Abstract Drug resistance and frequent tumor relapses are the major obstacles in glioblastoma therapy, and recurrent tumors are practically incurable. We previously reported that insulin receptor substrate 1 (IRS-1), which is a typical signaling molecule for insulin and insulin-like growth factor 1 receptors, can translocate to nucleus, and that nuclear IRS-1 (nIRS-1) was found in different tumor cells, including glioblastomas. To unravel its function, we employed glioblastoma cell culture, animal models, and clinical samples. Using confocal imaging, molecular cloning, subcellular fractionation, mass spectrometry, gene expression analysis, and different approaches to verify protein-protein interactions, we demonstrate for the first time that nIRS-1 can form complex nuclear structures in a restricted number of cancer cells in glioblastoma biopsies and in intracranial glioblastoma xenografts. We also demonstrated the formation of highly organized ring-like structures in several cell lines, following ectopic expression of IRS-1 cloned in frame with nuclear localization signal (NLS-IRS-1). In these nuclear structures IRS-1 localizes at the periphery, and the core of the structure harbors a key autophagy protein, LC3; however, other autophagy proteins or biological membranes were not detected. In living cells expressing NLS-IRS-1-GFP fusion protein, IRS-1/LC3 structures are highly dynamic. They rapidly exchange IRS-1 molecules with nucleoplasm and interact with other nuclear complexes including BMI1-positive Polycomb bodies, PML bodies and Cajal bodies. Importantly, clones and mixed populations of cells expressing the NLS-IRS-1 and capable of forming the IRS-1/LC3 ring-like structures undergo extensive remodeling of gene expression, which suggests a transition to stem-like phenotype and associated resistance to several different anticancer drugs, including temozolomide. This is the first demonstration of IRS-1/LC3 nuclear complexes, which are highly dynamic and may play a role in epigenetic remodeling of glioblastoma cells towards stemness. Further studies are required to determine detailed molecular composition and to explain how these new nuclear structures function. Citation Format: Adam Lassak, Dorota Wyczechowska, Anna Wilk, Adriana Zapata, Mathew Dean, Luis DelValle, Jann N. Sarkaria, Augusto Ochoas, Francesca Peruzzi, Krzysztof Reiss. IRS-1/LC3 nuclear structures and glioblastoma drug resistance. 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 2520.