Combining the constitutive TRAIL-secreting induced neural stem cell therapy with the novel anti-cancer drug TR-107 in glioblastoma

Abstract

Tumor-homing neural stem cell (NSC) therapy is emerging as a promising treatment for aggressive cancers of the brain. Despite their success, developing tumor-homing NSC therapy therapies that maintain durable tumor suppression remains a challenge. Herein, we report a synergistic combination regimen where the novel small molecule TR-107 augments NSC-tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) therapy (hiNeuroS-TRAIL) in models of the incurable brain cancer glioblastoma (GBM) in vitro. We report that the combination of hiNeuroS-TRAIL and TR-107 synergistically upregulated caspase markers and restored sensitivity to the intrinsic apoptotic pathway by significantly downregulating inhibitory pathways associated with chemoresistance and radioresistance in the TRAIL-resistant LN229 cell line. This combination also showed robust tumor suppression and enhanced survival of mice bearing human xenografts of both solid and invasive GBMs. These findings elucidate a novel combination regimen and suggest that the combination of these clinically relevant agents may represent a new therapeutic option with increased efficacy for patients with GBM.

Keywords: MT: Regular Issue; TNF-related apoptosis inducing ligand; TR-107; TRAIL; TRAIL resistance; apoptosis; glioblastoma.

Graphical abstract

Figure 1

Combining TR-107 and hiNeuroS-TRAIL demonstrates synergistic tumor growth suppression in various GBM cell lines (A–C) TR-107 dose-response curve in (A) LN229 (B) MS21 and (C) GBM8 (log scale) (n = 6). (D–F) hiNeuroS-TRAIL dose-response curve in (D) LN229 (E) MS21 and (F) GBM8 (log scale) (n = 6). (G–I) Combining IC₅₀ doses of both drugs demonstrate synergistic inhibition of tumor growth in (G) LN229 (n = 18) (H) MS21 (n = 6) and (I) GBM8 (log scale) (n = 12–18 for all cell lines). The data are represented as mean ± SEM (∗∗∗∗p < 0.0001).

Figure 2

TR-107 treatment activates caspase-mediated apoptotic signaling pathway in TRAIL-sensitive cell line, GBM8 (A) GBM8 tumor growth curve in response to TR-107 treatments (n = 12). (B) Extrinsic-mediated caspase 8 regulation (n = 6–8). (C) Intrinsic-mediated caspase 9 regulation (n = 6–8). (D) Extrinsic and intrinsic-mediated caspase 3/7 regulation (n = 5–6). The data are represented as mean ± SEM (∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns = not significant).

Figure 3

Combining TR-107 with hiNeuroS-TRAIL significantly upregulated caspase markers in TRAIL-resistant LN229 in vitro (A) The 24- and 48-h synergistic tumor killings with CDI values of 0.89 and 0.57, respectively in LN229 cell line (n = 7–12). (B) Extrinsic-mediated caspase 8 regulation from 24 to 48 h in response to single agents and combination treatments (n = 6–7). (C) Caspase 9 regulation from 24 to 48 h in response to single agents and combination treatments (n = 6–7). (D) Extrinsic and intrinsic-mediated caspase 3/7 regulation from 24 to 48 h in response to single agents and combination treatments (n = 6–7). The data are represented as mean ± SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns = not significant).

Figure 4

Combination treatment alters protein expression in the apoptotic pathways favoring the pro-apoptotic pattern in TRAIL-resistant LN229 cell line (A) A schematic for intrinsic and extrinsic mediated apoptotic pathways. Molecular markers highlighted in red are differentially regulated in the experiment in response to treatments. (B–F) Proteomic differential expressions for (B) cleaved caspase 3 (C) p53 phosphorylation at S15 site (D) p53 phosphorylation at S46 site (E) survivin and (F) XIAP (n = 4–6). The data are represented as mean ± SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns = not significant).

Figure 5

Single agent dose optimizations in solid LN229 GBM model in mice (A–C) Individual mouse tumor growth curves across all control and TR-107 treatment groups. (D and E) (D) Fold change in tumor growth curve and (E) survival outcome across all TR-107 treatment groups (n = 4). (F and G) Individual mouse tumor growth curves across all control and TR-107 treatment groups. (H and I) (H) Fold change in tumor growth curve and (I) survival outcome between control and hiNeuroS-TRAIL treatment groups (n = 3). The data are represented as mean ± SEM (∗p < 0.05).

Figure 6

Combining TR-107 and hiNeuroS-TRAIL significantly reduced tumor burden and improved survival outcome in a solid murine model of GBM (A–D) Individual mouse tumor growth data across all treatment groups from LN229 in vivo efficacy experiment. (E) Fold change in tumor growth curves across all treatment groups indicating that the combination therapy significantly reduced tumor burdens compared with the control group. (F) Kaplan-Meier survival curve of mice treated with control, TR-107, hiNeuroS-TRAIL, or combination at indicated doses (n = 7). The data are represented as mean ± SEM (∗p < 0.05, ∗∗p < 0.01, ns = not significant).

Figure 7

Combining TR-107 and hiNeuroS-TRAIL significantly improved survival outcome in an invasive murine model of GBM (A–D) Individual mouse tumor growth data across all treatment groups from GBM8 in vivo efficacy experiment. (E) Fold change in tumor growth curves across all treatment groups. (F) Kaplan-Meier survival curve of mice treated with control, TR-107, hiNeuroS-TRAIL, or combination at indicated doses (n = 6–7). The data are represented as mean ± SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns = not significant).