Abstract C10: High-throughput functional annotation of somatic driver aberrations in cancer
Authors: Dogruluk Turgut , Dogruluk Armel , Tsang Yiu-Huen , Nair Nikitha , Minelli Rosalba , Wu Ping , Scott Kenneth L. 
Published: August 2014

Tumor sequencing projects such as The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) have revealed the high complexity of cancer genomes that are comprised of both pathogenic “driver” aberrations and many neutral “passenger” events. The cancer research community is faced with the daunting challenge of discriminating between drivers and passengers to inform the most promising therapeutic targets and diagnostic markers. Much of these efforts are focused on identifying new oncogenes given that such factors have served as successful therapeutic targets to date.

To expedite oncogene discovery based on large-scale sequencing datasets, we established a target discovery pipeline involving a high-throughput mutagenesis and molecular barcoding (HiTMMoB) platform permitting (1) efficient site-directed DNA mutagenesis to model tumor somatic mutations into our collection of &gt;32,000 human open reading frames (ORFs or genes), (2) integration of a 24 nucleotide DNA barcode sequence into ORF clones to facilitate pooled genetic screens and (3) their simultaneous recombination into lentiviral expression backbones enabling their expression in cancer cell models. Cells infected with barcoded ORF libraries can be entered into a variety of individual or pooled in vitro and in vivo genetic screens to identify ORF drivers of cancer-related activities (e.g., proliferation, tumor growth, invasion, metastasis among others).

We have successfully used HiTMMoB to generate numerous wild-type and mutant ORF clones currently being used in multiple screening projects in our laboratory. One such project involves a gene focused screen designed to functionally characterize numerous lower frequency somatic tail mutations within the PIK3CA oncogene found mutated in breast and other cancers. Here, we have generated 36 mutated clones for PIK3CA that have been entered into in vitro and in vivo screens for drivers of (1) anchorage-independent growth (2) growth factor-independent growth (3) drug sensitivity/resistance and (4) orthotopic tumor formation in a pooled fashion. The goal of these screens is to functionally annotate these low frequency PIK3CA mutations, which may ultimately lead to tailored patient therapies based on the PIK3CA mutation status of an individual's tumor.

In summary, we have developed a prioritization pipeline being used in a variety of target screens across diverse cancer types, and this platform promises to provide the cancer research community functional annotation on the most promising cancer aberrations for downstream biomarker and drug development.

Citation Format: Turgut Dogruluk, Armel Dogruluk, Yiu-Huen Tsang, Nikitha Nair, Rosalba Minelli, Ping Wu, Kenneth L. Scott. High-throughput functional annotation of somatic driver aberrations in cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C10.