MYCN Research Articles

Multi-omics revealed that ELAVL3 regulates MYCN in neuroblastoma via immunogenic cell death: Risk stratification and experimental research.

Neuroblastoma (NB), a common and highly lethal malignant disease in pediatrics, still lacks an effective therapeutic approach that addresses all conditions. Immunogenic Cell Death (ICD) plays a crucial role in tumor cell death and triggers a potent anti-tumor immune response. In this study, we report an ICD-related index (ICDR-Index) in NB through various machine learning methodologies, utilizing bulk transcriptome data from 1244 NB samples and 16 scRNA-seq datasets. Our results showed that the ICDR-Index could accurately identify different risk subtypes of patients with NB and provide predictive value for prognosis. Importantly, we found that high-risk patients with NB exhibited significantly poor overall survival (OS) rates, adverse clinical phenotypes, poor immune cell infiltration, and low sensitivity to immunotherapy. Furthermore, we identified ELAVL3 as a key gene within the ICDR-Index, where high expression levels were associated with malignancy and poor OS in NB. Additionally, targeted silencing of ELAVL3 down-regulated MYCN gene expression and reduced the malignancy of NB cells. Notably, the si-ELAVL3-transfected NB cells enhanced the anti-tumor activity of NK cells. Collectively, this study offers avenues for predicting the risk stratification of patients with NB and reveals a potential mechanism by which ELAVL3 regulates NB cell death.

Unraveling the glycosphingolipid metabolism by leveraging transcriptome-weighted network analysis on neuroblastic tumors

BackgroundGlycosphingolipids (GSLs) are membrane lipids composed of a ceramide backbone linked to a glycan moiety. Ganglioside biosynthesis is a part of the GSL metabolism, which involves sequential reactions catalyzed by specific enzymes that in part have a poor substrate specificity. GSLs are deregulated in cancer, thus playing a role as potential biomarkers for personalized therapy or subtype classification. However, the analysis of GSL profiles is complex and requires dedicated technologies, that are currently not included in the commonly utilized high-throughput assays adopted in contexts such as molecular tumor boards.MethodsIn this study, we developed a method to discriminate the enzyme activity among the four series of the ganglioside metabolism pathway by incorporating transcriptome data and topological information of the metabolic network. We introduced three adjustment options for reaction activity scores (RAS) and demonstrated their application in both exploratory and comparative analyses by applying the method on neuroblastic tumors (NTs), encompassing neuroblastoma (NB), ganglioneuroblastoma (GNB), and ganglioneuroma (GN). Furthermore, we interpreted the results in the context of earlier published GSL measurements in the same tumors.ResultsBy adjusting RAS values using a weighting scheme based on network topology and transition probabilities (TPs), the individual series of ganglioside metabolism can be differentiated, enabling a refined analysis of the GSL profile in NT entities. Notably, the adjustment method we propose reveals the differential engagement of the ganglioside series between NB and GNB. Moreover, MYCN gene expression, a well-known prognostic marker in NTs, appears to correlate with the expression of therapeutically relevant gangliosides, such as GD2. Using unsupervised learning, we identified subclusters within NB based on altered GSL metabolism.ConclusionOur study demonstrates the utility of adjusting RAS values in discriminating ganglioside metabolism subtypes, highlighting the potential for identifying novel cancer subgroups based on sphingolipid profiles. These findings contribute to a better understanding of ganglioside dysregulation in NT and may have implications for stratification and targeted therapeutic strategies in these tumors and other tumor entities with a deregulated GSL metabolism.

Short-term results of a multicenter study based on a modified N7 induction regimen combined with arsenic trioxide in the treatment of children with high-risk neuroblastoma

Objective: To analyze the short-term clinical efficacy and safety of arsenic trioxide (ATO) combined with a modified N7 induction regimen in the treatment of children with high-risk neuroblastoma (NB). Methods: This study was a prospective, single-arm, multicenter phase Ⅱ clinical study. Sixty-seven high-risk NB children from eight units of Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Wuhan Children's Hospital of Tongji Medical College of Huazhong University of Science and Technology, First Affiliated Hospital of Guangxi Medical University, Hainan General Hospital, Affiliated Hospital of Guangdong Medical University, Kunming Children's Hospital, Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology, and Guangdong Provincial Agricultural Reclamation Center Hospital were enrolled from January 2019 to August 2023 and were treated with ATO combined with a modified N7 induction regimen. The efficacy and adverse effects at the end of induction chemotherapy were assessed and analyzed, and the differences in the clinical characteristics were further compared between the treatment-responsive and treatment-unresponsive groups by using the Fisher's exact test. Results: Among 67 high-risk NB children, there were 40 males (60%) and 27 females (40%), with the age of disease onset of 3.5 (2.6, 4.8) years. Primary NB sites were mostly in retroperitoneum (including adrenal gland) (56/67, 84%) and the common metastases sites at initial diagnosis were distant lymph node in 25 cases (37%),bone in 48 cases (72%),bone marrow in 56 cases (84%) and intracalvarium in 3 cases (4%). MYCN gene amplification were detected in 28 cases (42%). At the end of induction, 33 cases (49%) achieved complete remission, 29 cases (43%) achieved partial remission, 1 case (1%) with stable disease, and 4 cases (6%) were assessed as progressive disease (PD). The objective remission rate was 93% (62/67) and the disease control rate was 94% (63/67). The percentage of central system metastases at the initial diagnosis was higher in the treatment-unresponsive group than in the treatment-responsive group (2/5 vs. 2% (1/62), P=0.013), whereas the difference in MYCN gene amplification was not statistically significant between two groups (3/5 vs.40% (25/62), P=0.786). Grade Ⅲ or higher adverse reactions during the induction chemotherapy period were myelosuppression occurred in 60 cases (90%), gastrointestinal symptoms occurred in 33 cases (49%), infections occurred in 20 cases (30%), hepatotoxicity occurred in 4 cases (6%), and cardiovascular toxicity occurred in 1 case (2%). There were no chemotherapy-related deaths. Conclusion: ATO combined with N7-modified induction regimen had a superiority in efficacy and safety, which deserved further promotion in clinical practice.

DNp73 enhances tumor progression and immune evasion in multiple myeloma by targeting the MYC and MYCN pathways.

Multiple myeloma (MM) is an incurable hematological malignancy with high chromosome instability and heavy dependence on the immunosuppressive bone marrow microenvironment. P53 mutations are adverse prognostic factors in MM; however, clinically, some patients without P53 mutations also exhibit aggressive disease progression. DNp73, an inhibitor of TP53 tumor suppressor family members, drives drug resistance and cancer progression in several solid malignancies. Nevertheless, the biological functions of DNp73 and the molecular mechanisms in myelomagenesis remain unclear. The effects of DNp73 on proliferation and drug sensitivity were assessed using flow cytometry and xenograft models. To investigate the mechanisms of drug resistance, RNA-seq and ChIP-seq analyses were performed in MM cell lines, with validation by Western blot and RT-qPCR. Immunofluorescence and transwell assays were used to assess DNA damage and cell invasion in MM cells. Additionally, in vitro phagocytosis assays were conducted to confirm the role of DNp73 in immune evasion. Our study found that activation of NF-κB-p65 in multiple myeloma cells with different p53 mutation statuses upregulates DNp73 expression at the transcriptional level. Forced expression of DNp73 promoted aggressive proliferation and multidrug resistance in MM cells. Bulk RNA-seq analysis was conducted to assess the levels of MYCN, MYC, and CDK7. A ChIP-qPCR assay was used to reveal that DNp73 acts as a transcription factor regulating MYCN gene expression. Bulk RNA-seq analysis demonstrated increased levels of MYCN, MYC, and CDK7 with forced DNp73 expression in MM cells. A ChIP-qPCR assay revealed that DNp73 upregulates MYCN gene expression as a transcription factor. Additionally, DNp73 promoted immune evasion of MM cells by upregulating MYC target genes CD47 and PD-L1. Blockade of the CD47/SIRPα and PD-1/PD-L1 signaling pathways by the SIRPα-Fc fusion protein IMM01 and monoclonal antibody atezolizumab significantly restored the anti-MM activity of macrophages and T cells in the microenvironment, respectively. In summary, our study demonstrated for the first time that the p53 family member DNp73 remarkably induces proliferation, drug resistance, and immune escape of myeloma cells by directly targeting MYCN and regulating the MYC pathway. The oncogenic function of DNp73 is independent of p53 status in MM cells. These data contribute to a better understanding of the function of TP53 and its family members in tumorigenesis. Moreover, our study clarified that DNp73 overexpression not only promotes aggressive growth of tumor cells but, more importantly, promotes immune escape of MM cells through upregulation of immune checkpoints. DNp73 could serve as a biomarker for immunotherapy targeting PD-L1 and CD47 blockade in MM patients.

Application of the FISH method and high-density SNP arrays to assess genetic changes in neuroblastoma-research by one institute.

Neuroblastoma is the most common extracranial solid tumor in children. Amplification of the MYCN gene has been observed in approximately 20%-30% of tumors. It is strongly correlated with advanced-stage disease, rapid tumor progression, resistance to chemotherapy and poor outcomes independent of patient age and stage of advanced disease. MYCN amplification identifies high-risk patients. To assess neuroblastoma tumors with MYCN amplification we used paraffin-embedded tissue sections in 57 patients and intraoperative tumor imprints in 10 patients by fluorescence in situ hybridization (FISH). Positive results for MYCN amplification have been observed in twelve patients' paraffin-embedded tissue sections and in three patients' intraoperative tumor imprints, which represents 22.4% of all patients tested in the analysis. Fluorescence in situ hybridization is a highly sensitive and useful technique for detecting MYCN amplification on paraffin-embedded tissue sections of neuroblastoma tumors and intraoperative tumor imprints thus facilitating therapeutic decisions based on the presence or absence of this important biologic marker. The presence of structural changes, regardless of MYCN gene amplification status, influences the clinical behavior of neuroblastoma. High-Density SNP Arrays have emerged as the perfect tools for detecting these changes due to their exceptional accuracy, sensitivity and ability to analyze copy number and allele information. Consequently, they are proven to be highly valuable in the genomic diagnosis of immature neuroectodermal tumors.

Analysis of the relationship between MYCN gene and serum NSE and urinary VMA levels and neuroblastoma pathological features and prognosis.

The purpose of this study was to explore the relationship between the MYCN gene, serum neuron-specific enolase (NSE), urinary vanillylmandelic acid (VMA) levels, and neuroblastoma pathological features and prognosis. Ninety-four children with neuroblastoma treated in the hospital were selected to compare the differences in MYCN gene amplification, serum NSE, and urinary VMA levels in children with different clinicopathological features and prognoses. The proportion of children with MYCN gene copy number ≥10 in INSS stage 3-4 was higher than that of children with INSS stage 1-2 (P < 0.05); the proportion of children with MYCN gene copy number ≥10 in high-risk children in the COG risk stratification was higher than that of children with intermediate and low risk (P < 0.05); the serum NSE of children aged >12 months higher than that of children aged ≤12 months (P < 0.05); serum NSE of children with tumors >500 cm3 higher than that of children with tumors ≤500 cm3 (P < 0.05); serum NSE and urinary VMA of children with INSS staging of stages 3-4 were higher than that of children with stages 1 to 2 (P < 0.05); serum NSE and urinary VMA in children with lymph node metastasis were higher than that of children without lymph node metastasis (P < 0.05); serum NSE of children with MYCN gene copy number ≥10 was higher than that of children without lymph node metastasis (P < 0.05); the proportion of children with MYCN gene copy number ≥10 who died, and the percentages of serum NSE and urinary VMA were higher than those of the surviving children (P < 0.05). MYCN gene amplification and serum NSE and urinary VMA levels were related to the age, tumor size, INSS stage, COG stage, lymph node metastasis, and prognosis of the children with neuroblastoma.

Integrated analysis of transcriptome and genome variations in pediatric T cell acute lymphoblastic leukemia: data from north Indian tertiary care center

IntroductionT-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease with poor prognosis and inferior outcome. Although multiple studies have been perform on genomics of T-ALL, data from Indian sub-continent is scarce.MethodsIn the current study we aimed to identify the genetic variability of T-ALL in an Indian cohort of pediatric (age ≤ 12 years) T-ALL patients (n = 25) by whole transcriptome sequencing along with whole exome sequencing and correlated the findings with clinical characteristics and disease outcome.ResultsThe median age was 7 years (range 3 -12 years). RNA sequencing revealed a definitive fusion event in 14 cases (56%) (including a novel fusions) with STIL::TAL1 in 4 (16%), followed by NUP21::ABL1, TCF7::SPI1, ETV6::HDAC8, LMO1::RIC3, DIAPH1::JAK2, SETD2::CCDC12 and RCBTB2::LPAR6 in 1 (4%) case each. Significant aberrant expression was noted in RAG1 (64%), RAG2 (80%), MYCN (52%), NKX3-1 (52%), NKX3-2 (32%), TLX3 (28%), LMO1 (20%) and MYB (16%) genes. WES data showed frequent mutations in NOTCH1 (35%) followed by WT1 (23%), FBXW7 (12%), KRAS (12%), PHF6 (12%) and JAK3 (12%). Nearly 88.2% of cases showed a deletion of CDKN2A/CDKN2B/MTAP genes. Clinically significant association of a better EFS and OS (p=0.01) was noted with RAG2 over-expression at a median follow up of 22 months, while a poor EFS (p=0.041) and high relapse rate (p=0.045) was observed with MYB over-expression.ConclusionOverall, the present study demonstrates the frequencies of transcriptomic and genetic alterations from Indian cohort of pediatric T-ALL and is a salient addition to current genomics data sets available in T-ALL.

MYCN immunohistochemistry as surrogate marker for MYCN-amplified spinal ependymomas.

MYCN (master regulator of cell cycle entry and proliferative metabolism) gene amplification defines a molecular subgroup of spinal cord ependymomas that show high-grade morphology and aggressive behavior. Demonstration of MYCN amplification by DNA methylation or fluorescence-in situ hybridization (FISH) is required for diagnosis. We aimed to (i) assess prevalence and clinicopathological features of MYCN-amplified spinal ependymomas and (ii) evaluate utility of immunohistochemistry (IHC) for MYCN protein as a surrogate for molecular testing. A combined retrospective-prospective study spanning 8years was designed during which all spinal cord ependymomas with adequate tissue were subjected to MYCN FISH and MYCN IHC. Among 77 spinal cord ependymomas included, MYCN amplification was identified in 4 samples from 3 patients (3/74, 4%) including two (1st and 2nd recurrences) from the same patient. All patients were adults (median age at diagnosis of 32years) including two females and one male. The index tumors were located in thoracic (n = 2) and lumbar (n = 1) spinal cord. One of the female patients had neurofibromatosis type 2 (NF2). All four tumors showed anaplastic histology. Diffuse expression of MYCN protein was seen in all four MYCN-amplified samples but in none of the non-amplified cases, thus showing 100% concordance with FISH results. On follow-up, the NF2 patient developed widespread spinal dissemination while another developed recurrence proximal to the site of previous excision. To conclude, MYCN-amplified spinal ependymomas are rare tumors, accounting for ~ 4% of spinal cord ependymomas. Within the limitation of small sample size, MYCN IHC showed excellent concordance with MYCN gene amplification.

Visual conservation treatment dilemmas in neuroblastoma with bilateral blindness

ObjectiveTo investigate the clinical features, treatment strategies, and prognosis of neuroblastoma with bilateral blindness.MethodsThe clinical data of five patients with bilateral blindness neuroblastoma admitted to Beijing Children’s Hospital from April 2018 to September 2020 were retrospectively collected to summarize their clinical characteristics.ResultsAll patients were female and the median age at presentation was 25 (23, 41) months. The median intervention time from the onset of symptoms of bilateral blindness to the start of treatment was 10 (10, 12) days. All five cases were staged as stage M and grouped as high risk. Four cases were MYCN gene amplification and one case was MYCN acquisition. Five children were treated according to a high-risk neuroblastoma treatment protocol. Four children did not recover their vision after treatment, and one case improved to have light perception. All patients were effectively followed up for a median of 20 (12, 31) months, with three deaths, one tumor-free survival, and one recurrent tumor-bearing survival.ConclusionNeuroblastoma with bilateral blindness is rare in the clinic, mostly in children of young age, and is often associated with MYCN amplification and multiple metastases. Early hormone shock therapy and optic nerve decompression are beneficial for preserving the child’s vision. A joint multi-disciplinary treatment may help in the formulation of treatment decisions. Achieving a balance between good visual preservation and survival within the short optic nerve neurotherapeutic window is extremely challenging.

Evaluating Genetic Alterations in Metastatic Pancreatic Neuroendocrine Tumors with Potential Therapeutic and Prognostic Applications: A Comparative In Silico Study with Metastatic Neuroendocrine Tumors Originating from the Small Intestine and Rectum

Abstract Introduction/Objective Pancreatic neuroendocrine tumors (P-NET) are diverse tumors with slow growth and varying levels of potential malignant behavior. The overall 5-year survival rates for P-NET are approximately 95%, 72%, and 23% if localized, and in cases of regional and distant metastasis respectively. These tumors exhibit a low rate of mutations but show varied genomic abnormalities, contributing to their unpredictable nature. In this study, we aimed to find the genetic alterations observed in metastatic P-NET for their potential therapeutic and prognostic applications and compared these tumors to metastatic neuroendocrine tumors originating from the small intestine (SI-NET) and rectum (R-NET). Methods/Case Report We initially extracted RNA-seq datasets from the GEO database. Next, we analyzed the datasets in six defined groups and differentially expressed genes were obtained by comparison to controls. Either of the groups of P-NET, SI-NET, or R-NET cases were evaluated individually and altogether (primary vs metastatic; 4 groups) and in 2 groups comparing primary/metastatic P-NET cases to primary/metastatic cases of SI-NET plus R- NET. Using a Venn diagram, we isolated the commonality between gene expression profiles. Afterward, we obtained the ontology of genes and signaling pathways from KEGG and Enrichr. Finally, we drew the protein network and evaluated the proteins in GEPIA and TCGA clinical databases. Results (if a Case Study enter NA) A total of 212 cases including 113 P-NET (83 primary and 30 metastatic), 81 SI- NET (44 primary and 37 metastatic), and 18 R-NET (3 primary and 15 metastatic) were selected. We found 8 genes (ORM1, HP, APOA2, HRG, KNG1, ALB, SERPINC1, and AHSG) are mutually markedly upregulated in cases of metastatic P-NET and SI-NET but were not identified in metastatic R-NET. The products of these genes were predominantly involved in complement cascades. CABYR, MYCN, and CALU genes were among the most upregulated genes specifically isolated in the metastatic P-NET that played roles in activating intracellular signaling pathways, transcription, and protein folding. Conclusion The shared upregulated genes in metastatic P-NET and SI-NET may support the relatively better prognosis of these tumors in comparison to the metastatic R-NET due to the activation of the complement cascade (immune system). Furthermore, the identified genetic alterations in the metastatic P-NET cases may have potential therapeutic and prognostic applications for future use.

Enhanced Expression of DNp73 Promotes Drug Resistance and Immune Evasion in Multiple Myeloma By Targeting the MYC and MYCN Pathways

Background Multiple myeloma (MM) is an incurable haematological malignancy with high chromosome instability and is highly dependent on the immunosuppressive bone marrow microenvironment. TP53 mutations and deletions are uncommon in MM patients, with other types of mechanisms involved in the abrogation of TP53 pathway activity. DNp73, an inhibitor of the p53 tumour suppressor family, drives drug resistance and cancer progression in several solid malignancies. However, the biological functions of DNp73, the molecular mechanisms underlying these actions and their correlations with aberrant TP53 expression in myelomagenesis remain unclear. In this study, we investigated the role of DNp73 in the drug resistance of MM, and disclosed the corresponding mechanism of how DNp73 promotes the immune escape of MM cells. Aims To disclose the role of DNp73 in promoting drug resistance and immune evasion in MM, and explore the therapeutic strategy based on the mechanism research. Methods We constructed DNp73 overexpression and sh-RNA interference plasmids to obtain stable cell lines. The effects of DNp73 on proliferation and drug sensitivity were determined by CCK8, flow cytometry and xenotransplantation model. To identify mechanisms of drug resistance, we here perform RNA-seq and CHIP-seq analyses in MM cell lines, while Western blot and RT-PCR were used to confirm the results. The DNA damage repair and invasion ability of MM cells were detected by immunofluorescence and transwell assay. To validate the role of DNp73 in immune escape, we also performed phagocytosis assays in vitro. Results Our previous study revealed that the miRNA-15a is a tumour suppressor involved in aggressive MM cell proliferation and drug resistance. Here, we found that miRNA-15a downregulation in MM cells activated NF-κB-p65, which upregulated the expression of DNp73 at the transcriptional level. In addition, the level of miR-15a was negatively correlated with the expression of DNp73 in MM cells of patients (r=-0.672, p&amp;lt;0.05). Enforced expression of DNp73 promoted aggressive proliferation and multidrug resistance in MM cells (p&amp;lt;0.001). Bulk RNA sequencing data showed that the levels of MYCN, MYC and CDK7 were increased in cells with enforced DNp73 expression. Furthermore, a ChIP-qPCR assay revealed that DNp73 bound to the MYCN gene promoter and upregulated MYCN expression. The MM cell immune evasion rate was notable when DNp73 expression was high because of CD47 and PD-L1 upregulation. Blockade of the CD47/SIRPα and PD-1/PD-L1 signalling pathways by the SIRPα-Fc fusion protein IMM01 and monoclonal antibody atezolizumab significantly re-established the anti-MM function of macrophages and T cells in the MM microenvironment, respectively (p&amp;lt;0.01). Notably, DNp73 dysregulation and its pathogenetic function in MM cells were found to be independent of TP53 expression. Conclusion Altogether, our study demonstrates that DNp73 is a potential oncogene involved in MM pathogenesis and promotes MM cell proliferation and immune evasion by targeting the MYC and MYCN pathways.

Novel Auger-Electron-Emitting 191Pt-Labeled Pyrrole-Imidazole Polyamide Targeting MYCN Increases Cytotoxicity and Cytosolic dsDNA Granules in MYCN-Amplified Neuroblastoma.

Auger electrons can cause nanoscale physiochemical damage to specific DNA sites that play a key role in cancer cell survival. Radio-Pt is a promising Auger-electron source for damaging DNA efficiently because of its ability to bind to DNA. Considering that the cancer genome is maintained under abnormal gene amplification and expression, here, we developed a novel 191Pt-labeled agent based on pyrrole-imidazole polyamide (PIP), targeting the oncogene MYCN amplified in human neuroblastoma, and investigated its targeting ability and damaging effects. A conjugate of MYCN-targeting PIP and Cys-(Arg)3-coumarin was labeled with 191Pt via Cys (191Pt-MYCN-PIP) with a radiochemical purity of >99%. The binding potential of 191Pt-MYCN-PIP was evaluated via the gel electrophoretic mobility shift assay, suggesting that the radioagent bound to the DNA including the target sequence of the MYCN gene. In vitro assays using human neuroblastoma cells showed that 191Pt-MYCN-PIP bound to DNA efficiently and caused DNA damage, decreasing MYCN gene expression and MYCN signals in in situ hybridization analysis, as well as cell viability, especially in MYCN-amplified Kelly cells. 191Pt-MYCN-PIP also induced a substantial increase in cytosolic dsDNA granules and generated proinflammatory cytokines, IFN-α/β, in Kelly cells. Tumor uptake of intravenously injected 191Pt-MYCN-PIP was low and its delivery to tumors should be improved for therapeutic application. The present results provided a potential strategy, targeting the key oncogenes for cancer survival for Auger electron therapy.

Diffuse Pediatric-Type High-Grade Glioma H3-/IDH-wildtype with MYCN Deletion and Constitutional Mismatch Repair Deficiency: Case Presentation

Diffuse pediatric-type high-grade glioma H3-wildtype and IDH-wildtype (pHGG H3/IDH WT) is a heterogeneous entity that is currently defined by a combination of highly malignant morphology, glial or primitive neuroectodermal differentiation, and a number of molecular features. Depending on the DNA methylation profile in pHGG H3/IDH WT, three molecular subgroups are distinguished, one of which (pHGG MYCN) is characterized by amplification of the indicated gene. We report a unique case of pHGG H3/IDH WT in a 19-year-old girl with a deletion of the MYCN gene and constitutional mismatch repair deficiency syndrome.

Necroptosis-related genes are associated with prognostic features of kidney renal clear cell carcinoma

IntroductionRenal clear cell carcinoma is a common type of cancer in the adult urological system. It has a high mortality rate, with 30% of patients developing metastasis and 60% dying within 1–2 years of diagnosis. Recent advancements in tumor immunology and necroptosis have provided new insights into kidney cancer therapy. Therefore, it is crucial to identify potential targets for combining immunotherapy with necroptosis.Materials and methodsUsing the GSE168845 dataset and necroptosis-related genes, we identified genes that are differentially expressed in relation to necroptosis. We analyzed the prognostic value of these genes through differential expression analysis, prognostic analysis, and Cox regression analysis. The expression levels of the MYCN and CDKN2A genes were verified using the GSE53757 dataset. We also examined the association between the differentially expressed genes and clinicopathological features, as well as overall survival in our cohorts. In addition, we constructed a lasso Cox regression model to assess the correlation between these genes and immune score, ICP, and OCLR score. We conducted qRT-PCR to detect the expression of MYCN, CDKN2A, and ZBP1 in different samples of kidney renal clear cell carcinoma (KIRC). The expression levels of these genes were verified in a normal kidney cell line (HK-2 cells) and two KIRC cell lines (786-O, ACHN). The protein levels of MYCN and CDKN2A were detected using immunohistochemistry (IHC). SiRNA was used to silence the expression of MYCN and CDKN2A in the ACHN cell line, and wound healing assays were performed to measure cell migration.ResultsMYCN, CDKN2A, and ZBP1 were identified as necroptosis-related genes with independent prognostic value, leading to the development of a risk prognostic model. The expression of the CDKN2A gene was significantly higher in KIRC tissues compared to normal tissues, while the expression of the MYCN gene was significantly lower in KIRC tissues. The expression of MYCN and CDKN2A was associated with tumor stage, metastasis, and overall survival in our cohort. Furthermore, MYCN, CDKN2A, and ZBP1 were significantly correlated with immune score, ICP, and OCLR score. The expression levels of CDKN2A and ZBP1 were higher in KIRC cells compared to normal kidney cells, while the expression of MYCN was lower in KIRC cells. The protein expression of MYCN and CDKN2A was also higher in KIRC tissues, as confirmed by IHC. The results of the wound healing assay indicated that silencing CDKN2A inhibited cell migration, while silencing MYCN enhanced cell migration.ConclusionsMYCN and CDKN2A are potential targets and valuable prognostic biomarkers for combining immunotherapy with necroptosis in kidney renal clear cell carcinoma. CDKN2A promotes the migration of renal cancer cells, while MYCN inhibits their migration.

Chelators as Antineuroblastomas Agents.

Neuroblastoma represents 8-10 % of all malignant tumors in childhood and is responsible for 15 % of cancer deaths in the pediatric population. Aggressive neuroblastomas are often resistant to chemotherapy. Canonically, neuroblastomas can be classified according to the MYCN (N-myc proto-oncogene protein) gene amplification, a common marker of tumor aggressiveness and poor prognosis. It has been found that certain compounds with chelating properties may show anticancer activity, but there is little evidence for the effect of chelators on neuroblastoma. The effect of new chelators characterized by the same functional group, designated as HLZ (1-hydrazino phthalazine), on proliferation (WST-1 and methylene blue assay), cell cycle (flow cytometry), apoptosis (proliferation assay after use of specific pharmacological inhibitors and western blot analysis) and ROS production (fluorometric assay based on dichlorofluorescein diacetate metabolism) was studied in three neuroblastoma cell lines with different levels of MYCN amplification. The molecules were effective only on MYCN-non-amplified cells in which they arrested the cell cycle in the G0/G1 phase. We investigated the mechanism of action and identified the activation of cell signaling that involves protein kinase C.

P20.10.B CHARACTERIZATION OF NEW PROGNOSTIC MARKERS IN A COHORT OF ADULT SPINAL EPENDYMOMAS

Abstract BACKGROUND Spinal ependymomas (SP-EPN) are group of primary central nervous system tumors recently re-classified by the WHO (CNS5 2021), located in spinal cord. SP-EPN are grade 2-3 tumors, found mainly in cervical and thoracic region, characterized mostly by NF2 mutations and classical histology: grade 3 tumors present also microvascular proliferation, necrosis and high mitotic rate. Recently, a novel subtype was discovered with a unique methylation profile (SP-EPN-MYCN) and characterized by the amplification of MYCN gene, grade 3 histology and a very aggressive behavior. Treatment of these tumors remains challenging: gross-total resection is not often achieved and radiation failed to give sacceptable results. Besides, no prognostic factors except the mitotic index and tumor location, are available, thus, new diagnostic and prognostic biomarkers would be very useful for their accurate classification and management. MATERIAL AND METHODS We have collected 170 patients operated at our Institute from 1997 to 2019 of spinal ependymomas located in the cervical and thoracic region, excluded conus/filum mixopapillary ones. All tumors were revised according to the new WHO CNS5 2021 classification. In a cohort of 50 grade 2/3 SP-EPN we have evaluated by immunohistochemistry the expression of MYCN, H3K27me3, CDK4 and EZH2: the latter is a component of the PRC2 complex, which regulates the histone H3 methylation at Lys27 (K27) and recently described as a transcription factor which controls the expression of MYCN and CDK4 genes. We have also evaluated the amplification of MYCN and CDK4 genes by Copy Number Variation Assay and MLPA techniques. Clinical data were collected and related to the results obtained. RESULTS We have found strong overexpression of MYCN only in grade 3 SP-EPN (3/50), while grade 2 tumors showed low or no expression; amplification of MYCN gene was similarly confirmed by CNV assay and MLPA only in grade 3. Interestingly, also CDK4 overexpression was found only in grade 3 ependymomas (1/50), but it is expressed oppositely to MYCN: in tumors were MYCN is overexpressed, CDK4 was completely absent. Amplification of CDK4 gene was confirmed only in those with CDK4 overexpression. These results were both related to an interesting overexpression of EZH2 only in grade 3 SP-EPN (4/50), while H3K27me3 is expressed in all grades without differences. Finally, in our study both MYCN and CDK4-amplified ependymomas were related to a very aggressive behaviour and poor outcome with high recurrence rate, dissemination and metastases. CONCLUSION In this study we confirmed the prognostic role of MYCN as indicated by WHO classification and we have showed that CDK4 and EZH2 overexpression could be considered as new promising prognostic and predictive factors in SP-EPN. Since many trials with EZH2 and CDK4 inhibitors have been set up in the last years, these analyses could suggest a new therapeutic approach.

Novel protein products encoded by upstream open reading frames of the MYCN gene in pediatric embryonal tumors.

The MYCC and MYCN loci are each associated with two upstream open reading frames (uORFs) potentially encoding small proteins (9-21 kDa). We previously demonstrated that uORFs mrtl and MYCHEX1 of MYCC are translated, and their protein products may function to regulate the expression of the "parent" oncogene. We hypothesized that a similar relationship might exist between MYCN and its two uORFs: MYCNOT and MNOP, and investigated the uORF-encoded proteins associated with MYCN to confirm their expression and intracellular location in neuroblastoma and medulloblastoma cells and tissues. MNOP, MYCNOT, mrtl, and MYCHEX1 were readily detected via reverse transcription polymerase chain reaction and Western blot analysis in tumor cell lines. In tumor tissue, MNOP protein expression was confirmed; however, MCYNOT generated from alternative splicing MYCNΔ1b mRNA was not detected. Immunofluorescence staining of MYCNOT displayed multiple bright foci in the nucleus and diffuse staining in the cytoplasm, suggesting that this small protein may function in both the nucleus and cytoplasm. Upon JQ1 treatment, MYCN, MYCNOT, and mrtl decreased substantially or disappeared completely in three different tumor cell lines. Significant levels of apoptosis were observed in each pediatric embryonal tumor cell line but not T47D breast carcinoma cells, suggesting that response to JQ1 transcriptional inhibition is greatest in tumor cells, which depend on MYC to maintain an undifferentiated phenotype. In conclusion, both MYCN uORF-encoded proteins MNOP and MYCNOT, together with the two MYCC uORF-encoded proteins mrtl and MYCHEX1 were detected simultaneously in tumor cell lines and tumor tissues. These four distinct proteins are translated from the "5'-untranslated region" of MYCN or MYCC mRNAand display consistent distribution patterns within the cell. Additional studies to further elucidate the physiological and pathological roles of these uORF-encoded proteins are warranted, as insights gained could inform new strategies for modulating MYC-family oncogenes by targeting their uORFs.

Triple Therapy with Metformin, Ketogenic Diet, and Metronomic Cyclophosphamide Reduced Tumor Growth in MYCN-Amplified Neuroblastoma Xenografts.

Neuroblastoma (NB) is a childhood cancer in which amplification of the MYCN gene is the most acknowledged marker of poor prognosis. MYCN-amplified NB cells rely on both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) for energy production. Previously, we demonstrated that a ketogenic diet (KD) combined with metronomic cyclophosphamide (CP) delayed tumor growth in MYCN-amplified NB xenografts. The anti-diabetic drug metformin (MET) also targets complex I of the OXPHOS system. Therefore, MET-induced disruptions of mitochondrial respiration may enhance the anti-tumor effect of CP when combined with a KD. In this study, we found that MET decreased cell proliferation and mitochondrial respiration in MYCN-amplified NB cell lines, while the combination of KD, MET, and low-dose CP (triple therapy) also reduced tumor growth and improved survival in vivo in MYCN-amplified NB xenografts. Gene ontology enrichment analysis revealed that this triple therapy had the greatest effect on the transcription of genes involved in fatty acid ß-oxidation, which was supported by the increased protein expression of CPT1A, a key mitochondrial fatty acid transporter. We suspect that alterations to ß-oxidation alongside the inhibition of complex I may hamper mitochondrial energy production, thus explaining these augmented anti-tumor effects, suggesting that the combination of MET and KD is an effective adjuvant therapy to CP in MYCN-amplified NB xenografts.

Linked-read based analysis of the medulloblastoma genome.

Medulloblastoma is the most common type of malignant pediatric brain tumor with group 4 medulloblastomas (G4 MBs) accounting for 40% of cases. However, the molecular mechanisms that underlie this subgroup are still poorly understood. Point mutations are detected in a large number of genes at low incidence per gene while the detection of complex structural variants in recurrently affected genes typically requires the application of long-read technologies. Here, we applied linked-read sequencing, which combines the long-range genome information of long-read sequencing with the high base pair accuracy of short read sequencing and very low sample input requirements. We demonstrate the detection of complex structural variants and point mutations in these tumors, and, for the first time, the detection of extrachromosomal DNA (ecDNA) with linked-reads. We provide further evidence for the high heterogeneity of somatic mutations in G4 MBs and add new complex events associated with it. We detected several enhancer-hijacking events, an ecDNA containing the MYCN gene, and rare structural rearrangements, such a chromothripsis in a G4 medulloblastoma, chromoplexy involving 8 different chromosomes, a TERT gene rearrangement, and a PRDM6 duplication.

Integrative analysis of multi-omics data for discovery of ferroptosis-related gene signature predicting immune activity in neuroblastoma.

Immunotherapy for neuroblastoma remains unsatisfactory due to heterogeneity and weak immunogenicity. Exploring powerful signatures for the evaluation of immunotherapy outcomes remain the primary purpose. We constructed a ferroptosis-related gene (FRG) signature by least absolute shrinkage and selection operator and Cox regression, identified 10 independent prognostic FRGs in a training cohort (GSE62564), and then verified them in an external validation cohort (TCGA). Associated with clinical factors, the signature accurately predicts overall survival of 3, 5, and 10years. An independent prognostic nomogram, which included FRG risk, age, stage of the International Neuroblastoma Staging System, and an MYCN status, was constructed. The area under the curves showed satisfactory prognostic predicting performance. Through bulk RNA-seq and proteomics data, we revealed the relationship between hub genes and the key onco-promoter MYCN gene and then validated the results in MYCN-amplified and MYCN-non-amplified cell lines with qRT-PCR. The FRG signature significantly divided patients into high- and low-risk groups, and the differentially expressed genes between the two groups were enriched in immune actions, autophagy, and carcinogenesis behaviors. The low-risk group embodied higher positive immune component infiltration and a higher expression of immune checkpoints with a more favorable immune cytolytic activity (CYT). We verified the predictive power of this signature with data from melanoma patients undergoing immunotherapy, and the predictive power was satisfactory. Gene mutations were closely related to the signature and prognosis. AURKA and PRKAA2 were revealed to be nodal hub FRGs in the signature, and both were shown to have significantly different expressions between the INSS stage IV and other stages after immunohistochemical validation. With single-cell RNA-seq analysis, we found that genes related to T cells were enriched in TNFA signaling and interferon-γ hallmark. In conclusion, we constructed a ferroptosis-related gene signature that can predict the outcomes and work in evaluating the effects of immunotherapy.