A Novel In Vivo Gain-Of-Function Screen For Metastasis Drivers In Non-Small Cell Lung Cancers

Abstracts: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TXMetastatic lung cancer is the leading cause of cancer related mortality in the world. Insights on specific genetic causes are limited and novel approaches are necessary to elucidate specific genes that drive lung cancer progression and metastasis. Extensive genomic and transcriptomic profiling of human lung cancers by consortia like ‘The Cancer Genome Atlas’ (TCGA) and the ‘International Cancer Genome Consortium’ (ICGC) has revealed a complex genetic landscape marked by abundant aberrations. In addition to acquiring mutations or amplification of well documented drivers like Kras, p53 or EGFR, lung cancers also demonstrate an enrichment of hundreds of low frequency gene aberrations which are a mix of key driver mutations and nascent passenger alterations caused by progressive genomic instability that could have definite role in cancer progression and metastasis. Our goal was to establish a robust screening platform to selectively identify these functionally critical “driver” genes in lung cancers. To identify a prioritized list of potential candidates we performed a multi-level cross-species comparison of our published high confidence transcriptome data from genetically-engineered mouse models of lung cancers and human lung cancer genomic data from TCGA, focusing on elevated gene expression and/or gene amplification. We identified an enriched list of 225 putative driver genes in lung cancers which were used to construct a lentiviral based cDNA expression library with unique molecular barcoding of individual cDNAs. Using a non-metastatic syngeneic mouse lung cancer model we generated individually transduced stable over-expressing lines for each of the 225 genes. These candidate lines were then entered into parallel in vitro and in vivo screens. In vitro testing includes cell invasion assays in a 96-well format, with positive hits defined as those scoring above 3x standard deviations relative to the control cells expressing mCherry. Positive hits were revalidated using standard 24-well invasion assays and also studied for growth phenotypes in 3D cultures. To selectively identify genes with functional role in metastasis, we performed a unique in vivo positive selection screen to test individual and combinatorial effects of lung cancer driver aberrations. For this we used pools of 20 cDNA expressing lines transplanted into syngeneic mice and observed for primary tumor growth and occurrence of metastases in lungs and other organs. Metastatic lesions and primary tumors were collected and genomic DNA from these was used to identify the unique individual barcodes by NGS barcode enrichment analysis. Metastatic drivers were identified by the relative enrichment of the unique barcode sequences for all the respective pool cDNAs in the metastatic tissue. We have identified both known (e.g. MYC) and several novel (e.g. THRA, TMEM106B, GNAS) potential oncogenic and metastatic drivers from the pooled screen which are currently being revalidated for their individual in vivo metastatic potencies. We are performing gain-of-function/loss-of-function studies for the identified driver genes to determine the pathways which they regulate. We are also analyzing the clinical relevance of these genes based upon their expression pattern and prognostic utility across TCGA and other public datasets, along with in-house patient samples and tissue microarrays of resected and biopsy specimens. Our unique high through-put approach will significantly advance the field of cancer target discovery by identifying new drug targets and biomarkers, essential for novel effective treatment options for lung cancer patients.Citation Format: Samrat T. Kundu, Caitlin Grzeskowiak, Chad J. Creighton, Kenneth L. Scott, Don L. Gibbons. A novel in vivo gain-of-function screen for metastasis drivers in non-small cell lung cancers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B25.