidase 13) could significantlyupregulate NFE2L2/NRF2 (NFE2 like bZIP transcription factor 2)protein levels. In addition, in 39 cases of KRAS-mutated lungadenocarcinoma (LUAD), we found that approximately 76% of USP13overexpression is positively correlated with NFE2L2 overexpression.USP13 interacts with and catalyzes the deubiquitination of thetranscription factor NFE2L2. Additionally, USP13 depletion promotesan autophagy-to-ferroptosis switch invitro andin xenograft tumor mouse models, through the activation of theNFE2L2-SQSTM1/p62 (sequestosome 1)-KEAP1 axis in KRAS mutant cellsand tumor tissues. Hence, targeting USP13 effectively switchedautophagy-to-ferroptosis, thereby inhibiting KRAS (KRASproto-oncogene, GTPase) mutant LUAD, suggesting the therapeuticpromise of combining autophagy and ferroptosis in the KRAS-mutantLUAD. Abbreviation: ACSL4: acyl-CoA synthetase long-chain family member 4; ACTB: actin beta; AL: autolysosomes; AP: autophagosomes; BCL2L1/BCL-xL: BCL2 like 1; CCK8: Cell Counting Kit-8; CQ: chloroquine; CUL3: cullin 3; DMSO: dimethyl sulfoxide; DOX: doxorubicin; DUB: deubiquitinating enzyme; Ferr-1: ferrostatin-1; GPX4: glutathione peroxidase 4; GSEA: gene set enrichment analysis; 4HNE: 4-hydroxynonenal; IKE: imidazole ketone erastin; KEAP1: kelch like ECH associated protein 1; KRAS: KRAS proto-oncogene, GTPase; LCSC: lung squamous cell carcinoma; IF: immunofluorescence; LUAD: lung adenocarcinoma; Lys05: Lys01 trihydrochloride; MAPK1/ERK2/p42: mitogen-activated protein kinase 1; MAPK3/ERK1/p44; MTOR: mechanistic target of rapamycin kinase; NFE2L2/NRF2: NFE2 like bZIP transcription factor, 2; NQO1: NAD(P)H quinone dehydrogenase 1; PG: phagophore; RCD: regulated cell death; RAPA: rapamycin; ROS: reactive oxygen species; SLC7A11/xCT: solute carrier family 7 member 11; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TUBB/beta-tubulin: tubulin, beta; UPS: ubiquitin-proteasome system; USP13: ubiquitin specific peptidase 13.

CASTOR1 phosphorylation predicts poor survival in male patients with KRAS-mutated lung adenocarcinoma

BackgroundLung cancer, a leading global cause of cancer-related mortality, necessitates enhanced prognostic markers for improved treatment outcomes. We have previously shown a tumor suppressive role of cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1), which is targeted for degradation upon phosphorylation at S14 (pCASTOR1) in multiple types of cancer. This study focuses on the predictive value of pCASTOR1 in lung adenocarcinoma (LUAD) patients with KRAS mutations.ResultsEmploying a newly developed pCASTOR1 specific antibody, we found that tumor cells exhibited significantly elevated pCASTOR1 scores compared to non-tumor cells (P < 0.05). Higher pCASTOR1 scores predicted poorer overall survival (OS) (HR = 3.3, P = 0.0008) and relapse-free survival (RFS) (HR = 3.0, P = 0.0035) in male patients with KRAS mutations. pCASTOR1 remained an independent predictor for OS (HR = 4.1, P = 0.0047) and RFS (HR = 3.5, P = 0.0342) after controlling for other factors. Notably, in early-stage LUAD, elevated pCASTOR1 scores were associated with significantly worse OS (HR = 3.3, P = 0.0176) and RFS (HR = 3.1, P = 0.0277) in male patients with KRAS mutations, akin to late-stage patients.ConclusionElevated pCASTOR1 scores serve as biomarkers predicting poorer OS and RFS in male LUAD patients with KRAS mutations, offering potential clinical utility in optimizing treatment strategies for this subgroup.

Exploring the effects of pemetrexed on drug resistance mechanisms in human lung adenocarcinoma and its association with PGRMC1

According to the 2022 cancer statistics of the World Health Organization, lung cancer ranks among the top ten causes of death, with lung adenocarcinoma being the most prevalent type. Despite significant advancements in lung cancer therapeutics, many clinical limitations remain, primarily due to the development of drug resistance. The present study investigated the effects of pemetrexed on the drug resistance mechanisms in human lung adenocarcinoma and its association with progesterone receptor membrane component 1 (PGRMC1) expression. Given that KRAS-mutant lung adenocarcinoma cell lines (e.g., A549) exhibit a high folate synthesis activity, pemetrexed, which is structurally similar to folate, was selected as the therapeutic drug. The present study used a lung adenocarcinoma cell line (A549) and established a drug-resistant lung adenocarcinoma cell line (A549/PEM). The findings demonstrated that PGRMC1 expression was elevated in the A549/PEM cells. It has been hypothesized that PGRMC1 regulates iron absorption through heme binding, resulting in a preference for iron-related cell death pathways (ferroptosis). Our findings indicate that drug-resistant lung adenocarcinoma cells with high PGRMC1 levels exhibit elevated antioxidant activity on the cell membrane and increased reliance on iron-dependent cell death pathways. This suggests a correlation between PGRMC1 and pemetrexed-induced iron-dependent cell death. Our study contributes to the development of more effective therapeutic strategies to improve the prognosis of patients with lung adenocarcinoma, particularly those facing drug resistance challenges.

Abstract C171: Identifying cancer driver mutation signatures in lung adenocarcinoma from Black/African American individuals: Addressing cancer health disparities

Abstract Lung cancer remains the leading cause of cancer-related mortality globally and continues to contribute significantly to cancer health disparities. Black/African American (B/AA) men exhibit a higher risk of lung cancer compared to White men; B/AA individuals also exhibit lower 5-year survival and stage-specific survival rates than White subjects. Biological factors, including genetic differences in cancer driver mutation, may influence this disparity. Lung adenocarcinoma (LUAD) is the most common histological subtype of lung cancer across all ethnic groups, including B/AA individuals. Our study aims to characterize the main driver and co-mutation signatures of LUAD in B/AA individuals, who are significantly underrepresented in current mutational studies. We hypothesize that B/AA individuals will have a unique repertoire of cancer driver genes, which could contribute to genetic differences and response to targeted therapies compared to LUAD of White subjects. We are obtaining 100 de-identified archival formalin-fixed, paraffin-embedded (FFPE) LUAD samples from B/AA subjects from cancer registries in Florida and California. Extraction of DNA from the FFPE samples is in progress, and samples have been sent for whole exome sequencing (WES) and ancestry determination. This data will then be analyzed and compared with known LUAD mutation profiles from other ethnicities. Identification of cancer driver mutations will then inform the development of in vitro models for therapeutic testing. To date, the limited information on cancer driver genes in LUAD from B/AA subjects suggests that KRAS is the most common cancer driver gene, just like in LUAD from White subjects, and is found in ∼34% of cases. Therefore, we anticipate identifying KRAS mutations similar to this proportion based on the collection of previously published literature analyses. One key goal of this study is to obtain more information about the exact spectrum of KRAS mutations in LUAD from B/AA subjects. In addition, our work may unveil new variants of known cancer driver mutations such as KRAS, EGFR, BRAF, etc., and finally, “new” driver genes. Our efforts will address critical gaps in the understanding of lung cancer driver genes, a key step to developing personalized treatment of lung cancer and mitigating cancer health disparities between B/AA and White individuals. Supported by grants U54CA233396, U54CA233444, and U54CA233465 from the National Institutes of Health (NIH)/National Cancer Institute (NCI), the Norris Comprehensive Cancer Center core grant, award number P30CA014089 from the NIH/NCI, and by the Undergraduate Research Associates Program supported by the USC Provost. Citation Format: Davin Pan, Daniel J. Mullen, Colton Stensrud, Jose Aparicio, Christina M. Gobin, Sofia Lugo, Kweku Ofosu-Asante, Justin K. Mensah-Mamfo, Kyle R Phillips, Yong Huang, Nazarius S. Lamango, William D Wallace, Suhn K Rhie, Kristinanna M. Fredenburg, Ite A. Offringa. Identifying cancer driver mutation signatures in lung adenocarcinoma from Black/African American individuals: Addressing cancer health disparities [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C171.

Abstract PR-15: Identifying synergistic combinations with KRAS inhibition in PDAC

Abstract Background: To date, targeted therapies have largely failed in eliciting sustained responses in advanced pancreatic ductal adenocarcinoma (PDAC). KRAS-targeted therapies have the potential to drastically transform clinical management. Emerging data indicate that only a subset of patients respond, and that acquired resistance is common. Here, we used a combination of in vivo CRISPR activation (CRISPRa) screening and in vitro/preclinical models to identify synergistic combinations with KRAS inhibition (KRASi) in pancreatic and lung cancer. Material and Methods: We used our CRISPRa-competent PPKS (P53(F/F), Kras(LSL-G12D/+), R26(LSL-SAM)) model of adenocarcinoma to conduct targeted screening for drivers of resistance to MRTX133. Our library consisted of ∼450 frequently mutated and amplified putative oncogenes across human adenocarcinoma based on TCGA data. Mice were treated with 1e8 TU of lentiviral library through nasal instillation. After 6 weeks, treatment was initiated with 3 mg/kg MRTX1133 daily, 5 days/week (i.p.). Mice were sacrificed after 4 weeks of treatment and the guide distribution determined through bioinformatic analysis. Genes statistically significantly enriched (FDR&amp;lt;0.05) in the treatment group were determined. Cell lines with acquired resistance to MRTX1133 derived from the KPC PDAC model and the KPP lung adenocarcinoma (LUAD) model were generated using long-term, low-dose treatment. Resistance was confirmed with MTT drug response assays. In vitro combinatorial drug response was determined using a 72h, 96-well MTT assay, and clonogenic response using a 2 week, 384-well FACS assay. Mice were treated with 20 mg/kg MRTX1133 and/or 20mg/kg Temuterkib once daily, 7 days/week (i.p.) for 2 weeks. Results: We performed targeted in vivo CRISPRa screening for drivers of resistance to KRASi in an autochthons model of lung adenocarcinoma (LUAD) using a library targeting 452 genes commonly amplified or mutated in cancer. We identified a set of genes that were specifically enriched in at least 50% of tumors treated with MRTX1133, including MAPK1/ERK2. We also generated 16 cell lines with acquired resistance to MRTX1133, derived from KPC PDAC or KPP LUAD tumors. Transcriptomic analysis identified a large set of genes differentially expressed in cell with acquired resistance relative to controls. GSEA indicated that reactivation of the MAPK cascade as a common trait in resistant cells. To test if ERK inhibition synergizes with KRASi, we quantified the combinatorial response to MRTX1133 and Temuterkib, a specific inhibitor of ERK1 and ERK2, in a set of KPC and KPP derived cell lines, revealing significant synergy and reduced clonogenic potential in a majority of cell lines. A majority of cell lines with acquired resistance to MRTX1133 remained sensitive to Temuterkib. Finally, we tested combinatorial treatment of MRTX1133 and Temuterkib in a syngenetic subcutaneous model of PDAC, which showed at minimum additive effect. In conclusion we have identified ERK inhibition as a promising combination with KRAS inhibition in KRAS-mutant PDAC and LUAD. Citation Format: Fredrik I Thege, Amber Hoskins, Sonja M Woermann, Anirban Maitra. Identifying synergistic combinations with KRAS inhibition in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-15.

Trametinib sensitizes KRAS-mutant lung adenocarcinoma tumors to PD-1/PD-L1 axis blockade via Id1 downregulation

BackgroundThe identification of novel therapeutic strategies to overcome resistance to the MEK inhibitor trametinib in mutant KRAS lung adenocarcinoma (LUAD) is a challenge. This study analyzes the effects of trametinib on Id1 protein, a key factor involved in the KRAS oncogenic pathway, and investigates the role of Id1 in the acquired resistance to trametinib as well as the synergistic anticancer effect of trametinib combined with immunotherapy in KRAS-mutant LUAD.MethodsWe evaluated the effects of trametinib on KRAS-mutant LUAD by Western blot, RNA-seq and different syngeneic mouse models. Genetic modulation of Id1 expression was performed in KRAS-mutant LUAD cells by lentiviral or retroviral transductions of specific vectors. Cell viability was assessed by cell proliferation and colony formation assays. PD-L1 expression and apoptosis were measured by flow cytometry. The anti-tumor efficacy of the combined treatment with trametinib and PD-1 blockade was investigated in KRAS-mutant LUAD mouse models, and the effects on the tumor immune infiltrate were analyzed by flow cytometry and immunohistochemistry.ResultsWe found that trametinib activates the proteasome-ubiquitin system to downregulate Id1 in KRAS-mutant LUAD tumors. Moreover, we found that Id1 plays a major role in the acquired resistance to trametinib treatment in KRAS-mutant LUAD cells. Using two preclinical syngeneic KRAS-mutant LUAD mouse models, we found that trametinib synergizes with PD-1/PD-L1 blockade to hamper lung cancer progression and increase survival. This anti-tumor activity depended on trametinib-mediated Id1 reduction and was associated with a less immunosuppressive tumor microenvironment and increased PD-L1 expression on tumor cells.ConclusionsOur data demonstrate that Id1 expression is involved in the resistance to trametinib and in the synergistic effect of trametinib with anti-PD-1 therapy in KRAS-mutant LUAD tumors. These findings suggest a potential therapeutic approach for immunotherapy-refractory KRAS-mutant lung cancers.Graphical

Genomic ancestry and cancer among Latin Americans

Latin American populations, characterized by intricate admixture patterns resulting from the intermingling of ancestries from European, Native American (NA) Asian, and African ancestries which result in a vast and complex genetic landscape, harboring unique combinations of novel variants. This genetic diversity not only poses challenges in traditional population genetics methods but also opens avenues for a deeper understanding of its implications in health. In cancer, the interplay between genetic ancestry, lifestyle factors, and healthcare disparities adds a layer of complexity to the varying incidence and mortality rates observed across different Latin American subpopulations. This complex interdependence has been unveiled through numerous studies, whether conducted on Latin American patients residing on the continent or abroad, revealing discernible differences in germline composition that influence divergent disease phenotypes such as higher incidence of Luminal B and Her2 breast tumors, EGFR and KRAS mutated lung adenocarcinomas in addition to an enrichment in BRCA1/2 pathogenic variants and a higher than expected prevalence of variants in colorectal cancer associated genes such as APC and MLH1. In prostate cancer novel risk variants have also been solely identified in Latin American populations. Due to the complexity of genetic divergence, inputs from each individual ancestry seem to carry independent contributions that interplay in the development of these complex disease phenotypes. By understanding these unique population characteristics, genomic ancestries hold a promising avenue for tailoring prognostic assessments and optimizing responses to oncological interventions.

Abstract 1746: KRAS mutant lung adenocarcinoma is associated with distinct mutational signature profiles

Abstract Background: Lung cancer is the 3rd most common cancer in the UK and leading cause of cancer mortality worldwide. Somatic RAS mutations are the most common oncogenes in human cancer, and KRAS mutations are the largest molecular subset of lung adenocarcinoma (LUAD). Each cancer genome accumulates a unique combination of somatic mutations, and these mutational signatures provide a genetic imprint of mutational processes that have occurred during tumorigenesis. Mutational signatures provide information on tumor aetiology/maintenance and could highlight potential therapeutic targets. This is the largest study to date to profile KRAS mutant LUAD mutational signatures using whole genome sequencing (WGS) data. Methods: Paired tumor and germline WGS data were obtained through the 100 000 Genomes Project (Genomics England). KRAS mutations were profiled using Ensembl Variant Effect Predictor (VEP) v109.0. Single base substitution (SBS), doublet base substitution (DBS), small insertion and deletions (ID) and copy number (CN) mutational signature extraction was performed using SigProfiler (COSMIC version 3.3). Statistical analysis and plotting were performed in R; a two-tailed p-value &amp;lt;0.05 was considered statistically significant. Results: 680 LUAD patients were included; 301 KRAS mutant (44%), 379 KRAS wild type (63%). Common KRAS variants were present, including G12C (38.9%), G12V (15.9%) and G12D (14.0%). 17 SBS, 9 DBS, 10 ID and 9 CN signatures were extracted and present in both KRAS mutant and wild type cohorts. KRAS mutant samples were enriched in smoking related signatures (SBS4, DBS2, ID3; odds (OR) ratio 5.54, 3.80, 4.97 respectively, p &amp;lt;0.001) and diploid CN signature (CN1, OR 1.41, p = 0.03). Conversely, KRAS mutant samples were less likely to have clocklike (ID5, OR 0.61, p = 0.012; ID8, OR 0.44, p &amp;lt;0.001), APOBEC (SBS2, OR 0.49, p &amp;lt;0.001; SBS13; 0.50, p &amp;lt;0.001), chromosomal instability (CN9, OR 0.63, p = 0.019), loss of heterozygosity (CN14, OR 0.47, p = 0.015), or homologous recombination deficiency (CN17, OR 0.58, p &amp;lt;0.001) signatures. Discussion: These results show distinct mutational signature profiles between KRAS mutant and wild type LUAD. As previously described, KRAS mutant LUAD is dominated by smoking related signatures; however, the majority of remaining signatures were more common in the KRAS wild type cohort where there was a statistically significant difference. This data suggests different mutational processes are observed in KRAS mutant compared to wild type LUAD. KRAS mutations are commonly clonal and thought to initiate tumorigenesis: timing signatures relative to KRAS mutations is necessary to further our understanding of KRAS cancer evolution and the subject of future work. Combined with clinical data integration, mutational signatures could provide prognostic information and inform predictive models. Citation Format: Laura Woodhouse, Aliah Hawari, Andreas Gruber, Colin Lindsay, David Wedge. KRAS mutant lung adenocarcinoma is associated with distinct mutational signature profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1746.

Abstract 2667: Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer

Abstract Lung cancer, including lung adenocarcinoma (LUAD), is the leading cause of cancer-related deaths in the United States. KRAS mutations are the most prevalent oncogenic driver alteration in human LUAD. We have previously shown that high expression of Mucin 5 AC (MUC5AC), a main airway secretory mucin, is significantly associated with poor prognosis. Mucins are essential for mucociliary clearance and the maintenance of airway homeostasis, however, their overproduction could negatively affect the lung immune microenvironment and has been shown to promote airway diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer. Previously, we have shown that genetic deletion of MUC5AC in a murine model of K-ras mutant lung adenocarcinoma (KM-LUAD) impedes tumor development and reduces pro-tumor inflammatory and immune phenotypes. These findings indicate that MUC5AC is a potential druggable target against KM-LUAD. In this study, we sought a translational approach and inhibited MUC5AC in our K-rasG12D mutant mouse model of LUAD via RNA interference (RNAi). Six-week-old mice were given RNAi (5mg/kg) or vehicle (saline) via biweekly intratracheal administration for 8 weeks. This approach led to a significant decrease in tumor burden (40%) and a trending increase in total white blood cell counts, predominantly macrophages in the lung tumor microenvironment which was associated with phenotypic changes in macrophages characterized by reduced expression of Fizz1, and M2 pro-tumor macrophage marker. Additionally, RNAi-treated mice had reduced expression of Siglec-E, a mucin receptor and known inhibitor of the TLR4 endocytosis and promoter of NF-ĸB signaling, as well as a significant decrease in IL-6, a product of the NF-ĸB pathway and promoter of STAT3 signaling which we have previously shown to have an essential role in promotion of KM-LUAD. In summary, these findings elucidate a potential mechanism by which MUC5AC protects tumor cells from apoptosis and immune-mediated cytotoxic activity but also highlights how MUC5AC RNAi is an effective indirect preventive and therapeutic strategy in high-risk individuals (smokers with COPD) and patients with early stage KM-LUAD, respectively. Citation Format: Melvin Zarghooni, Maria T. Grimaldo, Avantika Krishna, Michael J. Clowers, Linda Phan, Yasmina H. Rezai, Arnav Gaitonde, Carlos Rodriguez Reyna, Walter Velasco Torrez, Erik Bush, Seyed Javad Moghaddam. Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2667.

Abstract 2772: Mechanisms linking STK11 loss with metastatic potential in KRAS-mutated lung adenocarcinoma

Abstract Metastasis is one of the major determinants of worsened patient prognosis and occurs in roughly 50% of lung adenocarcinoma (LUAD) patients at the time of diagnosis. Late-stage diagnosis of LUAD often results in the accumulation of a wide range of mutations in tumor cells, with altered KRAS being the most frequently encountered oncogenic driver. Co-mutation of the tumor suppressor STK11 is also common and occurs in 10-15% of KRAS-driven LUAD. Retrospective patient studies have demonstrated that concurrent oncogenic KRAS and STK11 loss-of-function mutations are associated with metastatic disease, poorer patient survival, and inferior therapy response. The purpose of this study was to identify molecular mechanisms by which STK11 loss results in a greater metastatic potential in KRAS-driven LUAD in hopes of exploiting that knowledge to improve patient outcomes. For our studies, we utilized the KRAS-mutated, STK11-intact human LUAD cell line NCI-H2009. Both parental and ∆STK11 (generated by CRISPR/Cas9) H2009 cells were seeded in Transwell® inserts coated with fibronectin. As expected, H2009 ∆STK11 cells displayed a 1.6-fold increase in trans-well migration compared to the parental line (SEM 14393 ± 877.3 cells, n=6; p&amp;lt;0.0001). To assess the invasive properties of parental and ∆STK11 cells in a three-dimensional environment, spheroids generated by hanging drop culture were embedded in Matrigel and imaged by light microscopy at zero and 24-hours post-embedding. Analysis demonstrates a 2.4-fold increase in invasion in ∆STK11 cells compared to parental cells (SEM 0.03 ± 0.01 mm2, n=19-21; p&amp;lt;0.0001). Three-dimensional LUAD spheroids have been reported to more closely recapitulate in-vivo tumor transcriptomes compared to two-dimensional monolayer cultures. qRT-PCR analyses of our spheroids demonstrate that loss of STK11 promotes a significant increase in the expression of lung cancer stem cell markers CXCR4, NANOG, CD44, and OCT4 (n=3-6; p&amp;lt;0.002). These markers are known to support stem cell maintenance, chemoresistance, as well as metastatic potential, suggesting a mechanistic link between STK11 and their transcriptional regulation. To assess the impact of STK11 loss on early and late-stage metastasis in-vivo, we will use an embryonic zebrafish xenograft model. Zebrafish represent a tractable metastasis model due to the number of conserved human homologs - most notable being the CXCR4/CXCL12 axis which models cross-communication between zebrafish endothelium and cognate human tumor ligands. Current and future studies focus on investigating the role of STK11 in regulating stem cell markers; thereby identifying potentially novel therapeutic targets for STK11 and KRAS co-mutated LUAD patients. Citation Format: Cole M. Royer, Lauren K. Bialek, Hailey M. Sarausky, Shannon M. Prior, Gopika Nandagopal, Paula B. Deming, David J. Seward, Melissa N. Scheiber. Mechanisms linking STK11 loss with metastatic potential in KRAS-mutated lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2772.

Abstract 4048: Comparative effects of SOS1 inhibitor with IL-6 blockade and their combination on Kras mutant lung cancer

Abstract Despite recent advances in treatment, lung adenocarcinoma (LUAD), particularly harboring Kras mutations, is still the leading cause of cancer mortality. We and others have previously shown that Kras mutant LUAD development and progression is associated with intrinsic inflammatory characteristics driven by epithelial NF-κB activation, increased levels of IL-6 (which is transcriptionally regulated by NF-κB), and activation of the IL-6-responsive transcription factor STAT3. We further demonstrated that IL-6 blockade suppresses lung tumorigenesis by reducing tumor-associated immunosuppressive myeloid populations and induction of a T cell-mediated anti-tumor response. The recent discovery of a novel SOS1-targeting compound (BI-3406) that selectively inhibits the KRAS-SOS1 interaction and leads to anti-proliferative activity has opened up the possibility to more efficiently inhibit Kras mutant lung cancer. Accordingly, in this study we used a mouse model of KRAS mutant LUAD (CC-LR) to compare the therapeutic effects of BI-3406, IL-6 blockade, and MEK inhibitor (trametinib). We also tested whether inhibiting IL-6 driven pro-tumor immune response would provide a synergistic/additive response and increase the tumor inhibitory effect of either BI-3406 or Trametinib. Eight cohorts of 14-week-old female mice were treated with either anti-IL-6 antibody, trametinib, or BI-3406, or their combinations for 4 weeks, and their tumor burden was compared at the end of study. We observed a significant reduction in tumor multiplicity and tumor area in response to all monotherapies, with the highest effect in the trametinib treated cohort. Neither combination therapy led to decreased tumor burden over either of the monotherapy regimens in our model. However, we observed an increase in T helper 1 and CD8 T expressing IFNγ cell populations in the cohort treated with Trametinib and anti-IL-6 Ab combination. In addition, using qPCR analysis of RNA extracted from the whole lung we found a higher expression of IFNγ along with a reduced expression of IL-10 in the cohort treated with BI-3406 and anti-IL-6 Ab combination. This suggests that these combinations may have the potential to improve the anti-tumor immune responses. Hence, we conclude that further adjustment of the treatment regimen (doses, interval, duration) may allow to obtain improved anti-tumor efficacy with these combinations. Overall, this study provides a new research perspective for the improvement of currently available treatment modalities for patients with Kras mutant lung cancer that could be further explored preclinically. Citation Format: Katherine E. Larsen, Maria J. Arredondo Sancristobal, Melody Zarghooni, Avantika Krishna, Maria T. Grimaldo, Nastaran Karimi, Arnav Gaitonde, Michael J. Clowers, Seyed Javad Moghaddam. Comparative effects of SOS1 inhibitor with IL-6 blockade and their combination on Kras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4048.

Abstract 5326: Selective deletion of estrogen receptor β in lung epithelium suppresses K-ras mutant lung tumorigenesis

Abstract Although lung cancer is the most prolific cause of global cancer-related deaths, outcomes by sex have gained traction as a means to stratify patients and tailor therapies accordingly. In recent years, females have begun to shoulder a greater burden of lung cancer diagnoses, specifically within the lung adenocarcinoma (LUAD) subtype; conversely, mortality is worsened for males. However, other sex-dependent effects in lung cancer are harder to predict. For example, hormone replacement therapy for post-menopausal women has been shown to predict better survival in some cohorts while auguring worse outcomes in others. Similar controversy exists regarding menopausal status, gender affirming care, and castration/oophorectomy status. However, there is consensus that these differential effects relate to a patient’s sex hormone composition. Estrogen, a sex hormone abounding in females, boasts well-described pro-tumor signaling in breast cancer. Unfortunately, its role in lung cancer remains elusive. Estrogen is reported to exert inflammatory properties in the lung environment, to drive immune function, and to induce proliferation of lung cancer cells. This functional antagonism has led to questions about how these compartment-specific effects are balanced. In the lung, estrogen signaling occurs mainly via estrogen receptor beta (ERβ), a nuclear hormone receptor highly expressed in lung tissue and lung tumors. ERβ is understudied relative to its sibling ERα, a well-known oncogene in breast cancer. While it is tempting to analogize ERα oncogene function to ERβ, the literature lacks consensus on the role of ERβ in lung cancer, with some authors classifying it as an oncogene and others as a tumor suppressor. In LUAD, Kras mutations encompass a quarter of driver mutations, and interaction of Kras with ERβ is understudied. Here, we tested the effects of genetic ablation of ERβ in a murine model of Kras mutant LUAD (KM-LUAD) termed CCSPCre/LSL-KrasG12D (CC-LR). Esr2 floxed mice were crossed with CC-LR mice to create a lung epithelial-specific conditional knockout of ERβ (LR/Esr2Δ/Δ). LR/Esr2Δ/Δ mice show significant decrease in tumor burden and total lung immune infiltration, indicating reduced tumor development and tumor-promoting inflammation. Of note, this effect manifests regardless of sex. RNA and protein-level analysis further suggest that ERβ deletion alters the balance of inflammatory transcription factors, resulting in skewing from STAT3- to NF-κB-driven inflammation and a net anti-tumor response. These data suggest that ERβ offers a promising early target in nascent KM-LUAD tumors with therapeutic intervention possible regardless of sex-specific effects. Acknowledgement: supported by NIH/NCI grant awarded to Seyed Javad Moghaddam (R01 CA225977); Michael J Clowers partly supported by NIH/NCI (F31 CA261121); Annamarie L Thompson supported by CPRIT Research Training Award (RP210028). Citation Format: Michael Joseph Clowers, Annamarie L. Thompson, Carlos Ignacio Rodriguez Reyna, Shanshan Deng, Seyed Javad Moghaddam. Selective deletion of estrogen receptor β in lung epithelium suppresses K-ras mutant lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5326.

Abstract 6675: Dissecting the relationship between mucin 5AC and the lung/gut microbiome in the pathogenesis of K-ras mutant lung cancer

Abstract Non-small cell lung cancer, including its most common subtype, lung adenocarcinoma (LUAD), is one of the most prevalent and lethal forms of cancer worldwide. This is largely due to patients having progressed to an advanced stage of disease by the time of diagnosis. Thus, better understanding the pathogenesis of early LUAD is paramount for the identification of key targets and development of preventive and therapeutic strategies for clinical management of the disease. KRAS mutations are the most common oncogenic driver alteration in human LUAD. Previously, we have found that KRAS mutant LUAD patients whose tumors harbor high expression of Mucin 5 AC (MUC5AC), a main airway secretory mucin, have significantly lower survival and poor prognosis. Mucins are membrane-bound and secreted hydrophilic proteins essential in clearance of allergens, pathogens, and debris in the lung airway. However, overproduction of mucins, such as MUC5AC, plays roles in the pathogenesis of airway diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer. Given the known effect of mucin polymers on bacterial growth and the immune microenvironment in the context of other airway inflammatory diseases, in this study, our objective is to better understand the MUC5AC-to-microbiome relationship using a lung epithelial specific mouse model of KRAS-mutant LUAD (CC-LR) and test the combinatorial effects of genetic deletion of MUC5AC (CC-LR/Muc5ac−/−) and the pan-microbiome depletion on tumor progression, the lung immune contexture, and microbiome (lung and gut). Thus far, we found that genetic deletion of MUC5AC or pan-microbiome depletion significantly decreases the tumor burden (66% vs 50%, respectively). Additionally, both groups show to have a significantly decreased percentage of tumor-specific Ki67+ cells, a cell proliferation marker, and ERG+ cells, an angiogenesis marker, in comparison to the CC-LR controls. The combination of the lack of MUC5AC with pan-microbiome depletion has also shown to reduce tumor development that needs further investigation. These findings highlight the need to better understand the mechanistic interaction between mucins and the lung/gut microbiome in the context of KRAS-mutant lung cancer which we are currently exploring. It also suggests that their relationship could be a key target for modulation, potentially leading to the development of a minimally invasive alternative preventive and/or treatment modality against KRAS-mutant LUAD. Citation Format: Maria T. Grimaldo, Michael J. Clowers, Avantika Krishna, Katherine E. Larsen, Nastaran Karimi, Yasmina H. Rezai, Arnav Gaitonde, Farbod Khalaj, Haoyue Liu, Florencia McAllister, Seyed Javad Moghaddam. Dissecting the relationship between mucin 5AC and the lung/gut microbiome in the pathogenesis of K-ras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6675.

Abstract 6862: Loss of STAT1 promotes an immunosuppressive tumor microenvironment in KRAS-driven lung adenocarcinoma

Abstract Introduction: Lung cancer is the leading cause of death worldwide. One third of lung adenocarcinomas (LUADs), the most abundant form of lung cancer, harbors activating mutations in KRAS. Since human KRAS-mutant LUAD is correlated to an inflammatory phenotype, we hypothesized that the JAK-STAT pathway plays a crucial role in disease progression. Indeed, preliminary data demonstrated that human KRAS-mutant LUADs are associated with activation of the JAK-STAT signaling pathway, including upregulation of STAT1 expression. Therefore, we aimed to elucidate the tumorigenic functions of STAT1 in KRAS-mutant LUAD. Methods: To analyze the functions of STAT1 in KRAS-driven LUAD, we used C57BL/6 mice, which were genetically engineered in order to develop KRAS-driven autochthonous LUAD deficient for P53. Tumors additionally expressed ovalbumin (OVA) which acts as a neoantigen to induce immune infiltration (KPO mice). In this immunogenic mouse model, STAT1 was deleted in the tumors (KPOS mice) and overall survival was assessed by Kaplan Meier analysis. Tumor burden and infiltration was analyzed in lungs via H&amp;E, IHC, and IF staining 6 weeks after tumor induction. An inflammatory cytokine antibody array was performed with lung fluid and single cell sequencing with cells isolated from lungs at 10 weeks. Results and discussion: Expression of OVA in tumor cells increased infiltration by T cells and prolonged survival suggesting neoantigen presentation in this mouse model. When STAT1 was deleted in OVA expressing tumors the survival was significantly decreased. Further, tumor burden as well as tumor size and grades were increased in KPOS lungs. Interestingly, infiltration of CD4 T cells was diminished upon loss of STAT1 while the percentage of CD8 T cells in tumors did not change suggesting an immunosuppressive tumor microenvironment. Indeed, M2 macrophages were increased in tumors lacking STAT1 and less proliferating immune cells were found indicating immune cell inhibition. This was further supported by the increase of CCL9 in KPOS lungs which might induce macrophage recruitment and further T cell inhibition. Finally, scRNAseq analysis revealed that KPOS lungs were composed of distinct immune cell populations as well as states. Conclusion: This data suggests that loss of STAT1 in tumor cells promotes secretion of CCL9 that drives recruitment of immunosuppressive macrophages to the tumor microenvironment. As a result, T helper cells are excluded from tumors and cytotoxic T cells are inhibited or exhausted. Further experiments will delineate the immune cell states and the mechanisms leading to this interesting phenotype. Citation Format: Christoph Trenk, Maša Bereš, Katalin Deszo, Jaqueline Horvath, Monika Homolya, Andreea Corina Luca, Robert Eferl, Herwig Peter Moll, Emilio Casanova. Loss of STAT1 promotes an immunosuppressive tumor microenvironment in KRAS-driven lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6862.

Abstract 4047: Unraveling synergistic therapeutic strategies involving FAK inhibition and immunotherapy using anti-PD1 antibody in KRAS and LKB1-driven lung cancer

Abstract Lung cancer presents a global health challenge demanding innovative preclinical models forresearch and therapeutic advancement. Genetically engineered mouse models (GEMMs) have seenremarkable growth in complexity and application over the past two decades. Our work leveragesthese models and is specifically designed to address the pressing need for effectiveimmunochemotherapies in the context of KRAS and LKB1 (KL) mutant lung adenocarcinoma,which shows resistance to most immunotherapies. Our study centers around a preclinicalsyngeneic orthotopic mouse model we developed using mouse lung and metastatic tumor cell linesderived from KL-GEMM. This model, optimized to mimic human lung cancer closely, providesan ideal platform for exploring the intricate interplay between the immune system and tumorprogression. Furthermore, we have previously shown the LKB1 mutant cell lines and tumormodels are uniquely sensitive to FAK inhibitors, and therefore we wanted to test whether thecombination of a FAK inhibitor with anti-PD1 therapy can improve sensitivity. The combinatoryadministration of FAK inhibitor (VS-6063) with anti-PD1 mAb synergistically increased anti-tumor response and survival outcome compared with single-agent therapy in the KL-mediatedsyngeneic orthotopic lung cancer model. By using GEMM-derived syngeneic cell lines, we haveevaluated the potential for creating innovative immunochemotherapies, customized for KL co-mutant lung adenocarcinomas with promising clinical potential. Furthermore, using this modelenables a deeper understanding of the KL mutant disease's pathogenesis, the impact of sex-biasedcancer development, and the assessment of novel immunochemotherapy treatment strategies in theKL genetic sub-type of lung adenocarcinoma. Citation Format: Junghui Koo, Carol Tucker-Burden, Melissa Roy, Chunzi Huang, Wei Zhou, Melissa Gilbert-Ross, Haian Fu, Suresh S. Ramalingam, Adam I. Marcus. Unraveling synergistic therapeutic strategies involving FAK inhibition and immunotherapy using anti-PD1 antibody in KRAS and LKB1-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4047.

The ERK inhibitor LY3214996 augments anti-PD-1 immunotherapy in preclinical mouse models of BRAFV600E melanoma brain metastasis.

Immune checkpoint inhibitors (ICI) have revolutionized cancer treatment; however, only a subset of patients with brain metastasis (BM) respond to ICI. Activating mutations in the mitogen-activated protein kinase signaling pathway are frequent in BM. The objective of this study was to evaluate whether therapeutic inhibition of extracellular signal-regulated kinase (ERK) can improve the efficacy of ICI for BM. We used immunotypical mouse models of BM bearing dual extracranial/intracranial tumors to evaluate the efficacy of single-agent and dual-agent treatment with selective ERK inhibitor LY3214996 (LY321) and anti-programmed death receptor 1 (PD-1) antibody. We verified target inhibition and drug delivery, then investigated treatment effects on T-cell response and tumor-immune microenvironment using high-parameter flow cytometry, multiplex immunoassays, and T-cell receptor profiling. We found that dual treatment with LY321 and anti-PD-1 significantly improved overall survival in 2 BRAFV600E-mutant murine melanoma models but not in KRAS-mutant murine lung adenocarcinoma. We demonstrate that although LY321 has limited blood-brain barrier (BBB) permeability, combined LY321 and anti-PD-1 therapy increases tumor-infiltrating CD8+ effector T cells, broadens the T-cell receptor repertoire in the extracranial tumor, enriches T-cell clones shared by the periphery and brain, and reduces immunosuppressive cytokines and cell populations in tumors. Despite the limited BBB permeability of LY321, combined LY321 and anti-PD-1 treatment can improve intracranial disease control by amplifying extracranial immune responses, highlighting the role of extracranial tumors in driving intracranial response to treatment. Combined ERK and PD-1 inhibition is a promising therapeutic approach, worthy of further investigation for patients with melanoma BM.

Abstract C063: Frequency and spectrum of KRAS mutations in lung adenocarcinoma from Black/African American and White/European patients

Abstract Lung cancer is the most common cause of cancer death in the United States and a major source of cancer health disparity. Lung cancer incidence and mortality rates are higher in Black/African American (B/AA) men than in men from other racial/ethnic groups. Lung adenocarcinoma, arising in the alveoli or air sacs of the lung, is the most common subtype of lung cancer in all ethnic/racial groups. Genetic driver mutations can differ among racial/ethnic groups, in the identity of the mutated genes, their mutation frequencies, and the spectrum of mutations for each given gene. In addition, the same mutation could have different effects depending on an individual patient’s genetic background, resulting in differential responses to therapies. Therefore, it is important to identify the frequency and spectrum of driver mutations in different population groups. Protooncogenes from the RAS family, KRAS, NRAS, and HRAS, play a crucial role in cell growth regulation, cell proliferation, and survival. KRAS is frequently mutated in numerous cancer types, including lung adenocarcinoma, and has been reported to be the driver gene in ~30% of lung adenocarcinomas from White/European (W/E) patients. Due to the much lower number of lung cancer samples analyzed from B/AA patients, it has been unclear if the KRAS mutational frequency and spectrum in B/AA lung adenocarcinoma patients differs from that in W/E patients. Here we collected available existing data sets with mutational information on KRAS muations in lung adenocarcinoma from B/AA and W/E patients. We obtained data for 1555 B/AA and 13840 White lung adenocarcinoma patients. While B/AA lung adenocarcinoma patients had a lower prevalence of KRAS mutations (27% vs. 35%) than W/E patients, (χ2 p-value= 5.877e-10), KRAS was still the most frequently mutated cancer driver gene in both groups. The most common KRAS mutations in both B/AA and W/E patients were alterations of glycine 12 to cysteine (G12C), valine (G12V), aspartic acid (G12D), and alanine (G12A), as well as glutamine 61 to histidine (Q61H) and glycine 13 to cysteine (G13C). Given that a substantial proportion of lung adenocarcinoma patients carry a mutated KRAS gene, investigating new potential KRAS-targeting therapies remains especially urgent. To this end, we are investigating polyisoprenylated cysteinyl amide inhibitors (PCAIs), a group of potential cancer therapy agents designed to specifically disrupt and suppress hyperactive G-protein signaling, as caused by mutated RAS proteins. We are testing PCAI efficiacy on B/AA and W/E lung adenocarcinoma cell lines carrying various KRAS mutations. Citation Format: Daniel J. Mullen, Pablo E. Puente, Christopher Leon, Diana I. Romero, Kweku Ofosu-Asante, Yong Huang, Nazarius S. Lamango, Ite A. Offringa. Frequency and spectrum of KRAS mutations in lung adenocarcinoma from Black/African American and White/European patients [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C063.

Abstract A043: Identifying critical vulnerabilities that sensitise Lkb1-mutant lung adenocarcinoma to T cell mediated killing

Abstract KRAS is the most frequently altered oncogene in lung adenocarcinoma (LUAD). While the clinical development of targeted therapies has improved the treatment of a subset of patients, the heterogeneity of KRAS mutant LUAD remains a clinical challenge. Identifying tumor intrinsic dependencies through common co-occurring mutations are one approach to more effectively target KRAS-mutant LUAD. Loss-of-function mutations in STK11/Lkb1, are frequently found co-mutated with KRAS, and comprise an aggressive form of the disease. Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis, which aims to unleash CD8 effector T cells against tumors, are currently used as a first-line treatment strategy for KRAS mutant patients. However, KRAS mutant tumors that harbour co-mutations in STK11 fail to respond to ICIs. A greater understanding of the tumor-intrinsic processes that allow STK11/Lkb1 mutant tumors to evade immune detection is required to enhance the response of ICIs and improve patient outcomes. Here, we have used an unbiased genome-wide CRISPR knockout screening approach in lung cancer cell lines generated from the Kras G12D/+/Lkb1 fl/fl (KL) mouse model to unveil tumor-intrinsic mechanisms involved in anti-tumor immunity. To identify gene targets that sensitise tumor cells to T cell mediated killing, a co-culture method using the OVA/OT-I system was employed. Importantly, sgRNAs targeting genes encoding components of interferon signalling (Jak1, Stat1, Ifngr1) and MHC-I presentation (B2m, H2-k1) were enriched following serial application of OT-I T cells, consistent with the role these pathways play in resistance to ICIs. Conversely, sgRNAs targeting several genes were found to sensitise KL tumor cells to T cell mediated killing. This included genes (Serpinb9, Ptpn2) previously implicated in increasing T cell-mediated tumor cell killing, supporting the robustness of our screening platform. Excitingly, sgRNAs targeting novel genes, not previously implicated in mediating anti-tumor immunity were also identified. Current investigations validating the ability of candidate genes to sensitise KL tumor cells to T cell killing will be presented. Together, these results demonstrate the power of whole genome CRISPR screens in identifying candidate genes that may serve as therapeutic targets to improve treatment responses in KRAS/STK11/Lkb1 mutant LUAD patients. Citation Format: Jackson A McDonald, Danielle Boyd, Sarah Diepstraten, Leanne Scott, Lin Tai, Andrew Kueh, Marian Burr, Sarah A Best, Marco J Herold, Kate D Sutherland. Identifying critical vulnerabilities that sensitise Lkb1-mutant lung adenocarcinoma to T cell mediated killing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A043.

Activation of KrasG12D in Subset of Alveolar Type II Cells Enhances Cellular Plasticity in Lung Adenocarcinoma.

Identification of progenitor like tumor-initiating cells in KRAS-mutant lung adenocarcinoma may allow development of novel targeted therapeutics.

Identification of LGR4 as a prognostic biomarker in KRAS-mutant lung adenocarcinoma: Evidence from integrated bioinformatics analysis.

Globally, lung cancer is the leading cause of cancer-related deaths, primarily non-small cell lung cancer. Kirsten Rat Sarcoma Oncogene Homolog (KRAS) mutations are common in non-small cell lung cancer and linked to a poor prognosis. Covalent inhibitors targeting KRAS-G12C mutation have improved treatment for some patients, but most KRAS-mutant lung adenocarcinoma (KRAS-MT LUAD) cases lack targeted therapies. This gap in treatment options underscores a significant challenge in the field. Our study aimed to identify hub/key genes specifically associated with KRAS-MT LUAD. These hub genes hold the potential to serve as therapeutic targets or biomarkers, providing insights into the pathogenesis and prognosis of lung cancer. We performed a comprehensive analysis on KRAS-MT LUAD samples using diverse data sources. This included TCGA project data for RNA-seq, clinical information, and somatic mutations, along with RNA-seq data for adjacent normal tissues. DESeq2 identified differentially expressed genes (DEGs), while weighted gene co-expression network analysis revealed co-expression modules. Overlapping genes between DEGs and co-expression module with the highest significance were analyzed using gene set enrichment analysis and protein-protein interaction network analysis. Hub genes were identified with the Maximal Clique Centrality algorithm in Cytoscape. Prognostic significance was assessed through survival analysis and validated using the GSE72094 dataset from Gene Expression Omnibus (GEO) database. In KRAS-MT LUAD, 3122 DEGs were found (2131 up-regulated, 985 down-regulated). The blue module, among 25 co-expression modules from weighted gene co-expression network analysis, had the strongest correlation. 804 genes overlapped between DEGs and the blue module. Among 20 hub genes in the blue module, leucine-rich repeats containing G protein-coupled receptor 4 (LGR4) overexpression correlated with worse overall survival. The prognostic significance of LGR4 was confirmed using GSE72094, but surprisingly, the direction of the association was opposite to what was expected. LGR4 stands as a promising biomarker in KRAS-MT LUAD prognosis. Contrasting associations in TCGA and GSE72094 datasets reveal the intricate nature of KRAS-MT LUAD. Additional explorations are imperative to grasp the precise involvement of LGR4 in lung adenocarcinoma prognosis, particularly concerning KRAS mutations. These insights could potentially pave the way for targeted therapeutic interventions, addressing the existing unmet demands in this specific subgroup.