Differentiated neuroblastoma cells remain epigenetically poised for de-differentiation to an immature state

Neuroblastoma is the most common extracranial solid tumor of childhood and accounts for a significant share of childhood cancer deaths. Prior studies utilizing RNA sequencing of bulk tumor populations showed two predominant cell states characterized by high and low expression of neuronal genes. Although cells respond to treatment by altering their gene expression, it is unclear whether this reflects shifting balances of distinct subpopulations or plasticity of individual cells. Using mouse and human neuroblastoma cell lines lacking MYCN amplification, we show that the antigen CD49b (also known as ITGA2) distinguishes these subpopulations. CD49b expression marked proliferative cells with an immature gene expression program, whereas CD49b-negative cells expressed differentiated neuronal marker genes and were non-cycling. Sorted populations spontaneously switched between CD49b expression states in culture, and CD49b-negative cells could generate rapidly growing, CD49b-positive tumors in mice. Although treatment with the chemotherapy drug doxorubicin selectively killed CD49b-positive cells in culture, the CD49b-positive population recovered when treatment was withdrawn. We profiled histone 3 (H3) lysine 27 acetylation (H3K27ac) to identify enhancers and super enhancers that were specifically active in each population and found that CD49b-negative cells maintained the priming H3 lysine 4 methylation (H3K4me1) mark at elements that were active in cells with high expression of CD49b. Improper maintenance of primed enhancer elements might thus underlie cellular plasticity in neuroblastoma, representing potential therapeutic targets for this lethal tumor.