Abstract A2-06: An in vivo shRNA functional genomics screen identifies transcriptional and epigenetic regulators that are essential in lung tumorigenesis
Authors: Hight Suzie K. , McMillan Elizabeth , Shao Chunli , Girard Luc , Canales Jaime Rodriguez , White Michael A. , Wistuba Ignacio I. , Mangelsdorf David , Minna John D. 
Published: November 2015

Cancer cells are characterized by the aberrant regulation of signaling pathways that govern response to external stimuli, resulting in uncontrolled cellular proliferation. The genomic alterations that accumulate to cause the malignant growth phenotype can also result in acquired vulnerabilities in the tumor. Loss-of-function screening using pooled short hairpin RNAs (shRNAs) is proving to be a powerful method by which new cancer therapeutic targets can be identified. A major limitation of these studies is that they are usually performed in vitro under low selection pressure, thus potentially missing key vulnerabilities that might be detected in a more clinically relevant setting. Here we tested the use of parallel in vitro and in vivo screens using a mini-library of shRNAs to identify previously unknown acquired vulnerabilities in lung cancer.

Using a mini-library of 1062 lentiviral shRNAs targeting nuclear hormone receptors and their coregulators (120 genes total), we screened the lung adenocarcinoma cell line NCI-H1819 for dependency on these genes during in vitro and in vivo growth. Briefly, cells were transduced with the library, allowed to undergo a 4 day period of puromycin selection, and then were either injected subcutaneously into NOD-SCID mice or maintained in 2D tissue culture. Tumors were harvested after ~2 months when reaching a volume of 300 mm3. In a parallel in vitro screen, transduced cells were cultured for 20 population doublings. The relative abundance of individual shRNAs was quantified by next generation sequencing, and candidate essential genes were nominated by filtering for depleted shRNAs that scored in a combination of two statistical methods (t-test and ranked KS-test).

We identified six genes required for survival of H1819 in vitro (BRCA1, CCND1, MED1, PHB, HNRNPU, and PELP1), and three genes that were required for tumor survival in vivo, but not in vitro: FOXA1, HDAC1, and NCOR2. None of these genes were mutated by full exome sequencing of H1819, however FOXA1 was found to be co-amplified with NKX2-1 on chromosome 14q. The NKX2-1 gene has previously been identified as one of the most frequent focally amplified genes in lung adenocarcinomas, and we found that FOXA1 is co-amplified with NKX2-1 in 80% of these cases. We also found that NSCLC cell lines and tumor samples have significantly higher FoxA1 expression than normal lung epithelial cells and tissues. Knockdown of Foxa1 in amplified NSCLC cell lines causes significant reduction of clonogenicity, anchorage-independent growth, and in vivo xenograft growth. FoxA1 is a pioneer transcription factor, able to interact with condensed chromatin and recruit other transcription factors to their target genes. Notably, both HDAC1 and NCOR2 are also known to play roles in epigenetic regulation of gene expression. The identification of FOXA1, HDAC1, and NCOR2 as genes that are specifically required for tumor growth in vivo suggests that there are transcriptional and epigenetic programs in lung cancer which depend on microenvironmental cues and/or regulation by paracrine signaling mechanisms. We conclude that our shRNA mini-library screen identifies unsuspected acquired vulnerabilities that are only detected in vivo, and thus new therapeutic targets in lung cancer.

Citation Format: Suzie K. Hight, Elizabeth McMillan, Chunli Shao, Luc Girard, Jaime Rodriguez Canales, Michael A. White, Ignacio I. Wistuba, David Mangelsdorf, John D. Minna. An in vivo shRNA functional genomics screen identifies transcriptional and epigenetic regulators that are essential in lung tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-06.