Abstract 3941: CRISPR screen of human stem cell derived MYC-driven medulloblastoma tumors reveal the mitochondria as a vulnerability

Abstract Background: Medulloblastoma (MB) is the most common malignant pediatric brain tumor and is derived from neuroepithelial stem cells (NESC). Amplification of each member of the MYC family of oncogenes (MYCN, CMYC, MYCL) has been found in MB tumors and amplified-MYCN or CMYC correlates with poor prognosis. Since transcription factors such as MYC are difficult to drug with small molecules, more druggable targets need to be identified. While genetic screens can be performed on human MB cell lines, there is a lack of a proper isogenic control to distinguish targets that also negatively affect healthy non-tumor cells. Here, we show the utility of a human induced pluripotent stem cell (iPSC) based model of MB to find therapeutic vulnerabilities. Methods: Human iPSC derived from a healthy adult were differentiated towards NESC and transduced with empty vector (EV), MYCN, CMYC or MYCL. We analyzed how each MYC protein influences the molecular profile (RNAseq and methylation), proliferation in vitro (EdU assay), and transformation in vivo (orthotopic implantation into the hindbrain of immunocompromised mice) compared to EV in NESC. Whole genome CRISPR interference (CRISPRi) screen was performed on MYCN and CMYC NESC-derived tumors and EV NESC. Viability in response to 72 hours of inhibitor treatment was analyzed qualitatively by transmitted light images of cells and quantitatively by LIVE/DEAD stain followed by flow cytometry. Results: MYCN and CMYC induced a higher percentage of NESC in S-phase (46%, 40%) compared to MYCL (35%) and EV (31%). Molecularly, MYCN and CMYC NESC had similar transcriptomic and methylation profiles compared to MYCL and EV. Gene ontology revealed the most enriched pathway in NESC with either MYCN or CMYC, but not MYCL, was mitochondria gene expression. In vivo, MYCN and CMYC were both 100% penetrant by day 98 while MYCL was only 30% penetrant and EV did not develop tumors. CRISPRi screen demonstrated MYCN and CMYC NESC-derived tumors, but not EV NESC, were dependent on subunits of Complex I, III, IV and V of the electron transport chain (ETC) in the mitochondria. In support of this, treatment with two different inhibitors of Complex I (Metformin, Mito-HNK) showed stronger toxicity against both MYC NESC-derived tumors than EV NESC. Strikingly, treatment with Mito-HNK resulted in higher cell death in CMYC-expressing MB cell lines D283 (30%), D425 (83%) and MB004 (82%) compared to healthy NESC (<10%). Conclusion: MYCN and CMYC induce molecular changes distinct from MYCL in NESC that lead to upregulation of mitochondria gene expression, faster growth and transformation. Both MYCN and CMYC driven MB tumors are more dependent on the ETC for viability than non-tumor NESC suggesting MYC tumors could be specifically targeted by disrupting their metabolism. Thus, we show the combination of human iPSC-based model of MB and CRISPR screening can uncover vulnerabilities in MB cell lines. Citation Format: Kenya Pleitez Valle, Miriam Velez, Yang Zhao, Bo Cheng, Sophia Estrada, Linwei Li, Tom O'Loughlin, Balaraman Kalyanaraman, Ming You, Luke A. Gilbert, David M. Hockenbery, David B. Shackelford, Peter J. Mullen, Miller Huang. CRISPR screen of human stem cell derived MYC-driven medulloblastoma tumors reveal the mitochondria as a vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3941.