Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression

Abstract

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNAdemethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genomewide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma.neuroblastoma | differentiation | retinoic acid | 5-Aza-dC | HIF2a

Wnt signalling drives context-dependent differentiation or proliferation in neuroblastoma

“…Our analysis of another MES line, GI-MEN, did not however support the notion that induction of proliferation by Wnt is solely attributable to a MES identity. We have previously shown that SK-N-AS cells are also quite different from SK-N-BE(2)-C and SH-SY5Y cells in terms of their genomewide DNA methylation pattern [69], and the epigenetic identity of SK-N-AS has been shown to influence their differentiation response [28,70]. Thus further analysis at the epigenetic and proteomic level with a broad range of neuroblastoma cell lines will be 2 1 necessary to precisely identify the determinants of the phenotypic responses to Wnt signalling by neuroblastoma cells.…”

“…We found genes encoding neuronal proteins were induced by Wnt3a/Rspo2, including several neurotransmitter receptors (HTR1E, HTR2B, NPY2R, ADRA2A and OPRM1), neurofilament protein M (NEFM), and transcription factors involved in differentiation of the neural crest, including MSX1 and MSX2 [41]. EPAS1/HIF-2α, which was very recently associated with differentiation of neuroblastoma [28] was 1 1 also upregulated after treatment. ASCL1, which is one of the few down-regulated genes amongst the DEGs, is also involved in neuronal differentiation [42], with mRNA downregulation also observed with retinoic acid induced differentiation of neuroblastoma cells [43].…”

“…To further evaluate the growth and differentiation variance of these cell lines at the protein level, we examined whether induction of two recently characterised inducers of differentiation, BMP4 [29] and EPAS1 [28], corresponded with the propensity of cells to differentiate. As shown in Figure 4A…”

Alternative splicing in neuroblastoma generates RNA-fusion transcripts and is associated with vulnerability to spliceosome inhibitors

“…FusionCatcher (18) was applied to detect the fusion transcripts in paired-end RNA-seq data. Reads were mapped to hg19 and differential expression analysis was performed as described in (42). For enrichment analysis in Figure 1C, only fusions occurring above ~3% in each risk group were included (1 case in Low/Intermediate-risk, 4 cases in High-risk and 5 cases for the common fusion transcripts shared by Low/Intermediate-risk and high-risk groups).…”

A Wnt-BMP4 signalling axis induces MSX and NOTCH proteins and promotes growth suppression and differentiation in neuroblastoma

“…These include EPAS1 (17) and MSX1 (18), both of which have been shown to inhibit neuroblastoma cell growth. Further analysis of our Wnt differentially expressed genes (DEGs) suggested that Wnt signalling is also a key regulator of mesenchymal and adrenergic differentiation states (19) which contribute to neuroblastoma cellular heterogeneity in both cell-lines and primary tumours (20,21).…”