Abstract The PI3K and MEK pathways are often implicated in many forms of human cancer. Aberration of upstream signaling molecules and receptor tyrosine kinases at the genetic level directly governs the dysregulation of the MEK and PI3K pathways. Both the pathways converge at various points along their cascade and possess a variety of feedback loops. Targeted inhibition of one of the pathways can lead to resistance via feedback activation of the other pathway. The inefficiency of single target inhibitors provides rationale for dual pathway inhibition. Clinical studies are currently evaluating the efficacy of dual pathway inhibition in several types of cancers such as KRAS ovarian cancer and BRAF melanoma. Poor responses and monitoring the toxicity have been major concerns. These two parallel pathways display different cross talks and feed-back loops across different tumour and tissue types, due to which deciding an optimal dosing schedule for combined inhibition has been a challenge. Current approaches include the use of two different targeted small molecule inhibitors for dual inhibition, however the probability of synergistic inhibition in the same cell is very low. Hence we engineered a new strategy to deliver both the drugs to the target cell and provide dual inhibition of the MEK and PI3K pathways in a sustained, controlled and synergistic manner via a supramolecular nanotherapeutic that carries both the drugs. The engineering of the supramolecular nanotherapeutic is facilitated by self-assembly driven by supramolecular interactions between various biocompatible lipids. A combination of PI103 and Selumetinib was chosen to inhibit PI3K, mTOR and MEK. These drugs were re-engineered by conjugating each of them to cholesterol and enabling them to self-assemble along with the other lipids to form a supramolecular fusion nanotherapeutic encapsulating them in the same nanostructure. The stability of the nanoparticle was measured in terms of changes in hydrodynamic size and zeta potential for a period of one month and was found to be stable. The loading of PI103 and Selumetinib into the nanostructure was confirmed using UV spectroscopy. The release of active PI103 and Selumetinib from the nanostructure was validated via release kinetics in cell lysate. In vitro studies for cytotoxicity in multiple tumor models confirmed that the supramolecular nanotherapeutic is as effective as or better than the free drug counterparts in most cases. Sustained inhibition of the PI3K and MEK pathways were confirmed by biological characterization. This study presents a strategy for overcoming acquired resistance and improving the efficacy of targeted therapeutics. Citation Format: Vineethkrishna Chandrasekar, Siva Kumar Natarajan, Shiladitya Sengupta, Ashish Kulkarni. Supramolecular fusion nanotherapeutic mediated synergistic inhibition of PI3K and MEK pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-059.