Non-small Cell Lung Cancer Cancer Cell Research Articles

Predictors of lung cancer subtypes and lymph node status in non-small-cell lung cancer: intravoxel incoherent motion parameters and extracellular volume fraction

ObjectiveTo determine the performance of intravoxel incoherent motion (IVIM) parameters and the extracellular volume fraction (ECV) in distinguishing between different subtypes of lung cancer and predicting lymph node metastasis (LNM) status in patients with non-small-cell lung cancer (NSCLC).MethodsOne hundred sixteen patients with lung cancer were prospectively recruited. IVIM, native, and postcontrast T1 mapping examinations were performed, and the T1 values were measured to calculate the ECV. The differences in IVIM parameters and ECV were compared between NSCLC and small-cell lung cancer (SCLC), adenocarcinoma (Adeno-Ca) and squamous cell carcinoma (SCC), and NSCLC without and with LNM. The assessment of each parameter’s diagnostic performance was based on the area under the receiver operating characteristic curve (AUC).ResultsThe apparent diffusion coefficient (ADC), true diffusion coefficient (D), and ECV values in SCLC were considerably lower compared with NSCLC (all p < 0.001, AUC > 0.887). The D value in SCC was substantially lower compared with Adeno-Ca (p < 0.001, AUC = 0.735). The perfusion fraction (f) and ECV values in LNM patients were markedly higher compared with those without LNM patients (p < 0.01, < 0.001, AUC > 0.708).ConclusionIVIM parameters and ECV can serve as non-invasive biomarkers for assisting in the pathological classification and LNM status assessment of lung cancer patients.Critical relevance statementIVIM parameters and ECV demonstrated remarkable potential in distinguishing pulmonary carcinoma subtypes and predicting LNM status in NSCLC.Key PointsLung cancer is prevalent and differentiating subtype and invasiveness determine the treatment course.True diffusion coefficient and ECV showed promise for subtyping and determining lymph node status.These parameters could serve as non-invasive biomarkers to help determine personalized treatment strategies.Graphical

Abstract LB008: Integrative dynamic MultiOMICs analysis identifies cell cycle and DNA damage response phenotypes as downstream consequences of using the PRMT5 inhibitor AZD3470

Abstract Background: PRMT5 is an epigenetic enzyme that catalyzes symmetric di-methylation of arginine (SDMA) of multiple substrates that regulate biological processes including RNA splicing and cell cycle. The role of PRMT5 in controlling chromatin accessibility has been investigated in some cancer contexts, but its role in non-small cell lung cancer (NSCLC) global chromatin regulation is not known. AstraZeneca has developed an MTA-cooperative PRMT5 inhibitor (AZD3470) that selectively inhibits PRMT5 in MTAP-null tumors and currently is in Phase I clinical trial (NCT06130553, NCT06130553). Here we applied integrative dynamic MultiOMICs analysis upon AZD3470 treatment in six MTAP-null NSCLC cancer cell lines to inform on PRMT5’s function in maintaining tumor cell growth and survival. Methods: Utilizing an integrative approach, our study explored the effects of AZD3470 on human MTAP-deleted NSCLC cells. MultiOMICs analysis including SDMA post-translational modification (PTM) scans, ATAC-seq, RNA-seq, and mass spectrometry proteomics were employed to delineate the immediate effects and downstream phenotypes of AZD3470 PRMT5 inhibition. Results: AZD3470 induced significant SDMA modification of splicing factors consistent with known roles for PRMT5. PRMT5 inhibition by AZD3470 led to changes in global chromatin accessibility in NSCLC cells. Specific changes in genes involved in the cell cycle pathway were observed including CDK6. Integrative analysis of transcriptomics and proteomics data consistently showed alterations in cell cycle, DNA damage repair and splicing related pathways upon treatment, consistent with the established biological roles of PRMT5. Alternative splicing analysis showed that AZD3470 treatment induced a unique retained intron within the ATM gene and resulted in reduced ATM protein levels. Conclusion: Here we identify AZD3470-induced changes to protein post-translational modification, chromatin accessibility, gene expression and RNA splicing in MTAP-deficient NSCLC cancer cell lines. Different area of MultiOMICs data together pointed to changes in cell cycle and DNA repair pathways. Further, we identified alternative splicing of ATM as a consequence of AZD3470 PRMT5i treatment resulting in significant reduction of ATM protein expression. Keywords: AZD3470, NSCLC, PRMT5 Inhibition, Chromatin Accessibility, Alternative Splicing, DNA Repair, ATM Gene. Citation Format: Ted Hong, Steven Criscione, Andrew Jarnuczak, Jelena Urosevic, Stephanie Ashenden, Ghaith Hamza, Praveen Kumar, Nevena Cvetesic, Lauren Bradshaw, Shaun Moore, Daniel Karl, Tianhui Zhang, Abel Sousa, Jonathan Cairns, Anthony Iannetta, Andrew Zhang, Eric Miele, John Reicha, Chris Chambers, Ramy Elgendy, Julia Lindgren, Daniel Jachimowicz, Maryam Clausen, Graham Belfield, Tony Cheung, Michael Grondine, James T. Lynch, Ho Man Chan, Susan Critchlow, Emma Dean. Integrative dynamic MultiOMICs analysis identifies cell cycle and DNA damage response phenotypes as downstream consequences of using the PRMT5 inhibitor AZD3470 [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 LB008.

Abstract 2617: BCG022, a novel HER3 × MET bispecific antibody-drug conjugate (bsADC), demonstrates promising anti-tumor efficacy

Abstract HER3 and MET expression act as bypass resistance mechanisms, conferring resistance to multiple therapeutic agents, such as EGFR TKI treatment. HER3 and MET are also co-expressed at high prevalence in multiple tumor types, including gastric, colorectal, breast, and non-small-cell lung cancer (NSCLC). In addition, HER3 and MET are frequently overexpressed in liver metastases from patients with colorectal cancer, indicating that targeting both targets may provide clinical benefit. We previously reported generation of a fully human bispecific antibody (bsAb) targeting HER3 and MET using RenLite® mice, which contain the full human heavy chain variable domain with a common human kappa light chain to facilitate bispecific antibody assembly. This bsAb demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including NSCLC, gastric, colorectal, and hepatocellular cancer cell lines. The bsAb also showed enhanced internalization activity compared to parental monovalent antibodies in the NUGC-4 cell line expressing HER3 and MET. Here, we evaluated the efficacy of the bsAb when conjugated to two distinct payloads: vcMMAE and BLD1102, Biocytogen’s novel, proprietary linker/payload system composed of a DNA Topo I inhibitor payload (BCPT02) and a highly hydrophilic protease-cleavable linker. The in vivo efficacy of BCG022-vcMMAE and BCG022-BLD1102 was evaluated in cell-derived NSCLC and gastric cancer xenografts, and in patient-derived gastric and pancreatic xenograft models. Both demonstrated strong anti-tumor activity, with efficacy greater than or comparable to benchmark ADCs. Taken together, these promising preclinical results suggest that the BCG022 bsADC could be a novel treatment for HER3 and MET co-expressing tumors. Citation Format: Zhuolin Li, Chengzhang Shang, Zhenyan Han, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. BCG022, a novel HER3 × MET bispecific antibody-drug conjugate (bsADC), demonstrates promising anti-tumor efficacy [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 2617.

Auranofin repurposing for lung and pancreatic cancer: low CA12 expression as a marker of sensitivity in patient-derived organoids, with potentiated efficacy by AKT inhibition

BackgroundThis study explores the repurposing of Auranofin (AF), an anti-rheumatic drug, for treating non-small cell lung cancer (NSCLC) adenocarcinoma and pancreatic ductal adenocarcinoma (PDAC). Drug repurposing in oncology offers a cost-effective and time-efficient approach to developing new cancer therapies. Our research focuses on evaluating AF's selective cytotoxicity against cancer cells, identifying RNAseq-based biomarkers to predict AF response, and finding the most effective co-therapeutic agents for combination with AF.MethodsOur investigation employed a comprehensive drug screening of AF in combination with eleven anticancer agents in cancerous PDAC and NSCLC patient-derived organoids (n = 7), and non-cancerous pulmonary organoids (n = 2). Additionally, we conducted RNA sequencing to identify potential biomarkers for AF sensitivity and experimented with various drug combinations to optimize AF's therapeutic efficacy.ResultsThe results revealed that AF demonstrates a preferential cytotoxic effect on NSCLC and PDAC cancer cells at clinically relevant concentrations below 1 µM, sparing normal epithelial cells. We identified Carbonic Anhydrase 12 (CA12) as a significant RNAseq-based biomarker, closely associated with the NF-κB survival signaling pathway, which is crucial in cancer cell response to oxidative stress. Our findings suggest that cancer cells with low CA12 expression are more susceptible to AF treatment. Furthermore, the combination of AF with the AKT inhibitor MK2206 was found to be particularly effective, exhibiting potent and selective cytotoxic synergy, especially in tumor organoid models classified as intermediate responders to AF, without adverse effects on healthy organoids.ConclusionOur research offers valuable insights into the use of AF for treating NSCLC and PDAC. It highlights AF's cancer cell selectivity, establishes CA12 as a predictive biomarker for AF sensitivity, and underscores the enhanced efficacy of AF when combined with MK2206 and other therapeutics. These findings pave the way for further exploration of AF in cancer treatment, particularly in identifying patient populations most likely to benefit from its use and in optimizing combination therapies for improved patient outcomes.

Investigations of the novel checkpoint kinase 1 inhibitor SRA737 in non-small cell lung cancer and colorectal cancer cells of differing tumour protein 53 gene status

Aim: In response to DNA damage the serine/threonine-specific protein kinase checkpoint kinase 1 (CHK1) is activated allowing cells to enter S phase (S) and G2 phase (G2) cell-cycle arrest. CHK1 inhibitors are expected to prevent cells from entering such arrest, thereby enhancing DNA damage-induced cytotoxicity. In contrast, normal cells with intact ataxia–telangiectasia mutated (ATM), CHK2 and tumour suppressor protein 53 (P53) signalling are still able to enter cell-cycle arrest using the functioning G1/S checkpoint, thereby being rescued from enhanced cytotoxicity. The main objective of this work is to investigate the in vitro effects of the novel CHK1 inhibitor SRA737 on pairs of non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines, all with genetic aberrations rendering them susceptible to replication stress but of differing tumour protein 53 (TP53) gene status, focusing on DNA damage induction and the subsequent effects on cell proliferation and viability. Methods: NSCLC cell lines H23 [TP53 mutant (MUT)] and A549 [TP53 wild-type (WT)] and CRC cell lines HT29 (TP53 MUT) and HCT116 (TP53 WT) were incubated with differing micromolar concentrations of SRA737 for 24 h and then analysed using alkaline comet and phosphorylated H2A.X variant histone (γH2AX)-foci assays to assess mostly DNA single strand break and double strand break damage, respectively. Cell-counting/trypan blue staining was also performed to assess cell proliferation/viability. Results: Clear concentration-dependent increases in comet formation and γH2AX-foci/cell were noted for the TP53 MUT cells with no or lower increases being noted in the corresponding TP53 WT cells. Also, greater anti-proliferative and cell killing effects were noted in the TP53 MUT cells than in the TP53 WT cells. Conclusions: This study’s data suggests that P53 status/functioning is a key factor in determining the sensitivity of NSCLC and CRC cancer cells towards CHK1 inhibition, even in circumstances conducive to high replicative stress.

Marine Antimicrobial Peptide Epinecidin-1 Inhibits Proliferation Induced by Lipoteichoic acid and Causes cell Death in non-small cell lung cancer Cells via Mitochondria Damage.

Non-small cell lung cancer (NSCLC) is among the deadliest cancers worldwide. Despite the recent introduction of several new therapeutic approaches for the disease, improvements in overall survival and progression-free survival have been minimal. Conventional treatments for NSCLC include surgery, chemotherapy and radiotherapy. Except for surgery, these treatments can impair a patient's immune system, leaving them susceptible to bacterial infections. As such, Staphylococcus aureus infections are commonly seen in NSCLC patients receiving chemotherapy, and a major constituent of the S. aureus cell surface, lipoteichoic acid (LTA), is thought to stimulate NSCLC cancer cell proliferation. Thus, inhibition of LTA-mediated cell proliferation might be a useful strategy for treating NSCLC. Epinecidin-1 (EPI), a marine antimicrobial peptide, exhibits broad-spectrum antibacterial activity, and it also displays anti-cancer activity in glioblastoma and synovial sarcoma cells. Furthermore, EPI has been shown to inhibit LTA-induced inflammatory responses in murine macrophages. Nevertheless, the anti-cancer and anti-LTA activities of EPI and the underlying mechanisms of these effects have not been fully tested in the context of NSCLC. In the present study, we demonstrate that EPI suppresses LTA-enhanced proliferation of NSCLC cells by neutralizing LTA and blocking its effects on toll-like receptor 2 and interleukin-8. Moreover, we show that EPI induces necrotic cell death via mitochondrial damage, elevated reactive oxygen species levels, and disrupted redox balance. Collectively, our results reveal dual anti-cancer activities of EPI in NSCLC, as the peptide not only directly kills cancer cells but it also blocks LTA-mediated enhancement of cell proliferation.

Use of a systems-biology informed machine learning model to predict drug response using clinically available NGS data.

3136 Background: Tremendous progress towards understanding the molecular underpinnings of cancer has ushered in an era of precision medicine, where actionable biomarkers inform rational design of novel drugs, repurposing of existing medications or new combination therapies, agnostic of tumor type. However less than 20% of cancer patients match to therapies using existing predictive biomarkers. There is therefore an acute need for new methods to expand the population of patients who could benefit from existing drugs and improve patient selection and design of clinical trials evaluating new or repurposed drugs. Methods: We developed systems biology-informed AI methods trained on vast multi-omics and multi-modal datasets from DepMap, TCGA and other publicly available sources, to uncover latent vulnerabilities existing in tumors. These vulnerabilities map to pharmacological sensitivities and can be assigned to individual patients using clinically available molecular data. To evaluate our model, we focused on predicting sensitivities to a third-generation tyrosine kinase inhibitor (TKI), which we assessed in non-small cell lung cancer (NSCLC) cell lines, patient derived xenografts (PDXs) and a clinicogenomics cohort from AACR Project GENIE (n=2004 patients, n=273 patients treated with TKI of interest). Where applicable, whole exome data used as input for validation model predictions were filtered to ~300 genes included on commercial NGS panels to reflect data available in RWD cohorts. Results: Our model accurately predicts response to the TKI across diverse cancer cell lines with high accuracy (r2= 0.768; rho= 0.83; mean absolute error = 0.065). Using NSCLC cancer cell lines and PDX model systems heterogeneous for this TKI’s current FDA molecular label (but within the scope of the drug’s current cancer indication), we were able to further validate our model’s predictions, show they were not dependent on obvious molecular confounders, and demonstrate a potential label expansion opportunity for this drug beyond the current narrow molecular label. While all NSCLC patients from AACR Project GENIE treated with this TKI were “on label”, NSCLC patients our model predicted to be sensitive to the TKI had a 4-month increased median PFS compared to patients predicted to be less sensitive (Log-rank test p-value&lt; 0.05; HR= 1.64; 1.23-2.22, 95% CI). The PFS partition was not due to obvious clinical confounders such as stage (p= 0.91), age (p=0.86), sex (p=0.63), smoking status (p=0.57) or on-target mutations (p=0.70). Biological networks associated with each prediction provide rich context and highlight a putative rational drug combination strategy for this drug. Conclusions: We have developed an ML model that uses clinically obtainable NGS data to accurately predict increased sensitivity to a third-generation TKI validated in NSCLC cell lines, PDX models and a RWD patient cohort.

TRIM58 Interacts with ZEB1 to Suppress NSCLC Tumor Malignancy by Promoting ZEB1 Protein Degradation via UPP.

Currently, how to successfully control refractory and metastatic diseases remains a fundamental goal for clinicians to improve therapeutic effects for patients with non-small cell lung cancer (NSCLC). Several studies have discovered that TRIM58, a member of tripartite motif protein family, shows antitumor effect in multiple types of cancer. In this study, we aimed to further clarify the molecular regulatory network of TRIM58 and corresponding targets for NSCLC patients. TRIM58 expression in clinical tumor tissue samples and cancer cell lines was examined. Functional experiments including cellular invasion, cell metastasis, chemoresistance assay, and ubiquitination evaluation experiments were conducted to investigate the interaction between TRIM58 and ZEB1, which is a prime element of transcription factor network that controls epithelial-to-mesenchymal transition. TRIM58 expression was characteristically decreased in NSCLC tumor tissues and cancer cell lines. Functional experiments demonstrated that TRIM58 suppression enhanced malignant biological behaviors including cellular survivability, migration, and invasion, as well as stem-like cellular phenotype of tumor cells. TRIM58 silencing also significantly enhanced the chemoresistance of NSCLC cells to chemoagents. TRIM58-ZEB1 interaction accelerated degradation of ZEB1 protein, thus further leading to the augment of tumor behaviors. Further detailed molecular experiments revealed that the interaction between TRIM58 and ZEB1 was mediated via ubiquitin-proteasome pathway (UPP). TRIM58 suppressed NSCLC through interacting with ZEB1 and promoting ZEB1 protein degradation via UPP. The present research sheds light on the interaction between TRIM58 and ZEB1, and TRIM58/ZEB1 axis might be the potential therapeutic targets of NSCLC.

Integrated network pharmacology and experimental analysis unveil multi-targeted effect of 18α- glycyrrhetinic acid against non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most malignant types of cancer with soaring incidence rates worldwide, attributed to its heterogeneity and complex etiology. Evidently, alternative anti-cancer therapies comprising traditional medicines and natural products have gained attention for their ability to act as chemopreventive agents with minimal toxicities, either alone or in combination. Accumulating studies have substantiated the inevitability of network pharmacology studies for effectively mapping molecular targets of natural products against multifaceted diseases, including cancer. The 18α-Glycyrrhetinic acid (18α-GA), a triterpenoid found in licorice plants, has shown promising medicinal properties, although, its mechanism of action against NSCLC yet remains elusive. The present study was conducted to explore the anti- NSCLC potential of 18α-GA, employing integrative network pharmacology, molecular docking, and experimental research. Initially, network analysis revealed 181 common targets of 18α-GA in NSCLC as shown in the “compound-target- disease” network employing Cytoscape 3.8.2. Further analyses identified EGFR, AKT1, PI3KR1, MAPK1, IGF1, and SRC as the most crucial hub targets of 18α-GA against NSCLC. Moreover, molecular docking simulations and functional enrichment analyses indicated the involvement of multiple signaling pathways in suppressing NSCLC. Subsequent in-vitro studies verified the antiproliferative effect of 18α-GA on two NSCLC cancer cell lines, H1299 and A549. Mechanistically, 18α-GA arrested cell cycle at the G1 phase, induced apoptosis, decreased migratory potential, and protein expression levels of EGFR-PI3K/AKT, as examined by flow cytometry, morphological assessment, RT-PCR, and western blot. In conclusion, this study delineates the therapeutic potential and underlying mechanism(s) of 18α-GA as a putative novel drug against NSCLC. However, further studies are warranted to elucidate the complete molecular mechanism(s) using animal models of NSCLC.

Circ_0000808 promotes the development of non-small cell lung cancer by regulating glutamine metabolism via the miR-1827/SLC1A5 axis

BackgroundCircular RNA (circRNA) has been proved to be an important molecular target for cancer treatment. However, the function and molecular mechanism of circ_0000808 in non-small cell lung cancer (NSCLC) are still unclear.MethodsQuantitative real-time PCR was used to detect the expression of circ_0000808, miR-1827, and solute carrier family 1 member 5 (SLC1A5). Cell proliferation, apoptosis, migration, and invasion were measured by cell counting kit 8 assay, colony formation assay, EdU staining, flow cytometry, wound healing assay, and transwell assay. The protein expression was measured by Western blot analysis. Dual-luciferase reporter assay and RIP assay were used to investigate the interactions between miR-1827 and circ_0000808 or SLC1A5. Cell glutamine metabolism was assessed by determining glutamine uptake, glutamate production, and α-ketoglutarate production. Xenograft mouse model was used to assess the in vivo effects of circ_0000808.ResultsCirc_0000808 expression was upregulated in NSCLC tissues and cancer cells, and its silencing inhibited NSCLC cell proliferation, migration, and invasion and led to apoptosis. Further results confirmed that circ_0000808 interacted with miR-1827 to positively regulate SLC1A5. The rescue experiments showed that miR-1827 inhibitor reversed the suppressive effect of circ_0000808 knockdown on the malignant behaviors of NSCLC cells. Also, SLC1A5 overexpression abolished the inhibition effect of miR-1827 on NSCLC cell progression. In addition, circ_0000808/miR-1827/SLC1A5 axis positively regulated the glutamine metabolism process in NSCLC cells. Moreover, circ_0000808 knockdown reduced the NSCLC tumor growth in vivo.ConclusionIn summary, our data showed that circ_0000808 enhanced the progression of NSCLC by promoting glutamine metabolism through the miR-1827/SLC1A5 axis.

185 (PB065) - NUC-3373 is a potent TS inhibitor and induces DNA damage in NSCLC cancer cells regardless of histological subtype

Background: Non-small cell lung cancer (NSCLC) represents >80% of lung cancer cases and is the leading cause of cancer mortality. It is comprised of three histological subtypes: adenocarcinoma (50–65%), squamous cell carcinoma (30–40%) and large cell carcinoma (5–15%). The majority of NSCLCs do not harbour actionable mutations, so treatment options include immune checkpoint inhibitors (ICIs) alone or in combination with chemotherapies. ICI/chemotherapy combinations have resulted in significant overall survival benefit compared to chemotherapy alone in both adenocarcinoma and squamous histology.

Oxysterol derivatives Oxy186 and Oxy210 inhibit WNT signaling in non-small cell lung cancer

BackgroundDevelopmental signaling pathways such as those of Hedgehog (HH) and WNT play critical roles in cancer stem cell self-renewal, migration, and differentiation. They are often constitutively activated in many human malignancies, including non-small cell lung cancer (NSCLC). Previously, we reported that two oxysterol derivatives, Oxy186 and Oxy210, are potent inhibitors of HH/GLI signaling and NSCLC cancer cell growth. In addition, we also showed that Oxy210 is a potent inhibitor of TGF-β/SMAD signaling. In this follow-up study, we further explore the mechanism of action by which these oxysterols control NSCLC cell proliferation and tumor growth.ResultsUsing a GLI-responsive luciferase reporter assay, we show here that HH ligand could not mount a signaling response in the NSCLC cell line A549, even though Oxy186 and Oxy210 still inhibited non-canonical GLI activity and suppressed the proliferation of A549 cells. Further, we uncover an unexpected activity of these two oxysterols in inhibiting the WNT/β-catenin signaling at the level of LRP5/6 membrane receptors. We also show that in a subcutaneous xenograft tumor model generated from A549 cells, Oxy186, but not Oxy210, exhibits strong inhibition of tumor growth. Subsequent RNA-seq analysis of the xenograft tumor tissue reveal that the WNT/β-catenin pathway is the target of Oxy186 in vivo.ConclusionThe oxysterols Oxy186 and Oxy210 both possess inhibitory activity towards WNT/β-catenin signaling, and Oxy186 is also a potent inhibitor of NSCLC tumor growth.

Abstract 4026: JDQ443, a covalent inhibitor of KRASG12C with a novel binding mode, shows broad antitumor activity in KRASG12C preclinical models as a single agent and in combination with inhibitors of SHP2, MEK or CDK4/6

Abstract Background: Oncogenic mutations occurring in the KRAS component of the RAS/MAPK pathway slow nucleotide cycling between its active (GTP-bound) and inactive (GDP-bound) states, shifting it towards the active state and increasing oncogenic signaling. Among these mutations, the glycine-to-cysteine mutation of amino acid 12 (KRASG12C), found in ~13% of non-small cell lung cancer (NSCLC) and ~4% of colorectal cancer (CRC), can be specifically targeted and irreversibly locked in the inactive state by covalent modification of Cys12. We have previously reported the discovery and preclinical profile of JDQ443, a selective, oral, covalent inhibitor of KRASG12C that binds under the Switch II loop. Here, we report its antiproliferative and antitumor activity against panels of cancer cell lines as well as cell- (CDX) and patient-derived (PDX) tumor xenografts. Methods: JDQ443 antiproliferative activity was assessed by a high-throughput cell viability assay in a large panel of KRASG12C (n=17) and non-G12C (n=233) cell lines. Single-agent antitumor activity was assessed against a panel of KRASG12C CDX models from NSCLC (LU99, H2122, H2030, and HCC44), pancreatic (Mia PaCa-2), and esophageal (KYSE410) cancer cell lines, plus one non-G12C lung line (H441; KRASG12V). JDQ443 in vivo activity against a panel of KRASG12C NSCLC (n=10) and CRC (n=9) PDX models was assessed either as a single agent or in combination with TNO155 (SHP2 inhibitor [i]), trametinib (MEKi), or ribociclib (CDK4/6i). In vivo combination studies with TNO155 were also performed in CDX models (LU99, H2030, HCC44, and KYSE410). Results: In vitro, JDQ443 demonstrated potent antiproliferative activity selectively towards KRASG12C cell lines. Dose-dependent tumor growth inhibition/regression was observed for all KRASG12C CDX models, but not for the H441 KRASG12V model, and was independent of once-daily (QD) or twice-daily (BID) dosing. Single-agent antitumor activity (best average response, duration of reduction in tumor doubling time) was observed across both PDX panels and was improved by all three combination treatments. The JDQ443/TNO155 combination improved single-agent activity across CDX models, with comparable antitumor benefit maintained at QD or BID TNO155 schedules in two of three models (LU99 and KYSE410). Combination with TNO155 allowed a reduced dose of JDQ443 to achieve similar target occupancy and antitumor activity versus JDQ443 alone. Conclusions: JDQ443 demonstrates significant activity against a broad range of KRASG12C solid tumor models, both in vitro and in vivo, that is increased when combined with agents targeting both upstream and downstream components of the RAS signaling pathway. The combination benefit of JDQ443 + TNO155 over JDQ443 alone is maintained at reduced doses for both agents. Citation Format: Andreas Weiss, Hans Voshol, Diana Graus Porta, Carmine Fedele, Dario Sterker, Ruben De Kanter, Rowan Stringer, Toni Widmer, Alice Loo, Daniel A. Guthy, Kim S. Beyer, Nils Ostermann, Catherine LeBlanc, Marc Gerspacher, Andrea Vaupel, Richard Sedrani, Frederic Zecri, Saveur-Michel Maria, Francesco Hofmann, Peter Hammerman, Jeff Engelman, Edwige Lorthiois, Simona Cotesta, Saskia M. Brachmann. JDQ443, a covalent inhibitor of KRASG12C with a novel binding mode, shows broad antitumor activity in KRASG12C preclinical models as a single agent and in combination with inhibitors of SHP2, MEK or CDK4/6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4026.

Abstract 6138: High Globo-H expression associated with poor survival of gastric cancer patients and enriched PD-L1 expression

Abstract Background: Globo series antigens have been implicated in playing important roles in enhancing tumor growth through protecting tumor cells from apoptosis, suppressing T cell activity in the tumor microenvironment and promoting endothelial cell angiogenesis. Globo-H (GH), a globo-series glycosphingolipid (GSL) antigen that is synthesized by key enzymes β1,3-galactosyltransferase V (β3GalT5), fucosyltransferase (FUT) 1 and 2, is highly expressed on a variety of epithelial cancers rendering it a promising target for cancer immunotherapy. GH is reported to associate the EGFR mutant and PD-L1 expression in non-small cell lung cancer (NSCLC) patients. In addition, GH-targeting antibody-drug conjugate (ADC) OBI-999 has been demonstrated an excellent tumor growth inhibition potency in animal models across multiple cancer types including gastric cancer (GC). This study aims to further investigate the GH roles in GC. Methods: Prognostic roles of β3GalT5, FUT1 and FUT2 in GC patients were accessed through the “Kaplan-Meier plotter” database. The level of GH expression was evaluated in clinical adenocarcinoma samples from 105 patients with GC by immunohistochemistry (IHC) using H-score. Microwestern protein array analysis was performed in sorted GC NCI-N87 cells. FACS methods were also employed to measure the levels of PD-L1 and phospho-EGFRs in multiple cancer cell lines. Results: Significant correlations were observed between high mRNA expression of synthesized key enzymes and worse overall survival (OS)/post-progression survival for all 498 GC patients. Positive GH expression (H score≥20; 33.3%) was significantly associated with a poor disease specific survival in all samples (at 15 years; p=0.029), a poor OS in poorly differentiated tumors (p=0.033), and invasiveness (p=0.013), indicating GH expression was a potential prognostic factor in GC. Sorted NCI-N87 cells with high level of endogenous GH expression showed a relatively greater proliferative activity compared with cells expressing low level of GH. Microwestern array analysis on sorted cells indicated upregulations of phospho-AKT/P38/JNK, Cyclin D1 and Cyclin E1 protein in high GH expression NCI-N87 cells, which implicated a mechanistic link between high GH and tumor promoting signals in GC. In addition, GH level was shown to be correlated with cell surface expression level of PD-L1 in NCI-N87, SNU-16, HCC-1428, ACHN, and TKI-resistant H1975 NSCLC cancer cells. Higher phospho-EGFRs (Tyr1068 and Tyr1173) were detected in high GH level of H1975 cells, which is consistent with the results from previous studies showing association between GH level and EGFR mutant/PD-L1 expression in NSCLC. Conclusions: GH level was associated with the survival of GC patients and positively correlated with cell surface PD-L1 expression in vitro. Therefore, GH-targeting therapy has a potential application for the treatment of GC patients. Citation Format: Tzer-Min Kuo, Hui-Ting Hsu, Cheng-Yen Wei, Ming-Tain Lai, Chun-Te Chen. High Globo-H expression associated with poor survival of gastric cancer patients and enriched PD-L1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6138.

Suppression of CX3CL1 by miR-497-5p inhibits cell growth and invasion through inactivating the ERK/AKT pathway in NSCLC cells

ABSTRACT Non-small cell lung cancer (NSCLC) is the most common lung cancer with a highest mortality rate. MiR-497-5p has been reported as tumor suppressor in many cancers, but the role and mechanism of miR-497-5p in regulating NSCLC progression are still largely unknown in vitro and in vivo. Here, miR-497-5p was significantly downregulated in human NSCLC tissues and cell lines, compared with matched adjacent tissues and normal lung epithelial cell line. Then, miR-497-5p mimic and inhibitor were, respectively, transfected into human NSCLC cells A549 and H460, CCK-8 assay, transwell assay, and flow cytometry were used to detect the capacities of cell proliferation, invasion and apoptosis. MiR-497-5p negatively regulated proliferation and invasion of NSCLC cancer cells. MiR-497-5p was demonstrated to directly bound to 3’-UTR of CX3CL1 mRNA and post-transcriptionally suppressed its expression thus inactivating its downstream oncogenic pathway ERK/AKT. Moreover, transfection with short hairpin RNA (shRNA) against CX3CL1 decreased capacity of cell proliferation and invasion and promoted cell apoptosis in NSCLC cells. In addition, ERK inhibitor U0126 attenuated the promotion effect of miR-497-5p inhibitor on activation of ERK/AKT and cell proliferation and migration. Finally, overexpression of miR-497-5p substantially suppressed activation of the ERK/AKT pathway and tumor growth in tumor-bearing mice in vivo. Taken together, our findings showed that miR-497-5p is downregulated in human NSCLC tissues and cell lines, and it inhibited tumor growth and cell invasion by targeting CX3CL1 gene to inactivate the ERK/AKT pathway in NSCLC cells.

LINC00852 is associated with poor prognosis in non-small cell lung cancer patients and its inhibition suppresses cancer cell proliferation and chemoresistance via the hsa-miR-145-5p/KLF4 axis.

Emerging evidence shows that long non-coding RNAs (lncRNAs) play important roles in human cancer. In the present study, we examined the expression, prognostic implications and functional roles of a lncRNA, LINC00852 in non-small cell lung cancer (NSCLC). LINC00852 expression was examined by quantitative real-time PCR (qRT-PCR) in both NSCLC clinical samples and in vitro NSCLC cell lines. In patients with NSCLC, postoperative overall survival was estimated according to endogenous LINC00852 expression in their cancerous lung tissues. In NSCLC cell line SW900 and H441 cells, LINC00852 was down-regulated to examine its effects on cancer proliferation, cisplatin chemoresistance and cell-cycle transition in vitro, as well as tumorigenicity in vivo. The potential downstream target of LINC00852, the axis of human microRNA-145-5p (hsa-miR-145-5p) and Kruppel-like factor 4 (KLF4) gene, was investigated in NSCLC, by dual-luciferase assay, qRT-PCR and genetic knockdown functional assays. LINC00852 is up-regulated in both NSCLC tumors and NSCLC cell lines. High LINC00852 expression was significantly correlated with NSCLC patients' short overall survival. In NSCLC cell lines, LINC00852 down-regulation had anti-cancer effects by suppressing cancer cell proliferation, cisplatin chemoresistance and cell-cycle transition in vitro, as well as explant growth in vivo. Moreover, the hsa-miR-145-5p/KLF4 axis was demonstrated to be directly regulated by LINC00852 in NSCLC. Inhibiting hsa-miR-145-5p or overexpressing KLF4 could reverse the LINC00852-down-regulation-induced anti-cancer effects on NSCLC cancer cell proliferation and chemoresistance. LINC00852 may be a prognostic biomarker for NSCLC. The epigenetic signaling pathway of LINC00852/hsa-miR-145-5p/KLF4 may be considered as a novel molecular target for fighting NSCLC.

Abstract 1263: Dual RAF/MEK inhibitor VS-6766 enhances antitumor efficacy of KRAS-G12C inhibitors through a vertical pathway inhibition strategy

Abstract KRAS is one of the most frequently mutated oncogenes in cancer, stimulating tumor growth through activation of the RAS/RAF/MEK/ERK pathway. G12C inhibitors (G12Ci), sotorasib (AMG 510) and adagrasib (MTRX849), have demonstrated promising antitumor activity in patients with KRAS G12C mutant (mt) non-small cell lung cancer (NSCLC). Several recent studies have shown that simultaneous targeting of multiple nodes in the RAS/RAF/MEK/ERK pathway may be optimal for durable pathway inhibition and response. VS-6766 is a unique dual RAF/MEK inhibitor. In contrast to other MEK inhibitors, VS-6766 is a potent allosteric inhibitor of MEK kinase activity which promotes a dominant negative RAF/MEK complex preventing phosphorylation of MEK by wildtype BRAF, BRAF V600E mt and CRAF. This unique mechanism allows VS-6766 to block MEK signaling without the compensatory activation of MEK that appears to limit the efficacy of other inhibitors. Clinical studies are ongoing evaluating VS-6766 alone or in combination with the focal adhesion kinase (FAK) inhibitor defactinib for the treatment of various solid tumors. Here, we tested the hypothesis that vertical pharmacological blockade of RAS, RAF and MEK with G12Ci in combination with VS-6766 might yield superior pathway blockade and antitumor efficacy. In 3D proliferation assays in vitro, VS-6766 was synergistic with both sotorasib and adagrasib in reducing viability of a panel of KRAS G12C mt NSCLC and colorectal cancer cell lines. Accordingly, VS-6766 effectively suppressed RAS pathway signaling (pMEK, pERK, p-p90RSK) across KRAS G12C mt NSCLC cell lines as a single agent, and the combination of VS-6766 + G12Ci showed improved depth and duration of RAS pathway signaling relative to G12Ci alone. Similarly, in the H2122 KRAS G12C mt NSCLC xenograft model, VS-6766 combination with sotorasib showed improved inhibition of pMEK and pERK in tumors relative to sotorasib alone. In KRAS G12C mt NSCLC xenograft models in vivo, combination with VS-6766 (0.3 mg/kg QD) augmented tumor growth inhibition by sotorasib (30 mg/kg QD) in the H2122 KRAS G12C mt NSCLC model, whereas trametinib (0.3 mg/kg QD) was much less effective in augmenting sotorasib efficacy. Sotorasib monotherapy induced &amp;gt;20% tumor regression in 0/10 mice, whereas combination of VS-6766, trametinib or a FAK inhibitor with sotorasib induced &amp;gt;20% tumor regression in 5/10, 1/10 and 4/10 mice, respectively, following 10 days of treatment. Strikingly, triple combination of VS-6766, sotorasib and a FAK inhibitor conferred tumor reductions of ≥35% in 10/10 mice. Similarly, in the H358 KRAS G12C mt NSCLC model, triple combination of VS-6766, sotorasib and a FAK inhibitor induced &amp;gt;35% tumor regression in 10/10 mice following 21 days of treatment. These results support the clinical evaluation of VS-6766 ± defactinib in combination with a G12C inhibitor for treatment of KRAS G12C mt solid tumors. Citation Format: Silvia Coma, Sanjib Chowdhury, Jonathan A. Pachter. Dual RAF/MEK inhibitor VS-6766 enhances antitumor efficacy of KRAS-G12C inhibitors through a vertical pathway inhibition strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1263.

Plumbagin suppresses non-small cell lung cancer progression through downregulating ARF1 and by elevating CD8+ T cells

Non-small cell lung cancer (NSCLC) is one of the most frequently diagnosed cancers and the leading causes of cancer death worldwide. Therefore, new therapeutic agents are urgently needed to improve patient outcomes. Plumbagin (PLB), a natural sesquiterpene present in many Chinese herbal medicines, has been reported for its anti-cancer activity in various cancer cells. In this study, the effects and underlying mechanisms of PLB on the tumorigenesis of NSCLC were investigated. PLB dose-dependently inhibited the growth of NSCLC cell lines. PLB promoted ROS production, activated the endoplasmic reticulum (ER) stress pathway, and induced cell apoptosis, accompanied by the decreased expression level of ADP-ribosylation factor 1 (ARF1) in NSCLC cancer cells, and those effects of PLB could be reversed by the pretreatment with N-acetyl-L-cysteine (NAC). More importantly, the calcium chelator (BM) significantly reversed PLB-induced cell apoptosis. Furthermore, PLB significantly inhibited the growth of both H1975 xenograft and LLC1 tumors and exhibited antitumor activity by enhancing the number and the effector function of CD8+ T cells in KRASLA2 mice model and the LLC1 xenograft. Our findings suggest that PLB exerts potent antitumor activity against NSCLC in vitro and in vivo through ARF1 downregulation and induction of antitumor immune response, indicating that PLB is a new novel therapeutic candidate for the treatment of patients with NSCLC.

Suppressing Lung Cancer Cell Migration and Invasion through Disruption of Rho GTPase Function by Diclofenac and Docosahexaenoic Acid

Background Low survival rates of lung cancer patients and high recurrence rate in non-small cell lung cancer (NSCLC) patients have been attributed to metastasis. The aberrant signaling of GTPases, such as the Rho family of GTPases (Cdc42, Rac1, and RhoA) have been implicated in lung cancer metastasis. Several studies indicate that cyclooxygenases (COX) inhibitors and omega-3 polyunsaturated fatty acids (PUFAs) may help prevent cell migration and invasion, which are critical steps in cancer cell metastasis. We previously reported the inhibitory effect of co-treatment with docosahexaenoic acid (DHA) and diclofenac on the expression of hyperactive small GTPases. This study aimed to investigate the potential anti-metastatic effects of such co-treatment on NSCLC cancer cells as well as to investigate the related mechanisms. Methods We conducted western blotting to detect Rho GTPases (RhoA, Rac1, and CDC42) in A549, NCI-H1299, and NCI-H1975 cells after exposure to DHA and diclofenac. We also conducted immunostaining analyses of F-actin organization and Rho GTPases in A549 cells. We further examined the effect of co-treatment on NSCLC cell migration and invasion. Expression of marker proteins in EMT and MMP-2/9 were detected by western blot analysis. Results Co-treatment with DHA and diclofenac disrupted actin filament assembly as well as inhibited cell migration and invasion. Exposure of A549 cells to DHA (5 μM) and diclofenac (25 μM) resulted in suppression of both the distance of migration by 58.1 ± 2.8% as well as the number of cells that migrated into the wounded area by 62.2 ± 1.8%. In addition, the co-treatment resulted in altered expression of marker proteins involved in EMT by increasing expression of epithelial marker, E-cadherin, while downregulating the expressions of mesenchymal markers, N-cadherin, Fibronectin and Vimentin, ZEB1, and β-catenin. Conclusions Our findings indicate that co-treatment with diclofenac and DHA suppressed NSCLC cell motility and invasion, and is associated with disruption of the actin cytoskeleton and inhibition of the Rho GTPase activity. Our findings suggest that combination of diclofenac and DHA could potentially prevent or control NSCLC metastasis.

Drug-Like Small Molecule HSP27 Functional Inhibitor Sensitizes Lung Cancer Cells to Gefitinib or Cisplatin by Inducing Altered Cross-Linked Hsp27 Dimers.

Relationships between heat shock protein 27 (HSP27) and cancer aggressiveness, metastasis, drug resistance, and poor patient outcomes in various cancer types including non-small cell lung cancer (NSCLC) were reported, and inhibition of HSP27 expression is suggested to be a possible strategy for cancer therapy. Unlike HSP90 or HSP70, HSP27 does not have an ATP-binding pocket, and no effective HSP27 inhibitors have been identified. Previously, NSCLC cancer cells were sensitized to radiation and chemotherapy when co-treated with small molecule HSP27 functional inhibitors such as zerumbone (ZER), SW15, and J2 that can induce abnormal cross-linked HSP27 dimer. In this study, cancer inhibition effects of NA49, a chromenone compound with better solubility, longer circulation time, and less toxicity than J2, were examined in combination with anticancer drugs such as cisplatin and gefitinib in NSCLC cell lines. When the cytotoxic drug cisplatin was treated in combination with NA49 in epidermal growth factor receptors (EGFRs) WT cell lines, sensitization was induced in an HSP27 expression-dependent manner. With gefitinib treatment, NA49 showed increased combination effects in both EGFR WT and Mut cell lines, also with HSP27 expression-dependent patterns. Moreover, NA49 induced sensitization in EGFR Mut cells with a secondary mutation of T790M when combined with gefitinib. Augmented tumor growth inhibition was shown with the combination of cisplatin or gefitinib and NA49 in nude mouse xenograft models. These results suggest the combination of HSP27 inhibitor NA49 and anticancer agents as a candidate for overcoming HSP27-mediated drug resistance in NSCLC patients.