It is still unclear what drives progression of childhood tumors. During Drosophila larval development, asymmetrically-dividing neural stem cells, called neuroblasts, progress through an intrinsic temporal patterning program that ensures cessation of divisions before adulthood. We previously showed that temporal patterning also delineates an early developmental window during which neuroblasts are susceptible to tumor initiation (Narbonne-Reveau et al., 2016). Using single-cell transcriptomics, clonal analysis and numerical modeling, we now identify a network of twenty larval temporal patterning genes that are redeployed within neuroblast tumors to trigger a robust hierarchical division scheme that perpetuates growth while inducing predictable cell heterogeneity. Along the hierarchy, temporal patterning genes define a differentiation trajectory that regulates glucose metabolism genes to determine the proliferative properties of tumor cells. Thus, partial redeployment of the temporal patterning program encoded in the cell of origin may govern the hierarchy, heterogeneity and growth properties of neural tumors with a developmental origin.

The mechanisms that govern the hierarchical organization of tumors are still poorly understood, especially in highly heterogeneous neural cancers. Previously, we had shown that aggressive neural 15 tumors can be induced upon dedifferentiation of susceptible intermediate progenitors produced during early development (Narbonne-Reveau et al., 2016). Using clonal analysis, stochastic modelling and single-cell transcriptomics, we now find that such tumors rapidly become heterogeneous, containing progenitors with different proliferative potentials. We demonstrate that tumor heterogeneity emerges from the deregulated transition between two antagonistic RNA-binding proteins, Imp and Syncrip, that switch 20 neural progenitors from a default self-renewing to a differentiation-prone state during development.Consequently, aberrant maintenance of Imp confers a cancer stem cell-like identity as Imp + progenitors sustain tumor growth while being able to continuously generate Syncrip + progenitors. The latter exhibit limited self-renewal likely due to Syncrip-mediated metabolic exhaustion. This study provides an example of how a subverted developmental transition establishes a hierarchical tumor. 25

COMPASS and Polycomb complexes are antagonistic chromatin complexes that are frequently inactivated in cancers, but how these events affect the cellular hierarchy, composition, and growth of tumors is unclear. These characteristics can be systematically investigated in Drosophila neuroblast tumors in which cooption of temporal patterning induces a developmental hierarchy that confers cancer stem cell (CSC) properties to a subset of neuroblasts retaining an early larval temporal identity. Here, using single-cell transcriptomics, we reveal that the trithorax/MLL1/2-COMPASS–like complex guides the developmental trajectory at the top of the tumor hierarchy. Consequently, trithorax knockdown drives larval-to-embryonic temporal reversion and the marked expansion of CSCs that remain locked in a spectrum of early temporal states. Unexpectedly, this phenotype is amplified by concomitant inactivation of Polycomb repressive complex 2 genes, unleashing tumor growth. This study illustrates how inactivation of specific COMPASS and Polycomb complexes cooperates to impair tumor hierarchies, inducing CSC plasticity, heterogeneity, and expansion.

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