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

Abstract

Medulloblastoma is the most common malignant brain tumor in children. It has WNT-driven, SHH-driven/TP53 mutant, SHH-driven/TP53 wildtype, and non-WNT/non-SHH subgroups. MAGMAS (Mitochondrial Associated Granulocyte Macrophage colony-stimulating factor Signaling molecules) encodes a mitochondrial import inner membrane translocase subunit and is responsible for the 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: DAOY (SHH driven/tp53 mutant) and D425 (non-SHH group 3) were treated with BT9. For in vitro analysis, cell proliferation, death, migration, invasion, and metabolic activity were assessed using MTT assay, TUNEL staining, scratch wound assay, Matrigel invasion chambers, and seahorse assay, respectively. A D425 orthotopic xenograft mouse model was used to evaluate BT9 efficacy in vivo.

Results: BT9 treatment resulted in a significant decrease in cell proliferation (DAOY, 24 hours IC50: 3.6 μM, 48 hours IC50: 2.3 μM, 72 hours IC50: 2.1 μM; D425 24 hours IC50: 3.4 μM, 48 hours IC50: 2.2 μM, 72 hours IC50: 2.1 μM) and a significant increase in cell death (DAOY, 24 hours p = 0.0004, 48 hours p<0.0001; D425, 24 hours p = 0.0001, 48 hours p = 0.02). In DAOY cells, 3 μM BT9 delayed migration and significantly reduced DAOY and D425 cell invasion (p < 0.0001). It also modified mitochondrial respiratory function in both medulloblastoma cell lines. Compared to control, however, BT9 administration did not improve survival in a D425 orthotopic xenograft mouse model.

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 in each condition were compared to control and are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig 2. MAGMAS inhibition induces medulloblastoma cell death.

Representative images show DAOY (A and B) and D425 (C and D) cells treated with vehicle control (A and C) or BT9 (B and D). Quantification of TUNEL-positive DAOY (E) or D425 (F) cells after 24 and 48 hours of BT9 treatment compared to control (*p < 0.05, ***p < 0.001, ****p < 0.0001, n = 9). 10x magnification.

Fig 3. MAGMAS inhibition reduces DAOY cells migration.

The scratch wound assay was used to compare the effect of control (A-D) or 3 μM BT9 (E-H) on DAOY cell migration. Representative photographs were taken at T0, T4, T8, and T12 after the scratch. 4X magnification.

Fig 4. BT9 reduces DAOY and D425 medulloblastoma cell invasion.

(A and B) Representative images show the invading DAOY cells (purple cells) in vehicle (A) or 3μM BT9-treated (B) cells. (C) Compared to control, BT9 treatment significantly decreased the number of invading DAOY and D425 cells (****p < 0.0001, BT9 vs vehicle control, n = 6). 4x magnification.

Fig 5. The effect of MAGMAS inhibition on mitochondrial respiration and acidification rate in D425 cells.

(A) Oxygen consumption rate (OCR) profile of D425 treated with 2 and 3 μM BT9 for 24 hours. The result showed a decline 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. (C) Extracellular acidification rate (ECAR) profile of D425 treated with 2 and 3μM BT9 for 24 hours (**p < 0.01, 3μM BT9 vs control (CNTR).

Fig 6. BT9 did not improve survival in the orthotopic xenograft model of D425 medulloblastoma.

(A) Experimental design. Immunodeficient (SCID) mice were surgically cannulated in the jugular vein. One week after, they were intracranially implanted with D425 cells. Seven days later, each animal started to receive intravenous injections of 50 mg/kg BT9 or vehicle control (Captisol) every 48 hours until they were euthanized. (B) Kaplan-Meier survival analysis. BT9 treatment did not increase survival in experimental group compared to vehicle-treated controls (27 days; n = 8 control vs. n = 9 treated group).