Neuroblastoma displays marked intratumoral heterogeneity and is composed of multiple subpopulations including the G-CSF receptor expressing (CD114+) cancer stem cells (CSC). These highly tumorigenic G-CSF responsive cells are in part distinguished by stem-cell like epigenetic alterations at microRNA and other oncogenic loci.
Since CSCs are well-known to be regulated by histone modifications, we evaluated the epigenetic regulation of the G-CSF receptor encoded by the CSF3R gene. In addition to elevated CSF3R expression, we found high levels of MLL1 (encoded by KMT2A), and Jumonji-D3 (JMJD3, encoded by KDM6B) in CD114+ NB CSCs. MLL1 is an H3K4 histone methyltransferase that catalyzes H3K4me1, H3K4me2 and H3k4me3 methylations, while JMJD3 is an H3K27me3 demethylase. Chromatin immunoprecipitation (ChIP)-qPCR analysis for H3K4me3 and H3K27me3 epigenetic marks at the CSF3R locus revealed increased activating H3K4me3 and reduced repressing H3K27me3 marks in CD114+ cells consistent with increased CD114 surface expression. In CD114- bulk tumor cells, we found opposite pattern of increased H3K27me3 and reduced H3K4me3 marks.
We hypothesized that inhibition of these epigenetic regulators would block CSF3R expression and inhibit CSC-mediated tumor growth and metastasis. Therefore, we evaluated the in vivo efficacy of MM-102 (MLL1/WDR5 inhibitor) and GSK-J4 (JMJD3 inhibitor) against NB xenografts. Remarkably, NB CD114+ cells proved highly sensitive to both compounds, leading to rapid apoptosis of CD114+ subpopulations and minimal death of CD114- cells. Apoptosis of CD114+ cells strongly and significantly correlated with decreased tumor size (p<0.001) and metastatic burden (p<0.001). Furthermore, exogenous G-CSF partially reversed these effects, and increased CD114+ percentages in resected tumors (p<0.01).
Overall, we demonstrate that specific histone modifications are essential for survival of the NB CSCs, and specific epigenetic inhibitors can be highly potent anti-tumor agents for high-risk neuroblastoma. Our findings have direct translational impact and support the development of epigenetic stem cell targeted therapies for neuroblastoma.