Patient-derived Non-small Cell Lung Cancer Research Articles

Predicting DNA damage response in non-small cell lung cancer organoids via simultaneous label-free autofluorescence multiharmonic microscopy

The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γ-H2AX), which is an indicator of DNA double-strand breaks. While γ-H2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γ-H2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γ-H2AX immunostained images confirmed strong correlation between SLAM image features and γ-H2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.

Abstract CT077: An open-label phase 1 study to investigate SGN-CEACAM5C in adults with advanced solid tumors (SGNCEA5C-001, Trial in Progress)

Abstract Background Patients (pts) with recurrent or metastatic solid tumors, including non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), colorectal cancer (CRC), gastric adenocarcinoma (GC), and pancreatic ductal adenocarcinoma (PDAC), have limited treatment options with short PFS and OS despite recent advances (Bordry 2021; Chakrabarti 2022; Merle 2022). Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is highly expressed across multiple solid tumors, such as CRC, GC, PDAC, and NSCLC. Restricted normal tissue expression and efficient lysosomal trafficking of CEACAM5 highlight its potential as an anticancer target. SGN-CEACAM5C is a novel investigational antibody-drug conjugate (ADC) directed to CEACAM5 composed of the humanized immunoglobulin G (IgG1) anti-CEACAM5 monoclonal antibody tusamitamab chemically conjugated to 8 molecules of the topoisomerase 1 inhibitor 7-aminomethyl-10,11-methylenedioxycamptothecin (AMDCPT). SGN-CEACAM5C selectively binds to CEACAM5 present on the cell surface and is internalized via the endo-lysosomal pathway, with subsequent release of the payload through enzymatic cleavage. Release of the cytotoxic payload induces DNA damage, cell cycle arrest in S phase, and apoptosis in tumor cells. In vitro bystander activity was observed against tumor cells without CEACAM5 surface expression. SGN-CEACAM5C is highly active in multiple patient-derived xenograft models, including CRC, GC, and NSCLC, across various levels of CEACAM5 expression (Baudat 2023; Baudat 2024). These preclinical findings justify evaluation of SGN-CEACAM5C in a clinical trial setting. Methods SGNCEA5C-001 (NCT06131840) is a phase 1, open-label, multicenter study designed to characterize the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary antitumor activity of SGN-CEACAM5C in adults with select advanced solid tumors. This study consists of dose escalation (Part A), dose and schedule optimization (Part B), and dose expansion (Part C). Eligible pts are adults ≥18 years of age with confirmed metastatic or unresectable solid tumor malignancy, measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1, an ECOG PS of 0-1, and 1 of the following tumor types: CRC, GC/gastroesophageal junction adenocarcinoma (GEJ), PDAC, NSCLC (squamous/non-squamous), or SCLC. Primary endpoints include incidence of AEs, laboratory abnormalities, dose modifications due to AEs, dose-limiting toxicities, and cumulative safety. Secondary endpoints include estimates of PK parameters, incidence of antidrug antibodies, objective response rate and best response per RECIST v1.1 by investigator, duration of response, PFS, and OS. Safety and antitumor activity endpoints will be summarized using descriptive statistics. Recruitment is ongoing for Part A in North America. Citation Format: Funda Meric-Bernstam, Cesar A. Perez, Nehal Lakhani, Meredith Pelster, Justin Call, Suzanne McGoldrick, Tianhua Wang, Brigitte Demers, Amita Patnaik. An open-label phase 1 study to investigate SGN-CEACAM5C in adults with advanced solid tumors (SGNCEA5C-001, Trial in Progress) [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 CT077.

Abstract 6909: KRASG12C inhibitor resistance in patient-derived non-small cell lung cancer models

Abstract Introduction: One quarter of lung adenocarcinomas (LUAD) harbor KRAS-mutations, with KRASG12C accounting for the majority (40%). GDP-KRASG12C inhibitors (G12Ci) have been developed for this patient cohort, and two of these are approved for clinical use in the US. While response rates to G12Ci tested in clinical trials have been between 35-50%, studies have identified mechanisms of adaptive resistance to these novel agents, limiting their use as monotherapies. However, primary (intrinsic) resistance to these compounds has not been explored extensively. We leveraged our patient-derived xenograft (PDX) and xenograft-derived organoid (XDO) development programs to study primary resistance to the novel G12Ci, AZD4625. Methods: Twelve KRASG12C PDX models were established which recapitulated patient tumor histology, and genomic, transcriptomic, and methylome profiles. From these 12 PDX, six long-term (passage >10) XDO were generated. PDX were treated with AZD4625 chronically for four weeks as well as in an acute dosing pharmacodynamic study, where tumors were harvested for further analysis. Results: The AZD4625 drug screen in our PDX models reproduced the G12Ci response rate observed in clinical trials. Four of twelve tumor models reduced in size on treatment, while the remainder were considered resistant as these tumors increased in size on treatment. Sensitive tumors became necrotic while those resistant remained proliferative. XDO models recapitulated their originating PDX genomic alterations, histology, as well as responses to AZD4625. During the acute pharmacodynamic screen in our PDX models, we observed a decrease in pERK1/2 and pS6 protein expression in sensitive but not resistant models, despite acute DUSP6 gene expression decreases in every model. Global proteomic analysis highlighted differences between each model. Conclusion: PDX and XDO are useful models to study resistance to this novel class of inhibitor, with the KRASG12C NSCLC PDX models exhibiting a treatment response rate that is similar to those observed in clinical trials. Global proteomics suggests uniqueness of each model in our cohort, hinting that a universal combination of specific targeted agents to treat KRASG12C LUAD tumors may be insufficient. Citation Format: Joshua C. Rosen, Nhu-An Pham, Quan Li, Pinjiang Cao, Katrina Hueniken, Takamasa Koga, Nikolina Radulovich, Alex Koers, Michael Niedbala, Sarah Ross, Adrian Sacher, Ming-Sound Tsao. KRASG12C inhibitor resistance in patient-derived non-small cell lung cancer models [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 6909.

Abstract 1974: Discovery of TRX-211-399, a potent, CNS-penetrant, highly selective inhibitor of EGFR exon 20 insertion mutations

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide, accounting for approximately 2 million new cases and 1.8 million deaths annually. Various types of EGFR-activating mutations are one of the most important oncogenic drivers in non-small cell lung cancer (NSCLC). Among NSCLC patients with EGFR mutations, 5-10% of patients have EGFR exon 20 insertion (ex20ins) mutations that are insensitive to clinically available EGFR tyrosine kinase inhibitors (TKIs). Recently, as mobocertinib (EXKIVITY®) failed to improve progression-free survival in Phase 3 clinical trial, there remains a high unmet medical need to develop new drugs with wider therapeutic window to treat NSCLC harboring EGFR ex20ins mutations. Herein, we report a novel EGFR-TKI, TRX-211-399, specifically designed to target ex20ins mutations. TRX-211-399 potently inhibited the proliferation of not only Ba/F3 cells overexpressing near-loop (A767dupASV, S768dupSVD) and far-loop (H773insNPH, H773dupH) mutations but also patient-derived NSCLC cell line. In particular, TRX-211-399 demonstrated high selectivity against wild-type EGFR. With favorable DMPK profiles, once-daily oral administration of TRX-211-399 showed significant tumor regression in multiple Ba/F3 xenograft models carrying different EGFR exon20ins mutations. Furthermore, in a PC-9-luc cell intracranial xenograft model, TRX-211-399 induced substantial anti-tumor effects and prolonged survival without body weight loss. TRX-211-399 is currently undergoing further preclinical evaluation as a potential candidate for clinical development for patients with EGFR exon20ins mutations. Citation Format: Kwangwoo Chun, Sumin Lim, Joohyun Lee, Seulgi Choi, Seongrak Kim, Youngil Choi, Areum Park, Bohee Kim, Yongseo Park, Eunhyun Choi, Sungwon Lee, Koo Lee. Discovery of TRX-211-399, a potent, CNS-penetrant, highly selective inhibitor of EGFR exon 20 insertion mutations [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 1974.

Immunofluorescence-Based Assay for High-Throughput Analysis of Multidrug Resistance Markers in Non-Small Cell Lung Carcinoma Patient-Derived Cells.

Lung cancer remains the leading cause of cancer death globally, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Multidrug resistance (MDR), often caused by ATP-binding cassette (ABC) transporters, represents a significant obstacle in the treatment of NSCLC. While genetic profiling has an important role in personalized therapy, functional assays that measure cellular responses to drugs are gaining in importance. We developed an automated microplate-based immunofluorescence assay for the evaluation of MDR markers ABCB1, ABCC1, and ABCG2 in cells obtained from NSCLC patients through high-content imaging and image analysis, as part of a functional diagnostic approach. This assay effectively discriminated cancer from non-cancer cells within mixed cultures, which is vital for accurate assessment of changes in MDR marker expression in different cell populations in response to anticancer drugs. Validation was performed using established drug-sensitive (NCI-H460) and drug-resistant (NCI-H460/R) NSCLC cell lines, demonstrating the assay's capacity to distinguish and evaluate different MDR profiles. The obtained results revealed wide-ranging effects of various chemotherapeutic agents on MDR marker expression in different patient-derived NSCLC cultures, emphasizing the need for MDR diagnostics in NSCLC. In addition to being a valuable tool for assessing drug effects on MDR markers in different cell populations, the assay can complement genetic profiling to optimize treatment. Further assay adaptations may extend its application to other cancer types, improving treatment efficacy while minimizing the development of resistance.

Intrinsic resistance to ROS1 inhibition in a patient with CD74-ROS1 mediated by AXL overexpression.

The vast majority of patients with ROS1 positive non-small cell lung cancer (NSCLC) derive clinical benefit from currently approved ROS1 therapies, including crizotinib and entrectinib. However, a small proportion of patients treated with ROS1 inhibitors fail to derive any clinical benefit and demonstrate rapid disease progression. The biological mechanisms underpinning intrinsic resistance remain poorly understood for oncogene-driven cancers. We generated a patient-derived cell line, CUTO33, from a ROS1 therapy naive patient with CD74-ROS1+ NSCLC, who ultimately did not respond to a ROS1 inhibitor. We evaluated a panel of ROS1+ patient-derived NSCLC cell lines and used cell-based assays to determine the mechanism of intrinsic resistance to ROS1 therapy. The CUTO33 cell line expressed the CD74-ROS1 gene fusion at the RNA and protein level. The ROS1 fusion protein was phosphorylated at baseline consistent with the known intrinsic activity of this oncogene. ROS1 phosphorylation could be inhibited using a wide array of ROS1 inhibitors, however these inhibitors did not block cell proliferation, confirming intrinsic resistance in this model and consistent with the patient's lack of response to a ROS1 inhibitor. CUTO33 expressed high levels of AXL, which has been associated with drug resistance. Combination of an AXL inhibitor or AXL knockdown with a ROS1 inhibitor partially reversed resistance. In summary, we demonstrate that AXL overexpression is a mechanism of intrinsic resistance to ROS1 inhibitors.

Circadian gene ARNTL initiates circGUCY1A2 transcription to suppress non-small cell lung cancer progression via miR-200c-3p/PTEN signaling

BackgroundAs a subclass of endogenous stable noncoding RNAs, circular RNAs are beginning to be appreciated for their potential as tumor therapeutics. However, the functions and mechanisms by which circRNAs exert protective functions in non-small cell lung cancer (NSCLC) remain largely elusive.MethodsThe prognostic role of circGUCY1A2 was explored in lung adenocarcinoma specimens. The overexpressed and knockdown plasmids were used to evaluate the effect of circGUCY1A2 on NSCLC cell proliferation and apoptosis efficacy. Luciferase reporter system is used to prove that circGUCY1A2 could bind to miRNA. Chip-PCR was used to prove that circGUCY1A2 could be initiated by transcription factors ARNTL. Subcutaneous tumorigenicity grafts models were established to validate findings in vivo.ResultsThe expression of circGUCY1A2 were significantly reduced (P < 0.001) and negatively correlated with tumor size (P < 0.05) in non-small cell lung cancer (NSCLC). CircGUCY1A2 upregulation promoted apoptosis and inhibits cell proliferation and growth of subcutaneous tumorigenicity grafts in nude mice (P < 0.01). In addition, intra-tumor injection of pLCDH-circGUCY1A2 inhibited tumor growth in patient-derived NSCLC xenograft models (PDX). Mechanism studies showed that circGUCY1A2 could act as a sponge to competitively bind miR-200c-3p, promote PTEN expression, and thereby inhibit PI3K/AKT pathway. In addition, we found that the circadian gene ARNTL, which was reduced in NSCLC and prolonged the overall survival of patients, could bind to the promoter of circGUCY1A2, thereby increasing its expression.ConclusionsThis study is an original demonstration that ARNTL can inhibit the development of lung adenocarcinoma through the circGUCY1A2/miR-200c-3p/PTEN axis, and this finding provides potential targets and therapeutic approaches for the treatment of lung adenocarcinoma.

OSGIN1 is a novel TUBB3 regulator that promotes tumor progression and gefitinib resistance in non-small cell lung cancer.

Oxidative stress induced growth inhibitor 1 (OSGIN1) regulates cell death. The role and underlying molecular mechanism of OSGIN1 in non-small cell lung cancer (NSCLC) are uncharacterized. OSGIN1 expression in NSCLC samples was detected using immunohistochemistry and Western blotting. Growth of NSCLC cells and gefitinib-resistant cells expressing OSGIN1 or TUBB3 knockdown was determined by MTT, soft agar, and foci formation assays. The effect of OSGIN1 knockdown on in vivo tumor growth was assessed using NSCLC patient-derived xenograft models and gefitinib-resistant patient-derived xenograft models. Potentially interacting protein partners of OSGIN1 were identified using IP-MS/MS, immunoprecipitation, PLA, and Western blottingassays. Microtubule dynamics were explored by tubulin polymerization assay and immunofluorescence. Differential expression of signaling molecules in OSGIN1 knockdown cells was investigated using phospho-proteomics, KEGG analysis, and Western blotting. We found thatOSGIN1 is highly expressed in NSCLC tissues and is positively correlated with low survival rates and tumor size in lung cancer patients. OSGIN1 knockdown inhibited NSCLC cell growth and patient-derived NSCLC tumor growth in vivo. Knockdown of OSGIN1 strongly increased tubulin polymerization and re-established gefitinib sensitivity in vitro and in vivo. Additionally, knockdown of TUBB3 strongly inhibited NSCLC cell proliferation. Mechanistically, we found that OSGIN1 enhances DYRK1A-mediated TUBB3 phosphorylation, which is critical for inducing tubulin depolymerization. The results of phospho-proteomics and ontology analysis indicated that knockdown of OSGIN1 led to reduced propagation of the MKK3/6-p38 signaling axis. We propose that OSGIN1 modulates microtubule dynamics by enhancing DYRK1A-mediated phosphorylation of TUBB3 at serine 172. Moreover, elevated OSGIN1 expression promotes NSCLC tumor growth and gefitinib resistance through the MKK3/6-p38 signaling pathway. Our findings unveil a new mechanism of OSGIN1 and provide a promising therapeutic target for NSCLC treatment in the clinic.

A novel platform to enrich for tumor-specific T cells in non–small-cell lung cancer.

2556 Background: To induce robust cellular immunity against non-small cell lung cancer (NSCLC), the leading cause of cancer-related death in the US, we have developed an innovative cell-based therapeutic platform technology to generate polyclonal, neoantigen-reactive T cells in vitro. Methods: Surgically resected NSCLC tissue and peripheral matched blood were collected from 17 patients under an IRB-approved protocol. From 3 of the patient-derived NSCLC specimens, methods were tested to enhance immunogenicity and stimulate neoantigen-specific immune responses. We then use a novel, human vascularized microtumor (VMT) platform to assess the sensitivity of tumor cells to T-cell mediated attack at the individual patient level. Results: The therapeutic platform induced a significant increase in T cell proliferation, interferon-gamma secretion, and blast phenotype of lymphocyte populations compared to T cells primed by standard methods. Interestingly, for one patient, immunosuppression of the T cell response with concomitant increase in CD96, an immune checkpoint receptor implicated in lung cancer progression, was observed in the tumor. Importantly, using sequencing data from one patient, an analytical and machine learning pipeline detected putative neoantigens using our therapeutic platform technology and not with standard methods. Conclusions: The therapeutic platform can be used to directly generate cell-based immunotherapies and to facilitate neoantigen discovery and development of targeted cancer immunotherapies. Our goal is to advance novel approaches to effectively mobilize anti-tumor immunity and overcome treatment resistance in NSCLC.

Abstract 3936: ALK fusion oncogene driven SERPINB4 expression enhances tumor survival in NSCLC

Abstract Anaplastic lymphoma kinase (ALK) fusion variants in non-small-cell-lung cancer (NSCLC) consist of numerous dimerising fusion partners, with the most common being EML4. Clinical data suggests that the degree of treatment benefit in response to ALK tyrosine kinase inhibitors (TKIs) differs among the variant present in the patient tumor. Therefore, a better understanding the oncogenic signaling networks driven by different ALK-fusion variants is important. Here, we developed highly controlled doxycycline-inducible cell models bearing four different ALK fusion proteins, namely EML4-ALK-V1, EML4-ALK-V3, KIF5B-ALK, and TFG-ALK, in the context of non-tumorigenic NL20 human bronchial epithelial cells. These were complimented by patient-derived NSCLC cell lines harboring either EML4-ALK-V1 or EML4-ALK-V3 fusions. RNA-seq and phosphoproteomics analysis were employed to identify dysregulated genes and hyper/hypo-phosphorylated proteins associated with ALK fusion expression. Among ALK fusion induced responses, we noted a robust inflammatory signature that included up-regulation of the Serpin B4 serine protease inhibitor in both NL20-inducible cell models and ALK-positive NSCLC patient-derived cell lines. We show that STAT3 is a major transcriptional regulator of SERPINB4 downstream of ALK fusions, along with NF-kB and AP1. The upregulation of SERPINB4 promotes survival of ALK fusion expressing cells and inhibits natural killer (NK) cell-mediated cytotoxicity. In conclusion, our study reveals a novel ALK downstream survival axis that regulates Serpin B4 expression and identifies a molecular target that has potential for therapeutic impact targeting the immune response together with ALK TKIs in NSCLC. Citation Format: Tzu-Po Chuang, Wei-Yun Lai, Jonatan L. Gabre, Dan E. Lind, Ganesh Umapathy, Abdulmalik A. Bokhari, Bengt Bergman, Linnea Kristenson, Fredrik B. Thorén, Anh Le, Robert Doebele, Jimmy V. Eynden, Ruth H. Palmer, Bengt Hallberg. ALK fusion oncogene driven SERPINB4 expression enhances tumor survival in NSCLC. [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 3936.

ALK fusion NSCLC oncogenes promote survival and inhibit NK cell responses via SERPINB4 expression

Anaplastic lymphoma kinase (ALK) fusion variants in Non-Small Cell Lung Cancer (NSCLC) consist of numerous dimerizing fusion partners. Retrospective investigations suggest that treatment benefit in response to ALK tyrosine kinase inhibitors (TKIs) differs dependent on the fusion variant present in the patient tumor. Therefore, understanding the oncogenic signaling networks driven by different ALK fusion variants is important. To do this, we developed controlled inducible cell models expressing either Echinoderm Microtubule Associated Protein Like 4 (EML4)-ALK-V1, EML4-ALK-V3, Kinesin Family Member 5B (KIF5B)-ALK, or TRK-fused gene (TFG)-ALK and investigated their transcriptomic and proteomic responses to ALK activity modulation together with patient-derived ALK-positive NSCLC cell lines. This allowed identification of both common and isoform-specific responses downstream of these four ALK fusions. An inflammatory signature that included upregulation of the Serpin B4 serine protease inhibitor was observed in both ALK fusion inducible and patient-derived cells. We show that Signal transducer and activator of transcription 3 (STAT3), Nuclear Factor Kappa B (NF-κB) and Activator protein 1 (AP1) are major transcriptional regulators of SERPINB4 downstream of ALK fusions. Upregulation of SERPINB4 promotes survival and inhibits natural killer cell-mediated cytotoxicity, which has potential for therapeutic impact targeting the immune response together with ALK TKIs in NSCLC.

RBIO-02. ACTIVATION OF GABAA RECEPTORS WITH A NON-TOXIC, BRAIN PENETRANT SMALL MOLECULE SENSITIZES LUNG ADENOCARCINOMA PRIMARY AND BRAIN METASTATIC TUMOR CELLS TO RADIATION VIA AUTOPHAGY INDUCTION

Abstract Most advanced-stage non-small cell lung cancer (NSCLC) patients have brain metastases that render a dismal prognosis. Treatment of metastatic brain lesions from NSCLC and other tumor types include radiation as part of a multimodal treatment regimen. Challenges in the application of radiotherapy include overcoming radiation resistance and reducing associated co-morbidities. Non-toxic therapeutics capable of sensitizing tumors to radiation are needed to improve survival and mitigate radiation side-effects. Many CNS and solid systemic tumors express ligand-gated ion channels, which may contribute to tumor growth. Leveraging ion channels is therefore a potential way of diminishing the spread of cancer. We find that NSCLC and its brain metastases express subunits of the type-A GABA-gated chloride channel or GABAA receptor. Importantly, patient-derived NSCLC cells have functional GABAA receptors. We identified a brain penetrant, small molecule activator of GABAA receptors (AMLAL-101), which alone impairs the viability of both primary NSCLC cells and brain metastatic cells. In addition, AMLAL-101 combined with radiation is a highly potent inducer of NSCLC cell death and clonogenic arrest. Using a human ex vivo model of NSCLC-on-chip, we assessed the efficacy and toxicity of AMLAL-101 relative to Docetaxel, an antimicrotubular agent used in treating advanced NSCLC. AMLAL-101 is as potent as Docetaxel but does not exhibit its toxic side effects. AMLAL-101 also potentiates radiation in vivo, significantly reducing lung adenocarcinoma xenograft tumor growth in mice, equivalent to docetaxel plus radiation. Mechanistically, AMLAL-101 activates GABAA receptors in NSCLC and synergizes with radiation by inducing an autophagic response that includes: (i) stabilization of Beclin-1, BNIP3L/NIX, and GABARAP; (ii) ATG7 upregulation; and (iii) utilization of ubiquitin-binding protein p62. Activating GABAA receptors in NSCLC and other tumor types may improve radiation efficacy and mitigate its toxic side effects in treating brain metastases.

EP16.04-010 Histological and Genetic Parental Tumor Characteristics are Conserved in Patient-derived Non-small Cell Lung Cancer Organoids

Recent innovations in the treatment of non-small cell lung cancer (NSCLC) require a personalized approach and representative in-vitro model systems that reflect the primary tumor both morphologically and genetically. Clonally expanded cell lines are fundamentally limited in representing the complexity of NSCLC as they do not generally maintain intratumoral heterogeneity and the three-dimensional structure. For clinical applications such as an ex-vivo evaluation of personalized cancer treatments, patient-derived organoids have been suggested as promising models.

Cisplatin-Resistant CD44+ Lung Cancer Cells Are Sensitive to Auger Electrons.

Cancer stem cells (CSCs) are resistant to conventional therapy and present a major clinical challenge since they are responsible for the relapse of many cancers, including non-small cell lung cancer (NSCLC). Hence, future successful therapy should also eradicate CSCs. Auger electrons have demonstrated promising therapeutic potential and can induce DNA damage while sparing surrounding cells. Here, we sort primary patient-derived NSCLC cells based on their expression of the CSC-marker CD44 and investigate the effects of cisplatin and a thymidine analog (deoxyuridine) labeled with an Auger electron emitter (125I). We show that the CD44+ populations are more resistant to cisplatin than the CD44− populations. Interestingly, incubation with the thymidine analog 5-[125I]iodo-2′-deoxyuridine ([125I]I-UdR) induces equal DNA damage, G2/M cell cycle arrest, and apoptosis in the CD44− and CD44+ populations. Our results suggest that Auger electron emitters can also eradicate resistant lung cancer CD44+ populations.

Abstract 336: Mechanism of action of MCLA-129, a bispecific antibody that targets EGFR and c-MET and impairs growth of EGFR exon 20 insertion mutant non-small cell lung cancer

Abstract Introduction: MCLA-129 is an ADCC-enhanced IgG1 bispecific antibody that targets epidermal growth factor receptor (EGFR) and c-MET. The HGF/c-MET pathway is frequently upregulated in tumors that are resistant to EGFR tyrosine kinase inhibitors (TKIs). This study investigated the MCLA-129 mechanism of action in vitro and effectiveness in vivo in an EGFR exon20 insertion (ex20ins) non-small cell lung cancer (NSCLC) model. Methods: To study the effect of MCLA-129 on c-MET and EGFR dimerization, β-galactosidase enzyme complementation assays were performed. Ligand-dependent phosphorylation of EGFR and c-MET was assessed in vitro on the NCI-H1650 NSCLC cell line by immunoblotting. Shotgun mutagenesis epitope mapping was used to identify the binding epitope of MCLA-129 to EGFR. ADCC activity of MCLA-129 was determined in NSCLC cells expressing different levels of EGFR and c-MET using an ADCC reporter assay with high affinity FcγRIII 158V-variant and low affinity 158F-variant effector cells. ADCP was measured with monocyte-derived macrophages incubated with pHrodo-labeled HCC827 NSCLC target cells with readout of tumor cell phagocytosis on the Incucyte® system. For the evaluation of efficacy in vivo in the EGFR exon20ins PDX model, MCLA-129 was administered at 2.5, 8 or 25 mg/kg i.p. weekly. Results: MCLA-129 blocks EGF and HGF binding to EGFR and c-MET, respectively, and prevents receptor dimerization. Consequently, MCLA-129 inhibits phosphorylation of EGFR and c-MET as demonstrated in NCI-H1650 cells. Mutagenesis analysis identified critical binding residues of domain III of EGFR for MCLA-129 binding that are distinct from cetuximab binding. MCLA-129 displayed potent ADCC activity on NSCLC cell lines using effector cells expressing either high or low affinity FcγRIII variant. MCLA-129 demonstrated potent dose-dependent ADCP of HCC827 NSCLC cells. Finally, MCLA-129 led to significant dose-dependent regression of a patient-derived EGFR exon20ins NSCLC tumor in a mouse PDX model. Conclusion: MCLA-129 is a Biclonics® common light chain bispecific antibody with multiple mechanisms of action including potent inhibition of c-MET and EGFR ligand binding, ligand-induced receptor dimerization and phosphorylation, ADCC and ADCP. MCLA-129 demonstrates significant growth inhibition of a patient-derived EGFR exon20ins tumor in a preclinical xenograft model. A phase 1/2 clinical trial of MCLA-129 in solid tumors is ongoing. These data support the further clinical development of MCLA-129 in patients with NSCLC, including NSCLC with EGFR exon20ins, and other solid tumors. Citation Format: David J. de Gorter, Marie O'Connor, Alexandre Deshiere, Martijn van Rosmalen, Szabolcs Fatrai, Jeroen Lammerts van Bueren, Cecile A. Geuijen. Mechanism of action of MCLA-129, a bispecific antibody that targets EGFR and c-MET and impairs growth of EGFR exon 20 insertion mutant non-small cell lung cancer [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 336.

The sodium/myo-inositol co-transporter SLC5A3 promotes non-small cell lung cancer cell growth

Identification of novel molecular signaling targets for non-small cell lung cancer (NSCLC) is important. The present study examined expression, functions and possible underlying mechanisms of the sodium/myo-inositol co-transporter SLC5A3 in NSCLC. The Cancer Genome Atlas (TCGA) database and local NSCLC tissue results demonstrated that SLC5A3 expression in NSCLC tissues (including patient-derived primary NSCLC cells) was significantly higher than that in normal lung tissues and lung epithelial cells. In primary NSCLC cells and immortalized lines, SLC5A3 depletion, using small hairpin RNA (shRNA) and CRSIRP/Cas9 methods, robustly impeded cell proliferation and migration, simultaneously provoking cell cycle arrest and apoptosis. Conversely, ectopic overexpression of SLC5A3 further enhanced proliferation and migration in primary NSCLC cells. The intracellular myo-inositol contents and Akt-mTOR activation were largely inhibited by SLC5A3 silencing or knockout (KO), but were augmented following SLC5A3 overexpression in primary NSCLC cells. Significantly, SLC5A3 KO-induced anti-NSCLC cell activity was largely ameliorated by exogenously adding myo-inositol or by a constitutively-active Akt construct. By employing the patient-derived xenograft (PDX) model, we found that the growth of subcutaneous NSCLC xenografts in nude mice was largely inhibited by intratumoral injection SLC5A3 shRNA adeno-associated virus (AAV). SLC5A3 silencing, myo-inositol depletion, Akt-mTOR inactivation and apoptosis induction were detected in SLC5A3 shRNA virus-injected NSCLC xenograft tissues. Together, elevated SLC5A3 promotes NSCLC cell growth possibly by maintaining myo-inositol contents and promoting Akt-mTOR activation.

LPIN1 Induces Gefitinib Resistance in EGFR Inhibitor-Resistant Non-Small Cell Lung Cancer Cells.

Simple SummaryHere, we attempted to identify targets that could be used to overcome resistance toward epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC). To accomplish this, we chose LPIN1 among the candidate targets that were identified from a previously performed genome-wide RNAi screening assay and validated it as a key factor regulating gefitinib resistance in EGFR-mutant NSCLC cells. We confirmed that LPIN1 depletion increased gefitinib sensitivity in drug-resistant H1650 NSCLC cells, as well as patient-derived YL05 lung cancer cells. Moreover, we found that LPIN1 expression was induced following gefitinib treatment, and activities of protein kinase C delta and nuclear factor kappa B, and lipid droplet formation were induced in an LPIN1-dependent manner. Additionally, we validated that targeting LPIN1 synergistically retarded tumor growth in an in vivo mouse xenograft model.Drug resistance limits the efficacy of targeted therapies, including tyrosine kinase inhibitors (TKIs); however, a substantial portion of the drug resistance mechanisms remains unexplained. In this study, we identified LPIN1 as a key factor that regulates gefitinib resistance in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) cells. Unlike TKI-sensitive HCC827 cells, gefitinib treatment induced LPIN1 expression and increased diacylglycerol concentration in TKI-resistant H1650 cells, followed by the activation of protein kinase C delta and nuclear factor kappa B (NF-κB) in an LPIN1-dependent manner, resulting in cancer cell survival. Additionally, LPIN1 increased the production of lipid droplets, which play an important role in TKI drug resistance. All results were recapitulated in a patient-derived EGFR-mutant NSCLC cell line. In in vivo tumorigenesis assay, we identified that both shRNA-mediated depletion and pharmaceutical inhibition of LPIN1 clearly reduced tumor growth and confirmed that gefitinib treatment induced LPIN1 expression and LPIN1-dependent NF-κB activation (an increase in p-IκBα level) in tumor tissues. These results suggest an effective strategy of co-treating TKIs and LPIN1 inhibitors to prevent TKI resistance in NSCLC patients.

Abstract 1710: A T-SIGn cancer gene therapy virus expressing an EpCAM-targeted bispecific T-cell activator stimulates T-cells and induces tumor cell death in patient-derived organotypic non-small cell lung cancer (NSCLC) spheroids ex vivo

Abstract Tumor-Specific Immuno-Gene (T-SIGn) cancer gene therapy vectors, which are based on the oncolytic adenovirus enadenotucirev, carry a gene cassette encoding combinations of human transgenes under control of the virus major late promoter (MLP) which restricts expression to tumor cells. These vectors can be dosed intravenously for delivery to tumors and selective expression of therapeutic proteins locally within the tumor microenvironment. Because these viruses only infect human tumor cells, in vivo studies are limited to human tumor xenografts in immunodeficient mice. Recently, we have optimized a 3D microfluidic system for evaluating response to immunotherapies using patient-derived organotypic tumor spheroids (PDOTS). PDOTS contain stromal as well as autologous myeloid and lymphoid cell populations and respond to PD-1 blockade in short-term ex vivo culture. As an alternative preclinical approach for evaluating activity and mechanistic underpinnings of T-SIGn viruses expressing human immunomodulatory transgenes, we have been developing the PDOTS ex vivo culture model. Here we report on the visualization of oncolytic virus uptake, replication in tumor cells and activation of T cells with a T-SIGn virus encoding an EpCAM-targeted bispecific T-cell activator (NG-611) within NSCLC PDOTS. Surgical NSCLC cases from St. Elizabeth's Medical Center, collected under an IRB approved protocol, were processed in PDOTS and assessed for EpCAM expression and number of T cells. Five different samples were treated with NG-611 (mixed with a GFP expressing vector) at concentrations ranging from 2x109 to 2x106 vp/mL final volume of culture. Virus infection was shown after 3 and 5 days of culture by dose-dependent expression of GFP reporter transgene driven by a CMV promoter, and virus replication in tumor cells was confirmed by using GFP expressed under the virus MLP promoter. Upregulation of IFNγ and Granzyme B in culture media indicated a strong activation of T-cells via expression of the bispecific T-cell activator. Cytotoxic T-cell activation also led to dose-dependent cell killing in NSCLC spheroids as shown by life/death analysis. Having established this proof of concept for functionality of T-SIGn viruses in PDOTS, ongoing studies are evaluating the immunomodulatory activities of a series of T-SIGn viruses expressing human IL-12 with or without other cytokine/chemokines. Citation Format: Moataz Noureddine, Katy West, Elizabeth Isaacson, Sylvia Alarcon, Christopher S. Lathan, John Wain, David A. Barbie, Cloud P. Paweletz, Elena Ivanova, Brian R. Champion. A T-SIGn cancer gene therapy virus expressing an EpCAM-targeted bispecific T-cell activator stimulates T-cells and induces tumor cell death in patient-derived organotypic non-small cell lung cancer (NSCLC) spheroids ex vivo [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 1710.

Abstract 1111: MYC promotes tyrosine kinase inhibitor resistance in ROS1 fusion positive lung cancers

Abstract Targeted therapy of ROS1 fusion-driven non-small cell lung cancer (NSCLC) with the tyrosine kinase inhibitor (TKI), crizotinib, has achieved notable clinical success. Despite these promising results, resistance to therapy poses a significant challenge. 47-92% of crizotinib-resistant tumors have a wild-type ROS1 kinase domain suggesting that concurrent aberrations in bypass signaling pathways contribute to sustained cancer growth even in the presence of ROS1 inhibition. Bypass resistance pathways are not well-characterized in ROS1 fusion-driven cancers; hence there is a dearth of secondary therapeutic strategies. To identify recurrent genomic alterations that co-occur with ROS1 fusions and could potentially impinge on sensitivity to crizotinib and other emerging ROS1 TKIs, we interrogated MSK-IMPACT datasets and found that 10% of patients with ROS1-fusion have concurrent MYC copy number amplification (CNA). We hypothesized that c-Myc overexpression compensates for the loss of ROS1 signaling due to ROS1 TKI treatment. Here we dissected the role of c-Myc overexpression in diminishing ROS1 TKI sensitivity using two models: 1) a MYC-amplified and crizotinib-resistant patient-derived ROS1-fusion NSCLC cell line (LUAD0006) and 2) a CRISPR-Cas9 engineered human bronchial epithelial EZR-ROS1 cell line (HBEC-ER) exhibiting sensitivity to ROS1 TKI. We validated LUAD0006 ROS1 TKI-resistance using cell viability assays and confirmed MYC CNA and protein overexpression with fluorescence in situ hybridization and immunoblotting, respectively. Live cell imaging assays revealed that ROS1-TKI or c-Myc knockdown via shRNA alone were insufficient to inhibit LUAD0006 cell growth; however combined ablation of ROS1 kinase activity and c-Myc expression significantly suppressed cell proliferation. Gain of function experiments using HBEC-ER revealed that these cells acquire functional resistance to multiple ROS1 TKIs upon expression of a degradation-resistant c-Myc T58A mutant. Notably, immunoblotting confirmed 50-fold overexpression of c-Myc T58A relative to endogenous c-Myc in unmodified cells. We also performed RNA sequencing and reverse-phase protein array (RPPA) analysis of LUAD0006 cells treated with or without crizotinib and found that targets related to cell cycle, apoptosis, and metabolism were differentially expressed. Finally, we found that CDK4/6 and BET bromodomain inhibitors re-sensitized LUAD0006 cells to crizotinib, demonstrating the importance of c-Myc to the survival of these TKI-resistant cells. Taken together, we demonstrate that CNA or post-translational stabilization that leads to c-Myc overexpression confers resistance to crizotinib and other emerging ROS1 TKIs. These patients will likely benefit from a combination therapeutic strategy that targets both ROS1 and c-Myc. Citation Format: Sudarshan R. Iyer, Igor Odintsov, Adam J. Schoenfeld, Hiroki Sato, Evan Siau, Alexander Drilon, Gregory J. Riely, Romel Somwar, Marc Ladanyi, Monika A. Davare. MYC promotes tyrosine kinase inhibitor resistance in ROS1 fusion positive lung cancers [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 1111.

Abstract 3184: Falcon-X 3D in vitro tumor model platform for screening patient-derived NSCLC, CRC, and RCC tumors with targeted therapy and immunotherapy

Abstract Significant challenges remain in developing oncology assays in vitro that correlate to the in vivo outcome, and in particular, including the immune compartment to effectively assay immunotherapy. Matrix-free systems such as 3D spheroids or 2D plastic plates fail to recreate critical features of the tumor microenvironment (TME) such as T cell infiltration through the extracellular matrix (ECM) stroma and desmoplastic T cell exclusion, and matrix-based systems such as basement membrane extract or collagen I suffer from batch variability and unwanted immunogenicity, as well as handling issues that impair their downstream analysis. Here, we aimed to solve these biological and technical challenges by utilizing a novel 3D hydrogel platform called Falcon-X to co-culture patient-derived xenograft (PDX) tumors of colo-rectal cancer (CRC), non-small cell lung cancer (NSCLC), or renal cell carcinoma (RCC) origin with other TME cell types such as fibroblasts and T cells in order to screen for targeted and immuno-therapy efficacy. High content confocal imaging enabled analysis of tumor size, killing, and T cell penetration. PDX tumors were cultured in a biocompatible and chemically-defined ECM hydrogel (Versagel, Hribar, 2019), layered in 96-well plates using a 3D gel patterning technology (Symphony, Hribar, 2019), and subsequently cultured in a standard cell culture medium. In some cases, fibroblasts were added to the culture in order to mimic stromal cues of the TME such as desmoplasia. For targeted therapy, compounds were added to the culture medium for 72 hours. For immunotherapy, pre-activated PBMCs were added and T cell infiltration and T-cell mediated tumor killing were quantitated in response to standard of care compounds. For CRC PDX tumors, Oxaliplatin, Cetuximab, 5-FU, Bevacizumab (anti-VEGF), and Pembrolizumab (anti-PD1) were assayed; for RCC: Sunitinib, Pazopanib, Temsirolimus, Bevacizumab, and Pembrolizumab; for NSCLC: Docetaxel, Cisplatin, Afatinib, Atezolizumab (anti-PDL1), and Pembrolizumab. Importantly, the IC50 values demonstrated a high degree of correlation to the in vivo PDX outcome. Differences in IC50s were noted with the addition of fibroblasts, suggesting active crosstalk between stromal, tumoral, and immune compartments.Together, these data reveal a novel Falcon-X 3D assay platform for culturing tumor, fibroblast, and immune cells in a 3D hydrogel and assaying critical features of the TME such as T cell infiltration and tumor killing. Such a platform is potentially suitable for cancer drug discovery and clinical decision making for screening cancer therapeutic efficacy. Further studies include identifying cytokines that modulate the immune response to fibroblast co-culture, expanding the approach to other solid tumor types such as breast, brain, sarcoma, and melanoma, as well as screening novel immunotherapy agents. Citation Format: Bin Xue, Julia Schueler, Timothy Jensen, Kolin C. Hribar. Falcon-X 3D in vitro tumor model platform for screening patient-derived NSCLC, CRC, and RCC tumors with targeted therapy and immunotherapy [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 3184.