A human neural stem cell model of MYC-driven medulloblastoma reveals glutamine metabolism as a potential therapeutic target
Allison Hanaford, Eric Raabe, Charles Eberhart
Johns Hopkins University School of Medicine, Baltimore, MD, USA
Medulloblastoma comprises four subgroups with different associated mutations and clinical prognoses. Group 3 tumors have increased MYC levels and the worst clinical prognosis. MYC over-expression in cancer can lead to increased glutamine metabolism and glutamine addiction. We hypothesized that human neural stem cells derived from the developing cerebellum transduced with MYC and cooperating oncogenes would create a genetically accurate model of group 3 medulloblastoma for pre-clinical testing. We further hypothesized that MYC-driven medulloblastoma would exhibit increased glutamine metabolism and be sensitive to inhibitors of glutamine metabolism.
We transduced cerebellar derived human neural stem cells with lentivirus coding for c-MYC, dominant negative R248W p53, constitutively active AKT, and hTERT. We also created neurospheres expressing subsets of these constructs as controls. We measured proliferation in vitro with BrdU and MTS assays and generated orthotopic xenografts using standard techniques. Tumors were processed for histology and stained by immunohistochemistry for markers of neuronal and glial differentiation. After treatment with the glutamine anti-metabolite (2S)-Amino[(5S)-3-chloro-4,5-dihydro-1,2-oxazol-5-yl]ethanoic acid (acivicin), we determined apoptosis by flow cytometry (cell cycle and annexin staining) and cleaved caspase 3 immunofluorescence. We determined glutaminase expression by western blotting.
R248Wp53/MYC/AKT/hTERT transformed cerebellar stem cells formed aggressive orthotopic xenograft tumors with large cell/anaplastic histology and spinal metastases. Control MYC-alone and R248Wp53/MYC/hTERT transduced cells formed tumors with increased latency and decreased penetrance. MYC expression positively correlated with increased expression of glutaminase. Acivicin decreased cell proliferation in MYC-containing neurospheres compared to those without MYC (p=0.012). Acivicin treatment of MYC expressing cells led to a 65% increase in apoptosis compared to MYC-negative cells. Forced MYC expression in the low-MYC UW228 medulloblastoma cell line also led to increased glutaminase expression and increased sensitivity to acivicin (p=0.03).
Human cerebellar stem cells can be transformed by c-MYC and cooperating oncogenes, resulting in a models that recapitulates group 3 medulloblastoma histologically and clinically. These cells and classical high-MYC medulloblastoma cell lines are sensitive to glutamine anti-metabolites, while non-MYC driven medulloblastoma and SV40 immortalized human cerebellar stem cells are resistant. Our group 3 human neural stem cell medulloblastoma model data suggest that glutamine metabolism may be a therapeutic target in MYC-driven medulloblastoma, and that glutamine anti-metabolites may be useful clinical agents.