Identify therapeutic vulnerabilities in MYC-driven medulloblastoma

Medulloblastoma (MB) is the most common malignant pediatric brain tumor derived from transformed neuroepithelial stem (NES) cells. A subset of MB tumors with poor prognosis harbor amplification of the MYC family of oncogenes, particularly CMYC and MYCN. Because MYC proteins are structurally difficult to target directly, alternative therapeutic vulnerabilities must be identified.

Human induced pluripotent stem cells (iPSCs) derived from a healthy adult were differentiated into NES cells and transduced with empty vector, MYCN, or CMYC and implanted orthotopically into hindbrain of immunocompromised mice. Resulting tumors were grown in vitro and we performed a whole-genome CRISPR interference (CRISPRi) screen on both MYC tumor lines and control NES cells. For each line, mitochondria activity was assessed by live-cell staining for mitochondria with active potential and evaluated their sensitivity to inhibitors of nucleotide synthesis (Mizoribine, H3B-120, or 5-FU) in CyQuant Direct proliferation assays. Lactic acid was measured in each MYC tumor line and healthy NES cells after treatment with H3B-120 or the Complex I inhibitor (IACS-010759).

CRISPRi screening revealed both MYC-driven tumors rely on genes encoding mitochondrial electron transport chain (ETC) subunits, particularly Complex I. Live-cell staining confirmed higher mitochondrial activity in MYC tumors compared to controls. Since a clinical trial demonstrated that inhibition of Complex I results in elevated levels of lactic acid, causing neurotoxicity, we instead targeted downstream effects of Complex I, specifically its role in nucleotide synthesis. Both MYC tumors were more sensitive than healthy NES cells to inhibition of pyrimidine synthesis (H3B-120), but not purine (Mizoribine) or pyrimidine/purine (5-FU). While IACS-010759 increased lactic acid in both tumor and healthy cells, H3B-120 only increased lactic acid in the tumor lines. 

Pyrimidine synthesis represents a key vulnerability for MYC-driven MB and offers a potential therapeutic alternative to Complex I inhibition with reduced systemic toxicity.