MYCN Amplification Research Articles

Genetic Risk Profiling Reveals Altered Glycosyltransferase Expression as a Predictor for Patient Outcome in Neuroblastoma

Background/Objectives: Neuroblastoma is a highly aggressive pediatric cancer that arises from immature nerve cells and exhibits a broad spectrum of clinical presentations. While low- and intermediate-risk neuroblastomas often have favorable outcomes, high-risk neuroblastomas are associated with poor prognosis and significant treatment challenges. The complex genetic networks driving these high-risk cases remain poorly understood. This study aims to investigate differences in gene expression patterns that may contribute to disease outcomes. Methods: We employed an in silico approach to analyze a cohort of 493 neuroblastoma tumor samples that underwent mRNA sequencing (GSE49711). This dataset was reanalyzed in depth with a non-hypothesis-driven approach to identify the expression patterns and regulatory mechanisms associated with a poor prognosis. Results: By exploring global gene expression and the integration of clinical parameters, we stratified the samples into two groups with highly distinct gene expression profiles. MYCN amplification emerged as a major driver not only of poor prognosis but also of specific gene regulatory patterns. Notably, tumors with MYCN amplification exhibited the strong regulation of immune response genes and less immune infiltration, suggesting potential immune evasion. However, while we observed only minor changes in immune checkpoint expression, there was a strong modulation of glycosyltransferase genes in MYCN-amplified tumors. Using this information, we were able to construct a risk profile based on 12 glycosylation-related genes, which correlates with the survival outcomes of neuroblastoma patients. Conclusions: This study highlights the role of MYCN amplification in driving a poor prognosis in neuroblastoma through the regulation of immune response and glycosylation-related genes. Based on this finding, we developed a genetic risk profile that correlates with survival outcomes in neuroblastoma patients.

Intradural extramedullary double primary ependymoma and meningioma rare condition: Case report and literature review.

Ependymomas are commonly prevalent intramedullary neoplasms in adults, with hardly any cases of exophytic extramedullary ependymoma being reported. Meningiomas, on the contrary, are one of the most common intradural extramedullary (IDEM) tumors. However, the occurrence of both IDEM tumors simultaneously is extremely rare. A 63-year-old female who presented with pain and numbness in both lower limbs, and symptoms rapidly progressed over the past 5 months. Based on the patient's clinical symptoms and imaging features, we conducted pathological examination and genetic testing, ultimately confirming that the patient had IDEM double primary ependymoma and meningioma. Surgery was performed to remove double spinal tumors, decompress spinal nerve roots, and perform laminectomy, and she was treated with electrocardiogram monitoring, antibiotics, hemostasis, and antiedema therapy. Histopathology confirmed World Health Organization grade II ependymoma at L2 and World Health Organization grade I meningioma at T12-L1. MYCN amplification and other genetic alterations were absent. Postoperative recovery was favorable, with no recurrence at 6-month follow-up. This is the first reported case of IDEM double primary ependymoma and meningioma, highlighting the rarity of such cases and the importance of thorough diagnostic workup and surgical excision for IDEM tumors. Genetic analysis adds to the understanding of these rare tumors and guides management strategies.

Switching Drivers: Epigenetic Rewiring to Genetic Progression in Glioma.

IDH-mutant low-grade gliomas (LGGs) are slow-growing brain tumors that frequently progress to aggressive high-grade gliomas that have dismal outcomes. In a recent study, Wu and colleagues provide critical insights into the mechanisms underlying malignant progression by analyzing single-cell gene expression and chromatin accessibility across different tumor grades. Their findings support a two-phase model: in early stages, tumors are primarily driven by oligodendrocyte precursor-like cells and epigenetic alterations that silence tumor suppressors like CDKN2A and activate oncogenes such as PDGFRA. As the disease advances, the tumors become sustained by more proliferative neural precursor-like cells, where genetic alterations, including PDGFRA, MYCN, and CDK4 amplifications and CDKN2A/B deletion, drive tumor progression. The study further highlights a dynamic regulation of interferon (IFN) signaling during progression. In low-grade IDH-mutant gliomas, IFN responses are suppressed through epigenetic hypermethylation, which can be reversed with DNMT1 inhibitors or IDH inhibitors, leading to reactivation of the IFN pathway. In contrast, higher-grade gliomas evade IFN signaling through genetic deletions of IFN gene clusters. These findings emphasize a broader epigenetic-to-genetic shift in oncogenic regulation that drives glioma progression, provides a valuable framework for understanding the transition from indolent tumors to lethal malignancies, and has implications for therapy and clinical management.

Clinical and Pathological Features of a Schwannoma Harboring a SH3PXD2A::HTRA1 Gene Fusion in a Pre-pubescent Patient

An 11-year-old girl presented with a soft tissue lesion on the dorsal aspect of the left middle finger. Ultrasound imaging demonstrated a 2.8 cm × 0.8 cm × 0.8 cm lesion overlying the dorsal aspect of the base of the digit near the metacarpophalangeal joint. The patient’s past medical history is remarkable for neuroblastoma, diagnosed at 9 months of age, with no MYCN amplification or 1p loss. We report a pediatric schwannoma harbouring a SH3PXD2A::HTRA1 gene fusion with a distinctive serpentine histology. The lesion consisted of well-circumscribed nodules surrounded by thin EMA-positive perineural capsules. Each nodule was composed of lesional cells arranged in short fascicles with occasional clefting and a distinct “serpentine” palisading pattern. The lesion demonstrated Antoni A regions with Verocay body formation. No significant Antoni B areas were seen. The lesional Schwannian cells were bland with elongated and tapered nuclei, showing strong and diffuse positivity for S100. This pre-pubescent girl (Tanner Stage 2) is currently the youngest reported case of fusion-positive schwannoma. In addition, she has a significant prior history of a malignant neoplasm, and the lesion arose in an appendicular location.

Application of modified Curie and SIOPEN skeleton scoring systems in 18F-AlF-NOTA-octreotide PET/CT for neuroblastoma.

The study aimed to explore the role of fluorine-18-aluminum fluoride-1,4,7-triazacyclononane-1,4,7-triacetic acid-octreotide (18F-OC) positron emission tomography/computed tomography (PET/CT) in neuroblastoma (NB) and compared it with Iodine-123 labeled metaiodobenzylguanidine (123I-MIBG) scintigraphy with single photon emission computed tomography/computed tomography (SPECT/CT), as well as to investigate the feasibility of the modified Curie scoring system and International Society of Pediatric Oncology Europe Neuroblastoma (SIOPEN) skeleton scoring system applied in 18F-OC PET/CT. Patients with pathologically confirmed NB underwent 123I-MIBG scintigraphy with SPECT/CT and 18F-OC PET/CT according the standard imaging protocols. The interval between the two imaging techniques ranged from 0 to 22days (median interval: 9days). The number of lesions in modified Curie scoring system and SIOPEN skeleton scoring system applied on 123I-MIBG SPECT/CT and 18F-OC PET/CT was compared. A total of 50 NB patients (male: female = 25:25) with a median age of 62-month-old were enrolled. 123I-MIBG and 18F-OC imaging were positive in 22 patients and negative in 27 patients. 1 patient had positive 18F-OC but negative 123I-MIBG results (p = 1.000). In lesion-based analysis, 18F-OC PET/CT revealed more positive lesions than 123I-MIBG scintigraphy with SPECT/CT (57 vs. 44, p < 0.001), regardless of bone/bone marrow lesions (43 vs. 37, p = 0.031) or soft tissue lesions (14 vs. 7, p = 0.016). The Curie scores of the two imaging techniques showed a significant difference (p = 0.047), whereas no statistic difference for SIOPEN scores (p = 0.688). The Curie and SIOPEN scores were significantly higher in patients with the presence of MYCN amplification or positive bone marrow puncture result (p < 0.05). 18F-OC could be used in the evaluation of NB, and the modified Curie scoring system could be used to semi-quantify the disease extent of NB in 18F-OC PET/CT.

Molecular diversity of embryonic-type neuroectodermal tumors arising from testicular germ cell tumors.

Embryonic-type neuroectodermal tumors (ENTs) arising from testicular germ cell tumors (GCTs) is a relatively common type of somatic transformation in GCTs with poor prognosis and limited therapeutic options, particularly when patients develop disease recurrence or metastasis. Knowledge of key events driving this transformation is limited to the paucity of comprehensive genomic data. We performed a retrospective database search in a CLIA- and CAP-certified laboratory for testicular GCT-derived ENTs that had previously undergone NGS-based comprehensive genomic profiling during the course of clinical care. Clinicopathological and genomic data were centrally re-reviewed. Here we report the molecular features of 10 ENTs of testicular GCT origin. All tumors harbored gain of chromosome 12p, often with KRAS, CCND2 and KMD5A co-amplification, supporting a germ cell origin. The tumors were microsatellite stable and exhibited low tumor mutational burden. Three tumors (30%) exhibited MYCN or MYC amplification with co-occurring inactivation of the p53 pathway via either TP53 mutations or MDM2 amplification in two tumors. Three additional tumors (30%) had activation of the PI3K pathway via PIK3CA and PIK3CG mutations or PIK3C2B amplification; one tumor with co-occurring CDK4 amplification. Gene rearrangements were detected in three tumors (30%), with novel BRD4-MAU2 and BCOR-CLIP2 fusions as well as an internal truncating ATRX rearrangement, respectively. In summary, ENTs arising from GCTs are molecularly heterogeneous; however, a large fraction of testicular ENTs could be stratified by two distinct sets of genetic alterations, including MYCN/MYC amplification with concurrent suppression of the p53 pathway, and activation of the PI3K pathway with co-occurring CDK4 amplification. Moreover, the novel gene fusions identified in a subset of testicular GCT-derived ENTs overlap with molecularly defined tumors of embryonic-type neuroectodermal features in the central nervous system, indicating the potential common driving events for tumorigenesis from different anatomic sites.

Multicellular model of neuroblastoma proposes unconventional therapy based on multiple roles of p53.

Neuroblastoma is the most common extra-cranial solid tumour in children. Over half of all high-risk cases are expected to succumb to the disease even after chemotherapy, surgery, and immunotherapy. Although the importance of MYCN amplification in this disease is indisputable, the mechanistic details remain enigmatic. Here, we present a multicellular model of neuroblastoma comprising a continuous automaton, discrete cell agents, and a centre-based mechanical model, as well as the simulation results we obtained with it. The continuous automaton represents the tumour microenvironment as a grid-like structure, where each voxel is associated with continuous variables such as the oxygen level therein. Each discrete cell agent is defined by several attributes, including its cell cycle position, mutations, gene expression pattern, and more with behaviours such as cell cycling and cell death being stochastically dependent on these attributes. The centre-based mechanical model represents the properties of these agents as physical objects, describing how they repel each other as soft spheres. By implementing a stochastic simulation algorithm on modern GPUs, we simulated the dynamics of over one million neuroblastoma cells over a period of months. Specifically, we set up 1200 heterogeneous tumours and tracked the MYCN-amplified clone's dynamics in each, revealed the conditions that favour its growth, and tested its responses to 5000 drug combinations. Our results are in agreement with those reported in the literature and add new insights into how the MYCN-amplified clone's reproductive advantage in a tumour, its gene expression profile, the tumour's other clones (with different mutations), and the tumour's microenvironment are inter-related. Based on the results, we formulated a hypothesis, which argues that there are two distinct populations of neuroblastoma cells in the tumour; the p53 protein is pro-survival in one and pro-apoptosis in the other. It follows that alternating between inhibiting MDM2 to restore p53 activity and inhibiting ARF to attenuate p53 activity is a promising, if unorthodox, therapeutic strategy. The multicellular model has the advantages of modularity, high resolution, and scalability, making it a potential foundation for creating digital twins of neuroblastoma patients.

Iwilfin (eflornithine) approved by the FDA as the first and only oral maintenance therapy for high-risk neuroblastoma in adult and pediatric patients: Narrative review.

Neural crest progenitor cells give rise to neuroblasts, the growing nerve cells of the sympathetic nervous system. These cells can undergo changes leading to neuroblastoma, a malignancy responsible for 15% of all pediatric cancer-related deaths. The molecular pathogenesis of this pediatric cancer involves complex genetic alterations, such as MYCN amplification, chromosomal abnormalities, and gene expression changes. Despite aggressive therapies, survival rates for children with high-risk neuroblastoma (HRNB) have not improved significantly compared to those with less severe forms of the disease. This highlights the challenge of managing HRNB and underscores the need for new, effective treatments. A comprehensive treatment regimen, including immunotherapy, radiation therapy, myeloablative chemotherapy, and surgical removal, has been employed to achieve remission in HRNB patients. While dinutuximab beta immunotherapy is an effective and widely used treatment, it has several potential side effects that must be carefully monitored. New drugs are being developed to reduce these side effects without compromising efficacy. One such drug is DL-alpha-difluoromethylornithine (DFMO), approved by the FDA under the brand name Iwilfin. Numerous clinical trials have shown that DFMO, when used as maintenance therapy, significantly improves event-free survival and overall survival in neuroblastoma patients. However, DFMO has adverse effects that require continuous monitoring. Further research is needed to minimize these side effects and improve its efficacy, particularly in addressing resistance caused by long-term use.

Development of Dual Aurora‐A and Aurora‐B Degrading PROTACs for MYCN‐Amplified Neuroblastoma

In neuroblastoma, MYCN amplification is associated with survival rates of &lt;50%. Overexpression of the mitotic kinases Aurora‐A and Aurora‐B are also associated with low survival and exacerbate the oncogenic effects of N‐Myc. As N‐Myc is stabilized by Aurora‐A, Aurora‐A targeting proteolysis targeting chimeras (PROTACs) have been developed that reduce Aurora‐A and N‐Myc levels. However, simultaneous degradation of N‐Myc, Aurora‐A, and Aurora‐B has not been previously achieved. Given the contributions of both Aurora kinases to MYCN‐amplified neuroblastoma, we designed PROTACs capable of degrading both Aurora‐A and Aurora‐B. Dual‐degrading PROTACs dAurAB2 and dAurAB5 potently degraded Aurora‐A (DC50 = 59 nM and 8.8 nM, respectively) and Aurora‐B (DC50 = 39 nM and 6.1 nM), eliminated 89% ‐ 97% of Aurora‐A and Aurora‐B, and reduced N‐Myc levels by 38% and 45% in MYCN‐amplified IMR32 neuroblastoma cells. Global proteomics screening revealed that while dAurAB2 demonstrated good selectivity, dAurAB5 downregulated additional targets including threonine tyrosine kinase (TTK). Interestingly, TKK is also associated with MYCN‐amplified neuroblastoma, and multi‐target PROTAC dAurAB5 reduced the viability of neuroblastoma IMR32 cells by 55% at 24 hours. The development of dAurAB2 and dAurAB5 generates new modalities for inhibiting the oncogenic activities of Aurora‐A, Aurora‐B, N‐Myc, and TTK in neuroblastoma and other cancers.

Abstract B015: CSF liquid biopsies reveal disease status and resistance mechanisms in children undergoing targeted therapy for malignant brain tumors

Abstract BACKGROUND: Patients with relapsed or refractory brain tumors face poor outcomes due to the aggressive nature of their disease. Recent studies suggest that administration of cell cycle inhibitors may help slow tumor progression. In the Phase I trial SJDAWN (NCT03434262) conducted at St. Jude Children’s Research Hospital, CDK4/6 inhibitor therapy (ribociclib) was assessed for safety and efficacy in such patients. To understand how tumors evolve in response to this treatment, longitudinal CSF liquid biopsies from SJDAWN were retrospectively analyzed by cell-free DNA (cfDNA) sequencing. METHODS: CSF samples (n=143) were collected by lumbar puncture from 34 of 68 (50%) patients before and during treatment. cfDNA was extracted and analyzed using an innovative DNA methylation-based platform that concurrently detects tumor-associated copy number alterations, classifies tumor subtypes, and distinguishes malignant from non-malignant methylation signatures. cfDNA inputs ranged from 50 to 500 pg. CSF profiles were compared to patient-matched tumor tissue DNA methylation arrays and subsequently correlated with MRI findings at corresponding timepoints. RESULTS: Circulating tumor DNA (ctDNA) was detected in 29 of 34 (85%) patients, yielding accurate tumor classification even in the setting of diploid genomes. CNV profiles from CSF liquid biopsies strongly correlated with patient-matched tumor tissue methylation arrays and often revealed genetic events not previously captured in the tumor tissue. Resistance to CDK4/6 inhibition was characterized by focal amplification of positive cell cycle regulators, including CDK6, CCND2, MYCN, and RRM2 that were detectable in CSF ∼6 months prior to radiographic progression. Remarkably, three high-risk Group 3/4-MB patients were progression-free for &amp;gt;1.5 years. The trend of their longitudinal cfDNA profiles varied: one had persistent ctDNA detected throughout therapy with resistance emerging at ∼1.5 years; another had undetectable ctDNA for ∼2 years before reemergence of tumor; and the third remained MRD-negative, potentially owing to reduced ctDNA release resulting from CDK4/6 inhibition. Methylation changes in response to treatment were also observed, providing unprecedented insights into epigenetic landscapes associated with tumor evolution concurrent with targeted therapy. CONCLUSIONS: Methylation-based analysis of CSF liquid biopsies provides an ultra-sensitive and comprehensive method for serial disease monitoring. Even with sub-nanogram cfDNA inputs, tumor reemergence and resistance mechanisms were detectable months before MRI changes, enabling timely clinical intervention. Resistance to CDK4/6 inhibition is mediated by focal amplification of cell cycle regulators, suggesting alternative approaches will be required to overcome this path of resistance. CDK4/6 inhibitor therapy offers clinical benefit to a specific subset of high-risk Group 3/4-MB patients, motivating expanded evaluation of this population in future trials. Citation Format: Katie Han, Kyle S Smith, Anna Kostecka, Sandeep Dhanda, Hong Lin, Margit Mikkelsen, Giles W Robinson, Paul A Northcott. CSF liquid biopsies reveal disease status and resistance mechanisms in children undergoing targeted therapy for malignant brain tumors [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B015.

EPCO-54. MULTIOMIC ANALYSIS OF SPINAL EPENDYMOMAS REVEALS MECHANISMS OF TUMOR AGGRESSIVENESS WITH MYCN AMPLIFICATION

Abstract Grade-3 spinal ependymomas, particularly those harboring MYCN amplification, have dismal prognoses despite optimal treatment regimens. MYCN functions as a transcription factor (TF) to drive the expression of key tumor driver genes. Our aim is to identify mechanisms underlying tumor aggressiveness and treatment resistance in MYCN-amplified ependymomas. MYCN amplification in spinal ependymomas was identified with custom next generation sequencing panels and bulk methylation analysis. We then conducted paired single-nucleus RNA (snRNA-seq) and chromatin accessibility (snATAC-seq) sequencing of grade-2 (n=2, 1 patient) and grade-3 spinal ependymomas (n=6, 6 patients, 4 MYCN-amplified). Comparator snRNA-seq datasets (grade-2/grade-3; 8/2) were included from a publicly available dataset (GSE163686; all non-MYCN-amplified). Canonical cell classes were identified with a combination of cluster- and cell-based strategies. Downstream analysis was conducted using R 4.3 (Seurat, Signac, clusterProfiler, CellChat), and Python 3.11 (scanpy) packages. We identified ependymoma tumor cells distinct from canonical immune cell classes with gene expression analysis and confirmed a distinct copy-number-variation pattern in these cells with chromosome 1 and 2p gain, and chromosome 6p and 22q loss. MYCN-amplified tumor cells (compared to non-MYCN-amplified grade2/3 tumor cells) showed increased differential accessibility at genes including VEGFA. Increased MYCN TF activity was confirmed with gene overexpression of downstream targets including NOTCH3, CREB5, PLK1, and VEGFA. Other genes that were highly accessible and overexpressed in MYCN-amplified tumor cells were related to oxidative stress, differentiation, and anti-apoptotic pathways. MYCN-amplified tumor cells modified the tumor microenvironment with increased VISTA signaling between MYCN-amplified tumor cells and tumor-associated macrophages, creating an immunosuppressive environment. We confirmed these effects by observing a higher proportion of M2-like macrophages in MYCN-amplified tumors (29.6% vs 18.2%). In conclusion, MYCN amplification in spinal ependymomas upregulates tumor cell proliferation, angiogenesis, and M2-like macrophage recruitment/programming.

EPCO-05. GENOMIC AND CLINICAL INSIGHTS FROM A LARGE-SCALE STUDY OF 172 RETINOBLASTOMAS

Abstract Retinoblastoma, a pediatric ocular cancer, is the only central nervous system tumor visible without specialized tools, readily detectable with the naked eye. This rare childhood cancer is mainly driven by mutations in RB1 or MYCN amplification, posing significant challenges in pediatric oncology. We conducted whole exome sequencing on 172 retinoblastoma tumors along with matched blood DNA from 171 patients, which included 119 unilateral, 49 bilateral, and three trilateral cases. Our analysis identified pathogenic variants in RB1 in 76 patients and MYCN duplication in 79 cases, highlighting their crucial roles in the disease. Additionally, we found mutations in RPTOR and TSC2, components of the mTOR signaling pathway, in two patients. Notably, our copy number alteration analysis revealed recurrent arm-level changes, including gains on 1q (23.3%), 2p (20.9%), and especially 6p (41.3%), along with other changes such as 5p gain (8.1%), 17q gain (8.1%), and 16q loss (5.8%). Our findings link these genetic alterations to specific clinical features; for instance, 6p gains were associated with Rubeosis, subretinal seeds, and tumor LC beyond, while 17q gains were linked to Rubeosis. Furthermore, gene collapsing techniques identified MATR3 and CYP4F2 as additional novel retinoblastoma-related genes, and module identification analysis pinpointed retinoblastoma-associated modules including HLA-DRB1-CBL, LPAR2, RB1, and TRIM28. Our study, the largest cohort of its kind, underscores the importance of next-generation sequencing from understudied populations in uncovering the complex genetic landscape of retinoblastoma, emphasizing the potential of precision medicine to enhance therapeutic strategies and improve outcomes for retinoblastoma patients.

PATH-47. AGE-, SEX-, AND GRADE-ASSOCIATED MOLECULAR DIFFERENCES IN A MULTI-INSTITUTIONAL COHORT OF CHOROID PLEXUS TUMORS

Abstract BACKGROUND Choroid plexus tumors (CPT) are central nervous system tumors where prognosis has been correlated to histologic grade, with aggressive clinical behavior and reduced survival rate in higher grade cases. Here, we present a genomic survey of a large multi-institutional cohort of CPTs across all grades. MATERIAL AND METHODS Fifty-eight CPT samples were analyzed by comprehensive genomic profiling (CGP) by a hybrid capture-based DNA sequencing platform (FoundationOne®CDx). Pathology reports, histopathology reviews, and patient clinical data were assessed. RESULTS Our cohort included 11 choroid plexus papillomas (CPP; grade 1), 16 atypical CPP (grade 2), and 31 choroid plexus carcinomas (CPC; grade 3). Males were slightly more affected than females (31 vs. 27 cases). Median patient age at tissue sampling was 11 years (range: 1–72 years). Median age of patients with CPC was younger at 3 years compared CPP at 34 years (p &amp;lt; 0.01). Genomic analyses identified three frequent, mutually exclusive mutations in TP53, TERTp, and DAXX. TP53 mutations (n = 15, 26%) were significantly more prevalent in CPC (n=14 CPC, p=0.0003), and were identified predominantly in children (n=12 CPC at &amp;lt;20 years). By contrast, TERT promoter mutations (TERTp; n = 5, 8.6%) were identified exclusively in CPP (p=0.019), predominantly grade 2 (4 of 5 cases), and exclusively in adult patients, with a median age of 48 years. DAXX mutations (n=4, 6.9%) were more common in CPC (n=3). Other alterations included PTEN (n=4, all adults) and MYCN amplification (n=3, all children &amp;lt;6 years), both exclusively in CPC. In 21 cases, including 9 out of the 11 Grade 1 CPP, no known cancer mutation was detected. CONCLUSION This study provides comprehensive genomic profiling of CPTs, highlighting distinct genetic alterations associated with different histological grades and patient demographics. These findings may inform future research and therapeutic strategies targeting specific molecular pathways in CPTs.

PATH-13. MOLECULAR AND BIOLOGY CORRELATIVE RESULTS FROM THE PHASE 1B TRIAL OF UNESBULIN (PTC596) WITH RADIOTHERAPY IN CHILDREN NEWLY-DIAGNOSED WITH DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) AND HIGH-GRADE GLIOMA (HGG): A REPORT FROM THE COLLABORATIVE NETWORK FOR NEURO-ONCOLOGY CLINICAL TRIALS (CONNECT)

Abstract BACKGROUND Within the CONNECT1702 trial of unesbulin (tubulin-binding agent causing BMI-1 modulation) with radiotherapy in patients with DIPG/HGG, tumor tissue was analyzed at diagnosis and/or autopsy for biology correlatives, enabling comprehensive, longitudinal molecular characterization. METHODS Whole genome sequencing, RNA-sequencing, and Western blot analyses were performed on tumor, normal brain, and peripheral blood mononuclear cells (PBMCs). RESULTS Tumor tissue from diagnosis (n=11) and/or autopsy (n=7) was analyzed for 16 patients (DIPG=9; HGG=7). Western blot analyses demonstrated 1) BMI-1 overexpression in baseline tumor compared to normal brain and 2) primary on-target effect of BMI1-modulation causing M-phase mitotic arrest when comparing pre- and post-treated PBMCs (increased H3S10-phosphorylation). Among 9 DIPG patients, alterations in the following genes were identified: H3-3A (H3.3) (n=5), H3C2 (H3.1) (n=2, both with co-occurring ACVR1 and PIK3CA mutations), TP53 (n=5), ATRX (n=2), and PPM1D (n=2). Tumors from two longer DIPG survivors were H3.1-, ACVR1-mutant (overall survival [OS]=26 months) and MET-amplified (OS=18 months); one patient with H3.3K27M-DIPG had germline MSH6 mutation (Lynch syndrome) (OS=12.6 months). Among 7 HGG patients, alterations in the following genes were identified: H3-3A (n=5), TP53 (n=3), NF1 (n=3), PDGFRA (n=1), EGFR (n=1), and PIK3CA (n=1). A BRAFV600E mutation was observed in the thalamic HGG patient (H3.3K27M) with greatest progression-free survival (&amp;gt;23 months). Early leptomeningeal metastases occurred in one DIPG patient with BCOR alteration and one HGG patient, with MYCN amplification identified in the spinal metastasis, but not intracranial disease. From gene set enrichment analyses, pathways enriched in patients with longer survival included telomerase regulation and ATP synthesis, whereas pathways enriched in patients with shorter survival included interneuron development, neuron semaphorin signaling, and gamma aminobutyric signaling. CONCLUSIONS Comprehensive molecular profiling of diagnostic and post-mortem tumor tissue provides valuable early biological insight, elucidating potential genomic biomarkers predictive of outcome and response to M-phase alteration and BMI-1 modulation in DIPG/HGG.

Simultaneous single-nucleus RNA sequencing and single-nucleus ATAC sequencing of neuroblastoma cell lines

Neuroblastoma is the most common extracranial solid tumor in children, and a leading cause of childhood cancer deaths. All neuroblastomas arise from neural crest-derived sympathetic neuronal progenitors, but numerous mutations, the most common of which is MYCN amplification, give rise to these lesions. Epigenetic aberrations also play a role in oncogenesis and tumor progression. To better understand biologic diversity of neuroblastomas, we performed joint single-nucleus ATAC sequencing and single-nucleus RNA sequencing on six neuroblastoma cell lines, three of which are MYCN amplified. After standard filtering for high-quality nuclei, we obtained chromatin accessibility and transcript abundance data from 41,733 neuroblastoma tumor cells. Preliminary analysis reveals significant diversity in chromatin landscape and gene expression across neuroblastoma cell lines. This dataset is a valuable resource for studying the transcriptional and epigenetic mechanisms of this deadly childhood disease.

Event-free survival in neuroblastoma with MYCN amplification and deletion of 1p or 11q may be associated with altered immune status

BackgroundNeuroblastoma exhibits substantial heterogeneity, which is intricately linked to various genetic alterations. We aimed to explore immune status in the peripheral blood and prognosis of patients with neuroblastoma with different genetic characteristics.MethodsWe enrolled 31 patients with neuroblastoma and collected samples to detect three genetic characteristics. Peripheral blood samples were tested for immune cells and cytokines by fluorescent microspheres conjugated with antibodies and flow cytometry. Event-free survival (EFS) was analyzed using the Kaplan‒Meier method.ResultsTwenty-two patients had genetic aberrations, including MYCN amplification in 6 patients, chromosome 1p deletion in 9 patients, and chromosome 11q deletion in 14 patients. Two genetic alterations were present in seven patients. The EFS was worse in patients with MYCN amplification or 1p deletion than in the corresponding group, whereas 11q deletion was a prognostic factor only in patients with unamplified MYCN. Changes in immune status revealed a decrease in the proportion of T cells in blood, and an increase in regulatory T cells and immunosuppression-related cytokines such as interleukin (IL)-10. The EFS of the IL-10 high-level group was lower than that of the low-level group. Patients with concomitant genetic alterations and a high level of IL-10 had worse EFS than other patients.ConclusionsPatients with neuroblastoma characterized by these genetic characteristics often have suppressed T cell response and an overabundance of immunosuppressive cells and cytokines in the peripheral blood. This imbalance is significantly associated with poor EFS. Moreover, if these patients show an elevated levels of immunosuppressive cytokines such as IL-10, the prognosis will be worse.

Golgi-localized Ring Finger Protein 121 is necessary for MYCN-driven neuroblastoma tumorigenesis

MYCN amplification predicts poor prognosis in childhood neuroblastoma. To identify MYCN oncogenic signal dependencies we performed N-ethyl-N-nitrosourea (ENU) mutagenesis on the germline of neuroblastoma-prone TH-MYCN transgenic mice to generate founders which had lost tumorigenesis. Sequencing of the mutant mouse genomes identified the Ring Finger Protein 121 (RNF121WT) gene mutated to RNFM158R associated with heritable loss of tumorigenicity. While the RNF121WT protein localised predominantly to the cis-Golgi Complex, the RNF121M158R mutation in Helix 4 of its transmembrane domain caused reduced RNF121 protein stability and absent Golgi localisation. RNF121WT expression markedly increased during TH-MYCN tumorigenesis, whereas hemizygous RNF121WT gene deletion reduced TH-MYCN tumorigenicity. The RNF121WT-enhanced growth of MYCN-amplified neuroblastoma cells depended on RNF121WT transmembrane Helix 5. RNF121WT directly bound MYCN protein and enhanced its stability. High RNF121 mRNA expression associated with poor prognosis in human neuroblastoma tissues and another MYC-driven malignancy, laryngeal cancer. RNF121 is thus an essential oncogenic cofactor for MYCN and a target for drug development.

A temporal (phospho-)proteomic dataset of neurotrophic receptor tyrosine kinase signalling in neuroblastoma

Neurotrophic receptor tyrosine kinases (TrkA, TrkB, TrkC), despite their homology, contribute to the clinical heterogeneity of the childhood cancer neuroblastoma. TrkA expression is associated with low-stage disease and is often seen with spontaneous tumour regression. Conversely, TrkB is present in unfavourable neuroblastomas that often harbour amplification of the MYCN oncogene. The role of TrkC is less clearly defined, although some studies suggest its association with a favourable outcome. Understanding the differences in activity of Trk receptors that drive divergent clinical phenotypes as well as the influence of MYCN amplification on downstream Trk receptor signalling remains poorly understood. Here, we present a comprehensive label-free mass spectrometry-based total proteomics and phosphoproteomics dataset (432 raw files with FragPipe search outputs; available on PRIDE with accession number PXD054441) where we identified and quantified 4,907 proteins, 16,744 phosphosites and 5,084 phosphoproteins, derived from NGF/BDNF/NT-3 treated TrkA/B/C-overexpressing neuroblastoma cells with differential MYCN status. Analysing our dataset offers valuable insights into TrkA/B/C receptor signalling in neuroblastoma and its modulation by MYCN status; and holds potential for advancing therapeutic strategies in this challenging childhood cancer.

Poly (ADP-ribose) polymerase inhibitor sensitized DNA damage caused by an alkylating pyrrole-imidazole polyamide targeting MYCN in neuroblastoma cells

MYCN amplification (MYCN-amp) is a significant prognostic factor and early genetic marker of high-risk neuroblastoma (NB). MYCN induces the DNA damage response (DDR) and modulates the insensitivity of NB cells to Poly (ADP-ribose) polymerase (PARP) inhibitors. We previously reported that CCC-002, a DNA-alkylating agent conjugated with pyrrole-imidazole polyamide targeting MYCN, inhibits NB cell proliferation and induces DNA damage signaling. In this study, we investigated the synergistic effects of CCC-002 and PARP inhibitors on MYCN-amp NB cells. Combination treatment with PARP inhibitors significantly enhanced the sensitivity of MYCN-amp NB cells to CCC-002. DNA damage signals, such as phosphorylation of H2AX and RPA32 elicited after CCC-002 treatment, were further enhanced by PARP inhibitors, as detected through western blotting and immunofluorescence analyses. The potent cytotoxicity of this combination treatment was confirmed by the significant increase in the subG0-G1 phase. Although MYCN knockdown showed no synergistic effect with PARP inhibitors, fluorescence in situ hybridization and quantitative PCR analyses indicated that PARP inhibitors enhanced the effect of CCC-002 to reduce MYCN copy number and suppress its expression. Overall, our study provides novel insights into a therapeutic approach that combines CCC-002 and PARP inhibition to effectively induce DNA damage and apoptosis in MYCN-amp NB cells.

Enhancing Retinoic Acid-mediated Effects Through Inhibition of CYP26A1, CYP26B1 and HGF Signaling in Neuroblastoma Cells.

Retinoic acid (RA) induces tumor cell differentiation in diseases like acute promyelocytic leukemia or high-risk neuroblastoma. However, the formation of resistant cells, which results from dysregulation of different signaling pathways, limits therapy success. The present study aimed to characterize basic regulatory processes induced by the application of RA in human neuroblastoma cells, to identify therapeutic targets independent of the often amplified oncogene MYCN. In MYCN-amplified Kelly and MYCN non-amplified SH-SY5Y cells, different assays were employed to quantify the viability and cytotoxicity, while RA-mediated expression changes were examined using genome-wide gene expression analysis followed by quantitative PCR. Enzyme-linked immunoabsorbent assays (ELISA) and western blots were used to determine the levels or activation of the examined proteins. In Kelly cells, treatment with 5 μM RA for 3 days significantly reduced the cell number due to attenuated proliferation, while SH-SY5Y cells were less responsive. An up-regulation of the RA-metabolizing enzymes CYP26A1 and CYP26B1 was observed in both cell lines, and co-treatment with the selective CYP26 inhibitor talarozole markedly decreased cell viability. When RA and ketoconazole, which inhibits CYP26 as well as RA-degrading CYP3A enzymes, were co-administered, not only cell survival was impaired in both cell lines, but also the release of hepatocyte growth factor (HGF). Accordingly, co-application of the c-Met inhibitor tepotinib and RA or ketoconazole substantially decreased cell viability. Independent of MYCN amplification, inhibitors of RA metabolism or HGF signaling might prevent the emergence of RA-resistant neuroblastoma cells when co-applied with RA.