Abstract B131: A prioritization pipeline for functionally annotating oncogenic somatic driver aberrations in cancer.

The Cancer Genome Atlas (TCGA) and similar projects are cataloging genomic aberrations across major cancer lineages with the goal of identifying the most promising therapeutic targets and diagnostic biomarkers. These efforts have revealed an extraordinary level of genome complexity made up of not only key “driver” events critical to pathogenesis, but also numerous biologically-neutral “passengers” that accompany unstable tumor genomes. The challenge now is to find ways to identify functional driver aberrations, as targeting such events or their activated pathways has great potential for improving patient outcomes. To complement the power of RNAi-based approaches, we are implementing a scalable gain-of-function screening infrastructure aimed at accelerating functional validation of oncogenic driver events. To do this, we devised a high-throughput mutagenesis and molecular barcoding (HiTMMoB) platform. The platform allows introduction of individual DNA mutations and small DNA insertions/deletions into a collection of over 32,000 sequence-verified human open reading frame (ORF) clones. In addition to permitting efficient modeling of gene aberrations, HiTMMoB also allows simultaneous "barcoding” of ORFs with unique 24-nuleotide DNA segments. Each barcode serves as a surrogate identifier for its associated ORF, thus permitting detection and quantitation of ORFs following pooled screens. We are employing HiTMMoB to functionally annotate gene aberrations across multiple cancer types, work that is funded in part by the NCI's Cancer Target Discovery and Development (CTD2) Network whose collective goal is to extract therapeutic targets and drug response markers from TCGA and similar efforts. ORFs processed through HiTMMoB are enlisted into a variety of in vitro screening platforms including those that annotate ORFs for their ability to drive colony formation in soft agar, cell survival and proliferation using the Ba/F3 myeloid cell system and drug resistance of sensitive cell models among other assays. While tractable, in vitro models do not recapitulate all hallmarks of tumorigenesis and metastasis. To address this challenge, we developed a Context-Specific Screen (CSS) platform that interrogates the tumorigenic and metastatic potency of candidate driver genes under the appropriate in vivo genetic and microenvironment contexts. These in vivo screens utilize genetically defined target cells and are performed at orthotopic sites in the mouse to ensure the correct microenvironment context. We identify putative driver ORFs from primary tumors and metastases by sequencing barcode pools from genomic DNA in parental (i.e., injected) cells, primary tumors and metastases. As expected, our data indicate that, relative to injected cells, primary tumors and metastases positively select driver genes and lose those with no role in driving tumorigenesis and metastasis in a given model (i.e., passengers). We have employed the CSS approach across tumors of multiple lineages that include pancreas, lung, breast and melanoma, work that has already revealed multiple novel drivers currently undergoing secondary validation and follow-up mechanistic studies. In summary, our aberration “prioritization pipeline” will provide the cancer research community information on the most promising somatic aberrations for downstream biomarker and drug development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B131. Citation Format: Turgut Dogruluk, Yiu Huen Tsang, Nikitha Nair, Jianhua Zhang, Timmothy Heffernan, Lynda Chin, Gordon Mills, Kenneth L. Scott. A prioritization pipeline for functionally annotating oncogenic somatic driver aberrations in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B131.