Non-small Cell Lung Cancer Cancer Cell Lines Research Articles

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.

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< 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.

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.

PAQR4 promotes chemoresistance in non-small cell lung cancer through inhibiting Nrf2 protein degradation.

Purpose: Lung cancer is the leading cause of cancer related deaths worldwide. We have previously identified many differentially expressed genes (DEGs) from large scale pan-cancer dataset using the Cross-Value Association Analysis (CVAA) method. Here we focus on Progestin and AdipoQ Receptor 4 (PAQR4), a member of the progestin and adipoQ receptor (PAQR) family localized in the Golgi apparatus, to determine their clinical role and mechanism in the development of non-small cell lung cancer (NSCLC).Methods: The protein expression profile of PAQR4 was examined by IHC using tissue microarrays, and the effects of PAQR4 on cell proliferation, colony formation and xenograft tumor formation were tested in NSCLC cells. Real-time RT-PCR, co-immunoprecipitation (co-IP) and GST-pulldown assays were used to explore the mechanism of action of PAQR4.Results: We provided evidence showing that PAQR4 is increased in NSCLC cancer cell lines (A549, H1299, H1650, H1975, H358, GLC-82 and SPC-A1), and identified many mutations in PAQR4 in non-small cell lung cancer (NSCLC) tissues. We demonstrated that PAQR4 high expression correlates with a worse clinical outcome, and that its knockdown suppresses cell proliferation by inducing apoptosis. Importantly, overexpressed PAQR4 physically interacts with Nrf2 in NSCLC cells, blocking the interaction between Nrf2 and Keap1.Conclusion: Our results suggest that PAQR4 depletion enhances the sensitivity of cancerous cell to chemotherapy both in vitro and xenograft tumor formation in vivo, by promoting Nrf2 protein degradation through a Keap1-mediated ubiquitination process.

MicroRNA-1197 downregulation inhibits proliferation and migration in human non- small cell lung cancer cells by upregulating HOXC11

ObjectivesIn this study, we assessed the expression and functional mechanism of microRNA-1197 (miR-1197) in human non-small cell lung cancer (NSCLC). MethodsIn both NSCLC cell lines and NSCLC human tumors, qRT-PCR was used to assess miR-1197 expression. Through lentiviral transduction, miR-1197 was downregulated in two NSCLC cell lines, H510A and A549 cells. The functional regulations of miR-1197 downregulation on cancer cellin vitro proliferation and migration, as well as in vivo development, were assessed by MTT, transwell and xenograft assays, respectively. The association of miR-1197 on its putative downstream target gene, Homeobox C11 (HOXC11), was assessed by dual-luciferase reporter assay and qRT-PCR, respectively. Finally, HOXC11 was further inhibited in miR-1197-downregulated H510A and A549 cells to assess its mechanistic correlation with miR-1197 in regulating NSCLC in vitro proliferation and migration. ResultsMiR-1197 was discovered to be predominantly upregulated in both NSCLC cancer cell lines and human tumors. MiR-1197 inhibition was able to suppress NSCLCin vitro proliferation and migration, as well as in vivo xenograft development. Biochemical analysis revealed that HOXC11 inversely regulated with miR-1197 in NSCLC. In double infected H510A and A549 cells, whose HOXC11 expression was further inhibited after miR-1197 downregulation, in vitro proliferation and migration were significantly augmented. ConclusionMiR-1197 was upregulated in NSCLC and its downregulation has tumor-suppressing effects in NSCLC, very likely through inverse regulation on downstream target gene of HOXC11.

Osteopontin promotes cancer cell drug resistance, invasion, and lactate production and is associated with poor outcome of patients with advanced non-small-cell lung cancer.

BackgroundOsteopontin (OPN), a member of the small integrin binding ligand N-linked glycoprotein family, has been analyzed in numerous types of human malignancy.PurposeThe present study detected the expression levels of OPN and evaluated its role in tumor progression in patients with non-small cell lung cancer (NSCLC).Patients and methodsOPN expression levels were detected using immunohistochemistry in 101 NSCLC tumors. The mRNA and protein levels have significant difference between advanced NSCLC and stage I/II NSCLC. The drug resistance, invasive ability and lactate production of NSCLC cancer cell lines (A549 and SK-MES-1) were detected in cancer cells with the disturbance of OPN.ResultsImmunostaining indicated that OPN was primarily expressed in the cytoplasm of NSCLC cells. Moreover, OPN correlates with NSCLC clinical traits. The results demonstrated that OPN expression levels significantly correlated with cancer differentiation, distant metastasis and the efficacy of platinum-based treatment. Notably, the results identified OPN expression levels as a potential factor for predicting the response of cells to first-line platinum-based chemotherapy using multivariate analysis, as well as predicting cancer differentiation and distant metastasis. Additionally, the abrogation of OPN levels reduced lactate production in NSCLC cells and occurred along side with the downregulation of lactate dehydrogenase A (LDHA).ConclusionThe results of the current study suggest that OPN may be able to predict poor prognosis and cisplatin resistance in patients.

LncRNA CCHE1 increased proliferation, metastasis and invasion of non-small lung cancer cells and predicted poor survival in non-small lung cancer patients.

Lung cancer is common cancer worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancers. Thus, we studied whether long non-coding RNAs influence malignancy in NSCLC to discover new therapeutic targets for increasing the survival rate of NSCLC patients. lncRNA has been proven to be important in different cancer progression. In this study, we mainly focused on the role of lncRNA CCHE1 (cervical carcinoma high expressed 1) expressed PCNA regulatory lncRNA in NSCLC cancer. qRT-PCR was used to detect the expression of CCHE1 in 126 cases of NSCLC tissue and adjacent tissue, and the clinical significance was also analyzed. Cell Counting Kit-8 (CCK8) assay was used to detect the proliferation ability of NSCLC cancer cell lines, and Western-blot was used to measure the expression of the protein when the expression of CCHE1 was suppressed. We found that the CCHE1 was highly expressed in the NSCLC tissue, compared with the adjacent tissue. We also found that the expression of CCHE1 was closely related to the size and survival time of patients. Moreover, CCHE1 could promote the proliferation, metastasis, and invasion of NSCLC cell line via increasing the expression of ERK/MAPK signaling pathway. According to the results, we found that CCHE1 was closely related the progression of NSCLC, which could be a potential target for treating NSCLC.

Mutant KRAS associated malic enzyme 1 expression is a predictive marker for radiation therapy response in non-small cell lung cancer.

BackgroundAdvanced non-small cell lung cancer (NSCLC) is an aggressive tumor that is treated with a combination of chemotherapy and radiation if the patient is not a candidate for surgery. Predictive biomarkers for response to radiotherapy are lacking in this patient population, making it a non-tailored therapy regimen with unknown outcome. Twenty to 30 % of NSCLC harbor an activating mutation in KRAS that may confer radioresistance. We hypothesized that mutant KRAS can regulate glutamine metabolism genes in NSCLC and maintain tumor redox balance through transamination reactions that generate cytosolic NADPH via malic enzyme 1 (ME1), which may contribute to radioresistance.FindingsA doxycycline-inducible mouse model of KRASG12D driven NSCLC and patient data was analyzed from multiple publicly accessible databases including TCGA, CCLE, NCBI GEO and Project Achilles. ME1 expression was found to be mutant KRAS associated in both a NSCLC mouse model and human NSCLC cancer cell lines. Perturbing glutamine metabolism sensitized mutant KRAS, but not wild-type KRAS NSCLC cell lines to radiation treatment. NSCLC survival analysis revealed that patients with elevated ME1 and GOT1 expression had significantly worse outcomes after radiotherapy, but this was not seen after chemotherapy alone.ConclusionsKRAS driven glutamine metabolism genes, specifically ME1 and GOT1 reactions, may be a predictive marker and potential therapeutic target for radiotherapy in NSCLC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0457-x) contains supplementary material, which is available to authorized users.

Selective expression and immunogenicity of the cancer/testis antigens SP17, AKAP4 and PTTG1 in non-small cell lung cancer: new candidates for active immunotherapy.

ABSTRACT BACKGROUND. Lung cancer is the leading cause of cancer deaths in both genders worldwide, with an incidence only second to prostate cancer in men and breast cancer in women. The lethality of the disease highlights the urgent need for innovative therapeutic options. Immunotherapy can afford efficient and specific targeting of tumor cells, improving efficacy and reducing the side effects of current therapies. We have previously reported the aberrant expression of cancer/testis antigens (CTAs) in tumors of unrelated histological origin. In this study we investigated the expression and immunogenicity of the cancer/testis antigens (CTAs) Sperm Protein 17 (SP17), A-kinase anchor protein 4 (AKAP4) and Pituitary Tumor Transforming Gene 1 (PTTG1) in human non-small cell lung cancer (NSCLC) cell lines and primary tumors. METHODS. We used RT-PCR, immunofluorescence, flow cytometry, ELISA and cytotoxicity assays to determine the expression levels and immunogenicity of SP17, AKAP4 and PTTG1 in human NSCLC cell lines and primary tumors. RESULTS. We found that SP17, AKAP4 and PTTG1 are aberrantly expressed in NSCLC cancer cell lines and primary tumor tissues from patients, compared to normal lung cell lines and tissues. We established the immunogenicity of these CTAs by measuring CTA-specific autoantibodies in patients' sera and generating CTA-specific autologous cytotoxic lymphocytes (CTLs) from patients' peripheral blood mononuclear cells (PBMCs). CONCLUSIONS. Our results provide proof of principle that the CTAs SP17/AKAP4/PTTG1 are expressed in both human NSCLC cell lines and primary tumors and can elicit an immunogenic response in NSCLC patients. Based on our findings, further studies are warranted to explore the feasibility of developing CTA-specific immunotherapeutic strategies for NSCLC patients.

178P - Tumor Endothelial Markers (TEMS) as Prognostic and Predictive Markers in Non-Small Cell Lung Cancer (NSCLC)

ABSTRACT Background Tumor angiogenesis plays a crucial role in tumor development and progression. Genome analyses of endothelial cells identified genes specifically expressed by tumor endothelial cells. These so called tumor endothelial markers (TEMs) are discussed as potential new therapeutic targets or as biomarkers for monitoring anti-angiogenic therapies. In non-small cell lung cancer (NSCLC) the role of TEMs is not yet investigated. Therefore, the present study aims to define the expression of TEMs in NSCLC cancer cell lines and tumor samples of NSCLC patients. Patients and methods The expression of different TEMs (Robo4, Clec14 and ECSCR) was evaluated by qPCR and Western blot analyses in several NSCLC cancer cell lines (A549, Calu1, Colo699) compared to human umbilical vein endothelial cells (HUVEC) and human bronchial epithelial cells (HBEpC). The expression of TEMs in resected tumor tissue of NSCLC patients (n = 64) was measured by qPCR and compared to adjacent lung tissue (n = 52). Further correlation analyses of patient samples with clinical course were performed using the data from the TYROL register. Results NSCLC cancer cell lines and HBEpC do not express TEMs neither on mRNA or on protein level compared to HUVECs (p = 0.001). Quantitative analyses of Robo4, ECSCR and Clec14 mRNA expression showed a significant up-regulation of CLEC14 in tumor samples (p = 0.01). Evaluating paired tissue samples from cancerous and corresponding non-cancerous tissues (n = 31) also Robo4 showed a significant upregulation in cancer specimens (p = 0.04). ECSCR showed a statistical trend towards an overexpression in tumor tissue (p = 0.09). Immunohistochemichal analyses of Robo4 revealed a higher expression in tumor tissue in comparison to the adjacent tissue (tumor vs. adjacent tissue; n = 20). Correlation with clinical data showed that increased TEM expression correlated with prolonged overall survival of NSCLC patients. Conclusion TEMs are overexpressed in NSCLC tissue specimens but not in cancer cells suggesting that TEMs are upregulated in stromal tissue including tumor vessels. Therefore, TEMs provide a potential new target structure for anti-angiogenic therapies. Increased TEM expression levels correlated with prolonged OS possibly due to vascular stabilization. Disclosure All authors have declared no conflicts of interest.

ALK amplification and crizotinib sensitivity in non-small cell lung cancer cell lines and patients report.

10556 Background: ALK high copy number (HCN) seems to be a frequent event, described in 13-17% of Non-small cell lung cancer (NSCLC). The goal of this study was to describe ALK genomic aberrations on NSCLC patients and cell lines, to explore the ALK HCN response to crizotinib through in vitro assays and to report three patients case. Methods: 191 Paraffin embedded specimens from advanced NSCLC patients and 27 NSCLC cancer cell lines were screened for ALK copy number by fluorescent in situ hybridization (FISH). Crizotinib sensitivity was evaluated in 9 cell lines through WST1 assays and clonogenic tests. Three patients exhibiting ALK HCN were assessed for response to crizotinib. Results: EML4-ALK translocation was present in 22 pts (11.5%). 21 pts (11%) exhibited over 6 copies of ALK. 6 (22%) cell lines displayed more than 5 copies of ALK, 19 (70%) presented a gain of 3 or 4 ALK copy number, only one cell line exhibited normal ALK copies and one harbored EML4-ALK translocation. FISH with CEP2 revealed a polysomy of chromosome 2 in cases with ALK HCN.Out of the 9 cell lines tested, 4 ALK HCN cell lines (H661, A427, BEN, H1299) exhibited increased sensitivity for crizotinib vs. 3 low ALK copy number (LCN) cell lines (H1975, H1651, H1650) with a low sensitivity. Median IC50 with crizotinib values was1750 nM [300-2800nM] in ALK HCN cell lines vs 4500 nM [800-8000nM] in ALK LCN cell lines, p=0.35. 3 patients with ALK HCN tumor received crizotinib ( in 4th , 5th and 6th -line therapy) for 2, 3 and 5 months with stable disease as best response and clinical benefit in 2 pts. Conclusions: ALK HCN may predict sensitivity to crizotinib. A clinical study is planned in ALK HCN pts.

Lentivirus-mediated siRNA interference targeting SGO-1 inhibits human NSCLC cell growth

Aneuploid is recognized as a hallmark of cancer and is caused by chromosome mis-segregation during mitosis. Recent studies have identified shugoshin (SGO-1) as an important player in proper chromosome segregation and the involvement in tumorigenesis. But, little is known about how SGO-1 is involved. The aim of this study was to obtain information about the role of SGO-1 in human lung cancer cells. In our study, real-time PCR and western blotting assay were performed to detect the expression of SGO-1 in nonsmall cell lung cancer (NSCLC) cell lines. Then, we successfully constructed a lentivirus vector mediating RNAi targeting of SGO-1 (LV-SGO-1 siRNA) and proved that it can suppress the expression of SGO-1 gene in H1299 cells. We found that SGO-1 was highly expressed in NSCLC cancer cell lines. RNA interference of SGO-1 by the LV-SGO-1 siRNA construct significantly decreased SGO-1 protein expression and inhibited the growth and ability of forming colonies of H1299 cells with more cells arrested in G2/M phase, but lentivirus vector control had no effect on H1299 cells. Moreover, suppression of SGO-1 by LV-SGO-1 siRNA increased the apoptosis of H1299 cells with down-regulated Bcl-2 and up-regulated Bax. SGO-1 may provide a novel therapeutic target for the treatment of lung cancer since it inhibits the growth and increases the apoptosis of lung cancer cells.