ATRT-10. SINGLE-CELL TRANSCRIPTOME ANALYSIS REVEALS CELLULAR HIERARCHIES AND THERAPEUTIC VULNERABILITIES OF ETMR

Embryonal tumors with multilayered rosettes (ETMR) are malignant brain tumors that occur predominantly in infants and young children. Most patients die within two years of diagnosis, and more effective, targeted therapies are urgently needed. To better characterize the oncogenic mechanisms of key driver alterations and to identify novel therapeutic targets, we set out to study the cellular heterogeneity of ETMR using single-cell RNA sequencing.

Analyses conducted on >4,000 high-quality cells collected from eleven primary and relapse specimens revealed a common cellular hierarchy across all tumors: A highly proliferative neural stem cell-like population (SOX2+) that gives rise to intermediate progenitors (NEUROD1/NEUROG1+) and more differentiated neuron-like cells (STMN2/4+). These malignant cell populations closely overlap with histological patterns of ETMRs, as confirmed by multiplexed immunofluorescence microscopy on patients’ tumors.

Comparison to single-cell datasets from human fetuses indicated high resemblance to normal cortical neurogenesis but also revealed key tumor-specific differences. These include expression of the chromosome 19 miRNA cluster (C19MC, the presumed driver in ~90% of ETMRs), which was restricted to the malignant stem cell-like population. Investigating oncogenic mechanisms of C19MC (comprising 46 miRNA genes) through transcriptome-wide RNA immunoprecipitation analysis, we identified extensive target gene regulation for most C19MC members, including distinct regulators of cell cycle, pluripotency, and neuronal differentiation. Silencing of C19MC families with antisense oligonucleotides resulted in pronounced reduction of ETMR cell line growth, indicating potential avenues for therapeutic targeting in the future.

To identify more immediately actionable targets, we investigated inter-cellular signaling between malignant cell populations of ETMRs. Interestingly, we identified marked FGFR and NOTCH receptor-ligand interactions common to all tumors. An in vitro screen of experimental and approved small molecule inhibitors designed to target these interactions nominated several promising candidates for clinical evaluation. Our unpublished results provide much needed insight into targeting ETMR cellular states using multiple modes of action.