Glioblastoma tedavisi için kinaz inhibitörleriyle kombine trifluoperazinin yeniden amaçlandırılması: ın vitro ve larval zebra balığı ın vivo modellerinden bulgular

Gliomas are classified into lower-grade gliomas and glioblastomas, with the latter representing the most aggressive and treatment-resistant form, highlighting the urgent need for more effective therapeutic strategies. Recently, the antipsychotic drug trifluoperazine emerged as a potent anticancer drug for multiple cancers, including gliomas. Our previous research identified a potent therapeutic combination of trifluoperazine with sorafenib, a well-established multi-kinase inhibitor with proven efficacy in hepatocellular carcinoma, in Hep3B cell line model. In the present study, the transcriptomic response to trifluoperazine alone or in combination with sorafenib was evaluated in U87-MG glioblastoma cells and revealed that the combinatorial treatment induced a synergistic effect characterized by growth arrest, reduced invasiveness, and transcriptional signatures of metabolic stress and cell fate reprogramming. While trifluoperazine alone promoted a proliferative gene expression profile through the upregulation of essential metabolic and cell cycle-associated genes, its combination with sorafenib counteracted these effects by suppressing oncogenic signaling and amplifying tumor-suppressive pathways. These findings highlight the potential of trifluoperazine as a repurposed agent in kinase-targeted glioblastoma therapy and underscore the benefit of combinatorial strategies to overcome adaptive resistance mechanisms. Although sorafenib treatment in glioblastoma cells provided mechanistic insights into its synergy with trifluoperazine, in vivo exposure to sorafenib caused severe morphological defects in zebrafish larvae over a 72-hour window from 2 to 5 dpf. Moreover, in a previous glial reporter imaging screen using Tg(gfap:GFP) line zebrafish larvae, sorafenib was observed to markedly reduce GFP fluorescence, whereas trifluoperazine alone did not produce any detectable adverse effects in either assay. This in vivo toxicity prompted the search for alternative targeted kinase inhibitors with more favourable safety profiles to combine with trifluoperazine. Other kinase inhibitors that had a growth inhibitory effect on U87-MG as well as A172 cells were identified using MTT assays. A screen of 157 kinase inhibitors resulted in 12 kinase inhibitors with IC50 values less than 5 μM for both cell lines. Among the most promising with full cytotoxic effect were volasertib, INK128, CAY10626, tamatinib, Torin1, bisindolylmaleimide IX, AZD7762, NH125, and BMS345541. Trifluoperazine was also combined with selected kinase inhibitors, and volasertib and INK128 were identified to elicit significant synergism. On the other hand, several kinase inhibitors with significant inhibitory effects alone exhibited significantly reversed impact by the addition of trifluoperazine. The PLK1 inhibitor volasertib was top prioritized due to its highest synergy score in combination with trifluoperazine, and RNA‑seq was performed on U87‑MG cells treated with 2.5 μM volasertib to investigate its translational relevance. The resulting drug‑induced gene signature was evaluated over the TCGA‑GBM cohort, where high volasertib signature scores correlated with significantly improved overall survival, thereby reinforcing the rationale for its repurposing approach in combination with trifluoperazine for glioblastoma therapy. Finally, the early larval toxicity profiles of prioritized single or combinatorial drug treatments were assessed in vivo, and neither induced developmental abnormalities or phenotypic signs of adverse effects. In this context, LDexplore, an R Shiny application was developed to enable multidimensional analysis of zebrafish larval locomotor behaviour under dark:light alternation, incorporating heatmaps and statistical analyses of velocity and acceleration in response to startle stimuli. The app was used to test whether the zebrafish larvae exposed to trifluoperazine alone or in combination with volasertib exhibited different locomotory behaviour and it was found that although trifluoperazine induced a heightened swimming pattern upon stimulus at higher concentrations, the combinatorial treatment was not significantly disruptive to light induced startle response. These findings indicate that the combination of trifluoperazine and volasertib exhibits strong therapeutic potential, with in vivo applicability supported by larval zebrafish assays. Additionally, other kinase inhibitors demonstrating lower toxicity and significant synergistic effects on glioblastoma cell growth were identified, providing insights for further investigation. Keywords: Glioblastoma, Cancer, Drug Screening, Drug Repurposing, Larval Zebrafish Model, Trifluoperazine, Volasertib.