Abstract The Rb (retinoblastoma) protein is highly functionally conserved between the Drosophila, and deregulation and mutations in the Rb pathway are common events across a wide range of cancers. Here, we identify two novel synthetic lethal interactions between Rb and mutant alleles of Nedd8 and Hrb27c that were identified through an unbiased genetic screen in Drosophila that resulted in apoptosis in both the wing disc pouch and the region of the eye disc posterior to the morphogenetic furrow. However, the molecular mechanisms behind the apoptotic phenotypes in both synthetic lethal double mutants have yet to be elucidated. It is known that wild-type Rb normally sequesters and can prevent the E2F1 transcription factor from promoting transcription of hid, the major pro-apoptotic regulator in imaginal discs. Synthetic lethality is therefore likely to be contingent on EGFR/hid signaling, and I will determine if such is the case through a series of genetic epistasis tests. Interestingly, preliminary evidence also suggests that EGFR/MAPK signaling may be ablated in both mutants of Nedd8 and Hrb27c. However, this observation is completely novel and therefore the molecular mechanisms behind downregulation of MAPK signaling have yet to be elucidated in both these mutants. I will again perform a series of epistasis experiments to determine how Nedd8 and Hrb27c mutations mechanistically affect EGFR/MAPK signaling. This will be achieved first by determining if signaling is downregulated either during EGFR ligand processing or downstream of the EGFR receptor. This will be followed by subsequent experiments to determine which specific element in either pathway is being affected by Nedd8 or Hrb27c ablation. I therefore hypothesize that inhibition of both nedd8 and hrb27c synergistically induce cell death in Rb-negative cells in a process that is dependent on both E2F1 activity and downregulation of MAPK signaling. Many existing treatment regimens fail to take into account the unique biological properties of cancer cells compared to their normal counterparts, thus often resulting in off-target effects and limitations in dosage. This proposal is therefore both novel and significant because the mechanisms behind both the Rb/Hrb27c and Rb/Nedd8 synthetic lethal interactions have yet to be elucidated and characterization of such interactions such as these have the potential to catalyze the development of new therapeutic strategies in the treatment of cancer. Note: This abstract was not presented at the meeting. Citation Format: Robin Zhang, Tianyi Zhang, Zhentao Sheng, Wei Du. Synergistic cell death in nedd8 and hrb27c mutant cells under an rbf-negative background is dependent on EGFR/MAPK signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4402. doi:10.1158/1538-7445.AM2014-4402