Abstract 1293: MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13-cisRA-mediated differentiation in neuroblastoma
Authors: Wei Sung Jen , Nguyen Thinh H. , Mook Dustin G. , Makena Monish R. , Verlekar Dattesh , Hindle Ashly , Martinez Gloria , Yang Shengping , Shimada Hiroyuki , Reynolds C. Patrick , Kang Min H. 
Published: September 2020

Abstract
MYCN genomic amplification is one of the risk factors in neuroblastoma. 13-cis retinoic acid (13-cisRA), a differentiating agent, down-regulates MYCN protein and is part of neuroblastoma maintenance therapy. Despite the improvement in clinical outcome with 13-cisRA, anti-GD2 monoclonal antibody plus cytokine immunotherapy given in first response ~40% of high-risk neuroblastoma patients still die of recurrent disease. Although MYC genomic amplification is rare in neuroblastoma (~1%), 11% of neuroblastoma primary tumors collected at diagnosis (Dx) have high c-MYC protein suggesting that MYC transcriptional activation rather than its gene amplification drives such tumors. Here, we sought to investigate the role of MYC oncogene in progressive disease (PD) and to molecularly characterize mechanisms of MYC expression in neuroblastoma. We report transcriptional activation of MYC medicated by the OCT4 (encoded by POU5F1), functionally replacing MYCN in 13-cisRA-resistant progressive disease neuroblastoma. In large panels of neuroblastoma patient-derived cell lines (19 Dx and 16 PD) and patient-derived xenograft PDX models (8 Dx and 9 PD), we confirmed that c-MYC expression levels were higher in PD relative to Dx lines (P = 0.0005). We identified OCT4 and TCF3 as transcription factors highly expressed in neuroblastoma cells with high c-MYC. Subsequently, we confirmed two novel OCT4-binding sites (including OBS1 and OBS2) located in the MYC promoter/enhancer region: -1209 to -1140 and found that OCT4 NH2-terminal domain (NTD) and POU specific domain (POUs) are critical for MYC transcriptional activation. To identify kinases that is associated with OCT4-induced c-MYC activation, we used mass spectrometry and PhosphoMotif Finder® and identified MAPKAPK2 (MK2) as one of the upstream kinases that can bind to and directly regulate the OCT4 biological function by phosphorylation at its amino acid Ser111 residue to transcriptionally activate MYC expression. The data in 175 MYCN non-amplified high-risk primary tumors (TARGET database) showed that MAPKAPK2 positively correlated with MYC expression (P &lt; 0.001) and overall survival was lower (P &lt; 0.001) for patients with high MAPKAPK2. Also, OCT4, MK2, and c-MYC were higher in PD relative to Dx neuroblastomas models. Functional studies by gene knockdown of the POU5F1 or MAPKAPK2 using shRNAs showed decreased c-MYC expression, inhibition of cell proliferation, and restoring neurite outgrowth in response to 13-cisRA. In conclusion, high c-MYC independent of genomic amplification, not MYCN amplification, is associated with disease progression in neuroblastoma. The MK2-mediated OCT4 transcriptional activation is a novel mechanism for MYC activation in PD neuroblastoma and provides a potential novel therapeutic target.
Citation Format: Sung Jen Wei, Thinh H. Nguyen, Dustin G. Mook, Monish R. Makena, Dattesh Verlekar, Ashly Hindle, Gloria Martinez, Shengping Yang, Hiroyuki Shimada, C. Patrick Reynolds, Min H. Kang. MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13-cisRA-mediated differentiation in neuroblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1293.