Characterizing and Targeting the Epigenetic Mechanisms Maintaining Neuroblastoma Tumor Initiating Cells

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.

Grant Number: 1R15CA277659-01
NIH Institute/Center: NIH
Principal Investigator: Saurabh Agarwal