Distinct molecular pathways leading to dosage-dependent temozolomide resistance in GBM stem cells

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, with a median survival of around 15 months despite complete therapy. A significant contributor to recurrence is the enduring presence of GBM stem cells (GSC), which exhibit remarkable self-renewal, adaptability, and resistance to treatment measures. Patient-derived cancer stem cells (GSC) were continuously exposed to temozolomide (TMZ) in vitro to create a model for investigating chemotherapy resistance. Transcriptomic profiling was conducted to investigate the molecular pathways associated with resistance, with Western blotting used to confirm the findings from RNA sequencing. The analyses focused on signaling pathways related to neurosynaptic transmission, stemness, pro-survival adaptability, ECM remodeling, and DNA repair. A convergent multi-pathway adaptation was noted in GSC treated with various dosages of TMZ. A transcriptomic analysis indicated that cells exposed to high-dose TMZ (TMZ-hc) displayed a specific activation of a neuroactive, synaptic-like expression program. This program included genes associated with neurotransmitter receptors as well as voltage-gated calcium and potassium channels, while simultaneously suppressing DNA mismatch repair mechanisms and negative feedback regulators. In contrast, an alternative resistance pathway was discovered in cells treated with low-dose TMZ (TMZ-Lc), which promoted a niche-dependent, dormant state marked by the expression of vascular mimicry markers and remodeling of the extracellular matrix. In addition, the protein levels of Survivin, Bcl-2, and Notch1 signaling were significantly elevated in TMZ-hc compared to TMZ-Lc and control cells. Our research underscores the translational significance of investigating GSC-specific resistance mechanisms, since GSC are recognized as the primary drivers of patient recurrence. Understanding the molecular mechanisms that enable TMZ resistance is crucial to developing new therapeutic options.