Lung Adenocarcinoma Cell Line Research Articles

Abstract B029: Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma

Abstract While KRAS is among the most frequently mutated oncogenes, our understanding of the mechanisms of KRAS-driven oncogenesis remains limited. A significant remaining gap is a lack of understanding of tissue-specific effectors of Ras activation and the role of specific KRAS mutations in determining downstream vulnerabilities. Previously, we used a combination of proteomics and CRISPR/Cas9 screens in human lung adenocarcinoma (LUAD) cells to identify a KRAS-specific vulnerability induced by the combined loss of RHOA and the long isoform of RAP1GDS1, suggesting a potentially novel approach for targeting KRAS-driven cancer. Here we use biochemical, proteomic, and genetic approaches to dissect the isoform-specific roles of RAP1GDS1 to elucidate its synthetic lethal interaction with RHOA in KRAS-driven LUAD. We performed AP/MS in KRAS-mutant A549 cells using both the long (RAP1GDS1-607) and the short (RAP1GDS1-558) isoforms of RAP1GDS1 as bait to identify overlapping and isoform-specific RAP1GDS1 interactors. We enriched this analysis with orthologous datasets from DepMap, published protein-protein interaction (PPI) data and RAP1GDS1-specific prenylome. This comprehensive analysis identified a cluster of RAB GTPases, proteins involved in vesicular transport and lysosomal function, as specific interactors of the long RAP1GDS1 isoform. We subsequently used the proximity ligation assay (PLA) to confirm the direct interaction between RAP1GDS1-607 and two of the strongest interactors - RAB7A and RAB22A – according to the PPI data. We also depleted either or both RAP1GDS1 isoforms in cells expressing GFP-tagged RAB7A, RAB22A or KRAS and used live cell imaging to determine how loss of RAP1GDS1 changes their subcellular localization. We found that depletion of both RAP1GDS1 isoforms and, to a lesser extent, single loss of the long RAP1GDS1 isoform, significantly decreased KRAS localization at the plasma membrane. We also used PLA to confirm that RAP1GDS1-607 has a stronger interaction with KRAS compared to the short isoform. Moreover, we demonstrated that loss of the long RAP1GDS1 isoform significantly decreased the membrane localization levels of GTP-bound KRAS, compared to the control or RAP1GDS1-558 knock-down. Finally, to identify the molecular mechanism of R1G1/RhoA synthetic lethality with KRAS, we performed a Genome Wide-CRISPR screen in R1G1-607 and RhoA knock-out cells. We identified the strongest hits and designed a focused CRISPR library of approximately 400 genes to screen a larger panel of KRAS-mutant LUAD cell lines (H23, A549 and H358). The aim is to identify other genes that are synthetic lethal in combination with RhoA or R1G1-607 loss and genes that could mediate the lethal effect of their combined loss. Citation Format: Marta Roman Moreno, Kaja Kostyrko, Kari Herrington, Michael Bassik, Peter Jackson, Eric Alejandro Sweet-Cordero. Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B029.

Regulation of VEGFR2 and AKT Signaling by Musashi-2 in Lung Cancer.

Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the diagnoses of lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, which are expressed on both endothelial and tumor cells, are one of the key proteins contributing to cancer development, and are involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer, which suggests that VEGFR2 protein is strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human lung adenocarcinoma cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR, which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting a direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human lung adenocarcinoma samples. We conclude that the MSI2/VEGFR2 axis contributes to lung adenocarcinoma progression and is worth further investigations and therapeutic targeting.

CircRNA ZKSCAN1 promotes lung adenocarcinoma progression by miR-185-5p/TAGLN2 axis.

Circ-ZKSCAN1 has been found to accelerate non-small cell lung cancer (NSCLC) progression; however, the role and mechanism of circ-ZKSCAN1 in lung adenocarcinoma (LUAD) cisplatin (DDP) resistance remain unclear. Levels of genes and proteins were examined using qRT-PCR. Functional experiments were performed using CCK-8 assay, flow cytometry, transwell assay and xenograft model assay, respectively. Glucose metabolism was calculated by detecting glucose consumption, lactate production, ATP and HK-2 levels. The interaction between miR-185-5p and circ-ZKSCAN1 or transgelin 2 (TAGLN2) was validated by dual-luciferase reporter assay. Circ-ZKSCAN1 was highly expressed in DDP-resistant LUAD tissues and cell lines, and circ-ZKSCAN1 knockdown weakened DDP resistance and suppressed cell viability, migration, invasion, and glycolysis in LUAD. Circ-ZKSCAN1 acted as a sponge of miR-185-5p, and the regulatory effects of circ-ZKSCAN1 knockdown on LUAD were reversed by miR-185-5p downregulation. Meanwhile, miR-185-5p directly targeted TAGLN2, and performed anticancer effects by regulating TAGLN2. Importantly, silencing of circ-ZKSCAN1 hindered tumor growth and promoted DDP sensitivity in vivo via regulating miR-185-5p and TAGLN2. Circ-ZKSCAN1 promoted LUAD tumorigenesis and DDP resistance by regulating miR-185-5p/TAGLN2 axis.

INHA acts as a novel and potential biomarker in lung adenocarcinoma and shapes the immune-suppressive tumor microenvironment

BackgroundINHA expression has been correlated with the development, growth, and progression of multiple cancer types. However, the biological role of INHA has not been investigated in patients with lung adenocarcinoma (LUAD). Here, we performed a comprehensive bioinformatics analysis of the LUAD dataset to determine the mechanisms underlying the regulation of tumorigenesis by INHA. Materials and methodsINHA expression and clinical information of patients with LUAD were obtained from The Cancer Genome Atlas (TCGA) database. Protein levels in LUAD cell lines and human lung epithelial cells were examined by western blotting. Next, the prognostic value of INHA in LUAD was assessed using Cox regression analysis, while the potential biological functions and the impact on the immune microenvironment of INHA were investigated using gene set enrichment analysis (GSEA) and single sample GSEA (ssGSEA). Finally, the effect of INHA on LUAD cell proliferation and invasion was determined in vitro and in vivo. ResultsWe found significantly high mRNA and protein expression levels of INHA in LUAD tissues and cell lines. Additionally, a higher expression of INHA was linked to a shorter overall survival (OS) and a worse pathological stage, while INHA expression was associated with immune cell infiltration and immune-related markers in the LUAD tumor microenvironment. LUAD with high INHA expression tends to be a cold tumor. Furthermore, GO and KEGG enrichment analysis indicated that INHA-related genes were enriched in the cell adhesion and immune signaling pathways of LUAD. INHA promoted LUAD cell proliferation and invasion, in vitro and in vivo, by inducing the EGFR pathway. ConclusionOur findings revealed that INHA is overexpressed in LUAD and is linked to a poor prognosis. Our study demonstrates the potential of INHA as an immunotherapeutic and predictive biomarker in LUAD.

Evolution of Gold and Iron Oxide Nanoparticles in Conjugates with Methotrexate: Synthesis and Anticancer Effects.

Au and Fe nanoparticles and their conjugates with the drug methotrexate were obtained by an environmentally safe method of metal-vapor synthesis (MVS). The materials were characterized by transmission and scanning electron microscopy (TEM, SEM), X-ray photoelectron spectroscopy (XPS), and small-angle X-ray scattering using synchrotron radiation (SAXS). The use of acetone as an organic reagent in the MVS makes it possible to obtain Au and Fe particles with an average size of 8.3 and 1.8 nm, respectively, which was established by TEM. It was found that Au, both in the NPs and the composite with methotrexate, was in the Au0, Au+ and Au3+ states. The Au 4f spectra for Au-containing systems are very close. The effect of methotrexate was manifested in a slight decrease in the proportion of the Au0 state-from 0.81 to 0.76. In the Fe NPs, the main state is the Fe3+ state, and the Fe2+ state is also present in a small amount. The analysis of samples by SAXS registered highly heterogeneous populations of metal nanoparticles coexisting with a wide proportion of large aggregates, the number of which increased significantly in the presence of methotrexate. For Au conjugates with methotrexate, a very wide asymmetric fraction with sizes up to 60 nm and a maximum of ~4 nm has been registered. In the case of Fe, the main fraction consists of particles with a radius of 4.6 nm. The main fraction consists of aggregates up to 10 nm. The size of the aggregates varies in the range of 20-50 nm. In the presence of methotrexate, the number of aggregates increases. The cytotoxicity and anticancer activity of the obtained nanomaterials were determined by MTT and NR assays. Fe conjugates with methotrexate showed the highest toxicity against the lung adenocarcinoma cell line and Au nanoparticles loaded with methotrexate affected the human colon adenocarcinoma cell line. Both conjugates displayed lysosome-specific toxicity against the A549 cancer cell line after 120 h of culture. The obtained materials may be promising for the creation of improved agents for cancer treatment.

Physicochemical characterization and cancer cell antiproliferative effect of silver-doped magnesia nanoparticles

Silver-doped magnesia nanoparticles (Ag/MgO) were synthesized using the precipitation method and characterized by various techniques such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA), Brunner-Emmett-Teller (BET) surface area measurements, and dispersive X-ray spectroscopy (EDX). The morphology of Ag/MgO nanoparticles was determined by transmission and scanning electron microscopy, which revealed cuboidal shaped nanoparticles with sizes ranging from 31 to 68 nm and an average size of 43.5 ± 10.6 nm. The anticancer effects of Ag/MgO nanoparticles were evaluated on human colorectal (HT29) and lung adenocarcinoma (A549) cell lines, and their caspase-3, -8, and -9 activities, as well as Bcl-2, Bax, p53, cytochrome C protein expressions were estimated. Ag/MgO nanoparticles showed selective toxicity towards HT29 and A549 cells while remaining relatively innocuous towards the normal human colorectal, CCD-18Co, and lung, MRC-5 cells. The IC50 values of Ag/MgO nanoparticles on the HT29 and A549 cells were found to be 90.2 ± 2.6 and 85.0 ± 3.5 μg/mL, respectively. The Ag/MgO nanoparticles upregulated caspase-3 and -9 activities, downregulated Bcl-2, upregulated Bax and p53 protein expressions in the cancer cells. The morphology of the Ag/MgO nanoparticle treated HT29 and A549 cells was typical of apoptosis, with cell detachment, shrinkage, and membrane blebbing. The results suggest that Ag/MgO nanoparticles induce apoptosis in cancer cells and exhibit potential as a promising anticancer agent.

MicroRNA-660 Enhances Cisplatin Sensitivity via Decreasing SATB2 Expression in Lung Adenocarcinoma.

Increasing evidence suggests that microRNAs' (miRNAs) abnormal expression is one of the main factors of chemotherapy resistance in various cancers. However, the role of miRNAs in lung adenocarcinoma (LUAD) resistance to cisplatin is still unclear. In this study, we analyzed a microarray dataset to investigate miRNAs related to cisplatin resistance in LUAD. The expression of miRNAs in LUAD tissues and cell lines was detected using real-time quantitative polymerase chain reaction (RT-qPCR). Special AT-Rich Sequence-Binding Protein 2 (SATB2) in LUAD cell lines was detected using RT-qPCR and Western blot. Cell proliferation was measured by CCK8 and colony formation assays, while cell cycle and apoptosis were measured by flow cytometry. A dual-luciferase reporter assay was performed to confirm that SATB2 is a target gene of microRNA-660 (miR-660). We showed that the expression of miR-660 was not only decreased in LUAD cells and tissues but also further decreased in the cisplatin-resistant A549 cell line. The overexpression of miR-660 increased cisplatin sensitivity in LUAD cells. In addition, we identified SATB2 as a direct target gene of miR-660. We also revealed that miR-660 increased cisplatin sensitivity in LUAD cells via targeting SATB2. In conclusion, miR-660/SATB2 axis is a key regulator of cisplatin resistance in LUAD.

Abstract LB259: Distinct microRNA signature and suppression of ZFP36L1 define ASCL1-positive lung adenocarcinoma

Abstract Background: Achaete-scute family bHLH transcription factor 1 (ASCL1) is a master transcription factor involved in neuroendocrine differentiation. ASCL1 is expressed in approximately 10% of lung adenocarcinomas and exerts tumor-promoting effects. However, microRNA (miRNA) profiles regulated by ASCL1 in lung adenocarcinoma cells remain unexplored. Method: We analyzed public database of gene expression profiling (RNA-sequencing and miRNA expression data). We also studied miRNA profiles in ASCL1-positive lung adenocarcinoma cells and identified a subset of miRNAs downregulated by ASCL1 knockdown. We examined functions of genes suppressed by miRNAs in ASCL1-positive lung adenocarcinoma cell line, VMRC-LCD. Result: We identified miRNA profiles in ASCL1-positive lung adenocarcinomas and found several miRNAs closely associated with ASCL1 expression, including miR-375, miR-95-3p/miR-95-5p, miR-124-3p, and members of the miR-17~92 family. Similar to small cell lung cancer, Yes1 associated transcriptional regulator (YAP1), a representative miR-375 target gene, is suppressed in ASCL1-positive lung adenocarcinomas. ASCL1 knockdown followed by miRNA profiling in a cell culture model further revealed that ASCL1 positively regulates miR-124-3p and members of the miR-17~92 family. Integrative transcriptomic analyses identified the RNA-binding protein zinc finger protein 36 like 1 (ZFP36L1) as a target gene of miR-124-3p, and immunohistochemical studies have demonstrated that ASCL1-positive lung adenocarcinomas are associated with low ZFP36L1 protein levels. Cell culture studies have shown that ectopic ZFP36L1 expression inhibits cell proliferation, survival, and cell cycle progression. Mechanistically, ZFP36L1 negatively regulated tumorigenic genes, including E2F transcription factor 1 (E2F1) and snail family transcriptional repressor 1 (SNAI1), indicating a tumor-suppressing action. Conclusion: Our study revealed that suppression of ZFP36L1 via ASCL1-regulated miR-124-3p could induce tumor-promoting effects, providing evidence that ASCL1-mediated regulation of miRNAs shapes malignant features of ASCL1-positive lung adenocarcinomas. Citation Format: Naoya Miyashita, Takayoshi Enokido, Masafumi Horie, Hiroshi I. Suzuki, Rei Matsuki, Hans Brunnström, Patrick Micke, Takahide Nagase, Akira Saito. Distinct microRNA signature and suppression of ZFP36L1 define ASCL1-positive lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB259.

DAP3-mediated cell cycle regulation and its association with radioresistance in human lung adenocarcinoma cell lines.

Mitochondria play important roles in the cellular response to various types of stress, including that triggered by ionizing radiation. We have previously reported that the mitochondrial ribosomal protein death-associated protein 3 (DAP3) regulates the radioresistance of human lung adenocarcinoma (LUAD) cell lines A549 and H1299. However, the underlying mechanism of this regulation remains to be elucidated. To this end, we have herein investigated the role of DAP3 in the cell cycle regulation after irradiation. Notably, the DAP3 knockdown attenuated the radiation-induced increase of the G2/M cell population. Furthermore, western blotting analysis has revealed that the DAP3 knockdown decreased the expression of proteins related to the G2/M arrest, such as those of the phosphorylated cdc2 (Tyr15) and the phosphorylated checkpoint kinase 1 (Ser296), in irradiated A549 cells and H1299 cells. Moreover, by using a chk1 inhibitor, we were able to demonstrate that chk1 is involved in the radiation-induced G2/M arrest in both A549 and H1299 cells. Notably, the chk1 inhibitor was able to enhance the radiosensitivity of H1299 cells, while both chk1 inhibitor-abolished G2 arrest and inhibition of chk2-mediated events such as downregulation of radiation-induced p21 expression were required for enhancing radiosensitivity of A549 cells. Collectively, our findings reveal a novel role of DAP3 to regulate G2/M arrest through pchk1 in irradiated LUAD cells and suggest that chk1-mediated G2/M arrest regulates the radioresistance of H1299 cells, whereas both the chk1-mediated G2/M arrest and the chk2-mediated events contribute to the radioresistance of A549 cells.

Inhibition of the mitochondria-shaping protein Opa1 restores sensitivity to Gefitinib in a lung adenocarcinomaresistant cell line

Drug resistance limits the efficacy of chemotherapy and targeted cancer treatments, calling for the identification of druggable targets to overcome it. Here we show that the mitochondria-shaping protein Opa1 participates in resistance against the tyrosine kinase inhibitor gefitinib in a lung adenocarcinoma cell line. Respiratory profiling revealed that oxidative metabolism was increased in this gefitinib-resistant lung cancer cell line. Accordingly, resistant cells depended on mitochondrial ATP generation, and their mitochondria were elongated with narrower cristae. In the resistant cells, levels of Opa1 were increased and its genetic or pharmacological inhibition reverted the mitochondrial morphology changes and sensitized them to gefitinib-induced cytochrome c release and apoptosis. In vivo, the size of gefitinib-resistant lung orthotopic tumors was reduced when gefitinib was combined with the specific Opa1 inhibitor MYLS22. The combo gefitinib-MYLS22 treatment increased tumor apoptosis and reduced its proliferation. Thus, the mitochondrial protein Opa1 participates in gefitinib resistance and can be targeted to overcome it.

Abstract 5752: Enhancer amplification defines lineage addiction in human lung adenocarcinoma

Abstract Lung cancer is the most common cause of death from cancer worldwide, and lung adenocarcinoma (LUAD) is the most common type of lung cancer. NKX2-1 (also known as TTF-1, TITF-1) is a lineage defining transcription factor for normal lung development. In LUAD, NKX2-1 is a highly specific and sensitive marker for both primary and metastatic LUADs, with 85-90% expressing NKX2-1. We and others identified that NKX2-1 as the most significantly amplified gene in LUAD, with 22.5% of primary LUADs exhibiting copy number gains. NKX2-1 is amplified as the earliest stages of LUAD development, and NKX2-1 amplification is a truncal event in multi-region LUAD evolution. Despite strong evidence for an oncogenic role for NKX2-1, little is known about the mechanisms of NKX2-1 activation, or how its oncogenic regulation drives LUAD. Here, we identify recurrent focal amplification targeting a super-enhancer (SE) of NKX2-1 as a driving event in LUAD. Using epigenomic data from LUAD cell lines and tumors, we identify this region as a alveolar lineage super-enhancer (NKX2-1 SE) that is specifically active in NKX2-1(+) LUAD cell lines and primary tumors. Notably, this region is co-amplified with NKX2-1 in 96-100% of NKX2-1-amplified samples, suggesting this region a critical component of the NKX2-1 amplicon. Using endogenous ChIP-seq and exogenous luciferase assays, we show the enhancer activity of the NKX2-1 SE is comprised of three constituent enhancers, and demonstrate that the key enhancer elements can activate transcription of the NKX2-1 promoter. Using CRISPR inhibition (CRISPRi) and CRISPR activation (CRISPRa) in LUAD cell lines expressing high or low levels of NKX2-1, we show that activity of the NKX2-1 SE defines endogenous NKX2-1 expression. Using RNA-seq, ChIP-seq, and ATAC-seq, we find that NKX2-1 controls the transcriptional and epigenomic landscape of lung adenocarcinoma. NKX2-1 knockdown activates an epithelial-mesenchymal transition (EMT) gene signature and downregulates an alveolar differentiation signature, including critical markers of LUAD differentiation, such as NAPSA, SFTPA1/2, and HOPX, at which NKX2-1 mediates lineage enhancer accessibility to regulate target gene expression. Global clustering of LUAD cell lines by RNA-seq or H3K27ac ChIP-seq identifies a distinct NKX2-1(+) cluster, and we find that NKX2-1 directly regulates the genes (57/97) and enhancers that distinguish this subpopulation. Using genome-wide shRNA and CRISPR screens, we identify a NKX2-1 dependency in NKX2-1(+) LUAD cell lines, and find that the majority of NKX2-1(+) LUAD cell lines (11/14) assayed are dependent on NKX2-1. Our data demonstrates that enhancer amplification is a hallmark of oncogenic NKX2-1 activation in LUAD, through which NKX2-1 drives a lineage addicted state and oncogenic cell proliferation. This suggests that NKX2-1 is a critical defining oncogene for LUAD, and that targeting of NKX2-1 or its enhancer may suppress LUAD. Citation Format: John Louis Pulice, Matthew Meyerson. Enhancer amplification defines lineage addiction in human lung adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5752.

Abstract 6116: Ablation of mitotic checkpoint kinase BUB1 sensitizes lung adenocarcinoma to different classes of chemotherapy, radiation, and chemo-radiation

Abstract Lung cancer causes the most cancer associated deaths worldwide. About 80 percent patients with inoperable non-small cell lung cancer (NSCLC) develop local recurrence and about 60 percent distant metastasis. NSCLC subtype, lung adenocarcinoma (LUAD) accounts for about 40% of all lung cancers. Recently, targeted therapy with chemotherapy combination has shown to enhance the pathologic complete response rate in a subgroup of NSCLC patients, but most patients do not benefit from therapy due to development of resistance. Thus, identifying molecular drivers of primary and adaptive chemotherapy resistance that can be effectively targeted represents a critical unmet clinical need. Here we aim to evaluate BUB1 (a mitotic checkpoint kinase) as a viable molecular target for increasing the effectiveness of radiotherapy and chemotherapy in LUAD. Bioinformatic analyses were conducted to evaluate BUB1 expression across lung cancer and normal tissues. BUB1 was found to be overexpressed in LUAD compared to normal tissues and BUB1 expression also correlated with cancer stage and tumor grade. LUAD cell lines A549, H1975 and H2030 were used to study the effect of BUB1 inhibition on chemo-radiosensitization. MTT cell proliferation and clonogenic cell survival studies were conducted with pharmacological (BAY1816032) and genomic (CRISPR) BUB1 ablation in combination with Platinum (Cisplatin), Taxol (Paclitaxel) or a PARP inhibitor (Olaparib) without/with radiation. BUB1 inhibition showed cytotoxicity in LUAD cell lines at micromolar concentration and enhanced the radiation mediated cell killing at nanomolar concentration. Moreover, BUB1 inhibition sensitized LUAD cell lines to Cisplatin, Paclitaxel and Olaparib in double or triple chemotherapy combinations. Additionally, BUB1 inhibition significantly increased LUAD cell death when combined with different classes of chemotherapies with radiation (chemo-radiation). Our preliminary mechanistic studies revealed prolonged presence of γH2AX foci, an indicator of DNA double stand breaks. BLRR DNA damage reporter assay indicated that the non-homologous end joining (NHEJ) pathway was inhibited upon BUB1 ablation. Detailed mechanistic studies combining BUB1 ablation with chemo-radiation are underway to delineate signaling cascades involved in BUB1 driven chemo-radiation sensitization. Our data provides evidence that BUB1 inhibition sensitizes lung adenocarcinoma to radiotherapy, different classes of chemotherapy and chemo-radiation through DNA-double strand break repair pathways and provides rationale for clinical trials that combine BUB1 inhibition with chemo-radiation in NSCLC. Citation Format: Shivani Thoidingjam, Sushmitha Sriramulu, Farzan Siddiqui, Benjamin Movsas, Shirish Gadgeel, Shyam Nyati. Ablation of mitotic checkpoint kinase BUB1 sensitizes lung adenocarcinoma to different classes of chemotherapy, radiation, and chemo-radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6116.

Abstract 2231: Epigenomic profiling uncovers distinct enrichment patterns of PBX1 super-enhancer among lung adenocarcinoma

Abstract Small cell lung cancers and squamous cell cancers can be epigenetically subtyped (Kong R et al 2022, Sato T et al, 2019) with focus on super-enhancer (SE) signals within or adjacent to transcription factors (TF). We reasoned that specific epigenomic subsets in lung adenocarcinomas (LUAD) operate with similar cellular programming showing TF dependency for lineage commitment and survival. We hypothesize that these dependencies on lineage programming are associated with a specific epigenomic landscape. Therefore, we investigated enrichment of SEs across the spectrum of LUADs to identify potential regulators for lineage commitment and survival. We obtained data for genome-wide H3K27ac enrichment by chromatin immunoprecipitation followed by sequencing (ChIP-seq) on a total of 53 LUAD cell lines. We performed unsupervised hierarchical clustering of signals on SEs at gene loci annotated with transcriptional regulation identified in two or more cell lines. To Uncover 3D contact information, we employed HiChIP, using antibody against SMC1, a cohesin complex, on NCI-H460. Three LUAD cell lines with distinct enhancer profiles were transfected with five enhancer segments of the SE at the PBX1 gene cloned into a Firefly luciferase plasmid. Lastly, four LUAD cell lines were treated with a PBX1 inhibitor, T417 for 72 hours in varying concentrations ranging from 20uM to 10nM and evaluated for sensitivity to the compound. Clustering analysis identified LUADs with differential enhancer enrichment for TFs. Among those, we focused on the PBX1 gene locus in LUADs harboring SEs proximal to its promoter. SMC1 HiChIP analysis on NCI-H460 suggest all enhancer segments are within a local topologically-associated domain with the PBX1 promoter. Luciferase reporter assay demonstrate activity of enhancer segments within the PBX1 SE, unique to each cell line (NCI-H460, PC-9 and NCI-H1437), with enhancers e4 and e5 rendered the highest level of activity while e2 being the most active in PC-9. The effects of these enhancer segments are congruent to H3K27ac enrichment patterns. Treatment with T417 showed reduction in cell proliferation at an IC50 of 2.5 uM and 0.97 uM for NCI-H460 and PC-9, respectively, whereas T417 did not affect cell viability in NCI-H1792 and NCI-H125. Data indicate a distinct subset of LUADs, enriched for the enhancer segments within the SE at the PBX1 gene locus, at which the HiChIP data displayed the enhancer segments lie within the same topologically-associated domain. The enhancer activity of enhancer segments correlated with H3K27ac enrichment pattern for each cell line, suggesting enrichment correlates with impact. Data with PBX1 T417 exposure suggests dependency of PBX1 in maintaining lineage state and survival for unique subsets of LUADs. Further investigation is needed to determine if T417 dependent inhibition is uniform to all PBX1 active LUADs and if other LUAD subsets are dependent on other TFs. Citation Format: Douglas M. Mansell, Maya Fridrich, Feng Jiang, Benjamin Chen, Ayushi Patel, Tian Li-Wang, Charles A. Powell, Hideo Watanabe. Epigenomic profiling uncovers distinct enrichment patterns of PBX1 super-enhancer among lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2231.

Abstract 48: Increasing the racial diversity of lung adenocarcinoma cell lines

Abstract Lung cancer is a prominent source of cancer health disparities. Black males tend to smoke less, but have a 37% higher chance of developing lung cancer. The three most common histological subtypes of lung cancer are lung adenocarcinoma (LUAD), squamous cell lung cancer (LUSQ), and small cell lung cancer (SCLC). To study lung cancer development, progression, and treatment, numerous tools are needed, including lung cancer cell lines. We undertook a literature and web-based investigation of lung cancer cell line availability from different racial/ethnic groups. We identified 188 cell lines from White individuals, 374 cell lines from Asian subjects, but only 26 cell lines from Blacks and none from Hispanic patients. We conclude that Black and Hispanic lung cancer cell lines are strongly underrepresented. Here we focus on LUAD cell lines since LUAD is the most common histological subtype in all racial/ethnic groups. While 67 White LUAD cell lines have been established, we only identified 5 Black LUAD lines. To remedy this lack of Black LUAD cell lines, we are collecting LUAD samples from Black patients to establish new cell lines (with full consent). In addition, we aim to increase the representation of Black cell lines by creating isogenic cell line variants of existing cell lines, like NCI-H23. NCI-H23 was derived from a Black male patient and carries a KRASG12C heterozygous mutation. We are using CRISPR/CAS9-based genome editing to introduce amino acid substitutions, including KRASG12V and KRASG12A. Our gene editing DNA includes a silent restriction site to help identify edited clones. A fluorescently labeled structured tracrRNA is included during the genomic editing process, allowing the electroporated cells to undergo fluorescence-activated cell sorting. We use the polymerase chain reaction to amplify the relevant genomic region, followed by restriction digestion to identify potential clones and sequencing to confirm successful editing. Several clones are currently under evaluation. Once new hetero- and homozygous KRAS mutants have been identified, we will test the efficacy of polyisoprenylated cysteinyl amide inhibitors (PCAIs). PCAIs have been shown to disrupt the RAS-mediated signaling pathway in racially/ethnically diverse cell lines (manuscript submitted). By generating newly-established and isogenic Black LUAD cell lines, we intend to ensure that potential therapeutics are tested on cell lines with the appropriate racial/ethnic genetic background. In the future, we hope to establish similar cell lines from Hispanic LUAD subjects. Citation Format: Christopher Leon, Matthew A. Gladstone, Chunli Yan, Diana I. Romero, Yong Huang, Nazarius S. Lamango, Ite A. Offringa. Increasing the racial diversity of lung adenocarcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 48.

Abstract 4039: RET overexpression is frequent in lung neuroendocrine tumors (NET) and associates with response to RET tyrosine kinase inhibitors (RET TKIs) in NET cell lines

Abstract Background: RET (rearranged during transfection) is a proto-oncogene encoding for a tyrosine kinase membrane receptor that promotes protein synthesis and cell proliferation. A small percentage of human tumors harbor RET fusions or mutations as oncogenic drivers and RET-TKIs have been developed. Two of them, selpercatinib and pralsetinib, have recently been approved. However, their effect on tumors overexpressing wild-type RET (wt-RET) has not been investigated. A large-scale mRNA analysis performed by our group revealed that ~2% of lung tumors present high expression of wt-RET in absence of fusions. Interestingly, ~60% of high wt-RET tumors presented neuroendocrine characteristics. Methods: Twenty large cell neuroendocrine (LCNEC) and 19 small cell (SCLC) lung carcinomas from two additional institutions were analyzed by nCounter® to validate the previous results. For in vitro experiments, a panel of cell lines derived from lung NETs (DMS-53, NCI-H82, NCI-H727) was used, together with a lung adenocarcinoma cell line (LC2/ad, a positive control harboring the CDCC6-RET translocation). RET fusions and mRNA expression were analyzed by nCounter®, RET protein expression by Western blotting and immunohistochemistry (IHC) and RET mutations by next generation sequencing. The inhibitory effects of selpercatinib and pralsetinib were analyzed in vitro by MTT in 2D and spheroid/3D cultures. Finally, in vivo experiments implanting tumor cells into athymic nude mice are ongoing. Results: Among the 20 LCNECs and 19 SCLCs analyzed, 3 (15%) and 3 (15%) presented very high levels of wt-RET mRNA by nCounter, respectively. Fusions were absent in all cases. The NCI-H82 and NCI-H727 NET cell lines showed very low RET mRNA and protein expression by nCounter, Western blotting and IHC. In contrast, DMS-53 cells presented levels of RET mRNA and protein higher than the observed in the RET-fusion positive LC2/ad. In 2D models, the three NET cell lines tested showed moderate resistance to pralsetinib and selpercatinib, with IC50s of 1.5 - 9.7 µM for pralsetinib and 4.6 - 6.1 µM for selpercatinib; while the RET-fusion positive LC2/ad cells showed an IC50 in the nM range, as expected. In 3D cultures, DMS-53 was found to be sensitive to RET TKIs, with IC50s of and 0.3 µM for praseltinib and 2.5 µM for selpercatinib. In contrast, spheroids of the two RET low cell lines showed complete resistance to both drugs (IC50>10 µM). Experiments in animal models are currently ongoing, final results will be presented at the meeting. Conclusions: Overexpression of RET is frequent in lung tumors with neuroendocrine features (LCNEC, SCLC). Selpercatinib and Pralsetinib show antitumor activity in lung NETs cells expressing high levels of wt-RET. Citation Format: Jaume Roca-Arias, Jordi Bertran-Alamillo, Ruth Román, Cristina Aguado, Laura López, Leticia Ferro-Leal, Beatriz Garcia-Pelaez, Rodrigo de Oliveira Cavagna, Silvia Teixeira, Rafael Rosell, Rui Manuel Reis, Miguel A. Molina-Vila, Ivana Sullivan. RET overexpression is frequent in lung neuroendocrine tumors (NET) and associates with response to RET tyrosine kinase inhibitors (RET TKIs) in NET cell lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4039.

Abstract 5027: Targeting ARAF p.S214C mutation with sorafenib in non-small cell lung cancer

Abstract Sorafenib is an FDA-approved multi-kinase inhibitor for the treatment of several cancer types, but is still under investigation for non-small cell lung cancer (NSCLC). Although sorafenib has not shown significant survival benefit in clinical trials for NSCLC patients, one study identified a patient with advanced lung adenocarcinoma harboring ARAF p.S214C mutation as an exceptional sorafenib responder. As NSCLC lacks clinically validated predictive biomarkers for drug responses, we aim to evaluate the potential of ARAF p.S214C mutation as a biomarker for sorafenib sensitivity and its mechanism of action. Constructs with ARAF p.S214C mutation, ARAF-wild type (WT) and EGFR-vector control were ectopically expressed in a lung adenocarcinoma cell line, H2023, by retroviral transduction. Western blot confirmed stable overexpression of ARAF proteins over time. MTT assay showed that sorafenib inhibited growth of cells expressing ARAF p.S214C more pronouncedly than cells expressing ARAF-WT or vector control. In addition, spheroids formed by cells expressing ARAF p.S214C were more sensitive to sorafenib inhibition, indicating sorafenib sensitivity of ARAF p.S214C in NSCLC in vitro. To evaluate whether such sorafenib sensitivity is dependent on the kinase activity of ARAF, phospho-MEK and MAPK levels in mutant cells will be determined. As ARAF belongs to the Raf protein family which also includes BRAF and RAF1, knockdown studies and co-immunoprecipitation will be performed to determine whether BRAF and RAF1 are essential for ARAF p.S214C activity. The in vivo sorafenib sensitivity of ARAF p.S214C will be validated in tumor xenografts established from cells expressing the above constructs upon sorafenib treatment. Tumor volume will be assessed and expression of the tumor marker cytokeratin will be evaluated by immunohistochemistry. This study may reveal ARAF p.S214C as a potential biomarker for sorafenib sensitivity and a new target for precision medicine in NSCLC. Citation Format: Carol Lee, Sze Man Chan, Sai Fung Yeung, Stephen Kwok-Wing Tsui. Targeting ARAF p.S214C mutation with sorafenib in non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5027.

Construction and validation of a T cell proliferation regulator-related signature for predicting prognosis and immunotherapy response in lung adenocarcinoma.

As the main executor of immunotherapy, T cells significantly affect the efficacy of immunotherapy. However, the contribution of the T cell proliferation regulator to the prognosis of lung adenocarcinoma (LUAD) and immunotherapy is still unclear. Based on T cell proliferation regulators, LUAD samples from The Cancer Genome Atlas (TCGA) were divided into two different clusters by consensus clustering. Subsequently, the T cell proliferation regulator (TPR) signature was constructed according to the prognostic T cell proliferation regulators. Combined with clinical information, a nomogram for clinical practice was constructed. The predictive ability of the signature was verified by the additional Gene Expression Omnibus (GEO) dataset. We also analyzed the differences of tumor microenvironment (TME) in different subgroups and predicted the response to immunotherapy according to the TIDE algorithm. Finally, we further explored the role of ADA (Adenosine deaminase) in the lung adenocarcinoma cell lines through the knockdown of ADA. According to the consensus clustering, there were differences in survival and tumor microenvironment between two different molecular subtypes. T cell proliferation regulator-related signature could accurately predict the prognosis of LUAD. The low-risk group had a higher level of immune infiltration and more abundant immune-related pathways, and its response to immunotherapy was significantly better than the high-risk group (Chi-square test, p<0.0001). The knockdown of ADA inhibited proliferation, migration, and invasion in lung adenocarcinoma cell lines. T cell proliferation regulators were closely related to the prognosis and tumor microenvironment of LUAD patients. And the signature could well predict the prognosis of LUAD patients and their response to immunotherapy. ADA may become a new target for the treatment of LUAD.

Abstract 5477: An investigation to study differential gene expression of lung cancer cells, tumor associated macrophages, and bone marrow derived mesenchymal stems cells in the tumor microenvironment

Abstract Cancer is a public health crisis affecting approximately 19.3 million globally, annually. Lung cancer has the highest percentage of incidence and is responsible for 27% of cancer-related deaths. The prevalence of lung cancer worldwide led us to investigate differential gene expression in lung adenocarcinoma and small cell lung cancer cell lines in comparison to a healthy lung cell line. We also investigated the effect of lung tumor microenvironment to bone marrow derived pluripotent mesenchymal stem cells (PMSC) and tumor associated macrophages. This research involved cancer and healthy cell line culture with appropriate medium. Whole transcriptomic and small non-coding RNA sequencing and analysis by Ingenuity Pathway Analysis (Qiagen, USA), Quantitative RT-PCR and ELISA assays. We further studied morphological changes and differential gene expression in extracellular vesicles(exosomes) isolated from the TAM and TME exposed PMCs by atomic force microscopy and RNA sequencing techniques. The results of our investigation showed differential gene expression and novel core canonical pathways in the lung cancer cells, TME exposed TAM, and PMSC. We also observed morphological changes in TME derived exosomes from TAM and PMSC. RNA sequencing results showed differential gene expression of both coding and non-coding RNAs including tRNA fragments. Thus, our findings of this investigation led to identification of multiple cancer related genes and small non-coding microRNA, long non-coding RNA, and tRNA fragments which will have potential diagnostic and therapeutic target value that demands further future research. Acknowledgement: This research is supported by a grant from US NIH-NIGMS- 5T34GM100831-08 and US-NIH Grant# U 54 MD007597-31-5959. Citation Format: Hirendra N. Banerjee, Joseph Hedley, Cheslsey Aurelus, Narendra Banerjee, Kuldeep Rawat, Quentin Reaves, Shennel Brown, Darla Gilmartin, Makieyah Liverman, Jazmine Cuffee, Elizabeth Cagle, Erik Armstrong, Brent Lake, Santosh Mandal, Somiranjan Ghosh, Stephen Beebe, Kamal Asadipour, Christopher Krauss, Santanu Bhattacharya, Tanmay Kulkarni, Zahidur Abedin. An investigation to study differential gene expression of lung cancer cells, tumor associated macrophages, and bone marrow derived mesenchymal stems cells in the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5477.

Abstract 2392: Multi-dimensional morphology analysis enables identification and label-free enrichment of heterogeneous tumor cell populations

Abstract Studying tumor heterogeneity at single cell resolution is crucial for elucidating tumor progression and treatment response. Classically, tumor cells are characterized with a limited set of biomarkers that cannot cover the full extent of the tumor cell heterogeneity. The advent of single cell genomics has enabled scientists to study heterogeneity at high resolution, but gene expression signatures may not translate to readily available biomarkers for functional studies. Multi-dimensional morphology analysis has the potential for higher resolution than cell surface biomarkers while also reducing cell manipulations. The Deepcell platform enables multi-dimensional morphology analysis of unlabeled single cells using artificial intelligence (AI), imaging, and microfluidics, allowing for higher resolution of population heterogeneity beyond protein expression markers. Using the Deepcell platform, we trained an AI model to identify and sort for malignant cells from non-small cell lung cancer cells (NSCLC) tumor biopsies based on morphology. scRNA-seq, CNV, and targeted mutation analysis verified enrichment of carcinoma cells. Sorted cells had gene expression signatures indicative of tumor cells (e.g. EpCAM expression), increased genomic alterations by CNV analysis, and increased allele frequency of P53 and KRAS mutations relative to pre-sorted samples. Overall, the data indicate brightfield images of cells can be used to detect macro-level changes in cell morphology resulting from the molecular events involved in tumorigenesis. In addition, we induced chemotherapeutic drug resistance of lung cancer cell lines in vitro, imaged the resulting cultures on the Deepcell platform, and used the bright-field cell images to generate multi-dimensional morphological embeddings that can be visualized by UMAP. The resulting UMAPs showed distinct clusters of cells for both the parent and resistant cell lines, indicating that there are morphological differences associated with drug resistance. We developed a random forest classifier to identify the top differential morphological features between parental and resistant cell lines. These top features can distinguish between the populations with high accuracy (87%). Finally, we performed multi-dimensional morphology analysis to compare lung adenocarcinoma (LA) and squamous cell carcinoma (SCC) cell lines. The classifier predicted the correct cell type with 75% accuracy, suggesting that differences between these two carcinomas are reflected in their morphology. Together, our data suggest that AI-detected morphological differences between cell populations may show a biologically significant link between morphology and phenotype. As such, multi-dimensional morphology analysis will bolster the understanding of complex disease states and tumor microenvironment, particularly in patient derived biopsies. Citation Format: Andreja Jovic, Kiran Saini, Ryan Carelli, Tiffine Pham, Christian Corona, Jeanette Mei, Michael Phelan, Stephane C. Boutet, Kevn Jacobs, Julie Kim, Manisha Ray, Chassidy Johnson, Nianzhen Li, Mahyar Salek, Maddison Masaeli, Matt Barnes, Cyril Ramathal. Multi-dimensional morphology analysis enables identification and label-free enrichment of heterogeneous tumor cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2392.

Abstract 502: ISM3091, a novel selective USP1 inhibitor as a targeted anticancer therapy

Abstract Ubiquitin-specific protease 1 (USP1), a well-characterized member of the deubiquitinating enzymes family, cleaves ubiquitin from various target proteins. USP-1 expression is dysregulated in many cancers where it acts as an oncogenic driver with roles in various DNA damage repair processes including translesion synthesis and the Fanconi anemia pathway. Previous data show that USP1 plays a critical role in protecting the replication fork in BRCA1-deficient cells and that its loss results in reduced cell survival and replication fork degradation, suggesting that USP1 inhibitors may be particularly useful in BRCA-deficient tumors. Though PARP inhibitors (PARPi) have shown clinical benefit in BRCA-mutated (BRCAm) populations, some patients either do not respond to therapy or develop resistance. USP1 inhibitors may have the potential to address this unmet clinical need. We evaluated ISM3091, a novel and selective USP1 inhibitor, against tumor cell lines and in vivo models with BRCAm and other homologous recombination repair deficiency (HRD), as well as in homologous recombination (HR)-proficient models.ISM3091 demonstrated potent anti-proliferation activity (IC50, 20 nM) in the BRCA1m triple negative breast cancer (TNBC) cell line, MDA-MB-436, with a ~1500-fold selectivity for BRCAm vs BRCA WT. Synthetic lethality between USP1 and HRD was due to persistent monoubiquitinated PCNA. In vitro data revealed that the combination of ISM3091 and olaparib, a PARPi, had synergistic activity in cell lines with HRD. Multiple in vivo CDX and PDX models confirmed the dose-dependent, single-agent inhibitory activity of ISM3091, with Tumor Growth Inhibition (TGI) of 66% at 30 mg/kg BID (TNBC) and 60% at 50 mg/kg BID (ovarian). The combination of ISM3091 and olaparib yielded a more robust and durable anti-tumor response, even at low doses of ISM3091, with TGI of 91% (TNBC; ISM3091, 3 mg/kg BID + olaparib, 50 mg/kg QD) and 110% (ovarian; ISM3091, 50 mg/kg BID + olaparib, 50 mg/kg QD). Notably, ISM3091 displayed strong single-agent activity (TGI 72% at 50 mg/kg BID) in an ovarian PDX model with acquired resistance to olaparib (BRCA WT), indicating that it has the potential to treat tumors with HRD beyond BRCAm, and also to overcome PARPi resistance. In vivo evaluation against the HR-proficient lung adenocarcinoma cell line, NCIH1792, showed potent efficacy (TGI 86% at 30 mg/kg BID) suggesting a broader ISM3091 potential. ISM3091also displayed very favorable ADME properties and PK profiles, and GLP toxicology studies indicated that it was well tolerated without significant gastrointestinal toxicity or hematological toxicity. These data support the future clinical development of ISM3091 as a potential best-in-class USP1 inhibitor not only for PARPi-resistant/responsive HRD-mutant cancers, both, as a single agent as well as in combination with PARPi, but also for subsets of HR-proficient cancers. Citation Format: Yangguang Li, Jianping Wu, Jinxin Liu, Luoheng Qin, Xin Cai, Junwen Qiao, Ling Wang, Sujata Rao, Feng Ren, Alex Zhavoronkov. ISM3091, a novel selective USP1 inhibitor as a targeted anticancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 502.