Next generation sequencing (NGS) technologies are rapidly being incorporated into the clinic to facilitate decisions on cancer patient care. However, successful translation of NGS data requires knowledge on which DNA aberrations represent actionable events, either for development or re-positioning of approved agents to target their activated pathways. Recognizing this, large-scale tumor profiling efforts by consortia such as The Cancer Genome Atlas (TCGA) are cataloging genomic aberrations across major cancer lineages. 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 do this, we have developed high-throughput approaches to construct molecularly-barcoded versions of gene aberrations for functional screens. Specifically, we developed technologies that include (1) high-throughput, accurate modeling of somatic DNA mutations (somatic missense mutations and small insertions/deletions) using our robotics-driven platform of >35,000 sequenced-verified open reading frame (ORF) clones, (2) a molecular barcoding strategy that permits rapid DNA tagging of wild-type and mutant ORFs, (3) multi-fragment recombineering methodologies allowing construction of cancer fusion genes, and (4) combining the use of these reagents for individual or pooled functional screens in vitro and in vivo using human and mouse systems. We are using these technologies, which are widely applicable to all cancer types, to identify the highest priority targets to enroll in deep mechanistic studies and drug discovery programs. We have scaled our pipelines to functionalize thousands of cancer gene aberrations. Importantly, we are now constructing entire somatically-mutated exomes from individual patients sequenced in the clinic. As an example of our “Personalized Functionalization” approach, we screened individual pancreatic ductal adenocarcinoma (PDAC) patient-derived aberration libraries for mutations capable of promoting tumorigenesis in vivo using a mouse xenograft model engineered with regulatable KRASG12D, an oncogene active in the majority of PDAC patient tumors. These studies revealed potent aberration drivers that are active as individual drivers as well as those that are contextual and are only active in the presence of KRASG12D. Based on these results, we have chosen NAD Kinase (NADK) for deep mechanistic studies and drug discovery programs. NADK catalyzes the conversion of cytoplasmic NAD+ to NADP+/NADPH and thus aids other modes of cellular NADPH production. Our validation studies indicate that the NADK mutation results in robust gain-of-function kinase activity leading to its hyper-phosphorylation of NAD+ accompanied by reduced accumulation or reactive oxygen species and increased tumor formation and growth. Interestingly, recent work by others report other mechanisms by which KRAS rewires PDAC tumors to maximize energy production and promote NADPH accumulation to maintain redox state and tumor growth. Even though NADK is mutated at low frequency in PDAC, its selection demonstrates that the discovery of rare, functional aberrations may intersect or otherwise lead us to important pathways and potential therapeutic liabilities. Our ultimate goal is to functionally annotate thousands of somatic aberrations in cancer, the vast majority of which have not been previously recognized or assayed for clinical relevance. These systems will reveal high priority edited targets to enroll in deep mechanistic biology studies, drug discovery and development programs ultimately leading to personalized treatment strategies. Citation Format: Kenneth Scott, Yiu Huen Tsang, Turgut Dogruluk, Philip Tedeschi, Joseph Bertino, Gordon Mills. Functional prioritization of rare gene aberration drivers of cancer. [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 PR06.