Identification of MYCN non-amplified neuroblastoma subgroups points towards molecular signatures for precision prognosis and therapy stratification.

BACKGROUND: Despite the extensive study of MYCN-amplified neuroblastomas, there is a significant unmet clinical need in MYCN non-amplified cases. In particular, the extent of heterogeneity within the MYCN non-amplified population is unknown. METHODS: A total of 1566 samples from 16 datasets were identified in Gene Expression Omnibus (GEO) and ArrayExpress. Characterisation of the subtypes was analysed by ConsensusClusterPlus. Independent predictors for subgrouping were constructed from the single sample predictor based on the multiclassPairs package. Findings were verified using immunohistochemistry and CIBERSORTx analysis. RESULTS: We demonstrate that MYCN non-amplified neuroblastomas are heterogeneous and can be classified into 3 subgroups based on their transcriptional signatures. Within these groups, subgroup_2 has the worst prognosis and this group shows a 'MYCN' signature that is potentially induced by the overexpression of Aurora Kinase A (AURKA); whilst subgroup_3 is characterised by an 'inflamed' gene signature. The clinical implications of this subtype classification are significant, as each subtype demonstrates a unique prognosis and vulnerability to investigational therapies. A total of 420 genes were identified as independent subgroup predictors with average balanced accuracy of 0.93 and 0.84 for train and test datasets, respectively. CONCLUSION: We propose that transcriptional subtyping may enhance precision prognosis and therapy stratification for patients with MYCN non-amplified neuroblastomas.

P300 Interacted With N-Myc and Regulated Its Protein Stability via Altering Its Post-Translational Modifications in Neuroblastoma.

MYCN amplification is an independent risk factor for poor prognosis in neuroblastoma (NB), but its protein product cannot be directly targeted because of protein structure. Thus, this study aimed to explore novel ways to indirectly target N-Myc by regulating its post-translational modifications (PTMs) and therefore protein stability. N-Myc coimmunoprecipitation combined with HPLC-MS/MS identified 16 PTM residues and 114 potential N-Myc-interacting proteins. Notably, both acetylation and ubiquitination were identified on lysine 199 of N-Myc. We then discovered that p300, which can interact with N-Myc, modulated the protein stability of N-Myc in MYCN-amplified NB cell lines and simultaneously regulated the acetylation level and ubiquitination level on lysine-199 of N-Myc protein in vitro. Furthermore, p300 correlated with poor prognosis in NB patients. Taken together, p300 can be considered as a potential therapeutic target to treat MYCN-amplified NB patients, and other identified PTMs and interacting proteins also provide potential targets for further study.

15,16-dihydrotanshinone I inhibits EOMA cells proliferation by interfering in posttranscriptional processing of hypoxia-inducible factor 1.

Infantile hemangioma (IH), which threatens the physical and mental health of patients, is the most common benign tumor in infants. Previously, we found that 15,16-dihydrotanshinone I (DHTS) was significantly more effective at inhibiting hemangioma proliferation in vitro and in vivo than the first-line treatment propranolol. To investigate the underlying mechanism of DHTS, we used EOMA cells as a model to study the effect of DHTS. We compared the transcriptomes of control and DHTS-treated EOMA cells. In total, 2462 differentially expressed genes were detected between the groups. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed downregulated activity of the hypoxia-inducible factor 1 alpha (HIF-1α) signaling pathway in EOMA cells following treatment with DHTS. Thus, we investigated HIF-1α expression at protein and mRNA levels. Our results revealed that DHTS downregulated HIF-1α expression by interfering in its posttranscriptional processing, and the RNA-binding protein HuR participated in this mechanism. Our findings provide a basis for clinical transformation of DHTS and insight into pathogenic mechanisms involved in IH.

XPO1/CRM1 is a promising prognostic indicator for neuroblastoma and represented a therapeutic target by selective inhibitor verdinexor.

BACKGROUND: High-risk neuroblastoma patients have a 5-year survival rate of less than 50%. It's an urgent need to identify new therapeutic targets and the appropriate drugs. Exportin-1 (XPO1), also known as chromosomal region maintenance 1, plays important roles in the progression of tumorigenesis. However, the prognostic and therapeutic values of XPO1 in neuroblastoma have not been reported. METHODS: Correlations between XPO1 expression level and clinical characteristics were analyzed using the Neuroblastoma Research Consortium (NRC) dataset and tissue microarray analysis. Cell proliferation assays, colony formation assays, apoptosis assays, cell cycle analysis were performed to analyze the anti-tumor effects of verdinexor (KPT-335) in vitro. Western blot and mRNA sequencing were performed to explore underlying mechanism. In vivo anti-tumor effects of verdinexor were studied in a neuroblastoma xenograft model. RESULTS: Higher XPO1 levels were associated with advanced stage and poor prognosis in neuroblastoma patients. The specific inhibitor of XPO1 verdinexor suppressed the neuroblastoma cell growth both in vitro and in vivo. Specifically, inhibition of XPO1 suppressed the neuroblastoma cell proliferation and induced cell apoptosis by nuclear accumulation of FOXO1 and RB1 in the neuroblastoma due to the inhibition of the PI3K/AKT pathway, and induced G0/G1 phase cell cycle arrest by activation of P53 function. CONCLUSIONS: XPO1 is a promising prognostic indicator for neuroblastoma and a novel target for antitumor treatment with selective inhibitor verdinexor.

MYT1 attenuates neuroblastoma cell differentiation by interacting with the LSD1/CoREST complex.

Impaired neuronal differentiation is a feature of neuroblastoma tumorigenesis, and the differentiation grade of neuroblastoma tumors is associated with patient prognosis. Detailed understanding of the molecular mechanisms underlying neuroblastoma differentiation will facilitate the development of effective treatment strategies. Recent studies have shown that myelin transcription factor 1 (MYT1) promotes vertebrate neurogenesis by regulating gene expression. We performed quantitative analysis of neuroblastoma samples, which revealed that MYT1 was differentially expressed among neuroblastoma patients with different pathological diagnoses. Analysis of clinical data showed that MYT1 overexpression was associated with a significantly shorter 3-year overall survival rate and poor differentiation in neuroblastoma specimens. MYT1 knockdown inhibited proliferation and promoted the expression of multiple differentiation-associated proteins. Integrated omics data indicated that many genes involved in neuro-differentiation were regulated by MYT1. Interestingly, many of these genes are targets of the REST complex; therefore, we further identified the physical interaction of MYT1 with LSD1/CoREST. Depletion of LSD1 or inhibition of LSD1 by ORY-1001 decreased MYT1 expression, providing an alternative approach to target MYT1. Taken together, our results indicate that MYT1 significantly attenuates cell differentiation by interacting with the LSD1/CoREST complex. MYT1 is, therefore, a promising therapeutic target for enhancing the neurite-inducing effect of retinoic acid and for inhibiting the growth of neuroblastoma.

Zhi Zhen Fang formula reverses Hedgehog pathway mediated multidrug resistance in colorectal cancer.

Zhi-Zhen-Fang (ZZR), a Traditional Chinese Medicine (TCM) formula, has been clinically used in China to treat drug-resistant colorectal cancer (CRC) patients as an adjuvant. In this study, the efficacy of ZZR in suppressing multidrug resistance (MDR) on CRC was evaluated in vitro and in vivo. We observed that ZZR enhanced the sensitivity of chemotherapeutic drugs and induced apoptosis in a dose- and time-dependent mannner in CRC MDR cells. Interestingly, signaling of Hedgehog pathway, particularly Gli1, was also inhibited by ZZR. This effect of ZZR in reversing drug resistance and suppressing Gli1 was attenuated by a Hedgehog activator (SAG). Furthermore, ZZR inhibited MDR CRC tumor growth in a xenograft mouse model as well as downregulated Gli1 levels. This study provided the first direct evidence demonstrating ZZR can attenuate MDR by repressing Hedgehog signaling in human CRC.