TLK1 as a therapeutic target in TMZ resistant glioblastoma using small molecule inhibitor

Abstract

The acquired resistance to existing therapies poses a grave concern in achieving successful therapeutic outcomes. Temozolomide (TMZ), a widely used alkylating chemotherapeutic in Glioblastoma therapy, often encounters resistance, necessitating the investigation of the underlying mechanisms of TMZ-acquired resistance. To study TMZ resistance, a cell-based model system was generated by intermittently exposing glioblastoma cells to increasing concentrations and time of TMZ over six months. The survival response of cells at higher concentrations confirmed TMZ-resistant cells, which exhibited a phenotypic shift toward a mesenchymal-like state, with decreased epithelial traits, indicating mesenchymal-epithelial transition (MET). This transition likely facilitates the stabilization and clonal growth of TMZ-resistant cells. Subsequent analysis revealed elevated expression of TLK1, a DNA repair protein, thus reinforcing its potential involvement in mechanisms associated with acquired resistance. To explore the therapeutic aspect of TLK1 inhibition, we utilized an in-house developed TLK1 inhibitor, J54. The inhibition of TLK1 in TMZ-resistant cells enhanced cytotoxicity, indicating TLK1 as a potential target to combat TMZ resistance. Moreover, TLK1 inhibition reduced cell migration and invasion, implying its role in promoting metastasis. In conclusion, our study sheds light on the role of TLK1 in the context of TMZ resistance, highlighting its potential as a valuable target for therapeutic intervention.

Keywords: Blood-brain-barrier; DNA damage response; Glioblastoma; Phenothiazine; Temozolomide; Tousled-like kinase-1.

Fig. 1

(a) Overview of generation of TMZ-resistant GBM Cells in this study. LN229 and LN18 cells were intermittently treated with increasing concentrations of TMZ (50–200 µM). (b) A clonogenic assay was performed to assess if the LN229 cells grown intermittently in an increasing TMZ media have acquired resistance, LN18 cells were used as control. (C) Quantification of colony formation assay.

Fig. 2

(a) Morphology of LN229 and LN229-TMZ-R cells observed under confocal and bright-field microscope. Cells are stained with DAPI and β-catenine. Quantification of cell length shows a two-fold increase in cell length. (b) Immunoblot images and quantification of E-M markers show an increase in epithelial markers and a decrease in mesenchymal markers. Statistical significance was determined by student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.0001 compared to the control group (n = 3, independent replicates).

Fig. 3

(a) Western blot analysis shows an increase in the level of TLK1 and its splice variant TLK1b along with p-Nek1 in LN229 TMZ-R compared to LN229 TMZ-S cells. Statistical significance was determined by student’s t-test, *p < 0.05, ***p < 0.001, compared to the control group (n = 2, independent replicates). (b) LN229 TMZ-R cell showing a decrease in cell viability in response to J54. On the other hand, no significant cytotoxicity was observed against TMZ, even at 100µM TMZ concentration. Statistical significance was determined by student’s t-test, **p < 0.01 compared to the control group (n = 3, independent replicates). (c) Representative images of cell colonies formed in untreated control and cells treated with increasing concentrations of J54 compared. Statistical significance was determined one-way ANOVA, ****p < 0.0001, compared to the untreated control (n = 3, independent replicates).

Fig. 4

(a) Western blot analysis demonstrating a dose-dependent decrease in the phosphorylation levels of TLK1 substrate, namely, NEK1 (p-NEK1) and RAD9 (p-RAD9) in response to J54 treatment. Statistical significance was determined by one-way ANOVA, **p < 0.01, ***p < 0.001, compared to the untreated control (n = 3, independent replicates). (b) Immunocytochemistry analysis demonstrating an increase in the γH2AX levels in response to J54. Statistical significance was determined one-way ANOVA, **p < 0.01, compared to the untreated control (n = 3, independent replicates).

Fig. 5

Representative images of live and dead staining using propidium iodide (PI) and Hoechst stain in untreated control cells and J54 treated cells. Cells treated with J54 demonstrate increased cell death as evidenced by the presence of red-stained dead cells (PI positive), which are significantly lower in the untreated control. Quantification of cell death reveals a significant increase in the percentage of dead cells in response to J54 treatment compared to the untreated control. Statistical significance was determined by Student’s t-test, ***p < 0.001, compared to the control group (n = 3, independent replicates).

Fig. 6

(a) The Figure shows the results of the wound healing assay performed on cell monolayers treated with J54. Representative images at t = 0 and t = 24 h reveal the inhibition of cell migration by J54. The wound gap closure is delayed in the presence of the J54, indicating its anti-migratory effect. (b) Transwell Invasion Assay demonstrating the anti-invasive property of J54. The upper chamber was coated with a geltrex, and cells treated with J54 were placed inside. J54 exhibited a notable reduction in cell invasion through the geltrex, as indicated by the reduced number of invaded cells in the lower chamber. Statistical significance was determined by student’s t-test, **p < 0.01, ***p < 0.001, compared to the control group (n = 3, independent replicates).