Piepers, J. | bioRxiv Science

bioRxiv2025

Adaptive Kinase Signaling Enables Escape from Small-Molecule Inhibition in Glioblastoma

Patients with glioblastoma (GB) have a median survival of 15 months. Despite an intensive treatment schedule with resection, radio- and chemotherapy, recurrence is inevitable. A significant challenge to overcome GB treatment resistance involves intratumoral heterogeneity, characterized by molecular, phenotypic, and clinical distinctive GB subtypes. Different small molecule inhibitors (SMI) have been designed to inhibit signalling proteins in oncogenic driver pathways in GB. However, SMIs have been unsuccessful in improving patient outcomes. Here, we investigate whether crosstalk between signalling pathways and signalling pathway redundancy are responsible for single-agent resistance using a primary patient-derived glioblastoma organoid (PGO) platform. This study used nine FDA-approved small-molecule inhibitors, based on their ability to cross the blood-brain barrier, targeting key GB driver genes. Although inhibition of downstream effector proteins reduced cell viability more effectively (IC50 70nM-1M) than inhibiting upstream membrane-bound tyrosine kinase receptors (IC50 1-15M), remaining cell proliferation was seen in all six PGOs. To uncover resistance mechanisms, we analysed phosphokinase activity following monotherapy in various PGOs and identified compensatory pathway activation, leading to the discovery of effective small-molecule inhibitor combinations, most notably CHIR99021 (GSK-3 inhibitor) with trametinib (MEK inhibitor). In conclusion, our findings highlight the potential of combination therapy targeting compensatory pathways to overcome single-agent resistance in GB, emphasizing the utility of patient-derived glioblastoma organoids as a platform for personalized therapeutic development.

cancer biology↗

bioRxiv2025

Disruption of Iron Metabolism Resulting from Dmt1/Slc11A2 Deficiency Compromises Notch Protein Degradation and Transcriptional Activation

Notch receptor activation requires {gamma}-secretase-mediated release of Notch intracellular domain 1(NICD1) to regulate gene transcription. Here, we identify the proton-driven Solute carrier 11A2 (Slc11A2) or divalent metal transport protein Dmt1 as an inhibitor of Notch signaling via regulating iron homeostasis and lysosomal integrity. Dmt1 loss reduces ferritin levels and increases labile Fe2+, causing elevated reactive oxygen species (ROS) and lipid peroxidation. These changes compromise lysosomal function and impair degradation of S3-Val1744 cleaved NICD1, resulting in its accumulation. Dmt1 has isoforms with or without an iron response element (IRE): Re-expressing Dmt1+IRE robustly increases ferritin heavy-chain (FTH), whereas Dmt1-IRE moderately elevates FTH and ferritin light-chain (FTL), with co-expression further enhancing FTL levels. Restoration of Dmt1 expression rescues ferritin levels, lysosomal activity, and NICD1 degradation while reducing oxidative stress and lipid peroxidation. Notably, Dmt1 deficiency decreases NICD1 binding to RBP-J{kappa}/CSL and its recruitment to Notch target gene promoters Hes1 and Hey1. Collectively, our findings demonstrate that Dmt1 regulates lysosomal function through iron homeostasis and that lysosomal dysfunction from Dmt1 loss impairs NICD1 degradation and disrupts Notch signaling, linking cellular iron metabolism and Notch pathway activity.

cell biology↗