KRAS-mutant Adenocarcinoma Research Articles

Unlocking therapeutic potential: IL-1β as a target in non-small cell lung cancer with oncogenic mutations—Prognostic and predictive insights.

8030 Background: Preclinical studies have demonstrated heterogeneity in interleukin-1β (IL-1β) expression and its potential implications in non-small cell lung cancer(NSCLC). The phase II CANTOS trial incidentally showed reduced lung cancer incidence and mortality with the IL-1β inhibitor canakinumab. However, the phase III CANOPY-1/2 trials failed to demonstrate survival benefit when combining canakinumab with chemo/immunotherapy. The impact of IL-1β inhibition in NSCLC with actionable mutations remains unknown. This study aims to evaluate the prognostic and predictive role of IL-1β in NSCLC and its potential as a target for combination therapy. Methods: 34,960 NSCLC tumors underwent next-gen sequencing of DNA (592-gene or whole exome) and RNA (whole transcriptome) at Caris Life Sciences (Phoenix, AZ). Tumors were stratified by IL-1β transcriptional expression quartiles (Q1: low expression and Q4: high expression). Quartiles were established within each subpopulation. Significance was calculated using chi-square, Fisher’s exact, or Mann-Whitney U test, with p-values adjusted for multiple comparisons (p < 0.05). Overall survival (OS) from either time of tissue collection to last contact or time on treatment (TOT) was estimated from insurance claims data using the Cox proportional hazards model to calculate hazard ratio (HR) and log-rank tests to calculate p-values. Results: In the entire cohort, low IL-1β expressors showed improved OS compared to high expressors (20.0 months (m) vs 17.3 m, HR 0.88, 95%CI 0.88-0.95, p < 0.0001). For NSCLC without driver mutations, IL-1β expression did not correlate with OS difference. IL-1β expression was positively associated with TP-53 mutations (Q4 76% vs Q1 57%), high TMB (Q4 44% vs Q1 37%) and PD-L1+ expression by IHC (Q4 67% vs Q1 44%), while negatively associated with mutations in KRAS(Q4 24% vs Q1 31%) , EGFR(Q4 8% vs Q1 14%) , ERBB2 (Q4 1% vs Q1 2%) , BRAF (Q4 4% vs Q1 5%) ,and STK11 (Q4 10% vs Q1 18%) (p < 0.05). In EGFR-mutant NSCLC, Q1 showed superior OS than Q4 (34.6 m vs 25.1 m, HR 0.79, 95% CI 0.68-0.92, p = 0.002). This survival benefit was amplified in adenocarcinoma (Q1 41.2 m vs Q4 27.1 m, HR 0.74, 95% CI 0.63-0.87, p < 0.001). For ALK fusion-positive NSCLC, low expressors demonstrated improved OS (Q1 63.4 m vs Q4 27.0 m, HR 0.53, 95% CI 0.37-0.76, p < 0.001). In KRAS-mutant adenocarcinoma, high expressors showed improved immune-checkpoint inhibitor (ICI) TOT (Q4 6.4 m vs Q1 5.4 m, HR 0.85, 95% CI 0.74-0.98, p = 0.026); however, this did not correlate with improved OS. Conclusions: Our study shows promising prognostic value of IL-1β expression in NSCLC, associating low expression with improved OS across NSCLC subtypes. IL-1β expression is closely linked to key driver mutations in NSCLC and may have predictive value for ICIs. The findings suggest the potential benefit of targeting IL-1β in high IL-1β expressing NSCLC with driver mutations.

Abstract LB231: The mutagenic forces shaping the genomic landscape of lung cancer in never smokers

Abstract While most lung cancers are associated with tobacco smoking, prior studies have indicated that 15-20% of cases are found in never smokers. Lung cancer in never smokers (LCINS) shows demographic variations, being more common in females and Asian populations. Epidemiological studies have also identified environmental risk factors for LCINS, including exposure to secondhand tobacco smoke and air pollution. As part of the Sherlock-Lung project (current data freeze of 1217 samples), we generated deep WGS data from a large international cohort of 871 treatment-naïve LCINS recruited from 28 locations across four continents. Our cohort allowed mapping the genomic landscape operative in lung cancer histologies commonly found in never smokers, including adenocarcinomas (n=737) and carcinoids (n=61), as well as other histologies rarely attributed to never smokers (n=73). Information on passive smoking was collected for 458 patients, with 250 being exposed to secondhand smoke. We identified driver alterations and performed mutational signature analyses for single base substitutions (SBS), indels, doublet base substitutions (DBS), copy number alterations, and structural variants. We detected eighteen SBS signatures, with eight not previously linked to LCINS, including SBS4, associated with smoking but also observed in lifelong non-smokers exposed to different environmental mutagens, such as indoor pollution. Several additional signatures were detected in other variant types, including a novel indel signature. Notably, secondhand smoke exposure corresponded to increased SBS burden, although it was not associated with specific signatures. In contrast, we observed a signature-specific association of air pollution (measured as PM2.5 exposure) with SBS, indel, and DBS burden. SBS4 and correlated signatures ID3 and DBS2 were positively associated with PM2.5 exposure. Similarly, clock-like signature SBS5 was highly correlated with PM2.5, potentially acting as a readout of a promotion mechanism where lung cells undergo more cell divisions in individuals living in highly polluted areas. We also observed variability across histologies and ancestries for signatures and driver mutations, including enrichment of signatures SBS17b and SBS40a as well as KRAS mutations in adenocarcinomas from Europeans; aristolochic-acid associated signature SBS22a, and EGFR and TP53 mutations in adenocarcinomas from East Asians; and signature SBS8 and ARID1A mutations in carcinoids from Europeans. The unprecedented size of our cohort allowed us to refine the prevalence and intensity of the driver mutations and mutational signatures involved in LCINS, as well as their variability across ancestries, geographical locations, and histologies. Our results indicate that air pollution may act as both initiator and promoter of neoplastic expansion of LCINS. Citation Format: Marcos Diaz-Gay, Tongwu Zhang, Azhar Khandekar, Burçak Otlu, Shuvro Nandi, Christopher D. Steele, Nathaniel Rothman, Stephen J. Chanock, Qing Lan, Ludmil B. Alexandrov, Maria Teresa Landi. The mutagenic forces shaping the genomic landscape of lung cancer in never smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB231.

Abstract C116: Oncogenic Kras signaling shapes the tumor microenvironment in lung adenocarcinoma

Abstract Despite advancements in targeted therapy and immunotherapy, lung cancer remains the leading cause of cancer-related deaths in both men and women worldwide, with an estimated 2.2 million deaths each year. At 85%, non-small cell lung cancer (NSCLC) is by far the most common subtype, comprising adenocarcinomas and lung squamous cell carcinoma. Mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR) and anaplastic lymphoma receptor tyrosine kinase (ALK) genes are common, with the worst overall survival in KRAS mutant adenocarcinoma patients. About 40% of lung tumors with KRAS mutations also harbor mutations in tumor protein p53 gene (TP53), a tumor suppressor gene. KrasG12C and KrasG12D are both prevalent mutations in lung cancer. Sotorasib, a KrasG12C inhibitor, was recently approved for treatment of NSCLC. However, only 37% of patients have an objective response to Sotorasib, with a median duration of just 11 months. The KrasG12D inhibitor MRTX1133 is entering clinical trials; elucidating the mechanism of action and potential resistance is imperative to design combination treatment approaches. While cell-intrinsic mechanisms of resistance in epithelial oncogenic KRASG12D (Kras*) are well studied, our group and others have shown that KRASG12D also extrinsically regulates the tumor microenvironment (TME) in pancreatic cancer. Whether and how this oncogene regulates the TME of lung cancer is less understood. To study the crosstalk between epithelial lung cells expressing oncogenic KRAS and the surrounding TME, we have established a triple transgenic murine model of Kras*-driven NSCLC, wherein both lung epithelial-specific expression of Kras* and expression of mutant TP53R172H (Tp53*) can be controlled in an inducible and reversible manner. Our preliminary data show that expression of Kras* drives tumor growth, while Kras* depletion results in regression, even in the presence of Tp53*. Here we interrogate how Kras* modulates the TME in lung adenocarcinomas. Harnessing scRNA sequencing, immunohistochemistry, and multiple biochemical approaches, we will provide insight into changes in stromal and immune contexture upon Kras* depletion in mice. Citation Format: Rachael Baliira, Emily Lasse Opsahl, Ivana Barravecchia, Marina Pasca di Magliano, Stefanie Galban. Oncogenic Kras signaling shapes the tumor microenvironment in lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C116.

Abstract PR07: Oncogenic Kras signaling shapes the tumor microenvironment in lung adenocarcinoma

Abstract Despite advancements in targeted therapy and immunotherapy, lung cancer remains the leading cause of cancer-related deaths in both men and women worldwide, with an estimated 2.2 million deaths each year. At 85%, non-small cell lung cancer (NSCLC) is by far the most common subtype, comprising adenocarcinomas and lung squamous cell carcinoma. Mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR) and anaplastic lymphoma receptor tyrosine kinase (ALK) genes are common, with the worst overall survival in KRAS mutant adenocarcinoma patients. About 40% of lung tumors with KRAS mutations also harbor mutations in tumor protein p53 gene (TP53), a tumor suppressor gene. KrasG12C and KrasG12D are both prevalent mutations in lung cancer. Sotorasib, a KrasG12C inhibitor, was recently approved for treatment of NSCLC. However, only 37% of patients have an objective response to Sotorasib, with a median duration of just 11 months. The KrasG12D inhibitor MRTX1133 is entering clinical trials; elucidating the mechanism of action for potential resistance is imperative to design combination treatment approaches. While cell-intrinsic mechanisms of resistance in epithelial oncogenic KRASG12D (Kras*) are well studied, our group and others have shown that KRASG12D also extrinsically regulates the tumor microenvironment (TME) in pancreatic cancer. Whether and how this oncogene regulates the TME of lung cancer is less understood. To study the crosstalk between epithelial lung cells expressing oncogenic KRAS and the surrounding TME, we have established a triple transgenic murine model of Kras*-driven NSCLC, wherein both lung epithelial-specific expression of Kras* and expression of mutant TP53R172H (Tp53*) can be controlled in an inducible and reversible manner. Our preliminary data show that expression of Kras* drives tumor growth, while Kras* depletion results in regression, even in the presence of Tp53*. Here we interrogate how Kras* modulates the TME in lung adenocarcinomas. Harnessing scRNA sequencing, immunohistochemistry, and multiple biochemical approaches, we will provide insight into changes in stromal and immune contexture upon Kras* depletion in mice. Citation Format: Rachael K. Baliira, Emily Lasse Opsahl, Elyse McLintock, Katelyn L. Donahue, Ivana Barravacchia, Marina Pasca di Magliano, Stefanie Galbán. Oncogenic Kras signaling shapes the tumor microenvironment in lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR07.

Regulation and Therapeutic Targeting of MTHFD2 and EZH2 in KRAS-Mutated Human Pulmonary Adenocarcinoma.

Activating KRAS mutations occur in about 30% of pulmonary adenocarcinoma (AC) cases and the discovery of specific inhibitors of G12C-mutated KRAS has considerably improved the prognosis for a subgroup of about 14% of non-small cell lung cancer (NSCLC) patients. However, even in patients with a KRAS G12C mutation, the overall response rate only reaches about 40% and mutations other than G12C still cannot be targeted. Despite the fact that one-carbon metabolism (1CM) and epigenetic regulation are known to be dysregulated by aberrant KRAS activity, we still lack evidence that co-treatment with drugs that regulate these factors might ameliorate response rates and patient prognosis. In this study, we show a direct dependency of Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and Enhancer of Zeste Homolog 2 (EZH2) expression on mutationally activated KRAS and their prognostic relevance in KRAS-mutated AC. We show that aberrant KRAS activity generates a vulnerability of AC cancer cell lines to both MTHFD2 and EZH2 inhibitors. Importantly, co-inhibition of both factors was synergistically effective and comparable to KRASG12C inhibition alone, paving the way for their use in a therapeutic approach for NSCLC cancer patients.

Targeting the ERβ/HER Oncogenic Network in KRAS Mutant Lung Cancer Modulates the Tumor Microenvironment and Is Synergistic with Sequential Immunotherapy.

High ERβ/HER oncogenic signaling defines lung tumors with an aggressive biology. We previously showed that combining the anti-estrogen fulvestrant with the pan-HER inhibitor dacomitinib reduced ER/HER crosstalk and produced synergistic anti-tumor effects in immunocompromised lung cancer models, including KRAS mutant adenocarcinoma. How this combination affects the tumor microenvironment (TME) is not known. We evaluated the effects of fulvestrant and dacomitinib on murine bone marrow-derived macrophages (BMDMs) and CD8+ T cells, and tested the efficacy of the combination in vivo, using the KRAS mutant syngeneic lung adenocarcinoma model, FVBW-17. While this combination synergistically inhibited proliferation of FVBW-17 cells, it had unwanted effects on immune cells, by reducing CD8+ T cell activity and phagocytosis in BMDMs and inducing PD-1. The effects were largely attributed to dacomitinib, which caused downregulation of Src family kinases and Syk in immune cells. In a subcutaneous flank model, the combination induced an inflamed TME with increased myeloid cells and CD8+ T cells and enhanced PD-1 expression in the splenic compartment. Concomitant administration of anti-PD-1 antibody with fulvestrant and dacomitinib was more efficacious than fulvestrant plus dacomitinib alone. Administering anti-PD-1 sequentially after fulvestrant plus dacomitinib was synergistic, with a two-fold greater tumor inhibitory effect compared to concomitant therapy, in both the flank model and in a lung metastasis model. Sequential triple therapy has potential for treating lung cancer that shows limited response to current therapies, such as KRAS mutant lung adenocarcinoma.

Identification of a targetable KRAS-mutant epithelial population in non-small cell lung cancer

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencing in aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas.

MEKiAUTO: A phase I/II open-label study of combination therapy with the MEK inhibitor cobimetinib, Immune-checkpoint blockade with atezolizumab, and the AUTOphagy inhibitor hydroxychloroquine in KRAS-mutated advanced malignancies.

TPS450 Background: Pancreatic ductal adenocarcinoma (PDAC) and colorectal carcinoma (CRC) are aggressive diseases which account for the third and second leading causes of cancer-related death, respectively. Limited progress has been made towards effective treatments or cure. K RAS is mutated or amplified in nearly 30% of all cancers, including up to 95% of PDAC and 45% of CRC. Although no successful KRAS directed therapy has been approved to date, data has emerged demonstrating that inhibition of downstream targets of KRAS, namely MEK/ERK, increases autophagic flux in KRAS-mutated tumors. This catabolic process is used by many tumors to maintain viability and recent preclinical studies have shown that combining MEK/ERK and autophagy inhibitors in KRAS-mutated tumors can synergistically suppress cancer cell proliferation. Inhibition of autophagy has demonstrated an increase in antigen presentation which sensitize tumors to immune checkpoint inhibitors. Presented here is a trial-in-progress that will evaluate combination of cobimetinib (MEK inhibitor), atezolizumab (anti-PDL1), and hydroxychloroquine (autophagy inhibitor) in KRAS-mutated advanced malignancies. Methods: This is a phase 1/2 multicenter, open-label study of combination cobimetinib (40-60mg) orally once daily on days 1-21, hydroxychloroquine (600mg) orally twice daily on days 1-28, and atezolizumab 840 mg IV on days 1 and 15 of each 28 day cycle. Patients with histologically confirmed metastatic or unresectable KRAS-mutant adenocarcinoma for which standard curative or meaningful life-prolonging treatment options do not exist or are no longer effective will be enrolled. The primary objective of the phase I portion of this trial, which seeks to enroll 18 patients, is to estimate the maximum tolerated dose (MTD) of these agents using a two-stage time-to-event continual reassessment method (TITE-CRM). The primary objective of the phase II portion of this trial, which seeks to enroll approximately 66-157 patients, is to evaluate the preliminary efficacy of this combination, based on the objective response by 16 weeks. Secondary endpoints include PFS, OS, and safety. Correlative aims include analyses of pre- and on-treatment biopsies with quantitative multiplex immunofluorescence, RNA-sequencing, reverse phase protein array for association with clinical benefit and to determine mechanisms of action/resistance. An interim analysis will be performed at the conclusion of the phase I portion of the study. This study is open with 4 patients enrolled at the time of submission. Clinical trial information: NCT04214418 . This trial is being conducted as part of the imCORE collaboration. Clinical trial information: NCT04214418.

Sustained Partial Response to Inhibition of the Mitogen-Activated Protein Kinase and Autophagy Pathways in Combination With Immune Checkpoint Blockade in KRAS-Mutated Adenocarcinoma of the Small Bowel.

Sustained Partial Response to Inhibition of the Mitogen-Activated Protein Kinase and Autophagy Pathways in Combination With Immune Checkpoint Blockade in KRAS-Mutated Adenocarcinoma of the Small Bowel.

Abstract 3855: Oncogenic Kras-mediated regulation of the tumor microenvironment in lung cancer

Abstract Lung cancer remains the leading cause of cancer-related deaths worldwide, with an estimated 1.6 million deaths each year. Non-small cell lung cancer (NSCLC) is with 85% by far the most common subtype of lung cancer, comprising adenocarcinomas and lung squamous cell carcinoma. Mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR) and anaplastic lymphoma receptor tyrosine kinase (ALK) genes are common with the worst overall survival for KRAS mutant adenocarcinoma patients. We have established a murine model of lung cancer, wherein expression of oncogenic Kras can be controlled genetically, allowing activation of oncogenic KrasG12D (Kras*) to initiate tumor growth, tumor eradication upon Kras* depletion and re-activation as a means to model relapse. Oncogenic Kras depletion (deactivation) has previously been reported to result in tumor cell apoptosis even in the presence of tumor suppressor loss. However, the mechanisms of apoptosis, the role of the immune system on these changes, and the mechanisms allowing some tumor cells to escape apoptosis, which typically results in tumor relapse, are unknown. Here, we interrogated the immune response in mediating tumor regression and relapse using this genetically engineered models. Multiplex immunohistochemistry as well as CyTOF provided insight into the changes in immune contexture upon Kras* depletion in mice haploinsufficient for tumor suppressor p53 or mutant for p53 (R172H). Interestingly, total number of T cells including cytotoxic T cells (CTLs) was elevated in lung tumors from p53 mutant mice supporting findings of heightened immune activation and overall response to immune therapy with an increased mutational burden. Kras* inactivation and thus inhibition of MAPK signaling resulted in an overall decrease in abundance of CTLs and antigen presenting cells (APC) as well as engagement of CTL with tumor cells and APCs indicating a decrease in immune presence likely due to proceeding tumor cell kill and immune recruitment. However, intracellular distance of CTL with tumor cells indicated active tumor cell kill of the CTLs to eradicate remaining tumor cells. In summary, these findings support recent observation of increased immune activation in tumors with higher mutational load as well as changes mediated by inhibition of MAPK signaling which both maybe harnessed for enhancing future immunotherapies. Citation Format: Nina Steele, Kristena Y. Abdelmalak, Sarah F. Ferris, Jennifer M. Lee, Carlos Espinoza, Yaqing Zhang, Sundaresh Ram, Craig Galban, Nithya Ramnath, Timothy L. Frankel, Marina Pasca di Magliano, Stefanie Galbán. Oncogenic Kras-mediated regulation of the tumor microenvironment in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3855.

Targeting PKC\u03b9-PAK1 signaling pathways in EGFR and KRAS mutant adenocarcinoma and lung squamous cell carcinoma

Introductionp21-activated kinase 1 (PAK1) stimulates growth and metastasis in non-small cell lung cancer (NSCLC). Protein kinase C iota (PKCι) is an enzyme highly expressed in NSCLC, regulating PAK1 signaling. In the present study we explored whether the PKCι-PAK1 signaling pathway approach can be an efficient target in different types of NSCLC cell and mouse models.MethodsThe effect of IPA-3 (PAK1 inhibitor) plus auranofin (PKCι inhibitor) combination was evaluated by cell viability assay, colony formation and western blotting assay, using three types of NSCLC cell lines: EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma with PAK1 amplification. In addition, for clinical availability, screening for new PAK1 inhibitors was carried out and the compound OTSSP167 was evaluated in combination with auranofin in cell and mice models.ResultsThe combination of IPA-3 or OTSSP167 plus auranofin showed high synergism for inhibiting cell viability and colony formation in three cell lines. Mechanistic characterization revealed that this drug combination abrogated expression and activation of membrane receptors and downstream signaling proteins crucial in lung cancer: EGFR, MET, PAK1, PKCι, ERK1/2, AKT, YAP1 and mTOR. A nude mouse xenograft assay demonstrated that this drug combination strongly suppressed tumor volume compared with single drug treatment.ConclusionsCombination of IPA-3 or OTSSP167 and auranofin was highly synergistic in EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma cell lines and decreased tumor volume in mice models. It is of interest to further test the targeting of PKCι-PAK1 signaling pathways in EGFR mutant, KRAS mutant and squamous NSCLC patients.

The outcomes of an impaired powerhouse in KRAS mutant lung adenocarcinoma cells by Elesclomol.

Lung cancer stands out as the most common cancer type worldwide. The most common genetic alteration detected in adenocarcinoma patients is KRAS. KRAS mutated patients still cannot get benefit from precision medicine approaches and lack a targeted therapy. Elesclomol is an investigational agent for melanoma and other malignancies. In this study, we evaluated its effect on cellular apoptosis, survival, and metastasis mechanisms on KRAS mutant A549 and Calu-1 cell lines. The cytotoxic effects of Elesclomol on A549 and Calu-1 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability test. Cells were treated with IC50 concentration and then apoptosis-related (Casp-3, Casp-9, Bcl-2, and Bcl-xL), survival-related (Akt, p-Akt, Erk, and p-Erk), and metastasis-related (E-cadherin, Vimentin, MMP-2, and MMP-9) protein expressions were determined by Western blot analysis. Elesclomol's effect on cell migration was evaluated by wound healing. Total oxidant, malondialdehyde (MDA), and glutathione (GSH) levels after Elesclomol treatment were assessed. Elesclomol not only induced apoptotic proteins but also inhibited metastatic protein expressions and migration in both cells. Also, p-Erk activity was diminished by Elesclomol treatment as a reflection of decreased proliferation. However, p-Akt was enhanced as a cellular survival mechanism. Although Elesclomol's effects on oxidative stress parameters were puzzling, it induced total oxidant status (TOS), and MDA in Calu-1 cells. Elesclomol might provide an alternative treatment approach for patients with KRAS mutant lung adenocarcinoma and other solid tumor malignancies that harbor KRAS mutations. This would enable the development of biomarker-driven targeted therapy for KRAS mutant adenocarcinoma patients.

MTOR mediates a mechanism of resistance to chemotherapy and defines a rational combination strategy to treat KRAS-mutant lung cancer.

Oncogenic KRAS mutations comprise the largest subset of lung cancer defined by genetic alterations, but in the clinic no targeted therapies are available that effectively control mutational KRAS activation. Consequently, patients with KRAS-driven tumors are routinely treated with cytotoxic chemotherapy, which is often transiently effective owing to development of drug resistance. In this study, we show that hyperactivated mammalian target of rapamycin (mTOR) pathway is a characteristic hallmark of KRAS-mutant lung adenocarcinoma after chemotherapy treatment, and that KRAS-mutant lung cancer cells rely on persistent mTOR signaling to resist chemotherapeutic drugs. Coherently, mTOR inhibition circumvents the refractory phenotype and restores sensitivity of resistant KRAS-mutant lung cancer cells to chemotherapy. Importantly, drug combinations of clinically approved mTOR inhibitors and chemotherapy drugs synergize in inhibiting cell proliferation of KRAS-mutant cancer cells in vitro and in vivo, and the efficacy of this combination treatment correlates with the magnitude of mTOR activity induced by chemotherapy alone. These results pinpoint mTOR as a mechanism of resistance to chemotherapy in KRAS-mutant lung cancer and validate a rational and readily translatable strategy that combines mTOR inhibitors with standard chemotherapy to treat KRAS-mutant adenocarcinoma, the most common and deadliest lung cancer subset.

Requirement for MUC5AC in KRAS-dependent lung carcinogenesis.

With more than 150,000 deaths per year in the US alone, lung cancer has the highest number of deaths for any cancer. These poor outcomes reflect a lack of treatment for the most common form of lung cancer, non-small cell lung carcinoma (NSCLC). Lung adenocarcinoma (ADC) is the most prevalent subtype of NSCLC, with the main oncogenic drivers being KRAS and epidermal growth factor receptor (EGFR). Whereas EGFR blockade has led to some success in lung ADC, effective KRAS inhibition is lacking. KRAS-mutant ADCs are characterized by high levels of gel-forming mucin expression, with the highest mucin levels corresponding to worse prognoses. Despite these well-recognized associations, little is known about roles for individual gel-forming mucins in ADC development causatively. We hypothesized that MUC5AC/Muc5ac, a mucin gene known to be commonly expressed in NSCLC, is crucial in KRAS/Kras-driven lung ADC. We found that MUC5AC was a significant determinant of poor prognosis, especially in patients with KRAS-mutant tumors. In addition, by using mice with lung ADC induced chemically with urethane or transgenically by mutant-Kras expression, we observed significantly reduced tumor development in animals lacking Muc5ac compared with controls. Collectively, these results provide strong support for MUC5AC as a potential therapeutic target for lung ADC, a disease with few effective treatments.

Establishment of highly metastatic KRAS mutant lung cancer cell sublines in long-term three-dimensional low attachment cultures.

Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3–4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo. A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells. Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion.

A prospective examination of circulating tumor cell profiles in non-small-cell lung cancer molecular subgroups

We report the first study examining the clinical, numerical and biological properties of circulating tumor cells according to molecular subtypes of non-small-cell lung cancer. 125 patients with treatment-naïve stage IIIb-IV NSCLC were prospectively recruited for CellSearch analysis. Anti-vimentin antibody was included for examination of CTCs to assess their mesenchymal character. Associations of total CTCs and vimentin-positive (vim +) CTCs with clinical characteristics, tumor genotype, and survival were assessed. 51/125 patients (40.8%) were total CTC+ and 26/125 (20.8%) were vim CTC+ at baseline. Multivariate analysis showed patients with ≥5 total CTCs had significantly reduced OS (HR 0.55, 95% CI 0.33-0.92, P = 0.022) but not PFS (HR 0.68, 95% CI 0.42-1.1, P = 0.118) compared to patients with <5 total CTCs. No OS difference was evident between vim+ CTC and vim-negative CTC patients overall (HR 1.24, 95% CI 0.67-2.28, P = 0.494), but after subdivision according to NSCLC driver mutation, we found an increase of vim+ CTCs in the EGFR-mutated subgroup (N = 21/94 patients; mean 1.24 vs 1.22 vim+ CTCs, P = 0.013), a reduction of total CTCs in the ALK-rearranged subgroup (N = 13/90 patients; mean 1.69 vs 5.82 total CTCs, P = 0.029), and a total absence of vim+ CTCs in KRAS-mutated adenocarcinomas (N = 19/78 patients; mean 0 vs 1.4 vim+ CTCs, P = 0.006). We validate that the baseline presence of ≥5 total CTCs in advanced NSCLC confers a poor prognosis. CTCs from EGFR-mutant NSCLC express epithelial-mesenchymal transition characteristics, not seen in CTCs from patients with KRAS-mutant adenocarcinoma.

Abstract 3708: RAS-MAPK signal is required for enhanced PD-L1 expression in human lung cancers

Abstract Ectopic programmed cell death ligand 1 (PD-L1) expression in non-small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Since the precise mechanisms responsible for ectopic PD-L1 expression remains obscure, we analyzed the molecular mechanisms of ectopic PD-L1 expression in human lung cancers, focusing on the MAPK signal. Because we found a higher frequency of EGFR/KRAS mutations in NSCLC cell lines with high PD-L1 expression (p &amp;lt; 0.001), we evaluated the relationships between downstream signals and PD-L1 expression, particularly in three KRAS-mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 μM) significantly decreased the surface PD-L1 levels by 50-60% compared with dimethyl sulfoxide (p &amp;lt; 0.0001). Phorbol 12-myristate 13-acetate stimulation (100 nM, 15 min) increased (p &amp;lt; 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p &amp;lt; 0.05) PD-L1 expression. Post-transcriptional mechanism by miR-200s appears not to be in general under the downstream of MAPK in the PD-L1 expression. The transcriptional activity of the potential AP-1 site (+4785 to +5056 from the transcription start site) in the PD-L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP-1 site. Two STAT3 siRNAs decreased PD-L1 expression by 10-32% in two of the three KRAS-mutant lung adenocarcinoma cell lines (p &amp;lt; 0.05), while the PI3K inhibitor LY294002 (40 μM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD-L1-high and -low NSCLCs revealed a correlation between PD-L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD-L1 expression at a transcriptional level, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD-L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK is important for determining PD-L1 expression, which could be useful for targeted therapies against lung cancers. Citation Format: Hidetoshi Sumimoto, Atsushi Takano, Koji Teramoto, Yataro Daigo. RAS-MAPK signal is required for enhanced PD-L1 expression in human lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3708. doi:10.1158/1538-7445.AM2017-3708

Abstract 2627: A study of dynamic changes in PD-L1 expression in KRAS mutant adenocarcinoma of the lung exposed to signal transduction inhibitors

Abstract Aim MEK and AKT inhibitors inhibit signaling down steam of KRAS and are being evaluated as single agents or in combination with other anticancer drugs for the treatment of KRAS mutant adenocarcinoma of the lung (adeno-NSCLC). We investigated whether increased PD-L1 expression with functional T cell inhibitory consequences were a common mechanism of resistance to these drugs in this setting. Material and Methods A panel of 10 KRAS mutant adeno-NSCLC cell lines were studied. Cells were exposed to GI50 concentrations of a MEK (trametinib) and AKT (AZD5363) inhibitor for 6hrs, 24hrs and 3 weeks. PD-L1 expression on cell lines was studied by immunofluorescence. Immunofluorescence at various time points were expressed as a ratio of values in drug treated cells compared to untreated control. Functional consequences of PD-L1 expression were studied using a T cell cancer cell line (Jurkat) transfected with a luciferase reporter where co-culture of cancer cells expressing PD-L1 led to reduction in luminescence. Luminescence at various time points were expressed as ratio of luminescence values of drug treated cells compared to untreated controls. Results Following characterization of expression of PD-L1 in 10 KRAS mutant adeno-NSCLC cell lines, 5 cell lines with the highest expression of PD-L1 was chosen for functional assays (H441, H2291, H23, H2030 and A549). When exposed to trametinib for 3 weeks, 3/5 cell lines (H23, H2030 and A549) showed an statistically significant increase in expression of PD-L1 (range 1.1-2.4) however significant reduction of luciferase activity was only observed in H23 and H2030, 0.93 and 0.75, p= 0.018 and p=0.01 respectively. When exposed to AZD5363 for 3 weeks, 3/5 cell lines (H441, H23 and H2030) showed a statistically significant increase in PD-L1 expression (range 1.3-1.9) however significant reduction in luciferase reduction was seen in only H441 and H23, 0.86 and 0.76, p= 0.03 and p= 0.04 respectively. Conclusion Chronic exposure of KRAS mutant cell lines to MEK and AKT inhibitors cause minor increases in PD-L1 expression but this does not result in functional inhibition of T cells in all instances. Consequently, a functional increase in PD-L1 expression is unlikely to be a common mechanism of resistance to MEK and AKT inhibitors in KRAS mutant adeno-NSCLC. 1 Citation Format: Anna R. Minchom, Parames Thavasu, Zai Ahmad, Adam Stewart, Alexandros Georgiou, Mary ER O'Brien, Sanjay Popat, Jaishree Bhosle, Timothy A. Yap, Johann de Bono, Udai Banerji. A study of dynamic changes in PD-L1 expression in KRAS mutant adenocarcinoma of the lung exposed to signal transduction inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2627. doi:10.1158/1538-7445.AM2017-2627

RAS-Mitogen-Activated Protein Kinase Signal Is Required for Enhanced PD-L1 Expression in Human Lung Cancers.

Ectopic programmed cell death ligand 1 (PD-L1) expression in non-small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Although some altered signals in NSCLCs are responsible for ectopic PD-L1 expression, the precise mechanisms remain obscure. Because we found a higher frequency of EGFR/KRAS mutations in NSCLC cell lines with high PD-L1 expression (p < 0.001), we evaluated the relationships between downstream signals and PD-L1 expression, particularly in three KRAS-mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 μM) significantly decreased the surface PD-L1 levels by 50–60% compared with dimethyl sulfoxide (p < 0.0001). Phorbol 12-myristate 13-acetate stimulation (100 nM, 15 min) increased (p < 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p < 0.05) PD-L1 expression. The transcriptional activity of the potential AP-1 site (+4785 to +5056 from the transcription start site) in the PD-L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP-1 site. Two STAT3 siRNAs decreased PD-L1 expression by 10–32% in two of the three KRAS-mutant lung adenocarcinoma cell lines (p < 0.05), while the PI3K inhibitor LY294002 (40 μM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD-L1-high and -low NSCLCs revealed a correlation between PD-L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD-L1 expression, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD-L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK, along with STAT3, is important for determining PD-L1 expression, which could be useful for targeted therapies against lung cancers.

Abstract 1255: Loss of the ubiquitin protease USP18 represses KRAS mutant lung cancer tumorigenicity in mice by destabilizing KRAS protein

Abstract KRAS is frequently mutated in lung cancers. Innovative strategies are needed to combat KRAS mutant lung cancers because these tumors are often resistant to therapy. This study reports that loss of the deubiquitinase USP18 leads to destabilization of the KRAS oncogenic protein in panels of murine and human lung cancer cells. In marked contrast, engineered gain of USP18 expression using retroviral vectors introduced into the same panel of murine and human lung cancer cells stabilized KRAS protein. Loss of USP18 expression in KRAS mutant-expressing lung cancer cells inhibited their growth while gain of USP18 expression opposed this effect. We sought to identify mechanisms engaged in this KRAS protein destabilization. Intriguingly, immunoprecipitation assays established that KRAS conjugates with the ubiquitin-like protein ISG15. This leads to KRAS protein destabilization. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown was shown to destabilize mutant KRAS more prominently than wild-type KRAS protein. To independently confirm that KRAS directly complexes with ISG15, site-directed mutagenesis was performed to render the C-terminal domain of KRAS lysine-less. This led to stabilization of KRAS, despite knockdown of USP18. These studies were confirmed and extended in the in vivo setting of KRAS-driven lung cancers in KRASLA2/+ mice within the FVB strain background. We crossed these mice with USP18 null (USP18-/-) mice to obtain KRASLA2/+;USP18-/- compound mice. Strikingly, these compound mice had statistically significantly (P &amp;lt; 0.05) reduced lung cancers as compared to parental KRASLA2/+ mice. To establish the biological importance of this finding, USP18 immunohistochemical expression was compared in these engineered mice versus a second engineered mouse lung cancer model that was not driven by KRAS, but by aberrant cyclin E expression. USP18 immunohistochemical expression was substantially higher in the KRAS-driven than cyclin E-dependent lung cancers. To explore the translational relevance of this work, lung cancer tissue arrays were examined in 551 lung cancers where a clinical database was available. USP18 immunohistochemical expression was significantly (P &amp;lt; 0.001) higher in KRAS mutant adenocarcinomas as compared to wild-type cases. There was also a significant increase in USP18 expression in adenocarcinoma versus squamous cell carcinoma cases. Taken together, these studies broaden the role of USP18 as an antineoplastic target to combat lung cancers that harbor KRAS mutations. Citation Format: Lisa Maria Mustachio, Yun Lu, Laura J. Tafe, Angeline S. Andrew, Vincent Memoli, Jaime Rodriguez-Canales, Pamela A. Villalobos, Ignacio Wistuba, Jun Yu, Jack J. Lee, Fadzai Chinyengetere, David J. Sekula, Xi Liu, Sarah J. Freemantle, Ethan Dmitrovsky. Loss of the ubiquitin protease USP18 represses KRAS mutant lung cancer tumorigenicity in mice by destabilizing KRAS protein. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1255.