Abstract: Cancer cell heterogeneity and the existence of phenotypically distinct subpopulations of cells with enhanced tumor-initiating and drug-resistant capacities is a major challenge in cancer treatment. These drug-resistant cells are known to drive cancer relapse, which is a major cause of therapy failure and deaths from high-risk cancers, such as pediatric neuroblastoma (NB). Deregulated differential expression of genes due to epigenetic machinery malfunction is the primary cause of cancer cell heterogeneity. Determining the role of specific epigenetic modifiers in maintaining tumor-initiating cancer stem cell sub-populations and developing targeted therapies to block these modifiers is mandatory for effectively battling cancer and its relapse. Recently, we discovered a drug-resistant, highly tumorigenic, metastatic, and self-renewing cell sub-population with features of tumor-initiating cells (TICs) in neuroblastoma. This sub-population, characterized by surface expression of the G-CSF receptor (CD114), can escape initial therapy and cause refractory and aggressively invasive relapsed disease. These TICs can differentiate to bulk tumor cells (CD114-) and produce complex neuroblastoma tumors with as few as 10 cells in vivo. Our recent preliminary data show that the CSF3R gene that codes for CD114 is expressed specifically in CD114+ NB TICs but not in CD114- bulk tumor cells. We hypothesize that this differential gene expression is epigenetically regulated and supports the maintenance of heterogeneous NB TICs. We also found that the epigenetic regulator mixed-lineage leukemia-1 (MLL1; an H3K4me3 methyltransferase) is overexpressed in CD114+ cells and regulates the expression of the TIC specific genes including CSF3R. MLL1 forms a protein complex with WDR5, and Menin to act as an active histone methyltransferase epigenetic regulator. Our preliminary data show that targeting MLL1-WDR5 and MLL1-Menin interactions using specific small molecule inhibitors inhibit NB TICs and in vivo NB growth and metastasis. These data support our hypothesis that epigenetic regulators maintain NB TICs and point towards a novel therapeutic strategy of blocking MLL1 activation to inhibit NB TIC-mediated tumorigenicity, metastasis, and disease relapse. In the proposed research, we will determine: a) the role of MLL1 in maintaining NB TIC subpopulation, b) the effects of blocking epigenetic regulators via specific small molecule inhibitors on NB tumorigenesis and metastasis in vivo, c) the effects of concomitantly targeting both NB TICs and bulk tumor cells by combining epigenetic inhibitors with standard chemotherapy drugs, and d) further developing novel epigenetic inhibitors to target MLL1 epigenetic functions. As specific small molecule inhibitors of MLL1, WDR5, and Menin are currently under extensive pre-clinical trials for solid tumors and leukemias, our findings will provide insights into NB tumor biology and a new epigenetic based therapeutic approach for high-risk NB.

Project Narrative: The majority of deaths from high-risk neuroblastoma are due to relapsed refractory disease that may be caused by a drug-resistant cancer stem cell sub-population with tumor-initiating potential. The proposed research will characterize the epigenetic mechanisms that maintain these tumor-initiating cells and contribute to neuroblastoma tumorigenicity and metastasis. We will also develop and pre-clinically test effective targeted therapeutic approaches against these tumor-initiating cells to limit neuroblastoma pathogenesis and relapse.