Lung Cancer Driver Research Articles

Impad1 and Syt11 work in an epistatic pathway that regulates EMT-mediated vesicular trafficking to drive lung cancer invasion and metastasis.

Lung cancer is a highly aggressive and metastatic disease responsible for approximately 25% of all cancer-related deaths in the United States. Using high-throughput invitro and invivo screens, we have previously established Impad1 as a driver of lung cancer invasion and metastasis. Here we elucidate that Impad1 is a direct target of the epithelial microRNAs (miRNAs) miR-200 and miR∼96 and is de-repressed during epithelial-to-mesenchymal transition (EMT); thus, we establish a mode of regulation of the protein. Impad1 modulates Golgi apparatus morphology and vesicular trafficking through its interaction with a trafficking protein, Syt11. These changes in Golgi apparatus dynamics alter the extracellular matrix and the tumor microenvironment (TME) to promote invasion and metastasis. Inhibiting Impad1 or Syt11 disrupts the cancer cell secretome, regulates the TME, and reverses the invasive or metastatic phenotype. This work identifies Impad1 as a regulator of EMT and secretome-mediated changes during lung cancer progression.

Phenotypic drug screen uncovers the metabolic GCH1/BH4 pathway as key regulator of EGFR/KRAS-mediated neuropathic pain and lung cancer.

Increased tetrahydrobiopterin (BH4) generated in injured sensory neurons contributes to increased pain sensitivity and its persistence. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme in the de novo BH4 synthetic pathway, and human single-nucleotide polymorphism studies, together with mouse genetic modeling, have demonstrated that decreased GCH1 leads to both reduced BH4 and pain. However, little is known about the regulation of Gch1 expression upon nerve injury and whether this could be modulated as an analgesic therapeutic intervention. We performed a phenotypic screen using about 1000 bioactive compounds, many of which are target-annotated FDA-approved drugs, for their effect on regulating Gch1 expression in rodent injured dorsal root ganglion neurons. From this approach, we uncovered relevant pathways that regulate Gch1 expression in sensory neurons. We report that EGFR/KRAS signaling triggers increased Gch1 expression and contributes to neuropathic pain; conversely, inhibiting EGFR suppressed GCH1 and BH4 and exerted analgesic effects, suggesting a molecular link between EGFR/KRAS and pain perception. We also show that GCH1/BH4 acts downstream of KRAS to drive lung cancer, identifying a potentially druggable pathway. Our screen shows that pharmacologic modulation of GCH1 expression and BH4 could be used to develop pharmacological treatments to alleviate pain and identified a critical role for EGFR-regulated GCH1/BH4 expression in neuropathic pain and cancer in rodents.

Estimated sensitivity profiles of lung cancer specific uncommon BRAF mutants towards experimental and clinically approved kinase inhibitors

Current experimental and clinical data are inadequate to conclusively predict the oncogenicity of uncommon BRAF mutants and their sensitivity towards kinase inhibitors. Therefore, the present study aims at estimating sensitivity profiles of uncommon lung cancer specific BRAF mutations towards clinically approved as well as experimental therapeutics based on computationally derived direct binding energies. Based on the data derived from cBioportal, BRAF mutants displayed significant mutual exclusivity with KRAS and EGFR mutants indicating them as potential drivers in lung cancer. Predicted sensitivity of BRAF-V600E conformed to published experimental and clinical data thus validating the usefulness of computational approach. The BRAF-V600K displayed higher sensitivity to most inhibitors as compared to that of the BRAF-V600E. All the uncommon mutants displayed higher sensitivity than both the wild type and BRAF-V600E towards PLX 8394 and LSN3074753. While V600K, G469R and N581S displayed favorable sensitivity profiles to most inhibitors, V600L/M, G466A/E/V and G469A/V displayed resistance profiles to a variable degree. Notably, molecular dynamic (MD) simulation revealed that increased number of interactions caused enhanced sensitivity of G469R and N581S towards sorafenib. RAF kinase inhibitors were further classified into two groups as per their selectivity (Group I: BRAF-V600E-selective and Group II: CRAF-selective) based on which potential mutation-wise combinations of RAF kinase inhibitors were proposed to overcome resistance. Based on computational inhibitor sensitivity profiles, appropriate treatment strategies may be devised to prevent or overcome secondary drug resistance in lung cancer patients with uncommon mutations.

Identification of exosomal circRNA CD226 as a potent driver of nonsmall cell lung cancer through miR-1224-3p/high mobility group AT-hook 2 axis.

Circular RNAs (circRNAs) are crucial for the pathogenesis of nonsmall lung cancer (NSCLC). Here, we set out to unravel the precise function of circRNA CD226 (circCD226) in NSCLC pathogenesis. The exosomes from serum specimens were observed by transmission electron microscopy. CircCD226, miR-1224-3p and high mobility group AT-hook 2 (HMGA2) were quantified by qRT-PCR, western blot and immunohistochemistry. Actinomycin D and Ribonuclease (RNase) R treatments and subcellular localization assay were used for circCD226 characterization. Cell viability, proliferation, migration, invasion and sphere formation abilities were gauged by CCK-8, EDU, wound-healing, transwell and sphere formation assays, respectively. Directed relationships among circCD226, miR-1224-3p and HMGA2 were examined by RNA pull-down, dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The abundance of circCD226 was elevated in serum exosomes, tissues and cells of NSCLC. NSCLC serum exosomes enhanced NSCLC cell proliferation, migration, invasion and stemness. Loss of circCD226 impeded cell proliferation, migration, invasion and stemness in vitro , as well as tumor growth in vivo . Mechanistically, circCD226 sponged miR-1224-3p, and miR-1224-3p targeted HMGA2. CircCD226 involved the posttranscriptional regulation of HMGA2 through miR-1224-3p. Moreover, the miR-1224-3p/HMGA2 axis was identified as a functionally downstream effector of circCD226 in regulating NSCLC cell behaviors. Our study identifies circCD226 as a potential driver in NSCLC development depending on the regulation of miR-1224-3p/HMGA2 axis.

An Immunogenic Model of KRAS-Mutant Lung Cancer Enables Evaluation of Targeted Therapy and Immunotherapy Combinations.

This study develops a mouse model of immunogenic KRAS-mutant lung cancer to facilitate the investigation of optimal combinations of targeted therapies with immunotherapies.

SRC Family Kinase Inhibition Targets YES1 and YAP1 as Primary Drivers of Lung Cancer and as Mediators of Acquired Resistance to ALK and Epidermal Growth Factor Receptor Inhibitors.

PURPOSEThe identification of novel oncogenic driver alterations and novel mechanisms of acquired resistance (AR) is the key for further development of personalized therapy. The current study investigates the potential role of YES1 amplification as a primary driver of tumorigenesis and of YES1/YAP1 amplifications as mediators of AR to ALK and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs).METHODSModels of ectopic expression were established and characterized for YES1 and YAP1 in human bronchial epithelial cells and ALK fusion–positive (ALK+) and EGFR-mutant lung adenocarcinoma cell lines. MSK-IMPACT data for all lung adenocarcinoma cases and for ALK and EGFR TKI AR cases were surveyed for YES1 and YAP1 amplification.RESULTSWe report response to SRC family kinase (SFK) inhibition in a patient whose lung cancer exhibited YES1 amplification, without any well-established primary driver alteration, suggesting that YES1 amplification can also function as a primary oncogenic driver. To investigate the possibility of YES1 as a primary driver in tumorigenesis, we established preclinical models of YES1 overexpression using human bronchial epithelial cells and normal human breast epithelial cells. We showed that YES1 overexpression conferred sensitivity to SFK TKIs and promoted EGF-independent growth in a YAP1-dependent manner. Analysis of clinical genomic sequencing data from cases of AR to EGFR and ALK inhibitors revealed acquired amplification of YAP1 in four cases. EGFR-mutant and ALK fusion–positive cells overexpressing YES1 or YAP1 were resistant to EGFR and ALK TKIs, respectively, but were sensitive to dual inhibition of the primary driver and YES1.CONCLUSIONOur results demonstrate the therapeutic potential of SFK inhibition in primary tumorigenesis and AR driven by YES1/YAP1 signaling.

Oncogene Overlap Analysis of Circulating Cell-free Tumor DNA to Explore the Appropriate Criteria for Defining MET Copy Number–Driven Lung Cancer

IntroductionDefining clinically relevant MET amplification levels in non-small cell lung cancer (NSCLC) remains challenging. We hypothesize that oncogene overlap and MET amplicon size decline with increase in MET plasma copy number (pCN), thus enriching for MET-dependent states. Patients and MethodsWe interrogated cell-free DNA NGS results of 16,782 patients with newly diagnosed advanced NSCLC to identify those with MET amplification as reported using Guardant360. Co-occurring genomic mutations and copy number alterations within each sample were evaluated. An exploratory method of adjusting for tumor fraction was also performed and amplicon size for MET was analyzed when available. ResultsMET amplification was detected in 207 (1.2%) of samples. pCN ranged from 2.1 to 52.9. Of these, 43 (20.8%) had an overlapping oncogenic driver, including 23 (11.1%) METex14 skipping or other MET mutations. The degree of (non-MET) oncogene overlap decreased with increases in pCN. Patients with MET pCN ≥ 2.7 had lower rates of overlapping drivers compared to those with MET pCN < 2.7 (6.1% vs. 16.3%, P = .033). None of the 7 patients with pCN > 6.7 had an overlapping driver. After adjusting for tumor fraction, adjusted pCN (ApCN) was also lower for those with overlapping drivers than those without (median ApCN 4.9 vs. 7.3, P =.024). There was an inverse relationship between amplicon size and pCN. ConclusionsWe propose that a high MET pCN and/or ApCN, together with the absence of overlapping oncogenic drivers and small MET amplicon size, will enrich for patients most likely to derive benefit from MET targeted therapy.

Selective RET inhibitors shift the treatment pattern of RET fusion-positive NSCLC and improve survival outcomes

PurposeRearranged during transfection (RET) fusions are important genetic drivers in non-small cell lung cancer (NSCLC). Selective RET inhibitors are setting a new paradigm in RET-driven NSCLC. However, the real-world treatment patterns, outcomes and toxicity remain largely unknown.MethodsData from RET fusion-positive NSCLC patients treated in our centre were retrospectively analysed. Of them, patients diagnosed before and after August 2018 were included in analysis of treatment patterns; and patients received selective RET inhibitors were eligible for analysis of adverse events (AEs).ResultsPatients diagnosed before August 2018 (n = 30) predominantly received chemotherapy and immunotherapy (83%) as initial therapy, while patients diagnosed after August 2018 (n = 39) mainly received selective RET inhibitors (38.5% at first-line; 50.0% at second-line). In the total 69 patients, overall survival (OS) was prolonged in patients treated with selective RET inhibitors versus untreated patients (median 34.3 versus 17.5 months; p = 0.002) during a median follow-up of 28.7 months. But there was no difference between patients treated with immunotherapy versus untreated patients. In the 38 patients received selective RET inhibition, median progression-free survival (PFS) was 11.9 months. AEs ≥ grade 3 occurred in 42.1% patients and were not associated with PFS (p = 0.63) or OS (p = 0.60). Haematological toxicity ≥ grade 3 occurred in 31.6% patients and was the leading cause of drug discontinuation.ConclusionSelective RET inhibitors are increasingly being adopted into clinical practice and are associated with improved OS. However, treatment-related ≥ grade 3 AEs, especially haematologic AEs, occur frequently in real-world setting.

Plasma exosomes in obesity hypoventilation syndrome patients drive lung cancer cell malignant properties: Effect of long-term adherent CPAP treatment

OHS patients have higher morbidity and mortality rates. Exosomes are released into the bloodstream and play a role in tumor growth and metastasis. Twenty-four OHS patients (13 male and 11 female) were evaluated at diagnosis (Pre), and after CPAP treatment (T3, T12 and T24 months). Plasma exosomes were applied into naïve NCI-H23 lung cancer cells. Exosomes from OHS patients enhance PMI of human lung cancer cells, and these effects are at least partially reversible with adherent CPAP treatment. Our findings suggest that OHS-induced changes in exosome cargo promote cancer aggressiveness in NCI-H23 lung adenocarcinoma and exhibit OHS treatment-related reversibility.

Abstract 1540: Accumulation of the long noncoding RNA Malat1 is a central driver and therapeutic target in metastatic lung cancer

Abstract The identification and functional characterization of long noncoding RNAs (lncRNAs) have suggested a potential source of unexplored cancer therapy targets. The metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was initially identified as a transcript significantly overexpressed in metastatic lung adenocarcinoma (LUAD) and strongly correlated with poor patient outcomes. Despite decades of effort to elucidate Malat1 functions and mechanisms of action in cancer, two key questions have remained unresolved: 1) Is Malat1 overexpression a driver of LUAD progression? and 2) What is the mechanism by which Malat1 overexpression promotes LUAD progression? We utilized CRISPR activation to model Malat1 overexpression in patient-derived LUAD cell lines and in the autochthonous KrasG12D/p53-deficient murine model of LUAD. We observed that Malat1 overexpression cooperates with p53 loss to promote LUAD progression and metastatic dissemination in vivo. Compared to controls, Malat1-overexpressing KrasG12D/p53-deficient tumors appeared poorly differentiated and were characterized by excessive stromal infiltration. Single-cell RNA sequencing of dissociated tumors revealed that Malat1 overexpression triggers pleiotropic immune and stromal responses that favor a metastasis-permissive tumor microenvironment through the secretion of cytokines and metalloproteinases and the recruitment of CAFs and tumor-associated macrophages. Consistent with the in vivo findings, we observed that Malat1 overexpression in vitro does not affect the proliferation but promotes the migration of tumor cells and cancer-associated fibroblasts (CAFs) through secreted factors, including inflammatory cytokines. Importantly, targeting the stabilizing ENE domain of overexpressed Malat1 by two independent approaches in vitro - direct mutagenesis and non-degrading antisense oligonucleotide (ASO) - led to Malat1 destabilization and rescued the increased cellular migration. Similarly, cytokine-targeted blocking antibodies reversed the effects of Malat1 overexpression. In sum, our results represent the first successful efforts to model the increased Malat1 levels observed in human LUAD and to dissect the pro-metastatic molecular consequences of overexpressed Malat1. Our findings indicate that Malat1 is both necessary and sufficient to rewire metastatic pathways and our preliminary data support further efforts to therapeutically target Malat1 and its downstream mediators in metastatic LUAD. Citation Format: Elena Martínez-Terroba, Fernando J. de Miguel, Camila Robles-Oteiza, Katerina Politi, Nadya Dimitrova. Accumulation of the long noncoding RNA Malat1 is a central driver and therapeutic target in metastatic 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 1540.

Abstract 2972: Identification of USP9X as a novel regulator of RIT1 protein abundance and as a potential therapeutic target in RIT1-driven lung cancer

Abstract Standard care of lung cancer treatment has shifted away from non-specific, cytotoxic chemotherapy in favor of targeted therapies based on genetic mutations within tumors. In 2014, somatic mutations in the small GTPase RIT1 (Ras-like in all tissues) were discovered as oncogenic drivers of lung adenocarcinoma. Thousands of patients per year are diagnosed with RIT1-driven cancer, but treatment options are limited. A targeted therapy for RIT1-driven tumors would address a major unmet clinical need. Little is known about how RIT1 drives cellular transformation. To genetically dissect RIT1 function, we performed a genome-wide CRISPR/Cas9 screen in isogenic PC9 lung adenocarcinoma cells. This screen took advantage of the observation that RIT1-mutant cells are resistant to EGFR inhibition. We leveraged this drug resistance phenotype to identify genetic dependencies (gene knockouts that are detrimental to cell growth) and cooperating factors (gene knockouts that are beneficial to cell growth) in RIT1-mutant cells. From this screen, we found that one of the top essential genes was the deubiquitinase USP9X. This is intriguing given that the protein abundance of RIT1 is known to be important for its function. Therefore, we sought out to test the hypothesis that USP9X regulates RIT1 abundance and that inhibition of USP9X could be an effective therapeutic strategy for abrogating RIT1-driven tumor growth. Our model suggests that USP9X promotes proteasome-mediated degradation of RIT1. To test this, we assessed RIT1 abundance in the context of USP9X knockout (KO). We found that RIT1 protein abundance was decreased in USP9X KO PC9 cells compared to parental cells. Furthermore, cycloheximide (CHX)-chase experiments revealed that RIT1 stability was decreased in USP9X KO cells, and RIT1 degraded faster than in parental cells. The average half-life of RIT1 in USP9X KO cells was 3.4 hrs while the average half-life in parental PC9 cells was 12.3 hrs (95% CI = -12.5 to -5.4 hrs). Treatment with the proteasome inhibitor bortezomib (BTZ) rescued RIT1 degradation by 99% in parental cells and 190% in USP9X KO cells (95% CI = 57 to 126%). In addition to assessing protein abundance and stability, we performed co-immunoprecipitation experiments in RIT1-expressing HEK293T cells and found that RIT1 and USP9X physically interact. Taken together, these data support the hypothesis that RIT1 is a substrate of USP9X. In addition to providing better insight on the protein regulation of RIT1, this work has crucial therapeutic implications. The protein abundance of RIT1 is important for its function, and our model suggests that USP9X inhibition could be an effective means of reducing RIT1 protein abundance and abrogating tumor growth. Overall, this work is poised to significantly impact the field of RIT1 biology and address a major unmet clinical need for the treatment of RIT1-driven diseases. Citation Format: Amanda K. Riley, Athea Vichas, Naomi T. Nkinsi, Phoebe C. Parrish, Shriya Kamlapurkar, Alice H. Berger. Identification of USP9X as a novel regulator of RIT1 protein abundance and as a potential therapeutic target in RIT1-driven 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 2972.

Abstract 3328: Antitumor activity of BLU-945 and BLU-701 as single agents and in combination in EGFR L858R-driven models of NSCLC

Abstract BACKGROUND: Lung cancer is the leading cause of cancer death globally. EGFR mutations are the most common genomic drivers of non-small cell lung cancer (NSCLC), occurring in ~17% and up to 50% of Caucasian and Asian patients, respectively, with exon 19 deletions (ex19del) and L858R substitutions being the most prevalent activating EGFR mutations (EGFRm). Outcomes have improved in patients with EGFR-driven NSCLC since the introduction of EGFR tyrosine kinase inhibitors (TKIs); however, treatment resistance almost inevitably occurs. It has been reported that patients harboring the EGFR L858R mutation, who represent 3.4% of lung adenocarcinoma cases in North America and 23% in Asia, have poorer outcomes than those with EGFR ex19del, suggesting a significant unmet need in this population. BLU-945 and BLU-701 are investigational, reversible, selective, and orally available TKIs optimized for use as single-agent or combination therapy to effectively suppress activating and on-target resistance EGFR mutants while sparing WT EGFR, and have the potential to treat or prevent central nervous system metastases. In vivo studies previously demonstrated the antitumor activity of BLU-945 against EGFR L858R/T790M and EGFRm/T790M/C797S mutants, and BLU-701 against EGFR ex19del and EGFRm/C797S mutants. Because EGFR L858R-driven NSCLC constitutes a large population of patients who may not be receiving the same benefit from third-generation (3G) TKIs as those with EGFR ex19del, studies were conducted to evaluate the antitumor activity of BLU-945 and BLU-701, as single agents and in combination, in preclinical NSCLC tumor models driven by L858R in the absence of the T790M mutation. METHODS: The in vivo antitumor activities of BLU-945 and BLU-701, as single-agents or in combination, were evaluated in 2 EGFR L858R-driven patient-derived xenograft (PDX) subcutaneous tumor models and in an engineered EGFR L858R/C797S-driven Ba/F3 cell line-derived xenograft (CDX) subcutaneous tumor model. RESULTS: Oral daily administration of single-agent BLU-945 and single-agent BLU-701 resulted in sustained tumor regression in 2 PDX models of EGFR L858R NSCLC. Compared with single agents in an EGFR L858R/C797S Ba/F3 CDX tumor model, combination of BLU-945 with BLU-701 resulted in marked antitumor activity and prolonged tumor regression. CONCLUSION: The in vivo antitumor activities of BLU-945 and BLU-701 in preclinical tumor models suggest that both BLU-945 and BLU-701 have the potential to be used in patients with EGFR L858R-driven NSCLC, including those who are treatment naïve or previously treated with 3G TKI. Combining BLU-945 and BLU-701 may enable coverage of frequent on-target resistance mechanisms, including the EGFR C797S mutation, in addition to the common L858R activating mutation. Citation Format: Luz Tavera, Stefanie Schalm, John Campbell, Jian Guo, Clare Medendorp, Maxine Chen, Faris Albayya, Tom Dineen, Zhuo Zhang, Maria Iliou, Ebby Job, Nisha Perez, Yoav Timsit, Scott Wardwell, Katie McGinn, Richard Woessner, Chiara Conti. Antitumor activity of BLU-945 and BLU-701 as single agents and in combination in EGFR L858R-driven models of NSCLC [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 3328.

Abstract 533: A study of sequencing circulating cell free DNA from lung cancer patients

Abstract Precision oncology requires accurate cancer molecular information. While widely used to characterize tumors, tissue biopsy is limited in patient follow-up due to its invasive nature and the lack representation of the entire tumor bulk. To develop the method of circulating cell free DNA (cfDNA) for obtaining clinically valuable sequence information, we isolated cfDNA from the plasma samples of 10 advanced metastatic lung cancer patients and performed next generation sequencing (NGS) using a unique molecular marker (UMI) integrated 275-gene-target-panel at the average depth of 4,000 X coverage. We were able to identify common mutants of lung cancer driver genes, e.g. EGFR, KRAS, DNMT3 and TP53 with high confidence. In one example, the TKI resistant EGFR mutant T790M was found coexisting with EGFR 746-750del and TP53 splice variant in a baseline sample of a patient progressed from a first-generation EGFR inhibitor. And the dynamic changes of the allele frequency of EGFR and TP53 mutants were consistent with total cfDNA levels of the patients. Interestingly, an osimertinib resistance EGFR mutant C797S emerged at Day 355 post-treatment and followed by disease progression in 4 months. In addition to EGFR gene mutations, we further found increasing EGFR copy number associated with drug resistance and cancer progression of this patient. Our study demonstrated that it is feasible using cfDNA for patient follow-up to understand cancer resistance or disease progression through characterization of cancer mutations. Citation Format: Zhigang Kang, Liang Cao. A study of sequencing circulating cell free DNA from lung cancer patients [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 533.

Abstract 290: Derivation of immortalized human alveolar epithelial cells for lung adenocarcinoma modeling

Abstract Lung cancer, primarily caused by tobacco smoke exposure, kills more Americans than the next top three cancers combined. The two main histological subtypes are lung adenocarcinoma, which arises in the alveoli, and squamous cell lung cancer, which arises in the airways. In vitro models of squamous cell lung cancer have used human bronchial epithelial cells immortalized with Simian Virus 40 Large T antigen (SV40 LgT) or mutant CDK4R24C combined with telomerase. The derived immortalized human bronchial epithelial cells have been very useful to study the effects of tobacco smoke exposure and oncogene activation. However, to date it has not been possible to develop similar models for lung adenocarcinoma; immortalized human alveolar epithelial cells (AECs) were not available due to the difficulty in establishing alveolar cell lines that proliferate yet remain epithelial. Here we describe a method to derive AEC lines, providing a new resource for in vitro lung adenocarcinoma models. We purified human AECs from de-identified remnant human transplant lungs from deceased subjects and tested numerous conditions to establish immortalized cell lines. We found that the addition of Rho-associated kinase inhibitor (Y27632) to culture media allowed AECs to proliferate so that they could then be transduced with lentivirus. Transduction with CDK4R24C + telomerase did not yield cell lines that grew well. However, transduction with SV40 LgT resulted in well-proliferating cell lines that retained their epithelial morphology. In 2-dimensional (2D) culture the AEC LgT-expressing cell lines grow in cobblestone monolayers and do not express mature alveolar markers. However, in 3-dimensional (3D) culture in Matrigel with fibroblasts the cells form alveolar-like organoids of varying complexity. Immunofluorescence and single cell RNA sequencing indicate that the transition from 2D monolayer to 3D organoids is accompanied by the expression of mature alveolar markers such as aquaporin 5 (AQP5), G-protein-coupled receptor class C, member A (GPRC5A), lysosomal-associated membrane protein 3 (LAMP3) and ATP-binding cassette subfamily A member 3 (ABCA3, involved in surfactant production). In addition, while in 2D culture the cells form a single cluster with homogeneous gene expression patterns, in 3D diverse cell groups appear. Using a 3D culture medium that stimulates alveologenesis induces additional mature alveolar markers. We are using AEC lines to study the effects of environmental exposures such as tobacco smoke and the role of lung cancer driver genes. In addition, we intend to establish AEC lines from individuals of diverse ethnicities to investigate the effects of genetic background on lung cancer development. Our AEC lines provide a valuable new system to model and study the distal lung and associated diseases in vitro. Citation Format: Ite A. Offringa, Evelyn Tran, Tuo Shi, Laurence St.Pierre, Pablo E. Puente, Rong Lu, Crystal N. Marconett, Beiyun Zhou, Zea Borok. Derivation of immortalized human alveolar epithelial cells for lung adenocarcinoma modeling [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 290.

Abstract 5781: Molecular positive margin of surgical resections is a strong biomarker for tumor recurrence in pIIIA-N2 NSCLC patients

Abstract Background: The high recurrence rate of stage pIIIA-N2 NSCLC patients may reflect inadequate removal of all residual tumor cells despite conventional histologic negative surgery margin. The NGS-detected BRM status was possible to serve as a more accurate examination to identify those patients with a high-risk of postoperative recurrence. Methods: We retrospectively studied 119 stage IIIA-N2 NSCLC patients who received curative-intent surgery plus adjuvant chemotherapy/chemoradiotherapy, and all of the patients were classified with negative resection margin by the conventional histologic method. Each patient had paired tumor and bronchial resection margin (BRM) samples, which were analyzed using next-generation sequencing (NGS) of 474 cancer-related genes, and we explored the prognostic values of various clinical and molecular features. Results: Among the 119 stage IIIA-N2 NSCLC patients with histologic negative resection margins, the BRM samples from 56 patients (47.1%) were positive for tumor mutations, including multiple lung cancer driver mutations. The number and the variant allele frequency of genetic alterations in BRM samples were generally lower than that of the paired tumor samples. Notably, patients who were positive for NGS-detected BRM mutations had significantly shorter disease-free survival (DFS) than the negative patients (p=0.001, HR 2.18, 95%CI 1.37~3.47). Several other clinical/molecular features were also found to be significantly associated with DFS, including the metastatic lymph node ratio (MLNR), smoking history, and KRAS mutation status, and NGS-assessed BRM status remained to be statistically significant in multivariable analysis by including all of these prognostic factors (p =0.020, HR 1.82, 95%CI 1.10~3.02). Conclusions: We demonstrated that the molecular positive margin of surgical resections was a strong and independent biomarker for disease recurrence in pIIIA-N2 NSCLC patients. Our results emphasized the clinical utility of molecular diagnostic approaches to detect minimal residual disease after surgical resection, which could potentially facilitate the stratification of high-risk patients for personalized and appropriate treatments. Citation Format: Li Li, Kewen He, Jiaohui Pang, Yang Xu, Yang Shao, Shuanghu Yuan, Jinming Yu. Molecular positive margin of surgical resections is a strong biomarker for tumor recurrence in pIIIA-N2 NSCLC patients [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 5781.

Abstract 2197: Targeted cancer therapy induces APOBEC fueling the evolution of drug resistance

Abstract Introduction: Increasing our understanding of drivers of mutagenesis in lung cancer is critical in our efforts to prevent tumor reoccurrence and resistance. Results: Using the multi-region TRACERx lung cancer study, we uncovered that APOBEC3B is significantly upregulated when compared with other APOBEC family members in EGFR driven lung cancer and identified subclonal enrichment of APOBEC mutational signatures. To model APOBEC mutagenesis in lung cancer, several novel EGFR mutant mouse models containing a human APOBEC3B transgene were generated. Using these models, it was uncovered that APOBEC3B expression is detrimental at tumor initiation when expressed continuously in a p53 wildtype background. This detrimental effect is likely due to elevated chromosomal instability, which was observed to increase significantly with APOBEC3B expression in an EGFR mutant TP53 deficient mouse model. Induction of subclonal expression of APOBEC3B in an EGFR mutant mouse model with tyrosine kinase inhibitor (TKI) therapy resulted in a significant increase in resistant tumor development. Significant downregulation of the base excision repair gene uracil-DNA glycosylase (UNG) was also observed in APOBEC3B expressing mice, which paralleled findings in patient tumors and cell lines treated with TKI therapy. Finally, a mouse mutational signature was identified in APOBEC3B expressing cell lines, reinforcing the idea that APOBEC driven mutagenesis contributes to TKI resistance. Conclusion: This study demonstrates a unique principle by which targeted therapy induces changes within tumors ideal for APOBEC driven tumor evolution, fueling therapy resistance. Citation Format: Manasi Mayekar, Deborah Caswell, Natalie Vokes, Emily K. Law, Wei Wu, William Hill, Eva Gronroos, Andrew Rowan, Maise Al Bakir, Clare Weeden, Caroline E. McCoach, Collin M. Blakely, Nuri Alpay Temiz, Ai Nagano, Daniel L. Kerr, Julia K. Rotow, Oriol Pich, Franziska Haderk, Michelle Dietzen, Carlos Martinez Ruiz, Bruna Almeida, Lauren Cech, Beatrice Gini, Joanna Przewrocka, Chris Moore, Miguel Murillo, Bjorn Bakker, Brandon Rule, Cameron Durfee, Shigeki Nanj, Lisa Tan, Lindsay K. Larson, Prokopios P. Argyris, William L. Brown, Johnny Yu, Carlos Gomez, Philippe Gui, Rachel I. Vogel, Elizabeth A. Yu, Nicholas J. Thomas, Subramanian Venkatesan, Sebastijan Hobor, Su Kit Chew, Nicholas McGranahan, Nnennaya Kanu, Eliezer M. Van Allen, Julian Downward, Reuben S. Harris, Trever Bivona, Charles Swanton. Targeted cancer therapy induces APOBEC fueling the evolution of drug resistance [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 2197.

Abstract 3981: Targeting SRP receptor-beta reduces protein glycosylation and cell growth in lung cancer

Abstract PURPOSE: Lung cancer is a major cause of morbidity and mortality world wide. Signaling receptors, adhesion molecules, and surface antigens that drive lung cancer progression are regulated by N-glycosylation, a post-translational protein modification initiated in the ER. Strategies for inhibition of N-glycosylation have recently been refined through the discovery of small molecules that partially disrupt glycan transfer. Here we screened for the target of a novel small molecule that reduces protein glycosylation with the goal of identifying new therapeutic targets for lung cancer. METHODS: Pooled results from chemical library HTS efforts identified hit compounds with activity. Counter-screening was performed in A549 cells using a fluorescence-based reporter (Halo1N) that detects aberrant N-glycosylation. The cellular targets for one class of inhibitors were evaluated by combining a whole-genome CRISPR-Cas9 screen with FACS sorting for loss of pharmacologic function. Significant changes in gRNA between populations was determined by MAGeCK analysis. The interaction between a compound and its target was determined by CETSA, and mechanism of action was verified by gene knockout and rescue experiments. Glycosylation status of glycoproteins were measured by western blot and cell compartment fluorescence. Predicted compound binding sites on the protein were mutated using site-directed mutagenesis. RESULTS: The lead tool compound (CP-9) has an IC50 in uM range and was used as a probe for target identification. CP-9 activates Halo-1N fluorescence, and whole genome CRISPR-Cas9 screening was used to identify gRNAs associated with loss of CP-9 activity. MAGeCK analysis demonstrated significant enrichment of genes associated with the translocon and ribosome (FDR P &amp;lt; 0.01) in the non-fluorescent population. We tested components of the signal recognition particle-receptor complex for direct interactions using the CETSA and demonstrate CP-9 binding to SR-β but not SR-α. Knockout experiments confirm that inhibition of glycosylation by CP-9 is dependent on SR-β expression, and CP-9’s effect on glycosylation and was rescued by SR-β re-expression. Mutated SR-β diminished CP-9’s effects on glycosylation and reduced SR-β thermal stabilization. CP-9 disrupted an interaction between SR-β/SR-α and the ribosome-translocon-OST complex suggesting a mechanism of action. Interestingly, a transmembrane domain mutant (ΔTM) of SR-β reduced the CP-9 effect and glycoprotein expression. ΔTM-SR-β also significantly reduced cell proliferation in A459 lung adenocarcinoma cells. CONCLUSION: CP-9 partially inhibits N-glycosylation by interfering with SR-β/SR-α and the translocon complex, leading to the disruption of protein import and glycosylation in the ER. Transmembrane domain mutants of SR-β reduce lung cancer cell growth and suggest this protein as a potential target for lung cancer drug development in the future. Citation Format: Chatchai Phoomak, Joseph N. Contessa. Targeting SRP receptor-beta reduces protein glycosylation and cell growth in 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 3981.

Inspiratory hyperoxia suppresses lung cancer metastasis through a MYC/SLC1A5-dependent metabolic pathway.

The lack of knowledge about the effect of inspiratory hyperoxia on the lung-specific tumour microenvironment and progression of lung cancer has attracted considerable attention. This study proposes that inspiratory hyperoxia has special significance for the malignant phenotype of lung cancer cells. The effects of different oxygenation parameters on the proliferation, apoptosis, invasion and migration of lung cancer cells were systematically evaluated in vitro and in vivo Our results reveal that inspiratory hyperoxia treatment (60% oxygen, 6 h·day-1) not only has no tumour progression-promoting effects, but also suppresses lung cancer metastasis and promotes long-term survival. In addition, we combined transcriptome, proteome and metabolome analysis and found that hyperoxia treatment induced significant intracellular metabolic changes in lung cancer cells. Overall, we established that MYC/SLC1A5-induced metabolic reprogramming and glutamine addiction is a new mechanism that drives lung cancer metastasis, which can be significantly suppressed by inspiratory hyperoxia treatment. These findings are relevant to the debate on the perils, promises and antitumour effect of inspiratory hyperoxia, especially for patients with lung cancer.

Targeting ALK in Neuroendocrine Tumors of the Lung.

BackgroundAnaplastic lymphoma kinase (ALK) rearrangements are known oncogenic drivers in non-small cell lung cancer (NSCLC). Few case reports described the occurrence of such rearrangements in large cell neuroendocrine carcinomas (LCNECs) of the lung without information on clinical responses to ALK tyrosine kinase inhibitors (TKIs) in these cases. Currently, neuroendocrine tumors of the lungs are not screened for ALK rearrangements.MethodsTo illustrate the clinical impact of molecular characterization in LCNECs, we report the disease course in three patients with ALK-rearranged metastatic LCNEC from our clinical routine, as well as their treatment response to ALK TKIs (index cases). To gain insight into the prevalence of ALK rearrangements in neuroendocrine tumors of the lung, we analyzed a retrospective cohort of 436 tumor biopsies including LCNEC (n = 61), small cell lung cancer (SCLC) (n = 206), typical (n = 91) and atypical (n = 69) carcinoids, and mixed histology (n = 9) for the presence of ALK rearrangements using a sequential diagnostic algorithm. ALK immunohistochemistry (IHC) was evaluable in 362 cases; fluorescence in situ hybridization (FISH) was evaluable in 28 out of the 35 IHC-positive cases, followed by next-generation sequencing (NGS) that was available in 12 cases.ResultsWithin the retrospective cohort, ALK IHC was positive in 35 out of 362 (9.7%) evaluable samples. FISH was positive in 3 out of the 28 (10.7%) evaluable cases: 2 with atypical carcinoids and 1 with LCNEC. Additionally, the 3 index cases showed positive ALK IHC, which was confirmed by NGS. Within the retrospective cohort, NGS confirmed the presence of an ALK genomic rearrangement in one FISH-positive atypical carcinoid where material was sufficient for sequencing. Two out of three patients with metastatic ALK-rearranged LCNEC received up-front treatment with the ALK TKI alectinib and showed rapid tumor response at all metastatic sites, including multiple brain metastases.ConclusionsALK rearrangements represent rare but targetable oncogenic driver alterations in LCNEC. Contrarily to NSCLC, the detection of ALK rearrangements in neuroendocrine tumors of the lung is challenging, since ALK IHC can lead to false-positive results and therefore needs confirmation by FISH or NGS. Up-front comprehensive molecular profiling with NGS should be performed in metastatic LCNEC in order not to miss actionable genomic alterations.

LCMC LEADER neoadjuvant screening trial: LCMC4 evaluation of actionable drivers in early-stage lung cancers.

TPS8596 Background: Comprehensive genomic profiling (CGP) has transformed the care of patients with advanced non-small cell lung cancer (NSCLC), giving many patients access to precision targeted treatment and immunotherapy with remarkable improvements in outcomes. Studies show that patients with lung cancers with oncogenic drivers are the least likely group to benefit from checkpoint inhibitors and are better served by enrollment in studies of targeted therapies. Early-stage NSCLC is now poised to benefit from these precision approaches with the regulatory approval of the first tyrosine kinase inhibitors and checkpoint inhibitors for the adjuvant treatment of resected NSCLC, each requiring testing for precision biomarkers. Neoadjuvant precision therapy for NSCLC has the potential to further improve treatment outcomes. Methods: The LCMC4 Evaluation of Actionable Drivers in EaRly Stage Lung Cancer (LEADER) Neoadjuvant Screening Trial (NCT04712877) is a collaborative diagnostic study developed by the Lung Cancer Mutation Consortium (LCMC), supported by the Thoracic Surgery Oncology Group and the Lung Cancer Research Foundation. The primary objective is to determine the proportion of patients with stage IA2-III lung cancers who possess actionable oncogenic drivers, defined as 1 of 11 actionable genomic alterations: mutations in EGFR, BRAFV600E, MET exon 14, KRAS G12C, and HER2, rearrangements in ALK, RET, NTRK, and ROS1, and amplification of MET and HER2. The study will also assess the feasibility of CGP to detect actionable oncogenic drivers in patients with suspected early-stage lung cancers scheduled to undergo biopsies to establish the diagnosis of lung cancer. The protocol will enroll 1000 patients with operable stage IA2-III (TNM 8th edition) lung cancer who will undergo CGP utilizing the Foundation Medicine 324 gene assay as well as paired liquid biopsy analysis. Results will enable selection of neoadjuvant therapy and enrollment onto independent therapeutic trials with genomically matched neoadjuvant treatment, standard therapies, or other trials if no driver is detected. The approach will be considered feasible if &gt;35% of non-squamous NSCLCs have 1 of the 11 actionable alterations. Tumor mutational burden and PD-L1 IHC will be assessed. Plasma specimens collected pre- and post neoadjuvant treatment and post-surgery will be used for research to study the ability of circulating tumor DNA to assess neoadjuvant treatment response and minimal residual disease. 26 academic sites in the US plan to enroll patients. Clinical trial information: NCT04712877.