Abstract Loss of tumor suppressor activity is a near universal feature of cancer. Synthetic lethality, in which combined perturbation of two genes results in cell death, has been proposed as a strategy to identify drug targets that specifically attack cancers deficient in a tumor suppressor (TS) gene. To develop a catalog of such combinations, we screened for synthetic-lethal interactions among all yeast orthologs of known TS and druggable genes, resulting in quantitative growth rates for 130,000 pairwise genetic mutants. Similar interaction profiles were highly predictive of TS genes in the same pathway, identifying a novel role for NDNL2/NSE3 in the G1/S checkpoint. TS/drug combinations likely to induce cancer cell death were prioritized using multiple criteria, including conservation across divergent species. Among the top hits were putative interactions involving either BRCA1 or XRCC3 with histone deacetylases, as well as Rad17 with CHK1/2. These were confirmed using small molecule inhibitors and short-pin RNA silencing in human cancer cell lines. This work provides a resource of >300 deeply conserved interactions among tumor suppressors and matched drug targets. Citation Format: Rohith Srivas, John Paul Shen, Jian Feng Li, Katherine Licon, Ze Zhong Wang, Ana Bojoquez-Gomez, Lucy Xu, Andrew Gross, Gordon Bean, Robert Sobol, Trey Ideker. High-throughput synthetic lethal interaction screening in model organisms as a strategy for the identification of novel therapeutic targets in cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B34.