High-Risk Neuroblastoma(HR-NB) is characterized by dysregulation of sympathoadrenal progenitor cell’s self-renewal and a failure to implement a differentiation program. Determining pathways that contribute to this state will lead to a better understanding of disease pathogenesis and potentially identify new targets. The re-programming of embryonic stem cells to a state primed for lineage specific differentiation first requires treatment with all-trans retinoic acid(ATRA). To gain insights into events important in reprogramming NB cells for differentiation, we used RNAseq analyses to assess early changes in the transcriptome of HR-NB cells(KCNR:MYCN-amplified) after 24hrs ATRA. We find a significant(FDR<0.01) up-regulation(760) and down-regulation(548) of genes. IPA analyses indicate cAMP signaling pathway is the most significantly up-regulated pathway. GSEA indicated that gene-sets associated with high MYCN expression are down-regulated and those enriched in PRC2(Polycomb Repressor Complex2) target genes are increased(p-value:0.001). Biochemical assays confirmed RA-mediated increases in cAMP levels and activation of PKA within 2hrs. By 6hrs, activation of PKA causes increases in phosphorylation of EZH2 at ser21(P-EZH2ser21) which inhibits EZH2 binding to H3, resulting in a decrease in the epigenetic gene suppression mark-H3K27me3. Such decreases in H3K27me3 at EZH2 target genes result in increased RNA transcription, consistent with the GSEA analysis of the RNAseq data. siRNAs against PRKACB, a catalytic subunit of PKA blocked the RA mediated activation of cAMP pathway in NB cells, prevented P-EZH2ser21 and induction of EZH2 target genes such as RARß. In primary NB tumors, microarray studies indicate that low levels of PRKACB are associated with poor prognosis. The 8-year survival in Stage 4 NB patients is only 20% for patients with low PRKACB levels in their tumors compared to 70% survival for patients whose tumors have relatively high levels of PRKACB (P=3.6e-05; R2database-Kocak). This study identifies PKA activation as an early event that may be necessary for re-programming HR-NB cells to differentiate.