Retinoic acids (RAs) are the most successful therapeutics for cancer differentiation therapy used in high-risk neuroblastoma (NB) maintenance therapy but are limited in effectiveness. This study identifies a strategy for improving efficacy through disruption of cancer cell identity via BET inhibitors. Mutations that block development are theorized to cause NB through retention of immature cell identities contributing to oncogenesis. NB has two interchangeable cell identities, maintained by two different core transcriptional regulatory circuitries (CRCs): a therapy-resistant mesenchymal/stem cell state and a proliferative adrenergic cell state. MYCN amplification is a common mutation of high-risk NB and recently found to block differentiation by driving high expression of the adrenergic CRC transcription factor ASCL1. We investigated whether disruption of immature CRCs can promote RA-induced differentiation since only a subset of NB patients responds to RA. We found that silencing ASCL1, a critical member of the adrenergic CRC, or global disruption of CRCs with the BET inhibitor JQ1, suppresses gene expression of multiple CRC factors, improving RA-mediated differentiation. Further, JQ1 and RA synergistically decrease proliferation and induce differentiation in NB cell lines. Our findings support preclinical studies of RA and BET inhibitors as a combination therapy in treating NB.

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Neuroblastomas (NB) are embryonal childhood tumors that derive from the multipotent neural crest cells (NCCs) of the peripheral nervous system. NB accounts for more than 15% of all childhood cancer-related deaths. Despite the most intensive multimodal therapy, more than 50% of patients with high-risk NB relapse with often fatal, resistant disease. Finding novel treatments, especially for relapse disease, is desperately needed for high-risk NB. In cancers, distinct transcription factors TFs networks forming core regulatory circuitries (CRCs) control gene expression programs that drive cell identity. Recent studies reported the presence of two types of identity states in NB tumors: one establishing a more proliferative adrenergic (ADRN) cell state and a second establishing a more invasive, therapy-resistant mesenchymal (MES) cell state. While most studies are investigating the CRC members for targeting MYCN amplified NB (ADRN) or the MES/NCCs cell states, most primary tumors are heterogeneous, comprised of cells with both identities. Thus, our work is focused on identifying universal factors shared across different lineage states to determine whether targeting both identities could be a valuable strategy for NB treatment. NB was identified from a screen of 673 cancer cell lines as highly sensitive to the BET-inhibitor JQ1. In our work, we identified the class I basic helix-loop-helix transcription factor (bHLH) TCF4 as a critical target of JQ1 mediated cell death in NB. TCF4 functions as a transcriptional hub that heterodimerizes with class II (bHLH) TFs including the proneural gene ASCL1 (ADRN), and TWIST1 a master regulator of the MES state. Heterodimers formed between TCF4 and ASCL1, and TWIST1, have been demonstrated to provide lineage-specific differentiation from embryonic stem cells. Here, we hypothesize that TCF4 is a cell dependency gene in NB that is crucial for determining NB identity. Using RNA seq analysis of TCF4 dox-inducible shRNA stable cell lines, we found that the gene expression changes in TCF4-depleted cells indicate a role for TCF4 in differentiation, cell signaling, and neurodevelopment. Interestingly, gene set enrichment analysis (GSEA) indicates that both identity states are suppressed after silencing of TCF4. Our data also shows that inducible decrease of TCF4 protein resulted in a significant inhibition of NB cell growth, and impaired tumor growth in vivo. Furthermore, we identified TCF4 targets in NB by combined analysis of TCF4 ChIP-seq data and gene expression changes following TCF4 knockdown. ENRICHR analysis suggests that differentially expressed genes in TCF4-knockdown cells that also have a TCF4 ChIP-seq peak are targets of the TFs TWIST1 (MES) and HAND1 (ADRN) that interact directly with TCF4. In conclusion, our preliminary data supports the hypothesis that TCF4 is a master transcriptional regulator of NB oncogenic program that is crucial for maintaining NB identity states. Citation Format: Nour Aljouda, Dewan Shrestha, Megan Walker, Satyanarayana Alleboina, Evan Glazer, Yong Cheng, Kevin Freeman. Defining TCF4’s role as a novel key regulator in mediating neuroblastoma cell identity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5719.