Abstract 4781: Epigenetic regulation of neuroblastoma tumorigenicity through MLL1 and JMJD3 modulation in cancer stem cells

Abstract

Abstract Introduction: Increasing evidence suggest that epigenetic regulators play a critical role in cancer cell heterogeneity and in maintaining tumor sub-populations with enhanced tumor-initiating and drug-resistant capacities. Recently, we discovered a drug-resistant, highly tumorigenic, self-renewing cell sub-population with features of cancer stem cells (CSCs) in pediatric neuroblastoma (NB). This sub-population, characterized by surface expression of G-CSF receptor (CD114), can escape initial therapy and cause refractory and aggressively invasive relapsed disease. Recently, we found that the CSF3R gene that code for CD114 is expressed specifically in CD114+ sub-population (<1% of total cells) but not in CD114- bulk tumor cells. We hypothesize that this differential gene expression is epigenetically regulated and maintain heterogeneous NB sub-populations. Methods: Chromatin immunoprecipitation (ChIP)-qPCR for the histone marks H3K4me3 and H3K27me3 was performed in sorted NB sub-populations. Pathway qPCR arrays and orthotopic xenografts mouse model were used to determine the effects of epigenetic inhibitors in vitro and in vivo. Results: We found that the epigenetic regulators mixed-lineage leukemia-1 (MLL1; KMT2A; a H3K4me3 methyltransferase) and Jumonji D3 (JMJD3; KDM6B; a H3K27me3 demethylase) are overexpressed in CD114+ NB cells. These regulators maintain a reversible epigenetic switch of high H3K4me3 and low H3K27me3 histone marks at the CSF3R locus in CD114+ cells for active transcription. Targeting these epigenetic regulators with specific small-molecule inhibitors MM-102 (inhibit MLL1) and GSK-J4 (inhibit JMJD3) in NB, reverses the histone patterns at CSF3R promoter locus and block gene expression, induces apoptosis selectively in CD114+ cells, and inhibits overall NB proliferation in vitro. These inhibitors also inhibit the expression of cancer stem cell specific genes as determined by pathway arrays. Inhibiting MLL1 and JMJD3 in NB orthotopic xenografts significantly decreases tumor size (p<0.001) and metastatic burden (p<0.001) compared to controls. As expected, reduction in tumor size was significantly correlated with the reduction in tumor CD114+ cells. We also found that MM-102 and GSK-J4 sensitize NB tumors to genotoxic chemotherapy etoposide and further reduce tumor growth (p<0.01) in comparison to either drug alone. Interestingly, we do not observe any metastatic incidences in mice treated with MM-102+etoposide. Conclusion: Overall, we demonstrate that epigenetic regulators MLL1 and JMJD3 regulate the expression of critical genes and maintain NB CSC sub-population. Targeting these epigenetic regulators reduce NB tumorigenicity, metastasis and increase drug sensitivity. Further developing these strategies will pave the way for incorporating epigenetic inhibitors in current therapies for effectivity improving long-term cure rates. Citation Format: Saurabh Agarwal, Zaowen Chen, Julie A. Tomolonis, Sanjeev A. Vasudevan, Jason M. Shohet. Epigenetic regulation of neuroblastoma tumorigenicity through MLL1 and JMJD3 modulation in cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4781. doi:10.1158/1538-7445.AM2017-4781