Abstract 1293: MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13-cisRA-mediated differentiation in neuroblastoma
Sung‐Jen WeiThinh H. NguyenDustin G. MookMonish Ram MakenaDattesh VerlekarAshly HindleGloria Martı́nezShengping YangHiroyuki ShimadaC. Patrick ReynoldsMin H. Kang
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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 < 0.001) and overall survival was lower (P < 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.Keywords:
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N-Myc
Previous studies had revealed that DNA with partial similarity to the myc oncogene (N-myc) is frequently amplified in human neuroblastoma cell lines and neuroblastoma tumors. We show here for one patient that N-myc amplification is confined to the neuroblastoma tumor and is not present in normal tissue. N-myc mRNA approximately equal to 4.0 kilobases in size is detectable in neuroblastoma cell lines and tumors and in a retinoblastoma cell line. By contrast, appreciable amounts of this RNA were not present in a number of cell lines derived from other human tumors and in fibroblasts from a normal individual and from a neuroblastoma patient. Low levels of N-myc RNA were found in human and murine neuroblastoma cell lines lacking amplification of this gene, up to 80-fold greater levels in all cell lines carrying amplified N-myc. In situ hybridization to sections of neuroblastoma tumors revealed high expression of N-myc predominantly in undifferentiated neuroblasts. We hypothesize that amplification and consequent elevated expression of N-myc may be related to malignant progression.
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The expression of the endogenous CRF gene was examined in the human neuroblastoma cell line, BE(2)-M17. In this cell line, treatment with retinoic acid induces CRF mRNA transcription. We examined the requirement for the POU transcription factor, Brn-2, for this response. We confirmed that Brn-2 is expressed in retinoic acid-induced BE(2)-M17 cells. Expression of antisense Brn-2 message aborted the retinoic acid-mediated induction of CRF transcription. However, overexpression of Brn-2 was not sufficient for CRF expression in the absence of retinoic acid. These experiments support the hypothesis that Brn-2 is an intermediary for retinoic acid-induced CRF expression.
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The expression of the endogenous CRF gene was examined in the human neuroblastoma cell line, BE(2)-M17. In this cell line, treatment with retinoic acid induces CRF mRNA transcription. We examined the requirement for the POU transcription factor, Brn-2, for this response. We confirmed that Brn-2 is expressed in retinoic acid-induced BE(2)-M17 cells. Expression of antisense Brn-2 message aborted the retinoic acid-mediated induction of CRF transcription. However, overexpression of Brn-2 was not sufficient for CRF expression in the absence of retinoic acid. These experiments support the hypothesis that Brn-2 is an intermediary for retinoic acid-induced CRF expression.
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To investigate the relations between anaplastic lymphoma kinase (ALK) and v-myc myelocytomatosis viral related oncogene neuroblastoma derived homolog (MYCN) protein expression and their prognostic roles in neuroblastoma tumours.Sixty-one neuroblastoma tumours obtained at diagnosis were stained with anti-MYCN and anti-ALK antibodies by immunohistochemical staining. The correlations between protein expression of MYCN, ALK and clinicopathological and biological variables of neuroblastoma tumours were analysed.High expression of ALK protein could be detected in 25 (41%) and high expression of MYCN protein could be detected in 24 (39.3%) of the 61 neuroblastoma tumours, respectively. The majority of neuroblastoma tumours with evident of ALK or MYCN protein high expression exhibited undifferentiated or poorly differentiated histology (30/35, 85.7%). ALK or MYCN protein high expression in neuroblastoma tumours was associated with adverse clinical prognostic factors and ALK protein high expression was significantly associated with MYCN protein high expression. In addition, either ALK or MYCN protein high expression in neuroblastoma tumours was the independent adverse prognostic factor and also predicted worse survival outcomes for neuroblastoma patients with MYCN non-amplified status or non-high-risk Children's Oncology Group grouping.Our study showed a novel coordinately prognostic role of ALK and MYCN protein expression in neuroblastoma and is the first report to demonstrate the correlation between ALK and MYCN protein expression in primary neuroblastoma tumours.
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Abstract Amplification of MYCN plays a pivotal role in multiple types of tumors and correlates with poor prognosis in high-risk neuroblastoma. Despite recent advances in the treatment of neuroblastoma, no approaches directly target the master oncogene MYCN. Difficulties in targeting the MYCN protein inspired us to develop a new gene-level–inhibitory strategy using a sequence-specific gene regulator. Here, we generated a MYCN-targeting pyrrole-imidazole (PI) polyamide, MYCN-A3, which directly binds to and alkylates DNA at homing motifs within the MYCN transcript. Pharmacologic suppression of MYCN inhibited the proliferation of cancer cells harboring MYCN amplification compared with MYCN nonamplified cancer cells. In neuroblastoma xenograft mouse models, MYCN-A3 specifically downregulated MYCN expression and suppressed tumor progression with no detectable adverse effects and resulted in prolonged overall survival. Moreover, treatment with MYCN-A3, but not MYCN nontargeting PI polyamide, precipitated a copy number reduction of MYCN in neuroblastoma cells with MYCN amplification. These findings suggest that directly targeting MYCN with MYCN-A3 is a novel therapeutic approach to reduce copy number of the MYCN gene for MYCN-amplified neuroblastoma. Significance: This study presents a novel approach to drugging an amplified oncogene by showing that targeting gene amplification of MYCN suppresses MYCN expression and neuroblastoma growth.
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Purpose Amplification of the MYCN oncogene at chromosome 2p24-25 identifies an aggressive subtype of human neuroblastoma with a poor clinical outcome. Differences in amplicon structure and coamplification of genes telomeric and centromeric to the MYCN oncogene have previously been described. A relevant role of gene coamplification for neuroblastoma pathogenesis remains elusive. Patients and Methods We analyzed 98 primary neuroblastoma tumors with MYCN amplification for coamplification of seven additional genes at chromosome 2p24-25 (DDX1, NAG, NSE1, LPIN1, EST- AA581763 , SMC6, and SDC1). Two semiquantitative multiplex polymerase chain reactions were used to obtain the amplification status of the target genes in relation to a reference gene on chromosome 2q (Inhibin-beta-b). Furthermore, mRNA expression pattern of coamplified genes in a subset of tumors was analyzed. Results Our results show that the frequency of gene coamplification on 2p24-25 in neuroblastoma is correlated directly to the physical distance to MYCN. Coamplification is correlated to an upregulated gene expression for DDX1 and NAG. Coamplification of the DDX1 gene within 400kb telomeric to MYCN identifies a subgroup of advanced stage neuroblastoma tumors with a more favorable outcome (P = .027, log-rank test). A high expression level of DDX1 is associated with a trend towards a better survival probability (P = .058, log-rank test). Conclusion Our results indicate that DDX1 coamplification correlates with a better prognosis and improved patient survival in MYCN-amplified neurobastoma.
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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 < 0.001) and overall survival was lower (P < 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.
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Neuroblastoma is a pediatric tumor of the sympathetic nervous system. MYCN (V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived [avian]) is amplified in 20% of neuroblastomas, and these tumors carry a poor prognosis. However, tumors without MYCN amplification also may have a poor outcome. Here, we identified downstream targets of MYCN by shRNA-mediated silencing MYCN in neuroblastoma cells. From these targets, 157 genes showed an expression profile correlating with MYCN mRNA levels in NB88, a series of 88 neuroblastoma tumors, and therefore represent in vivo relevant MYCN pathway genes. This 157-gene signature identified very poor prognosis tumors in NB88 and independent neuroblastoma cohorts and was more powerful than MYCN amplification or MYCN expression alone. Remarkably, this signature also identified poor outcome of a group of tumors without MYCN amplification. Most of these tumors have low MYCN mRNA levels but high nuclear MYCN protein levels, suggesting stabilization of MYCN at the protein level. One tumor has an MYC amplification and high MYC expression. Chip-on-chip analyses showed that most genes in this signature are directly regulated by MYCN. MYCN induces genes functioning in cell cycle and DNA repair while repressing neuronal differentiation genes. The functional MYCN-157 signature recognizes classical neuroblastoma with MYCN amplification, as well as a newly identified group marked by MYCN protein stabilization.
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Amplified MYCN oncogene resulting in deregulated MYCN transcriptional activity is observed in 20% of neuroblastomas and identifies a highly aggressive subtype. In MYCN single-copy neuroblastomas, elevated MYCN mRNA and protein levels are paradoxically associated with a more favorable clinical phenotype, including disseminated tumors that subsequently regress spontaneously (stage 4s-non-amplified). In this study, we asked whether distinct transcriptional MYCN or c-MYC activities are associated with specific neuroblastoma phenotypes.We defined a core set of direct MYCN/c-MYC target genes by applying gene expression profiling and chromatin immunoprecipitation (ChIP, ChIP-chip) in neuroblastoma cells that allow conditional regulation of MYCN and c-MYC. Their transcript levels were analyzed in 251 primary neuroblastomas. Compared to localized-non-amplified neuroblastomas, MYCN/c-MYC target gene expression gradually increases from stage 4s-non-amplified through stage 4-non-amplified to MYCN amplified tumors. This was associated with MYCN activation in stage 4s-non-amplified and predominantly c-MYC activation in stage 4-non-amplified tumors. A defined set of MYCN/c-MYC target genes was induced in stage 4-non-amplified but not in stage 4s-non-amplified neuroblastomas. In line with this, high expression of a subset of MYCN/c-MYC target genes identifies a patient subtype with poor overall survival independent of the established risk markers amplified MYCN, disease stage, and age at diagnosis.High MYCN/c-MYC target gene expression is a hallmark of malignant neuroblastoma progression, which is predominantly driven by c-MYC in stage 4-non-amplified tumors. In contrast, moderate MYCN function gain in stage 4s-non-amplified tumors induces only a restricted set of target genes that is still compatible with spontaneous regression.
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