SAT-289 Rationally designed cholesterol coated peptide NS as a novel therapeutic strategy in the management of Adrenocortical Carcinoma

Abstract Disclosure: M. Hassany: None. R. Challapalli: None. O. Covarrubias-Zambrano: None. S. Feely: None. S. Fiachetti: None. M. Luconi: None. M. O'Halloran: None. C. Hantel: None. P. Prakash: None. S. Bossmann: None. M.C. Dennedy: None. Management of adrenocortical carcinoma (ACC) is challenging, with diagnosis often occurring at advanced stage. Surgical resection of localized disease offers potential cure, but recurrence is high (75-85%). Mitotane is the only approved drug for advanced ACC, but has a narrow therapeutic window, is poorly tolerated, and effective in less than 30% of cases. Nanotheranostics rationally designs nanomaterials for simultaneous diagnosis, drug-delivery, and treatment. Most FDA-approved nanomedicines rely on the enhanced permeability and retention (EPR), where nanoparticles passively accumulate in tumors. This improves drug delivery while reducing adverse effects but is subject to variability between patients and tumor types, which can be improved by specific nanoparticle- targeting to tumors. ACC cells rely on cholesterol, for steroid hormone synthesis, as a key component of cell membranes, and as fuel. Exploiting this, we designed tri-maleimide peptide-based nanosponges (NS) functionalized with two cholesterol molecules and a fluorescent tag (Rhodamine B). These rationally designed NS were tested for uptake and cytotoxicity on three ACC cell lines: H295R (chemotherapy-sensitive), HAC-15 (chemoresistant), MUC-1 (chemoresistant). Cell death was assessed using SYTOX Blue, and the role of SR-B1 in NS uptake was evaluated using BLT-1. Caspase-dependent/independent cytotoxicity was investigated using z-VAD (pan-caspase inhibitor), and measuring markers of apoptosis, ER stress and autophagy (Caspase 3 & 8, CHOP, LC3). LC-MS/MS was used to measure steroidogenesis. NS were rapidly taken up by all ACC cell lines, with significant cell death observed within 2 hours in the H295R cell line at the physiological concentration of 20 ug/ml (p<0.01), at 50 ug/ml in HAC-15 (p<0.01) and at 100 ug/ml in MUC-1 (p<0.5). NS uptake was not prevented by the Scavenger Receptor-B1 (SR-B1) inhibitor BLT-1, suggesting direct-membrane or LDL-R mediated uptake. Confocal microscopy and TEM revealed that NS accumulated in mitochondria. ACC cell death occurred through a caspase-independent pathway: (i) cleaved caspase-3 protein was not upregulated (ii) zVAD-fmk hand no significant effect on NS-induced cytotoxicity, while (iii) autophagy-related markers (LC3II-LC3I ratio) were upregulated. Forskolin-stimulated cortisol production was also reduced in H295R cells after NS treatment. Rationally designed cholesterol-functionalized nanosponges (NS) demonstrate rapid uptake and potent cytotoxicity in ACC, inducing caspase-independent cell death and disrupting steroidogenesis. These findings highlight the potential of NS as a novel theranostic approach for ACC, offering targeted drug delivery with reduced toxicity and improved efficacy compared to conventional therapies. Future studies will load NS with lipid-soluble cytotoxic drugs e.g. mitotane. Presentation: Saturday, July 12, 2025