Patient-derived Lung Cancer Cell Lines Research Articles

Preclinical Models for the Study of Lung Cancer Pathogenesis and Therapy Development.

Experimental preclinical models have been a cornerstone of lung cancer translational research. Work in these model systems has provided insights into the biology of lung cancer subtypes and their origins, contributed to our understanding of the mechanisms that underlie tumor progression, and revealed new therapeutic vulnerabilities. Initially patient-derived lung cancer cell lines were the main preclinical models available. The landscape is very different now with numerous preclinical models for research each with unique characteristics. These include genetically engineered mouse models (GEMMs), patient-derived xenografts (PDXs) and three-dimensional culture systems ("organoid" cultures). Here we review the development and applications of these models and describe their contributions to lung cancer research.

Mutation-oriented profiling of autoinhibitory kinase conformations predicts RAF inhibitor efficacies

Kinase-targeted therapies have the potential to improve the survival of patients with cancer. However, the cancer-specific spectrum of kinase alterations exhibits distinct functional properties and requires mutation-oriented drug treatments. Besides post-translational modifications and diverse intermolecular interactions of kinases, it is the distinct disease mutation which reshapes full-length kinase conformations, affecting their activity. Oncokinase mutation profiles differ between cancer types, as it was shown for BRAF in melanoma and non-small-cell lung cancers. Here, we present the target-oriented application of a kinase conformation (KinCon) reporter platform for live-cell measurements of autoinhibitory kinase activity states. The bioluminescence-based KinCon biosensor allows the tracking of conformation dynamics of full-length kinases in intact cells and real time. We show that the most frequent BRAF cancer mutations affect kinase conformations and thus the engagement and efficacy of V600E-specific BRAF inhibitors (BRAFi). We illustrate that the patient mutation harboring KinCon reporters display differences in the effectiveness of the three clinically approved BRAFi vemurafenib, encorafenib, and dabrafenib and the preclinical paradox breaker PLX8394. We confirmed KinCon-based drug efficacy predictions for BRAF mutations other than V600E in proliferation assays using patient-derived lung cancer cell lines and by analyzing downstream kinase signaling. The systematic implementation of such conformation reporters will allow to accelerate the decision process for the mutation-oriented RAF-kinase cancer therapy. Moreover, we illustrate that the presented kinase reporter concept can be extended to other kinases which harbor patient mutations. Overall, KinCon profiling provides additional mechanistic insights into full-length kinase functions by reporting protein-protein interaction (PPI)-dependent, mutation-specific, and drug-driven changes of kinase activity conformations.

Abstract 1732: Switch to MET oncogene dependence as a novel mechanism of drug resistance in EGFR-mutant lung cancer

Abstract Introduction: Non-small cell lung cancers harboring sensitizing gain-of-function mutations in the epidermal growth factor receptor (EGFR) are treated with small molecule EGFR kinase inhibitors including erlotinib and osimertinib. Unfortunately, the clinical efficacy of these inhibitors is limited by progression to drug resistance. Genomic amplification of the hepatocyte growth factor receptor (MET) is a prevalent mechanism of resistance to clinical EGFR inhibitors. EGFR mutant lung cancers with MET amplification are co-dependent on signaling through EGFR and MET kinases, whereby either oncogene can phosphorylate the EGFR family receptor ERBB3, activating downstream signaling and promoting survival. These EGFR/MET co-dependent cancers are characteristically resistant to treatment with individual drugs, but retain sensitivity to combination treatment with EGFR and MET inhibitors. Here, we describe three patient-derived EGFR mutant, MET amplified lung cancer models that exhibit a switch away from EGFR, to MET oncogene dependence. Methods: We established and characterized patient-derived lung cancer cell lines and xenograft models harboring concurrent EGFR activating mutations (EGFR L858R or EGFR Del19) and genomic MET copy number gain. Three patient-derived models—DFCI81, DFCI161, and DFCI307—were developed from pleural effusion and core biopsy specimens of patients whose tumors progressed on erlotinib. Clinical samples were immediately sorted and stably cultured in vitro or engrafted into immunodeficient mice. Results: DFCI81, DFCI161, and DFCI307 cell lines and xenograft models retained mutant EGFR expression, but exhibited resistance to clinical EGFR inhibitors and sensitivity to single-agent c-MET inhibitors, including crizotinib and savolitinib. Comparing our EGFR-mutant and MET-dependent cell lines to EGFR/MET-codependent models, we observed that EGFR expression was significantly reduced in our models compared to controls. Ectopic overexpression of EGFR Del19 and EGFR L858R in DFCI81 and DFCI161 cell lines, respectively, was sufficient to confer crizotinib resistance and induce EGFR/MET co-dependency. We demonstrated that MET-mediated ERBB3 phosphorylation drives downstream PI3K activation to promote cell proliferation and survival in DFCI81 and DFCI161 cells. In these contexts, ERBB3 reactivation by recombinant ligand treatment was sufficient to induce EGFR-mediated ERBB3 activation, conferring resistance to single-agent crizotinib treatment. Conclusions: We have identified and characterized three patient-derived models of treatment refractory EGFR mutant lung cancer that exhibit a switch to MET oncogene dependency. Clinically, we predict a subset of EGFR mutant, MET-dependent tumors exists, and can be identified de novo by a reduced EGFR to MET expression ratio: a potential biomarker predictive of sensitivity to single-agent MET inhibition. Citation Format: Pinar Ö. Eser, Raymond M. Paranal, Michael J. Poitras, Man Xu, Stephen Wang, Atsuko Ogino, Jihyun Choi, Pavlos Missios, Heidi M. Haikala, Jieun Son, Mika Lin, Masahiko Yanagita, Prafulla C. Gokhale, George Q. Daley, Pasi A. Jänne. Switch to MET oncogene dependence as a novel mechanism of drug resistance in EGFR-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1732.

Abstract A157: Antitumor activity of tarloxotinib, a hypoxia-activated EGFR TKI, in patient-derived lung cancer cell lines harboring EGFR exon 20 insertions

Abstract Tarloxotinib (TRLX) is a prodrug that releases an irreversible EGFR/HER2 tyrosine kinase inhibitor (TRLX-TKI) under pathophysiologically hypoxic conditions. Non-small cell lung cancer (NSCLC) has been characterized as a hypoxic disease and approximately 15% of lung adenocarcinomas harbor EGFR mutations. While most EGFR mutations predict for response to several FDA-approved tyrosine kinase inhibitors, in-frame insertions in exon 20 of EGFR are activating mutations in the tyrosine kinase domain that have significantly decreased sensitivity to EGFR inhibitors and currently have no approved targeted therapies. We derived and characterized three human lung adenocarcinoma cell lines with different EGFR exon 20 insertions in order to accelerate development of targeted therapies for this mutation class. Using these novel cell lines, we evaluated tarloxotinib as a therapeutic agent for tumors harboring this type of mutations. We demonstrate that our three patient-derived cell lines: CUTO14 (p.A767_V769dupASV), CUTO17 (p.N771_H773dupNPH), and CUTO18 (p.S768_770dupSVD) are dependent on EGFR for cell proliferation using shRNA-mediated knockdown. Our results show that EGFR exon 20 insertion cell lines are resistant to gefitinib; however, treatment with afatinib or TRLX-TKI reduces cell proliferation and signaling in a similar manner. The IC50 values for the three cell lines were 203nM, 89nM, and 709 nM for afatinib and 208nM, 33nM, and 345nM for TRLX-TKI, respectively. The prodrug form of tarloxitinib has minimal effect on cell proliferation in these models, consistent with the necessity for hypoxia-induced activation (to TRLX-TKI). Importantly, we evaluated the effect of tarloxotinib in vivo using murine xenograft models of CUTO14 and CUTO17. After four weeks of treatment, afatinib did not alter tumor growth compared to untreated tumors, whereas treatment with tarloxotinib induced significant tumor regression. The in vivo data suggest that the activated TKI of tarloxitinib is accumulating to biologically active concentrations in tumors following cleavage of tarlxoxotinib under hypoxic conditions. We conclude that our EGFR exon 20 insertions cell lines represent novel models for the investigation of therapeutic strategies for this mutation class. These cell lines have the ability to develop tumors in vivo and show reduced sensitivity to current EGFR TKIs, mimicking the lack of response in patients with these mutations. Finally, we demonstrate that tarloxotinib can overcome intrinsic EGFR exon 20 mutation resistance to standard EGFR TKIs. Citation Format: Adriana Estrada-Bernal, Andrea E. Doak, Anh T. Le, Hengbo Zhu, Nan Chen, Shevan Silva, Jeff B. Smaill, Adam V. Patterson, Robert C. Doebele. Antitumor activity of tarloxotinib, a hypoxia-activated EGFR TKI, in patient-derived lung cancer cell lines harboring EGFR exon 20 insertions [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A157.

Abstract PR14: MEK inhibitors block growth of Ataxia Telangiectasia Mutated (ATM) mutant lung tumors

Abstract Introduction: Lung cancer is the leading cause of cancer death worldwide. In the past decade, deep sequencing projects have shed light on the molecular drivers commonly found altered in NSCLC. As a result, the first molecularly targeted agents have been approved for the treatment of tumors presenting activating oncogenic events in EGFR or EML4/ALK. However, the translation into therapies for tumors presenting loss-of-function mutations has proven challenging and constitutes an unexplored and promising field. In order to narrow the gap between cancer genomics and effective treatments for tumors harboring mutations in well-defined tumor suppressor genes (such as PTEN, BRG1 or ATM), we have developed a genetically tractable lung cancer cell model. Focusing on lung adenocarcinoma, we have engineered a panel of isogenic cell lines capturing the molecular heterogeneity found in patients. This panel has been screened against a collection of drugs, comprising classical chemotherapeutics and kinase inhibitors, providing a comprehensive evaluation for hundreds of gene-drug interactions. Results: The screen confirmed known or highly expected interactions, proving the robustness of our approach. Among the unexpected and most relevant hits, we have found that ATM-deficient cells are exquisitely sensitive to drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Extensive validation in ATM mutant patient-derived lung cancer cell lines has confirmed the synthetic lethal interaction between ATM and MEK in vitro and in vivo. Mechanistically, the interaction may not be directly associated with the major role of ATM in DNA damage signaling. Rather, we have observed a deregulation of the crosstalk between the MAPK and AKT/mTOR signaling pathways as a consequence of ATM deficiency, resulting in an increased dependency on MEK kinase activity for cell survival. Perspectives: Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma (5- 10%) but directly targeting these with drugs remains challenging. MEK inhibitors are currently being tested in clinical trials for efficacy in KRAS or BRAF mutant lung cancer. However, these mutations alone do not adequately predict response to MEK inhibition. Our findings indicate that including ATM mutation status in lung cancer as a mechanistic biomarker for MEK inhibitors can improve patient stratification. Ultimately, extending the applicability of these drugs beyond KRAS or BRAF mutant tumors. Citation Format: Ferran Fece de la Cruz, Michal Smida, Sebastian Nijman. MEK inhibitors block growth of Ataxia Telangiectasia Mutated (ATM) mutant lung tumors [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR14.

Differential KrasV12 protein levels control a switch regulating lung cancer cell morphology and motility.

Oncogenic Kras mutations are important drivers of lung cancer development and metastasis. They are known to activate numerous cellular signaling pathways implicated in enhanced proliferation, survival, tumorigenicity and motility during malignant progression. Most previous studies of Kras in cancer have focused on the comparison of cell states in the absence or presence of oncogenic Kras mutations. Here we show that differential expression of the constitutively active mutation KrasV12 has profound effects on cell morphology and motility that drive metastatic processes. The study relies on lung cancer cell transformation models, patient-derived lung cancer cell lines, and human lung tumor sections combined with molecular biology techniques, live-cell imaging and staining methods. Our analysis shows two cell functional states driven by KrasV12 protein levels: a non-motile state associated with high KrasV12 levels and tumorigenicity, and a motile state associated with low KrasV12 levels and cell dissemination. Conversion between the states is conferred by differential activation of a mechano-sensitive double-negative feedback between KrasV12/ERK/Myosin II and matrix-adhesion signaling. KrasV12 expression levels change upon cues such as hypoxia and integrin-mediated cell-matrix adhesion, rendering KrasV12 levels an integrator of micro-environmental signals that translate into cellular function. By live cell imaging of tumor models we observe shedding of mixed high and low KrasV12 expressers forming multi-functional collectives with potentially optimal metastatic properties composed of a highly mobile and a highly tumorigenic unit. Together these data highlight previously unappreciated roles for the quantitative effects of expression level variation of oncogenic signaling molecules in conferring fundamental alterations in cell function regulation required for cancer progression.

Abstract 5102: Differential levels of mutated Kras drive lung cancer cell motility and morphology via a hypoxia-sensitive FAK/Erk/Myosin II signaling pathway

Abstract Kras controls many cellular processes such as cell migration and survival. Mutation of Kras to a constitutively active form is a common alteration in lung cancer. Being constantly active results in a hyper activation of downstream signaling cascades, which leads to enhanced tumorigenicity and high metastatic potential. How constitutive Kras activation translates into these different functions, however, remains unclear. Using patient-derived lung cancer cell lines expressing oncogenic KrasV12 we discovered that Kras expression, not transcription, gets up-regulated under long-term culture in 1% O2. We confirmed the in vivo relevance of this finding, staining mouse lung tumors for Kras and Hif1α, as an indicator for hypoxic areas, and found heterogeneous Kras levels throughout the tumors with hot-spots that overlaid with Hif1α expression. Up-regulation of KrasV12 also correlated with a significant increase in growth of tumor-like aggregates in soft-agar assays and a morphological change from adherent single-cells into sphere-like aggregates in cell culture. This morphology was reversible upon knock-down of Kras. Separation of single clones confirmed the relation between adherent and sphere-like morphologies and the level of KrasV12 expression. Interestingly, the expression level of Kras correlated with clonal migration behavior. Low Kras expressers were highly motile whereas high Kras expressers remained stationary in aggregates. In agreement, exogenous expression of KrasV12 in motile, low Kras expressers converted their morphology and migration behavior to sphere-like, stationary aggregates. Using Western blot analysis we found that these changes are due to differences in Erk activation down-stream of Kras. In high Kras expressers Erk is highly phosphorylated which leads to higher Myosin II activity and thus to a contractile, sphere-like morphology with low mobility. Low Kras levels result in less Erk activation and therefore lower Myosin II activity which leads to an adherent, motile phenotype. Using a specific Erk inhibitor, we could convert high Kras expressers into adherent, motile low Kras clones. At the same time we found an adhesion-dependent feedback mechanism which regulates cellular Kras levels. Protein analysis of cells cultured in low adhesion dishes formed spheres and dramatically increased their Kras content. Most interestingly, this increase was reversible by stimulation of adhesion. Additionally, blocking FAK activation in low Kras expressers resulted in up-regulation of Kras and Erk phosphorylation which went along with a switch to sphere-like morphology. Based on these findings we propose a novel mechanism for controlling cancer cell migration by a hypoxia-sensitive, adhesion-dependent Kras switch which makes the cell very adaptable to different environments during the whole process of metastasis. Citation Format: Anette C. Schafer, Meghan Discoll, Ashwathi Mohan, Andrew Ludlow, Wesley Burford, Jerry W. Shay, Gaudenz Danuser. Differential levels of mutated Kras drive lung cancer cell motility and morphology via a hypoxia-sensitive FAK/Erk/Myosin II signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5102. doi:10.1158/1538-7445.AM2015-5102

Identification of CCDC6-RET Fusion in the Human Lung Adenocarcinoma Cell Line, LC-2/ad

Rearranged during transfection (RET) fusions have been newly identified in approximately 1% of patients with primary lung tumors. However, patient-derived lung cancer cell lines harboring RET fusions have not yet been established or identified, and therefore, the effectiveness of an RET inhibitor on lung tumors with endogenous RET fusion has not yet been studied. In this study, we report identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line LC-2/ad. LC-2/ad showed distinctive sensitivity to the RET inhibitor, vandetanib, among 39 non-small lung cancer cell lines. The xenograft tumor of LC-2/ad showed cribriform acinar structures, a morphologic feature of primary RET fusion-positive lung adenocarcinomas. LC-2/ad cells could provide useful resources to analyze molecular functions of RET-fusion protein and its response to RET inhibitors.