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J.D. Minna



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    O12 - Lung Cancer Biology II (ID 87)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      O12.05 - Defining the role of ZEB1 in the pathogenesis of non-small cell lung cancer (NSCLC) using immortalized human bronchial epithelial cells (HBECs) (ID 1139)

      10:30 - 12:00  |  Author(s): J.D. Minna

      • Abstract
      • Presentation
      • Slides

      Background
      To study the role of common lung cancer mutations in transforming lung epithelial cells in an appropriate cellular context we used cdk4/hTERT-immortalized normal HBECs. We developed an isogenic series of HBECs by introducing genetic manipulations representing common lung cancer mutations (such as p53, KRAS[V12], cMYC, and LKB1). This defined in vitro system allows characterization of specific tumorigenic contributions as well as identification of acquired changes, likely representing tumor acquired vulnerabilities and novel therapeutic targets (Mol Cancer Res 2013). One acquired change observed with oncogenic transformation of HBECs is a spontaneous epithelial-to-mesenchymal transition (EMT), an important biologic process in cancer. This study sought to characterize the role of EMT in driving tumorigenesis in HBECs and, in turn, lung cancer to identify novel therapeutic targets.

      Methods
      Genetic manipulations were introduced into cell lines using siRNA/shRNA or over-expression constructs. Tumorigenicity was measured using in vitro (anchorage-dependent and -independent colony formation, proliferation, migration and transwell Matrigel invasion assays) and in vivo (subcutaneous or intravenous injection into NOD/SCID mice) methods. Genome-wide mRNA expression data from five independent datasets was obtained either in-house using Illumina HumanHT-12v4 BeadChips or from publicly available databases.

      Results
      Analysis of EMT-promoting transcription factors in our isogenic series of oncogenically-manipulated HBECs found ZEB1 expression highly correlated with mesenchymal-like HBECs. Functional studies confirmed ZEB1 was a significant driver of tumorigenic phenotypes in both oncogenic HBECs and human lung cancer cell lines where loss of ZEB1 resulted in decreased colony formation, migration and invasion in vitro and subcutaneous tumor growth and intravenous colonization in vivo. A set of ZEB1-associated genes was identified from analyzing five independent mRNA microarray datasets comprising both cell lines and lung adenocarcinomas. From this gene set we found ZEB1 directly represses ESRP1 by binding to its promoter, which leads to increased mesenchymal splicing of the ESRP1 target CD44. The mesenchymal isoform of CD44, CD44s, conferred a CD44[hi] flow cytometry profile which, in turn, could be used to select for a highly tumorigenic subpopulation in partially transformed HBECs. To identify candidate ZEB1-activated targets we screened ZEB1-upregulated genes in a siRNA invasion assay. Several genes including PMP22 and CD70 could phenocopy ZEB1 where siRNA-mediated loss of expression led to decreased invasiveness in multiple NSCLC cell lines. CD70 (also called TNFSF7, tumor necrosis factor ligand superfamily member 7) may represent a prime therapeutic target for anti-metastatic growth in lung cancer. The ligand for CD27, it is involved in immune regulation, upregulated in some cancers and is being studied as a potential target for antibody therapeutics. Importantly, an anti-CD70 monoclonal antibody inhibited invasion of NSCLC cell lines comparably to siCD70 and siZEB1.

      Conclusion
      We demonstrate in vitro models of defined oncogenic HBEC transformation provide an invaluable tool to study lung cancer progression where EMT is an important mediator. ZEB1 is spontaneously expressed with malignant transformation of HBECs and is a significant driver of oncogenic progression in both HBECs and NSCLC cells. Identification of CD70 and PMP22 as downstream targets of ZEB1 may represent novel therapeutic targets for lung cancer.

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    P1.02 - Poster Session 1 - Novel Cancer Genes and Pathways (ID 144)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.02-010 - Evaluation of the oncogenic ability of EML4-ALK to transform human bronchial epithelial cells (HBECs) (ID 1503)

      09:30 - 16:30  |  Author(s): J.D. Minna

      • Abstract

      Background
      Lung cancer is a highly lethal disease, and is believed to develop through a multistep carcinogenic process, which involves numerous genetic and epigenetic alterations. Among these alterations, mutations in “driver genes” such as KRAS and EGFR are found in non-small cell lung cancer (NSCLC) and they are demonstrated to contribute to a phenomenon, oncogene addiction. Recently, the EML4-ALK (echinoderm microtubule-associated protein–like 4 anaplastic lymphoma kinase) fusion gene has been discovered as a novel driver gene in a subset of NSCLC. We evaluated the oncogenic transformation ability of EML4-ALK by using an hTERT/CDK4-immortalized normal human bronchial epithelial cell (HBEC) model.

      Methods
      We used two HBEC lines, HBEC3 and HBEC4. Mutant KRAS[V12]-expressing HBEC was used as a positive control for oncogenic transformation. A lentiviral vector system was used to generate HBECs stably expressing EML4-ALK. EML4-ALK protein expression was confirmed by westernblotting, and downstream pathways were analyzed by westernblotting with phospho-specific antibodies. Malignant phenotypes of EML4-ALK-expressing HBECs were examined by WST-1 proliferation assay and liquid and soft agar colony formation assays.

      Results
      Westernblotting analysis showed that EML4-ALK was expressed in HBECs. Analysis of downstream pathways did not show significant differences between EML4-ALK-expressing and control HBECs. Introduction of EML4-ALK in HBECs increased the number of soft agar colonies but its effect was not as strong as KRAS[V12].Figure 1 A. Soft agar colony formation assay showing that EML4-ALK increased the number of colonies compared to control cells to a lesser extent than did KRAS[V12]. B. Cell proliferation assay (MTS-1) showing no significant difference between EML4-ALK-expressing and control HBECs.

      Conclusion
      EML4-ALK alone did not induce dramatic oncogenic changes in HBECs. To acquire more malignant phenotype, additional genomic alterations may be required and this is now under investigation.

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    PL03 - Presidential Symposium Including Top Rated Abstracts (ID 85)

    • Event: WCLC 2013
    • Type: Plenary Session
    • Track:
    • Presentations: 1
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      PL03.07 - Treatment with Therapies Matched to Oncogenic Drivers Improves Survival in Patients with Lung Cancers: Results from The Lung Cancer Mutation Consortium (LCMC) (ID 2444)

      08:15 - 09:45  |  Author(s): J.D. Minna

      • Abstract
      • Slides

      Background
      Detecting and targeting the oncogenic drivers EGFR and ALK have transformed the care of patients with lung adenocarcinomas. The LCMC was established to use multiplexed assays to test tumors for alterations in 10 genes and provide the results to clinicians to select treatments and clinical trials matched to the driver detected.

      Methods
      Fourteen LCMC sites enrolled patients with metastatic lung adenocarcinomas and tested their tumors in CLIA laboratories for activating mutations in 10 oncogenic driver genes.

      Results
      Tumors were tested from 1,007 patients for at least one gene and 733 for all 10 genes. An oncogenic driver was found in 466 (64%) of fully-genotyped cases. Among these 733 tumors, drivers found were: KRAS 182 (25%), sensitizing EGFR 122 (17%), ALK rearrangements 57 (8%), “other” EGFR 29 (4%), two genes 24 (3%), HER2 19 (3%), BRAF 16 (2%), PIK3CA 6 (1%), MET amplification 5 (1%), NRAS 5 (1%), MEK1 1 (<1%), AKT1 0. For cases with any genotyping, we used results to select a targeted therapy or trial in 275 (28%). Among 938 patients with follow-up, the median survivals were 3.5 years for the 264 with an oncogenic driver treated with genotype-directed therapy, 2.4 years for the 318 with an oncogenic driver with no genotype-directed therapy, and 2.1 years for the 360 with no driver identified (p<0.0001).

      Conclusion
      Individuals with lung cancers with oncogenic drivers receiving a corresponding targeted agent lived longer than similar patients who did not. An actionable driver was detected in 64% of tumors from patients with lung adenocarcinomas; more than one was present in 3%. Multiplexed testing aided physicians in choosing therapies and targeted trials in 28% of patients. This paradigm for care and research will expand as genotyping becomes more efficient with Next-Gen platforms, additional drivers are identified (i.e.ROS1 and RET), and more targeted drugs become available in the pharmacy and through clinical trials. Supported by HSS NIH NCI 1RC2CA148394-01. Trial Registered with Clinicaltrials.gov: NCT01014286.

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