CDK2/9 blockade remodels chromatin and drives phenotypic plasticity in glioblastoma organoids.

Abstract

Background: Glioblastoma (GBM) remains a highly lethal brain tumor with limited therapeutic options and pervasive resistance to standard treatments. Cyclin-dependent kinases (CDKs), key regulators of cell cycle progression and transcription, are frequently dysregulated in GBM and contribute to tumor pathogenesis.

Methods: We employed a range of preclinical GBM models-including 2D cultures, 3D spheroids, patient-derived organoids (PDOs), and multicellular 3D bioprints-to evaluate the effects of the CDK2/9 inhibitor fadraciclib in comparison to the standard-of-care temozolomide. Endpoints included cell viability, stress responses, immune-modulatory effects, and transcriptomic reprogramming.

Results: Fadraciclib robustly reduces cell viability and invasion in all patient-specific 2D- and 3D models. This effect was independent of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, demonstrating superior efficacy to temozolomide. Mechanistically, fadraciclib disrupted lysosomal, mitochondrial, and endoplasmic reticulum integrity and increased γH2AX levels, indicating enhanced DNA double-strand breaks. Seahorse analysis demonstrated impaired mitochondrial bioenergetics following fadraciclib treatment. In a triple-culture blood-brain barrier (BBB) model, fadraciclib demonstrated effective BBB penetration and antitumoral activity against GBM cells, while exhibiting minor cytotoxicity toward brain-specific normal cells. In semi-autologous co-culture systems, fadraciclib inhibited GBM invasion, promoted intercellular communication via Connexin 43 upregulation in microglia, and activated peripheral T cells. Transcriptomic analyses of treated patient-derived organoids revealed widespread transcriptional reprogramming, including downregulation of oncogenic long non-coding RNAs (MALAT1, POM121L9), suppression of immune-related pathways, and upregulation of the tumor suppressor LZTS1. Increased histone-associated gene expression suggests chromatin remodeling and induction of a quiescent tumor cell state. Subtype analysis indicated a shift toward a proneural molecular signature. Notably, the antitumoral effects of fadraciclib were preserved in multicellular biomimetic bioprints, whereas nonmalignant astrocytes and microglia were not affected. Therapeutic activity also coincided with altered secretion profiles of extracellular vesicles (EV). EVs from reactive astrocytes contained higher amounts of Fas, which may act as modulator of neuroinflammation.

Conclusion: CDK2/9 inhibition shows promise as a therapeutic strategy for GBM. Fadraciclib exhibits multifaceted anti-tumor activity and spares nonmalignant cells of the central nervous system, supporting its further development in therapies that target transcriptional and immune pathways, as well as quiescent tumor cell populations.