Abstract 2349: Identifying genes and microRNAs that when lost, can drive neoplastic transformation of non-cancerous lung cells

Abstract

Abstract Despite the fact that the death rate continues to drop over the last decades, lung cancer is still by far the leading cause of cancer mortalities due to lack of highly accurate prediction method and effective targeted therapeutics. Thus, this calls for identification of novel biomarkers and therapeutic targets, particularly those targeting critical genes that drive lung cancer development and malignancy. KRAS and TP53 are two of the most commonly mutated genes in non-small cell lung cancer (NSCLC) which represents 85% of all cases in lung cancer. However, aberrant expressions of many other genes that act as drivers of lung cancer are yet unidentified. Particularly, microRNAs which are genome-encoded small RNA molecules are globally downregulated in many cancers, and disrupting microRNA biogenesis has been shown to promote tumor formation. We utilize the power of the CRISPR-Cas9 gene knockout system to screen for critical tumor suppressor genes and microRNAs in the human and mouse genomes that when lost, can drive neoplastic transformation of lung cells. Two non-cancerous mammalian lung model systems are used for this study. (1) The human bronchial epithelial cells that stably express KRAS G12V and TP53-targeting shRNA (HBEC-KP) are used as the baseline in the first approach. Importantly, the HBEC-KP cells are anchorage dependent and incapable of forming tumor in in vivo. We have transiently transfected HBEC-KP tdTomato-expressing cells with Cas9 and transduced the cells with lenti-sgRNA human library (A). Cells were either passaged in two-dimensions for over two-months or were selected for growth in soft agar assays. Resulting cells were harvested to identify sgRNAs enriched in each of the individual condition. Several known tumor suppressor genes and microRNAs (such as BRCA2, let-7a-3, miR-34a) are present among top hits, while MYC and TP53 are highly depleted, which suggests the validity of this data. Top hits will be selected and validated. (2) The second model being used is the KrasLSL-G12D mouse model. Genetic recombination induces lung hyperplasia in these mice, which will serve as the baseline to identify gene and microRNA knockouts that drive advanced tumor progression. The KrasLSL-G12D; RosaLSL-Cas9/LSL-Cas9 (KC) mice have been generated, and will be validated for Cre-induced Cas9 stable expression. KC mice will be intratracheally injected with the mixture of adeno-Cre and lenti-sgRNAs, and eventually sgRNAs that are highly enriched in individual tumors that develop will be identified through deep sequencing and bioinformatic analysis, and targeted genes and microRNAs downregulated in the tumors will be validated in functional assays and for loss in human NSCLC tumor samples. Citation Format: Chennan Li, Sagar M. Utturkar, Andrea L. Kasinski. Identifying genes and microRNAs that when lost, can drive neoplastic transformation of non-cancerous lung cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2349.