Abstract PR09: Developing metabolic intervention strategies to reprogram neuroblastoma epigenome and overcome tumor resistance to differentiation therapy

Patients with solid tumor usually do not respond to retinoic acid (RA)-based differentiation therapy because RA-resistance often develops. The goal of this study is to develop metabolic intervention strategies that target the cancer epigenome and overcome tumor resistance to differentiation therapy. Experimental procedures: We classified our neuroblastoma (NB) cell line collection into two categories: RA-sensitive cell lines and RA-resistant cell lines. Metabolic intervention agents were combined with RA to treat the RA-resistance cells. Chromatin markers including histone acetylations and methylations were determined using immunoblot. Global DNA methylation were accessed using dot blot. NB cell differentiation were quantified with neurite counting and immunofluorescence staining for NB differentiation marker β-tubulin III. Retinoic acid receptor (RAR) and neuron differentiation marker expression were measured using q-PCR. Summary of findings: Unlike the RA-sensitive NB cells, the RA-resistant cells did not show morphological change or proliferation arrest upon RA treatment. Previously we discovered that glycerol triacetate (GTA) supplementation restored histone acetylation and chromatin accessibility to reactivate the expression of differentiation markers and the RA-induced neuron differentiation program under hypoxia. However, the combination of GTA and RA did not induce proliferation arrest or morphologic change in RA-resistant cells. Interestingly, the combination of RA and the substrate of DNA demethylase, α-ketoglutarate (αKG), significantly induced neuronal differentiation in RA-resistant cells. RAR and differentiation markers expression were also restored by RA+αKG combination. αKG treatment rapidly reduced global 5-methylcytosine (5mC) levels within 1 hour. Although GTA alone or RA+GTA did not induce differentiation, adding GTA in combination with RA and αKG further enhanced the differentiation morphology change in resistant cells. In addition, αKG and GTA supplementation had a synergistic effect in reducing the clonogenic expansion of RA-resistant cells when combined with RA, suggesting that the RA+αKG+GTA combination inhibits self-renewal of RA-resistance NB cells. However, in RA-sensitive cells, αKG could not restore RAR repression caused by hypoxia, probably due to the elevated NADH/NAD+ ratio that favors the conversion of αKG to 2-hydroxyglutarate (2HG), which causes DNA hypermethylation. Conclusions: (1) Unlike hypoxia-induced resistance, which was mainly due to histone deacetylation, DNA methylation is the major cause of resistance in RA-resistant NB cells. (2) αKG+GTA combination effectively reduces DNA methylation and increases histone acetylation to promote RA-sensitivity in RA-resistant NB cells. Citation Format: Haowen Jiang, Yang Li, Michela Yip, Joshua Gruber, Albert Li, Jiangbin Ye. Developing metabolic intervention strategies to reprogram neuroblastoma epigenome and overcome tumor resistance to differentiation therapy [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PR09.