Poster #32: Kelsey Keith MS

Targeted modulation of IGFBP5/IGF1, THPO, and P38 MAPK signaling are potent therapeutic strategies generalizable for mitochondrial respiratory chain disease

Kelsey Keith1#, Min Peng2#, Cristina Remes2, Victoria Miranda2, Nicholas Wachowski2, Melis Kose2, Shrikant Dhraskar2, Suraiya Haroon2,3, Agustin B. Velasco4, Priya Sivaramakrishnan3,4, Donna Iadarola2, Sundeep Dugar6, Marni J. Falk2,3,6* Affiliations: 1Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA 2Mitochondrial Medicine Frontier Program, Division of Genetic and Genomic Medicine, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 3Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 4Center for Computational and Genomic Medicine, Children’s Hospital of Philadelphia 5Department of Pathology and Laboratory Medicine, University of Pennsylvania 6Rarefy Therapeutics, Voorhees, NJ #, Equal Contribution *Corresponding Author

Primary mitochondrial diseases (PMD) are genetically heterogeneous disorders of oxidative phosphorylation with few disease-modifying options, each benefiting only a limited subset of patients. We previously found that cycloheximide, a cytosolic translation inhibitor used as a chemotherapeutic, unexpectedly improves cell viability and mitochondrial function across multiple PMD models. Because global suppression of protein synthesis precludes clinical use, we sought to identify specific mediators of cycloheximide’s protective effect to enable targeted therapeutic strategies. We performed an unbiased proteomic screen in metabolically stressed patient fibroblasts, followed by validation in cell-based assays and Caenorhabditis elegans models. Complex I–deficient patient fibroblasts and rotenone-exposed control lines were shifted from glucose to galactose to unmask mitochondrial dysfunction, then treated with cycloheximide. Proteome profiling (SomaLogic SOMAscan) and differential protein/pathway analyses identified stress-responsive proteins whose changes were reversed by cycloheximide. Two candidates—thrombopoietin (THPO) and insulin-like growth factor binding protein 5 (IGFBP5)—showed opposite regulation during galactose stress versus cycloheximide exposure specifically in PMD cells. Pathway analyses further implicated ERK/MAPK signaling. Functional studies demonstrated that suppressing THPO, via siRNA knockdown or pharmacologic inhibition with eltrombopag, improved viability across multiple complex I–deficient fibroblast lines under metabolic stress and extended survival in C. elegans models of complex I disease. In contrast, IGFBP5 overexpression promoted survival in complex I–deficient cells. Pharmacologic inhibition of IGF1 signaling (a pathway antagonized by IGFBP5) using OSI-906 recapitulated protection across diverse nuclear- and mitochondrial DNA–encoded complex I defects and additional PMD genotypes in both fibroblasts and C. elegans. Consistent with pathway-level findings, inhibition of p38-MAPK with talmapimod also increased viability. Protective effects were enhanced in the presence of glucose and/or with combination treatments. Together, these findings nominate THPO, IGFBP5/IGF1, and MAPK signaling as actionable targets that can mimic cycloheximide’s benefits in cellular and worm models of mitochondrial dysfunction while avoiding the toxicity of global translational inhibition. Mechanistically, these interventions may reduce p38-MAPK–driven autophagy and improve energy balance under mitochondrial stress, supporting further preclinical development.