EPEN-02. A gene regulatory network comprising clusterin and VEGFA drives group A posterior fossa ependymoma cell state transitions

Group A posterior fossa ependymoma (PFA) is a deadly cancer with few effective treatment options. Single cell profiling of PFA has identified several distinct cell states within the tumor. However, the relationship of these cell states, including their hierarchical organization and associated gene regulatory networks is not known. To more fully understand the nature of cell states within PFA, we leveraged cutting-edge analytical techniques of single cell RNA data including RNA velocity, vector field analysis, dynamical systems theory, and differential geometry to predict likely cell state transitions within PFA and infer underlying gene regulatory networks. This analysis showed that PFA tumors recapitulate classic cellular developmental and differentiation hierarchies. Specifically, a small population of relatively quiescent stem-like cells enter the cell cycle and give rise to a larger population of progenitor cells with high velocity (rapidly changing cell state) and two differentiated “ground” states. Analysis of the vector field showed that the transition from this stem-like population to more differentiated cell states is relatively unidirectional. Using machine learning to calculate the Jacobian (underlying gene regulatory relationships) nominated hypoxia and cell-injury related genes as the primary regulators of cell state transitions. Specifically, CLU and VEGFA were among the genes with the highest calculated Jacobian (regulatory effects). These genes exhibited mutual inhibition, particularly in the stem-like population, which exhibited high CLU expression and low VEGFA expression. In silico upregulation of CLU predicted cell de-differentiation toward the stem-like population. On the other hand, in silico upregulation of VEGFA predicted transition away from the stem-like state. Taken together, these data suggest that CLU is the central regulator of a hypoxia-associated gene network that maintains a stem-like population of PFA cells, and that perturbations of this gene network may hold the key to inducing therapeutic differentiation.