Non-small Cell Lung Cancer Models Research Articles

Graph Neural Network Model for Prediction of Non-Small Cell Lung Cancer Lymph Node Metastasis Using Protein-Protein Interaction Network and 18F-FDG PET/CT Radiomics.

The image texture features obtained from 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) images of non-small cell lung cancer (NSCLC) have revealed tumor heterogeneity. A combination of genomic data and radiomics may improve the prediction of tumor prognosis. This study aimed to predict NSCLC metastasis using a graph neural network (GNN) obtained by combining a protein-protein interaction (PPI) network based on gene expression data and image texture features. 18F-FDG PET/CT images and RNA sequencing data of 93 patients with NSCLC were acquired from The Cancer Imaging Archive. Image texture features were extracted from 18F-FDG PET/CT images and area under the curve receiver operating characteristic curve (AUC) of each image feature was calculated. Weighted gene co-expression network analysis (WGCNA) was used to construct gene modules, followed by functional enrichment analysis and identification of differentially expressed genes. The PPI of each gene module and genes belonging to metastasis-related processes were converted via a graph attention network. Images and genomic features were concatenated. The GNN model using PPI modules from WGCNA and metastasis-related functions combined with image texture features was evaluated quantitatively. Fifty-five image texture features were extracted from 18F-FDG PET/CT, and radiomic features were selected based on AUC (n = 10). Eighty-six gene modules were clustered by WGCNA. Genes (n = 19) enriched in the metastasis-related pathways were filtered using DEG analysis. The accuracy of the PPI network, derived from WGCNA modules and metastasis-related genes, improved from 0.4795 to 0.5830 (p < 2.75 × 10-12). Integrating PPI of four metastasis-related genes with 18F-FDG PET/CT image features in a GNN model elevated its accuracy over a without image feature model to 0.8545 (95% CI = 0.8401-0.8689, p-value < 0.02). This model demonstrated significant enhancement compared to the model using PPI and 18F-FDG PET/CT derived from WGCNA (p-value < 0.02), underscoring the critical role of metastasis-related genes in prediction model. The enhanced predictive capability of the lymph node metastasis prediction GNN model for NSCLC, achieved through the integration of comprehensive image features with genomic data, demonstrates promise for clinical implementation.

BLU-945, a potent and selective next-generation EGFR TKI, has antitumor activity in models of osimertinib-resistant non-small-cell lung cancer.

Despite the availability of several epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), most patients with non-small-cell lung cancer (NSCLC) eventually develop resistance to these agents. Notably, EGFR_C797S mutations confer resistance to the third-generation EGFR-TKI osimertinib and no approved post-osimertinib targeted pharmacology options are currently available. BLU-945 is a novel, reversible, and orally available next-generation EGFR-TKI that selectively targets EGFR-activating (EGFRm) and resistance mutations (including EGFR_C797S) with nanomolar potency while sparing wild-type EGFR in vitro. In vitro activity of BLU-945 as a single agent and in combination with osimertinib was tested in engineered EGFR-mutant cell lines as well as patient-derived cells and patient-derived organoids. In vivo activity was evaluated in osimertinib-resistant patient-derived xenograft mouse models. Three patient cases from the global, first-in-human, phase I/II SYMPHONY trial (NCT04862780) demonstrating the clinical efficacy of BLU-945 were reported. In vitro BLU-945 demonstrated inhibited cell viability and growth of EGFR-mutant/osimertinib-resistant cell lines. BLU-945 demonstrated in vivo tumor shrinkage in osimertinib-resistant models of NSCLC (osimertinib second line: EGFR_L858R/C797S and third line: EGFR_ex19del/T790M/C797S and L858R/T790M/C797S) both as monotherapy and in combination with osimertinib. BLU-945 also demonstrated tumor shrinkage in patients from the SYMPHONY trial. Our findings demonstrate the preclinical and early clinical activity of BLU-945 in EGFRm NSCLC progressing on previous EGFR-TKIs.

Tanreqing injection inhibits stemness and enhances sensitivity of non-small cell lung cancer models to gefitinib through ROS/STAT3 signaling pathway.

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) has emerged as a significant obstacle in managing patients with EGFR-mutant non-small-cell lung cancer (NSCLC), necessitating the exploration of novel therapeutic approaches. Tanreqing injection (TRQ) is a kind of Chinese patent medicine known for its heat-clearing and detoxifying properties. Studies have shown a correlation between tumor drug resistance and enrichment of cancer stem cells (CSCs). We aim to investigate the feasibility of TRQ enhancing sensitivity to gefitinib by targeting CSCs and reactive oxygen species (ROS). In our study, TRQ significantly inhibited cell proliferation in gefitinib-resistant non-small-cell lung cancer (NSCLC) models including 2D cell lines, 3D cell spheres, tumor-bearing animal and organoids. Compared with the gefitinib group alone, addition of TRQ elevated ROS levels, attenuated upregulation of the protein levels of sex-determining region Y-box 2 (SOX2) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) induced by gefitinib treatment, and inhibited the phosphorylation of signal transducer and activator of transcription 3 (STAT3). Scavenging ROS could restore tumor stemness, attenuate the inhibitory effect on the phosphorylation of STAT3, and promote cell proliferation. These results suggested that TRQ could enhance sensitivity of NSCLC models to gefitinib, providing a new combined treatment strategy.

Mechanism of non-small cell lung cancer cell-derived exosome miR-196b-5p promoting pyroptosis of tumor T cells and tumor cell proliferation by downregulating ING5.

In the world, lung cancer is one of the most common malignant cancers and has become the leading cause of death of cancers in China, among which non-small cell lung cancer (NSCLC) accounts for a relatively high proportion, but there is a lack of effective treatment at present. An animal model of NSCLC was established, and BEAS-2b, H1299, Lewis, and T cells were used for subsequent experimental verification. The level of miR-196b-5p was detected by quantitative real-time polymerase chain reaction. Growth inhibitor 5 (ING5), CD9, CD63, HSP70, Caspase-1, NLRP3, and GSDMD-NT were detected by western blot. The level of ING5 was confirmed by immunohistochemistry, the location of miR-196b-5p was analyzed by fluorescence in situ hybridization (FISH), cell viability was investigated by Cell Counting Kit-8 kit, and interleukin (IL)-1β and IL-18 were confirmed by enzyme-linked immunosorbent assay. Cell apoptosis was detected by flow cytometry. In addition, the binding site was verified by dual-luciferase reporter gene experiments. Tumor volume was measured. TUNEL staining was used to detect apoptosis. Flow cytometry was used to measure the levels of CD8 T, CD4 T, and Treg cells in tumors. miR-196-5p was highly expressed in exosomes secreted by tumor cells. miR-196-5p negatively targeted ING5 to promote the growth of tumor cells. Cancer-derived exosomes promote pyroptosis of T cells to further aggravate the development of cancer. Exosome-derived miR-196b-5p promoted pyroptosis of T cells. Exosome-derived miR-196b-5p inhibited the level of ING5 to promote tumor growth and accelerate the process of NSCLC.

Targeting lncRNA16 by GalNAc-siRNA conjugates facilitates chemotherapeutic sensibilization via the HBB/NDUFAF5/ROS pathway.

Chemoresistance is a significant barrier to effective cancer treatment. Potential mechanisms for chemoresistance include reactive oxygen species (ROS) accumulation and expression of chemoresistance-promoting genes. Here, we report a novel function of lncRNA16 in the inhibition of ROS generation and the progression of chemoresistance. By analyzing the serum levels of lncRNA16 in a cohort of 35 patients with non-small cell lung cancer (NSCLC) and paired serum samples pre- and post-treatment from 10 NSCLC patients receiving neoadjuvant platinum-based chemotherapy, performing immunohistochemistry (IHC) assays on 188 NSCLC tumor samples, using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) assays, as well as RNA immunoprecipitation (RIP) and RNA pull-down analyses, we discovered that patients with increased serum levels of lncRNA16 exhibited a poor response to platinum-based chemotherapy. The expression of hemoglobin subunit beta (HBB) and NDUFAF5 significantly increases with the development of chemoresistance. LncRNA16 binds to HBB and promotes HBB accumulation by inhibiting autophagy. LncRNA16 can also inhibit ROS generation via the HBB/NDUFAF5 axis and function as a scaffold to facilitate the colocalization of HBB and NDUFAF5 in the mitochondria. Importantly, preclinical studies in mouse models of chemo-resistant NSCLC have suggested that lncRNA16 targeting by trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA restores chemosensitivity and results in tumor growth inhibition with no detectable toxicity in vivo. Overall, lncRNA16 is a promising therapeutic target for overcoming chemoresistance, and the combination of first-line platinum-based chemotherapy with lncRNA16 intervention can substantially enhance anti-tumor efficacy.

Modeling RET-Rearranged Non-Small Cell Lung Cancer (NSCLC): Generation of Lung Progenitor Cells (LPCs) from Patient-Derived Induced Pluripotent Stem Cells (iPSCs).

REarranged during Transfection (RET) oncogenic rearrangements can occur in 1-2% of lung adenocarcinomas. While RET-driven NSCLC models have been developed using various approaches, no model based on patient-derived induced pluripotent stem cells (iPSCs) has yet been described. Patient-derived iPSCs hold great promise for disease modeling and drug screening. However, generating iPSCs with specific oncogenic drivers, like RET rearrangements, presents challenges due to reprogramming efficiency and genotypic variability within tumors. To address this issue, we aimed to generate lung progenitor cells (LPCs) from patient-derived iPSCs carrying the mutation RETC634Y, commonly associated with medullary thyroid carcinoma. Additionally, we established a RETC634Y knock-in iPSC model to validate the effect of this oncogenic mutation during LPC differentiation. We successfully generated LPCs from RETC634Y iPSCs using a 16-day protocol and detected an overexpression of cancer-associated markers as compared to control iPSCs. Transcriptomic analysis revealed a distinct signature of NSCLC tumor repression, suggesting a lung multilineage lung dedifferentiation, along with an upregulated signature associated with RETC634Y mutation, potentially linked to poor NSCLC prognosis. These findings were validated using the RETC634Y knock-in iPSC model, highlighting key cancerous targets such as PROM2 and C1QTNF6, known to be associated with poor prognostic outcomes. Furthermore, the LPCs derived from RETC634Y iPSCs exhibited a positive response to the RET inhibitor pralsetinib, evidenced by the downregulation of the cancer markers. This study provides a novel patient-derived off-the-shelf iPSC model of RET-driven NSCLC, paving the way for exploring the molecular mechanisms involved in RET-driven NSCLC to study disease progression and to uncover potential therapeutic targets.

Comprehensive analysis of mitophagy-related genes in NSCLC diagnosis and immune scenery: based on bulk and single-cell RNA sequencing data.

Lung cancer is the main cause of cancer-related deaths, and non-small cell lung cancer (NSCLC) is the most common type. Understanding the potential mechanisms, prognosis, and treatment aspects of NSCLC is essential. This study systematically analyzed the correlation between mitophagy and NSCLC. Six mitophagy-related feature genes (SRC, UBB, PINK1, FUNDC1, MAP1LC3B, and CSNK2A1) were selected through machine learning and used to construct a diagnostic model for NSCLC. These feature genes are closely associated with the occurrence and development of NSCLC. Additionally, NSCLC was divided into two subtypes using unsupervised consensus clustering, and their differences in clinical characteristics, immune infiltration, and immunotherapy were systematically analyzed. Furthermore, the interaction between mitophagy-related genes (MRGs) and immune cells was analyzed using single-cell sequencing data. The findings of this study will contribute to the development of potential diagnostic biomarkers for NSCLC and the advancement of personalized treatment strategies.

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.

Abstract A079: FHD286 is a BRG1/BRM ATPase inhibitor showing efficacy in NSCLC models and is applicable to NSCLC patients both as a single agent treatment and in combination with current standards of care

Abstract The BAF (BRG/BRM-associated factors) complex plays a critical role in shaping the chromatin landscape. The two mutually exclusive ATPases BRM/BRG1 (SMARCA2/4, respectively) drive chromatin remodeling activity in the BAF complex. Genetic alterations in the SMARCA4 gene, encoding BRG1, are found in approximately 10% of non-small cell lung cancer (NSCLC) patients, leaving this subset of BRG1-deficient tumors highly susceptible to inhibition of BRM (Hoffman, 2014; Oike, 2013). However, BRG1 can also have oncogenic activity in NSCLC (Li, 2019) and can serve as a predictive biomarker for chemotherapy outcome in patients (Bell, et al 2015), rationalizing the use of a dual BRG1/BRM ATPase inhibitor (FHD286) in NSCLC patients in combination with chemotherapy. Here, we demonstrate that a panel of in vitro and in vivo NSCLC models respond to BAF ATPase inhibition as a single agent treatment, irrespective of SMARCA4 mutation status. Analysis of gene expression and dependency data suggests that FHD286 sensitivity correlates with activity of genes related to DNA damage response and repair. We furthermore demonstrate an enhancing effect on tumor growth inhibition in vitro and in vivo driven by apoptosis and cell cycle arrest when combining dual FHD286 with current first and second line standards of care. Collectively, these results indicate a path forward for the development of FHD-286, a clinical stage BRM/BRG1 ATPase inhibitor, for the treatment of NSCLC patients both as a single agent treatment and in combination with current standards of care. Citation Format: Oliver Mikse, Ammar Adam, GiNell Elliott, Dave Lahr, Victoria Amaral, Priyadharshini Viswanathan, Liv Johannessen, Hafiz Ahmad, Jessica Wan, Steven Bellon, Murphy Hentemann. FHD286 is a BRG1/BRM ATPase inhibitor showing efficacy in NSCLC models and is applicable to NSCLC patients both as a single agent treatment and in combination with current standards of care [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A079.

Abstract B030: Characterizing MAIT cells in lung cancer: Insights from NSCLC and murine models

Abstract Background: Mucosal-associated invariant T cells (MAIT) are unconventional αβT cells with a semi-invariant TCR that recognizes microbially-derived riboflavin metabolites presented by MHC-related protein 1 (MR1). Upon TCR engagement or cytokine stimulation, MAIT cells can rapidly secrete pro-inflammatory cytokines and cytotoxic molecules. The precise role of MAIT cells in the tumor-immune microenvironment remains unclear, as different reports have described them as either beneficial or detrimental to patient prognosis. Considering that MAIT cells constitute a notable proportion (2-4%) of the lung T cell repertoire in both humans and mice, the lung serves as an appropriate setting for ex vivo and in vivo MAIT cell characterization. Objective: To characterize the phenotype of MAIT cells in lung tumor and uninvolved tissue from non-small cell lung cancer (NSCLC) specimens and orthotopic murine models. Results: Surgical resections of primary tumor and normal adjacent tissue from 29 treatment-naïve NSCLC patients were profiled using a comprehensive 31-marker spectral flow cytometry panel. MAIT cells expressing activating and inhibitory receptors including CD69, PD-1, CTLA-4, and CD39, were enriched in tumor tissue. Conversely, cytotoxic MAIT cells expressing granzyme B, granulysin, and CD57, were enriched in normal adjacent tissue. For in vivo studies, lungs of B6-MAITCAST mice, which harbor higher frequencies of MAIT cells compared to conventional mouse strains, were orthotopically injected with CMT167 tumor cells or vehicle control. MAIT cells from lungs injected with CMT167 tumors also expressed higher levels of activating and inhibitory receptors, and decreased levels of granzyme B. Additionally, there was a slight decrease in IFNγ and IL-17A production upon stimulation in vitro with PMA/ionomycin compared to vehicle controls. Conclusion: In the context of lung cancer, MAIT cells are activated but may lose functionality within the tumor-immune microenvironment. Rationale: There is growing interest in the immunotherapeutic potential of unconventional T cells due to their rapid effector functions and non-MHC-restricted nature. By investigating the role of MAIT cells in the tumor microenvironment, we can comprehensively identify their biological advantages and limitations. Citation Format: Stephanie WY Wong, Carolina de Amat Herbozo, Adriana Vukosich-Pennell, Ben X Wang, Pamela S Ohashi, Adrian G Sacher. Characterizing MAIT cells in lung cancer: Insights from NSCLC and murine models [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 B030.

Abstract C026: Initial safety, pharmacokinetics, and recommended Phase 2 dose from RAMP 203: A Phase 1/2 study of Avutometinib + Sotorasib in KRAS G12C Mutant Non-Small Cell Lung Cancer

Abstract Background: KRAS mutations occur in ~30% of non-small cell lung cancer (NSCLC), KRAS G12C mutations being the most common (40%). KRAS G12C inhibitors (G12Ci) are FDA approved for KRAS G12C mutant (mt) NSCLC. Acquired mutations and amplifications occurring upon clinical progression may limit the benefit of G12Ci. Simultaneous targeting of multiple nodes in the RAS/MAPK pathway may improve response depth and durability. Avutometinib is a first-in-class oral RAF/MEK clamp that potently inhibits MEK kinase activity while also blocking the compensatory reactivation of MEK by upstream RAF. In preclinical G12Ci-naïve NSCLC models, addition of avutometinib improved depth of MAPK pathway inhibition and substantially enhanced tumor regression relative to sotorasib alone. Initial clinical efficacy of avutometinib in KRAS G12C NSCLC was observed in the FRAME study [NCT03875820]: 4/6 patients (pts) demonstrated tumor reduction including 1 PR. Methods: RAMP 203 is a phase 1/2, multicenter, open label, dose evaluation/expansion study evaluating the efficacy and safety of avutometinib + sotorasib in pts with KRAS G12C NSCLC [NCT05074810]. Pts with unresectable stage 3B-C and stage 4 KRAS G12C NSCLC and 1-3 prior systemic regimens were enrolled. Previous treatment with MEK inhibitors was excluded, but prior G12Ci treatment was allowed. Up to 3 dose levels (DL) of avutometinib (DL1, 3.2 mg PO BIW 21/28 days; DL2, 4.0 mg PO BIW 21/28 days; DL-1, 2.4 mg PO BIW 21/28 days) + sotorasib (960 mg PO QD 28/28 days) were evaluated in the Part A (dose evaluation, standard 3+3 design) to determine the Recommended Phase 2 Dose (RP2D, based on dose limiting toxicities [DLTs], other safety data, and pharmacokinetics) for Part B (dose expansion). Results: At the time of data cut, 10 pts (9 DLT evaluable) with KRAS G12C NSCLC (all stage 4) were enrolled to DL1 (n=3) and DL2 (n=7). Pts received a median of 3 prior systemic regimens (5 pts with previous G12Ci). No DLTs were observed in DL1 or DL2, leading to the selection of DL2 as RP2D. The majority of DL2 TRAEs observed were grades 1-2. The most common TRAEs of any grade with a frequency of ≥20% of pts included nausea, AAT increase, diarrhea, fatigue, and pruritus. The mean exposures (AUC) of avutometinib and sotorasib in DL1 and DL2 were within range of past single agent studies, indicative of minimal drug-drug interactions (DDIs). Preliminary efficacy data will be available at the time of presentation. Conclusions: The combinability of avutometinib and sotorasib was demonstrated in heavily-pretreated, advanced KRAS G12C NSCLC. Avutometinib 4.0 mg PO BIW 21/28 days + sotorasib PO 960 mg QD 28/28 days was established as the RP2D with no indications of DDIs. No new safety signals were observed and most TRAEs were mild to moderate. Enrollment in RAMP 203 Part B is ongoing. Citation Format: Mark M Awad, Jerome Goldschmidt, Alexander I Spira, Rajat Malhotra, Jeffrey T Yorio, Vijeta Bhambhani, Yaofeng Cheng, Pablo S Lee, Ramaswamy Govindan. Initial safety, pharmacokinetics, and recommended Phase 2 dose from RAMP 203: A Phase 1/2 study of Avutometinib + Sotorasib in KRAS G12C Mutant Non-Small Cell Lung Cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C026.

Abstract C052: Circumventing EGFR inhibitor resistance in NSCLC using transomics

Abstract Drug discovery continues to be refined to enhance efficiency, yet &amp;gt;80% of new drugs fail in the clinical trial stage. It is apparent that a novel approach is needed. COMPASS is a transomic analysis platform that integrates data across multiple omics layers; genomics, transcriptomics, proteomics, and phosphoproteomics. This analysis allows us to interpret complex biological relationships and provide a functional map of the biochemical drivers of disease. We have used EGFR inhibitor (EGFRi) resistance as a case study. EGFRi have been successful in treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (EGFRm), however, this success is temporary. The emergence of resistance is a problem and limits long-term treatment for many patients, even with osimertinib, a third-generation inhibitor and current standard of care. As resistance to EGFRi occurs through mutations in EGFR we adopted a novel strategy using COMPASS to identify and validate alternative, non-EGFR targets that could potentially recapitulate the pharmacological effects of EGFRi in EGFRm NSCLC. The transomic signatures of EGFRi were generated using a NSCLC cell line containing the activating mutation ex19del. The drug concentration required to inhibit cell growth (IC50) was first determined. For transomic analyses, cells were incubated for 24 h with the drug IC50 concentration and harvested for genomic, transcriptomic, proteomic, and phosphoproteomic analyses. We have previously shown transomics analysis can differentiate between EGFRi that have been designed to treat EGFRm NSCLC with the transomics signature for osimertinib being significantly different from comparator drugs that failed during drug development. We employed our proprietary target prioritization algorithm to further analyze the omics data to identify and rank novel targets. Targets were then filtered to select those with an available pharmacological tool compound (PTC). The PTCs were used to evaluate the targets in a xenograft model of osimertinib resistant NSCLC, PC9-Del19/T790M/C797S; which contains the activating EGFR mutation (ex19del), and two mutations conferring EGFRi resistance, T790M gatekeeper mutation, and C797S which confers resistance to osimertinib. Of the targets tested 82% (9/11) were associated with &amp;gt;25% inhibition of tumor growth. Furthermore, 36% (4/11) targets were associated with &amp;gt;50% inhibition by the PTCs, with inhibition of one target giving &amp;gt;85% inhibition resulting in stasis of tumor growth. Future work will investigate targets using gene silencing, as PTCs were not available for many of the novel high-ranked targets. These data show that using COMPASS transomic analysis, it is possible to identify novel drug targets to treat difficult-to-treat cancers such as EGFRi-resistant NSCLC and provides validation of this discovery platform by disease-relevant in vivo pharmacology. Further validation studies are ongoing against other difficult-to-treat cancers to identify novel clinical targets and drug candidates. Citation Format: Christopher J Nicholson, Samuel J Roth, Arudhir Singh, Caitlin Brown, Simon P Fricker, Jon Hu, Samantha Dale Strasse. Circumventing EGFR inhibitor resistance in NSCLC using transomics [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C052.

Abstract B025: The next generation farnesyltransferase inhibitor, KO-2806, blocks oncogenic signaling at multiple nodes to enhance the antitumor efficacy of KRASG12C inhibitor adagrasib in KRASG12C non-small cell lung carcinoma

Abstract Emerging clinical and preclinical data have shown that responses to KRASG12C inhibition are limited by the activation of compensatory signaling proteins, including receptor tyrosine kinases (RTKs) and mTOR. Combination therapeutic strategies co-targeting these nodes are needed to fully realize the potential of breakthrough KRASG12C-selective inhibitors. We propose that, given its multipronged mechanism of action, KO-2806, a next-generation farnesyltransferase inhibitor (FTI) with increased potency and improved pharmacokinetic properties relative to earlier FTI candidates that is poised to enter a first-in-human clinical trial, is a uniquely suited combination partner for KRASG12C inhibitors. Here, we evaluate the therapeutic potential of combined KO-2806 and adagrasib in preclinical models of KRASG12C non-small cell lung cancer (NSCLC). Our previous work indicated that FTIs amplify the antitumor effects of PI3Kα inhibition by blocking compensatory mTOR signaling. As activation of compensatory signaling is a well-described bypass mechanism to escape KRAS inhibition, we asked whether KO-2806 could improve the in vivo efficacy of adagrasib. Combination of KO-2806 with adagrasib led to markedly enhanced antitumor effects in KRASG12C-mutant NSCLC cell line- and patient-derived xenograft models compared to adagrasib alone. Tumor regression in the combination group corresponded to increased apoptosis, as evidenced by higher cleaved caspase 3 staining in combination-treated tumors compared to single agent groups. Intriguingly, adagrasib induced expression of the RTK HER3, a potential driver of bypass signaling, while HER3 expression in combination-treated tumors was lower than in vehicle-treated tumors. To further interrogate the mechanistic basis of the observed in vivo synergy, we cultured KRASG12C-mutant cell lines as 3D tumor spheroids and exposed them to KO-2806 and/or adagrasib. Consistent with the xenograft tumor data, spheroids exposed to the combination expressed lower levels of the upstream receptor HER3 (total and phosphorylated forms) and higher levels of apoptotic markers cleaved PARP and cleaved caspase 3. Concurrently, activity of the downstream effectors ERK and mTOR was reduced. Co-exposure to KO-2806 and adagrasib potently inhibited phosphorylation of ERK and its substrate p90 RSK as well as phosphorylation of mTOR substrates p70 S6K, 4EBP1, and S6. The mTOR signaling reduction paralleled defarnesylation of the farnesylation-dependent mTOR activator RHEB, so we depleted RHEB expression in spheroids and assayed their growth. Genetic depletion of RHEB phenocopied the antiproliferative effects of KO-2806 in combination with adagrasib, implicating RHEB as an important farnesylated target in KRASG12C NSCLC. These preclinical results suggest that KO-2806 is an effective combination partner for KRASG12C inhibitors by simultaneously targeting an upstream activator (HER3) and a downstream effector (RHEB/mTOR) of KRAS, leading to improved antitumor activity in KRASG12C-mutant NSCLC models. Citation Format: Hetika V Patel, Alison Smith, Stacia Chan, Linda Kessler, Francis Burrows, Shivani Malik. The next generation farnesyltransferase inhibitor, KO-2806, blocks oncogenic signaling at multiple nodes to enhance the antitumor efficacy of KRASG12C inhibitor adagrasib in KRASG12C non-small cell lung carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B025.

Abstract C104: Targeted epigenomic inhibition of MYC enhances responses to immune checkpoint and EGFR inhibitors in preclinical models of NSCLC

Abstract Aberrant expression of the transcription factor MYC is found in most cancers including non-small cell lung cancer (NSCLC) and has critical roles in a wide range of oncogenic processes including immune evasion and EGFR inhibitor resistance. Despite MYC’s therapeutic attractiveness, potent inhibitors of MYC activity have been elusive, due to its intrinsically disordered protein structure and tightly autoregulated expression. We have developed NSCLC-specific programmable epigenomic mRNA therapy, termed a MYC Omega epigenomic controller (NSCLC MYC-OEC) that is designed to target the insulated genomic domain of MYC. We have previously shown that NSCLC MYC-OEC effectively downregulates MYC expression pre-transcriptionally and have demonstrated that NSCLC MYC-OEC inhibits NSCLC patient-derived organoid viability in vitro and abrogates xenograft tumor growth in vivo. To identify combination treatments with NSCLC MYC-OEC, we retrospectively evaluated baseline MYC expression and real-world progression-free survival (PFS) in a cohort of 283 NSCLC patients treated with pembrolizumab, an anti-PD1 monoclonal antibody. This analysis revealed that NSCLC patients with MYC-overexpressing tumors exhibited shorter PFS when treated with pembrolizumab. Moreover, analysis of PD1 or PD-L1 transcript levels in tumors from 13,230 lung adenocarcinoma patients revealed a significant positive correlation between PD1 or PD-L1 and MYC mRNA levels, consistent with a role for MYC in regulation of the immune response. In vivo evaluation of NSCLC MYC-OEC activity and immune checkpoint immunotherapy (ICI) treatment of preclinical Lewis lung carcinoma syngeneic tumor models demonstrated that the combination of NSCLC MYC-OEC with anti-PD-L1 antibody enhanced inhibition of tumor growth and prolonged survival over administration when compared to either NSCLC MYC-OEC or anti-PD-L1 antibody monotherapy. Retrospective analysis of a cohort of 301 NSCLC patients also showed that patients with MYC-overexpressing tumors exhibited significantly shorter PFS when treated with osimertinib, a third generation EGFR inhibitor. To further understand this, we investigated the effects of NSCLC MYC-OEC and osimertinib treatment alone or in combination in EGFR mutant cells, H1975 and PC9. While NSCLC MYC-OEC and osimertinib monotherapy treatments resulted in downregulation of MYC protein expression in both cell lines, combination treatment led to a significantly greater decrease in MYC expression. Furthermore, the combination synergistically reduced H1975 and PC9 cell viability in vitro and led to a significantly greater inhibition of H1975 xenograft growth relative to each single agent in vivo. Together, these data provide preclinical proof-of-concept in NSCLC for development of OEC-mediated pre-transcriptional epigenomic inhibition of MYC expression in combination with immune checkpoint and EGFR inhibitors. Furthermore, these findings suggest that NSCLC MYC-OEC combination with ICI or EGFR-targeted therapy may improve patient outcomes. Citation Format: Defne Yarar, Eugine Lee, Padraich Flahardy, Cameron Vergato, Graeme Hodgson, Thomas McCauley. Targeted epigenomic inhibition of MYC enhances responses to immune checkpoint and EGFR inhibitors in preclinical models of NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C104.

Synergistic Effects of 2-Deoxy-D-Glucose and Cinnamic Acid with Erlotinib on NSCLC Cell Line

Background: In spite of all efforts, Non-small cell lung cancer (NSCLC) is a fatal solid tumor with a poor prognosis as of its high metastasis and resistance to present treatments. Tyrosine kinase inhibitors (TKI) such as erlotinib are efficient in treating NSCLC but the emergence of chemoresistance and adverse effects substantially limits their single use. Objective: in this study, the combination treatments of either 2-deoxy-D-glucose (2DG) or cinnamic acid (CINN) with erlotinib (ERL) were tested for their possible synergistic effect on the proliferation and migration capacity of NSCLC cells. Methods: In this study, NSCLC model cell line A549 was used to investigate the effects of single compounds and their combination on cell growth inhibition, clonogenic potential, and migration capacity. Next, the Combination index (CI) and the Dose-Reduction Index (DRI) were determined to determine the nature of the drug’s combination and to measure how many folds the dose could be lowered for each drug in a synergistic combination. Results: the combination treatment demonstrated more significant inhibition of viability of A549 cells compared to individual therapy. Our data show that CINN augments the sensitivity to erlotinib in all doses tested. The combination of 2DG or CINN with erlotinib also reduced the clonogenicity of NSCLC cells up to 67% and 85%, respectively, as compared to the erlotinib single treatment. Furthermore, CINN +ERL decreased the migratory ability of A549 cells by 3-fold and further induced much more apoptotic cell death phenotypes. Conclusion: in summary, co-administration of 2DG or cinnamic acid with erlotinib increases the inhibitory effects of erlotinib on NSCLC cell tumorigenicity and migration.

IL-18 serves as a main effector of CAF-derived METTL3 against immunosuppression of NSCLC via driving NF-κB pathway

ABSTRACT Background: N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNA. However, its role in non-small cell lung cancer (NSCLC) has not been completely elucidated. Objective: To explore whether methyltransferase like 3 (METTL3) in cancer associated fibroblasts (CAFs) affects the secretion of IL-18, which drives NSCLC cells to regulate PD-L1-mediated immunosuppression via the nuclear factor kappa B (NF-κB) pathway. Methods: Histopathological features of NSCLC tissues were identified by H&E and IHC staining. The levels of m6A writers (METTL3), IL-18 and NF-κB pathway related genes were assessed. The quantity of CD8+ T cells was evaluated by flow cytometry (FCM). The direct binding relationship between METTL3 and IL-18 mRNA was detected by RIP assay and RNA pulldown and confirmed by dual – luciferase reporter assay. The level of RNA m6A was detected by RNA m6A dot blot and meRIP assays. A heterotopic implantation model of NSCLC was established in NOD-SCID mice for further explore the effect of CAF derived METTL3 on immunosuppression of NSCLC in vivo. Results: Our results illustrated that METTL3 was down-regulated in CAFs, and CAF derived METTL3 alleviated PD-L1-mediated immunosuppression of NSCLC through IL-18. Subsequently, we found that IL-18 was main effector of CAF-derived METTL3 against immunosuppression of NSCLC, and IL-18 accelerated immunosuppression of NSCLC by driving NF-κB pathway. In vivo, METTL3 knockdown-derived CAFs accelerated immunosuppression of NSCLC. Conclusion: IL-18 served as a main effector of CAF-derived METTL3 against immunosuppression of NSCLC via driving NF-κB pathway.

A redox-responsive nanosystem to suppress chemoresistant lung cancer through targeting STAT3

Cancer stem cells (CSCs) have been demonstrated to be involved in tumor initiation and relapse, and the presence of CSCs in the tumor tissue often leads to therapeutic failure. BBI608 has been identified to eliminate CSCs by inhibiting signal transducer and activator of transcription 3 (STAT3). In this study, we confirm that BBI608 can efficiently suppress the proliferation and migration of non-small cell lung cancer (NSCLC) cells, and specifically kill the stemness-high population in chemoresistant NSCLC cells. To improve its bioavailability and tumor accumulation, BBI608 is successfully encapsulated into redox-responsive PEGylated branched N-(2-hydroxypropyl) methacrylamide (HPMA)-deoxy cholic acid (DA) polymeric nanoparticles (BBI608-SS-NPs). The BBI608-SS-NPs can release the drug in response to high concentrations of intracellular glutathione, and exhibit cytotoxicity against lung cancer cells and CSCs comparable to the free drug BBI608. Furthermore, the BBI608-SS-NPs preferentially accumulate in tumor sites, resulting in a superior anti-tumor efficacy in both cisplatin-resistant cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models of NSCLC. Mechanistic studies demonstrate that BBI608-SS-NPs not only directly inhibit the downstream genes of the STAT3 pathway, but also indirectly inhibit the Wnt pathway. Overall, this stimuli-responsive polymeric nanoformulation of BBI608 shows great potential in the treatment of chemoresistant NSCLC by targeting CSCs.

C-MYC/METTL3/LINC01006 positive feedback loop promotes migration, invasion and proliferation of non-small cell lung cancer

BackgroundThis study aims to clarify the N6-methyladenosine (m6A) modification of LINC01006, which is involved in migration, invasion and proliferation of non-small cell lung cancer (NSCLC). Materials and methodsLINC01006 and METTL3 expressions were analyzed in TCGA-LUAD cohort. Colony formation assay, wound-healing assay and transwell assay were performed to evaluate the ability of colony formation, migration and invasion. Q-PCR and western blot analysis determined gene expressions. M6A-RNA immunoprecipitation and m6A quantification assay were used to evaluate m6A modification. qChIP assay was used to validate transcriptional target. Luciferase assay validated the miRNA targets and transcriptional targets. In-situ xenograft model were included to evaluate tumor proliferation in vivo. ResultsLINC01006 and METTL3 expressions were elevated in NSCLC cells and tissues. LINC01006 promoted the migration and invasion of NSCLC via epithelial – mesenchymal transition (EMT). The expression of LINC01006 was positively correlated to the expression of METTL3. METTL3 promoted tumor formation and proliferation in the in-situ xenograft model of NSCLC. The expression of LINC01006 was increased by METTL3 via m6A modification. c-MYC directly induced METTL3. Both c-MYC and LINC01006 were commonly targeted by miR-34a/b/c and miR-2682, and thereby c-MYC/METTL3/LINC01006 formed a positive feedback loop through miRNA targets in NSCLC. ConclusionsLINC01006 is an oncogenic lncRNA, which induces migration, invasion and proliferation of NSCLC. METTL3 increases LINC01006 expression through stabilizing LINC01006 mRNA. c-MYC, as a transcription factor, activates METTL3, which results in an elevated level of LINC01006. c-MYC, METTL3 and LINC01006 form a positive feedback loop through multiple miRNA targets in NSCLC.

An antioxidant feedforward cycle coordinated by linker histone variant H1.2 and NRF2 that drives nonsmall cell lung cancer progression

Nonsmall cell lung cancer (NSCLC) is highly malignant with limited treatment options, platinum-based chemotherapy is a standard treatment for NSCLC with resistance commonly seen. NSCLC cells exploit enhanced antioxidant defense system to counteract excessive reactive oxygen species (ROS), which contributes largely to tumor progression and resistance to chemotherapy, yet the mechanisms are not fully understood. Recent studies have suggested the involvement of histones in tumor progression and cellular antioxidant response; however, whether a major histone variant H1.2 (H1C) plays roles in the development of NSCLC remains unclear. Herein, we demonstrated that H1.2 was increasingly expressed in NSCLC tumors, and its expression was correlated with worse survival. When crossing the H1c knockout allele with a mouse NSCLC model (KrasLSL-G12D/+), H1.2 deletion suppressed NSCLC progression and enhanced oxidative stress and significantly decreased the levels of key antioxidant glutathione (GSH) and GCLC, the catalytic subunit of rate-limiting enzyme for GSH synthesis. Moreover, high H1.2 was correlated with the IC50 of multiple chemotherapeutic drugs and with worse prognosis in NSCLC patients receiving chemotherapy; H1.2-deficient NSCLC cells presented reduced survival and increased ROS levels upon cisplatin treatment, while ROS scavenger eliminated the survival inhibition. Mechanistically, H1.2 interacted with NRF2, a master regulator of antioxidative response; H1.2 enhanced the nuclear level and stability of NRF2 and, thus, promoted NRF2 binding to GCLC promoter and the consequent transcription; while NRF2 also transcriptionally up-regulated H1.2. Collectively, these results uncovered a tumor-driving role of H1.2 in NSCLC and indicate an "H1.2-NRF2" antioxidant feedforward cycle that promotes tumor progression and chemoresistance.

CCL21-DC in situ vaccination in murine NSCLC overcomes resistance to immunotherapy and generates systemic tumor-specific immunity

BackgroundDespite recent advances in immunotherapy, many patients with non-small cell lung cancer (NSCLC) do not respond to immune checkpoint inhibitors (ICI). Resistance to ICI may be driven by suboptimal priming...