Preclinical assessment of MAGMAS inhibitor as a potential therapy for pediatric medulloblastoma

Abstract

Medulloblastoma, the most common pediatric brain malignancy, has Sonic Hedgehog (SHH) and non-SHH group3 subtypes. MAGMAS (Mitochondrial Associated Granulocyte Macrophage colony-stimulating factor Signaling molecules) encode for mitochondrial import inner membrane translocase subunit and is responsible for translocation of matrix proteins across the inner membrane. We previously reported that a small molecule MAGMAS inhibitor, BT9, decreases cell proliferation, migration, and oxidative phosphorylation in adult glioblastoma cell lines. The aim of our study was to investigate whether the chemotherapeutic effect of BT9 can be extended to pediatric medulloblastoma.

Methods: Multiple in vitro assays were performed using human DAOY (SHH activated tp53 mutant) and D425 (non-SHH group 3) cells. The impact of BT9 on cellular growth, death, migration, invasion, and metabolic activity were quantified using MTT assay, TUNEL staining, scratch wound assay, Matrigel invasion chambers, and seahorse assay, respectively. Survival following 50mg/kg BT9 treatment was assessed in vivo in immunodeficient mice intracranially implanted with D425 cells.

Results: Compared to control, BT9 treatment led to a significant reduction in medulloblastoma cell growth (DAOY, 24hrs IC50: 3.6uM, 48hrs IC50: 2.3uM, 72hrs IC50: 2.1uM; D425 24hrs IC50: 3.4uM, 48hrs IC50: 2.2uM, 72hrs IC50: 2.1uM) and a significant increase in cell death (DAOY, 24hrs p=0.0004, 48hrs p<0.0001; D425, 24hrs p=0.0001, 48hrs p=0.02). In DAOY cells, 3uM BT9 delayed migration, and significantly decreased DAOY and D425 cells invasion (p < 0.0001). Our in vivo study, however, did not extend survival in xenograft mouse model of group3 medulloblastoma compared to vehicle-treated controls.

Conclusions: Our in vitro data showed BT9 antitumor efficacy in DAOY and D425 cell lines suggesting that BT9 may represent a promising targeted therapeutic in pediatric medulloblastoma. These data, however, need to be further validated in animal models.

Fig 1.

BT9 exhibits a dose- and time-dependent cytotoxic effect on medulloblastoma. DAOY (A) and D425 (B) were incubated for 24, 48, and 72 hours with increasing concentrations of BT9. Cell viability was measured by MTT assay. The relative numbers of proliferating cells compared with control are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig 2.

MAGMAS inhibition promotes medulloblastoma cell death. Apoptosis– based quantification of TUNEL-positive cells (dead cells) following BT9 treatment in DAOY (A, B, and E) and D425 (C, D, and F) cells after 24 and 48 hours (E and F). BT9 treatment significantly increases cell death in both cells (*p < 0.05, ***p < 0.001, ****p < 0.0001, BT9 vs vehicle control, n = 9). 10x magnification.

Fig 3.

MAGMAS inhibition reduces DAOY cells migration. Migration of DAOY cells treated with vehicle control (A-D) or 3 μM BT9 (E-H) was assessed using the scratch wound assay. Representative photographs were taken immediately after the scratch (T0) and 4 hours intervals (T4, 8, and 12). 4X magnification.

Fig 4.

Invasion of medulloblastoma cells treated with vehicle or BT9 (3μM) was assessed using inserts coated with Matrigel matrix. (A and B) Representative images show the invading DAOY cells (purple cells). (C) BT9 treatment significantly decreased the number of both DAOY and D425 cells invading through the Matrigel matrix (****p < 0.0001, BT9 vs vehicle control, n = 6). 4x magnification.

Fig 5.

MAGMAS inhibition impaired mitochondrial respiration in D425 cells. Oxygen consumption rate (OCR) profile of D425 treated with 2μM and 3μM BT9 for 48 hours. (A) The result showed a significant reduction in oxygen consumption of D425 cells treated with 3μM BT9. (B) Bar chart showing the results of mitochondrial respiration changes in BT9-treated cells, which were analyzed with basal respiration, ATP production, maximal respiration, and spare respiratory capacity. (*p < 0.05, **p < 0.01, 3μM BT9 vs control.

Fig 6.

BT9 does not change survival in an orthotopic xenograft model of group3 medulloblastoma. A: Experimental setup. Immunodeficient (SCID) mice were intracranially implanted with D425 cells and treated every other day with an intravenous injection of 50 mg/kg BT9 or vehicle control (Captisol). BT9 treatment started 7 days after implantation. B: Kaplan-Meier survival analysis for in vivo experiment. BT9 treatment did not lead to a statistically significant increase in median survival compared to vehicle-treated controls (27 days; n = 8 control vs n= 9 treated group).