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KPNA3 drives temozolomide resistance in glioblastoma by upregulating MGMT and activating STAT3 to sustain glioma stem cells

ABSTRACT: Glioblastoma (GBM) invariably develops resistance to temozolomide (TMZ), the frontline chemotherapeutic agent, leading to treatment failure. The molecular mechanisms underlying this resistance remain incompletely understood. Here, we identify karyopherin subunit alpha 3 (KPNA3) as a novel and critical driver of TMZ resistance. Through integrated bioinformatics analysis of temozolomide-resistant glioma cells (SF126R) and patient databases, we found KPNA3 expression is elevated in TMZ-resistant contexts and correlates with poor prognosis in TMZ-treated patients. Functional studies demonstrated that KPNA3 knockdown in resistant cells impaired malignant behaviors, re-sensitized cells to TMZ-induced apoptosis, and suppressed glioma stem cell (GSC) properties, including sphere formation and expression of stemness factors (OCT4, ALDH1A1). Mechanistically, KPNA3 promotes resistance via a dual pathway: it upregulates the expression of the DNA repair enzyme MGMT and activates the STAT3 signaling pathway. KPNA3 knockdown reduced both MGMT levels and STAT3 phosphorylation (Tyr705). Furthermore, pharmacological inhibition of STAT3 mirrored the effect of KPNA3 knockdown by potently inhibiting GSC generation. Clinically, KPNA3 and p-STAT3 protein levels were positively correlated in high-grade glioma tissues. Collectively, our findings unveil KPNA3 as a central regulator that concurrently enhances DNA repair and sustains the GSC population to foster TMZ resistance, nominating it as a promising therapeutic target for overcoming chemoresistance in GBM.

ORGANISM(S): Homo sapiens

PROVIDER: GSE327029 | GEO | 2026/07/17

REPOSITORIES: GEO

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Project description:Background. Glioblastoma multiforme (GBM) exhibits a cellular hierarchy with a subpopulation of stem-like cells known as glioblastoma stem cells (GSCs), that drive tumor growth and contribute to treatment resistance. NAD(H) emerged as a crucial factor influencing GSCs. Methods. A multistep process of machine learning algorithms was implemented to construct the glioma stemness-related score (GScore). Further in silico and patient tissue analyses validated the predictive ability of the GScore and identified a potential target, CYP3A5. Loss-of-function or gain-of-function genetic experiments were performed to assess the impact of CYP3A5 on the self-renewal and chemoresistance of GSCs both in vitro and in vivo. Mechanistic studies were conducted using nontargeted metabolomics, RNA-seq, seahorse, transmission electron microscopy, immunofluorescence, flow cytometry, ChIP‒qPCR, RT‒qPCR, western blotting, etc. The efficacy of pharmacological inhibitors of CYP3A5 was assessed in vivo. Results. Based on the proposed GScore, we identify a GSC target CYP3A5, which is highly expressed in GSCs and temozolomide (TMZ)-resistant GBM patients. This elevated expression of CYP3A5 is attributed to transcription factor STAT3 activated by EGFR signaling or TMZ treatment. Depletion of CYP3A5 impairs self-renewal and TMZ resistance in GSCs. Mechanistically, CYP3A5 maintains mitochondrial fitness to promote GSC metabolic adaption through the NAD⁺/NADH-SIRT1-PGC1α axis. Additionally, CYP3A5 enhances the activity of NAD-dependent enzyme PARP to augment DNA damage repair. Treatment with CYP3A5 inhibitor alone or together with TMZ effectively suppresses tumor growth in vivo. Conclusion. Together, this study suggests that GSCs activate STAT3 to upregulate CYP3A5 to fine-tune NAD⁺/NADH for the enhancement of mitochondrial functions and DNA damage repair, thereby fueling tumor proliferation and conferring TMZ resistance, respectively. Thus, CYP3A5 represents a promising target for GBM treatment.

Project description:Temozolomide (TMZ) is an important first-line treatment for glioblastoma (GBM), but there are limitations to TMZ response in terms of durability and dependence on the promoter methylation status of the DNA repair gene O6-methylguanine DNA methyltransferase (MGMT). MGMT-promoter-hypermethylated (MGMT-M) GBMs are more sensitive to TMZ than MGMT-promoter-hypomethylated (MGMT-UM) GBMs. Moreover, TMZ resistance is inevitable even in TMZ-sensitive MGMT-M GBMs. Hence, epigenetic reprogramming strategies are desperately needed in order to enhance TMZ response in both MGMT-M and MGMT-UM GBMs. In this study, we present novel evidence that the epigenetic reactivation of Tumor Suppressor Candidate 3 (TUSC3) can reprogram sensitivity of GBM stem cells (GSCs) to TMZ irrespective of MGMT promoter methylation status. Interrogation of TCGA patient GBM datasets confirmed TUSC3 promoter regulation of TUSC3 expression and also revealed a strong positive correlation between TUSC3 expression and GBM patient survival. Using a combination of loss-of-function, gain-of-function and rescue studies, we demonstrate that TUSC3 reactivation is associated with enhanced TMZ response in both MGMT-M and MGMT-UM GSCs. Further, we provide novel evidence that the demethylating agent 5-Azacitidine (5-Aza) reactivates TUSC3 expression in MGMT-M GSCs, whereas the combination of 5-Aza and MGMT inhibitor Lomeguatrib is necessary for TUSC3 reactivation in MGMT-UM GSCs. Lastly, we propose a pharmacological epigenetic reactivation strategy involving TUSC3 that leads to significantly prolonged survival in MGMT-M and MGMT-UM orthotopic GSCs models. Collectively, our findings provide a framework and rationale to further explore TUSC3-mediated epigenetic reprogramming strategies that could enhance TMZ sensitivity and outcomes in GBM. Mechanistic and translational evidence gained from such studies could contribute towards optimal design of impactful trials for MGMT-UM GBMs that currently do not have good treatment options.

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KPNA3 drives temozolomide resistance in glioblastoma by upregulating MGMT and activating STAT3 to sustain glioma stem cells

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