Peripheral neuroblastic tumors consist of a variety of neoplasms ranging from benign to highly malignant, with significant intertumoral and intratumoral heterogeneity. The current gold standard in classification, the International Neuroblastoma Pathology Committee classification, relies on morphologic features and patient age to assign favorable or unfavorable histology for each patient. The International Neuroblastoma Risk Group uses a subset of this pathologic data, along with patient age and several key molecular features, to assign one of several prognostic categories.

Peripheral neuroblastic tumors occur on a histologic spectrum from benign ganglioneuromas to malignant neuroblastomas, but even within the latter category, there is extensive heterogeneity in morphologic appearance and genetic composition. The International Neuroblastoma Pathology Committee classification has traditionally been used to successfully categorize tumors with favorable or unfavorable histology, but morphology must be supplemented with the results of additional testing. While MYCN amplification, diploid DNA content, and 11q loss have long been known to be negative prognostic factors, a new group of molecular biomarkers has emerged that define discrete high-risk categories. These include MYCN/MYC overexpression, dysregulated telomere maintenance mechanisms (both increased expression of telomere reverse transcriptase and alternate lengthening of telomeres), and ALK aberrations. Testing for these biomarkers and an integrated classification scheme may lead to improved risk stratification and selection of emerging targeted therapies.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that acts as an oncogenic driver in solid and haematological malignancies in both children and adults. Although ALK-expressing (ALK+) tumours show strong initial responses to the series of ALK inhibitors currently available, many patients will develop resistance. In this Review, we discuss recent advances in ALK oncogenic signalling, together with existing and promising new modalities to treat ALK-driven tumours, including currently approved ALK-directed therapies, namely tyrosine kinase inhibitors, and novel approaches such as ALK-specific immune therapies. Although ALK inhibitors have changed the management and clinical history of ALK+ tumours, they are still insufficient to cure most of the patients. Therefore, more effort is needed to further improve outcomes and prevent the tumour resistance, recurrence and metastatic spread that many patients with ALK+ tumours experience. Here, we outline how a multipronged approach directed against ALK and other essential pathways that sustain the persistence of ALK+ tumours, together with potent or specific immunotherapies, could achieve this goal. We envision that the lessons learned from treating ALK+ tumours in the clinic could ultimately accelerate the implementation of innovative combination therapies to treat tumours driven by other tyrosine kinases or oncogenes with similar properties.

Cancer stem cells (CSCs) are described as a subpopulation of cells with capabilities of self-renewal, chemoresistance, and invasiveness. CSCs reside in tumor niches and can be studied in vitro through their enrichment in spheroids (Stem). Molecular iodine (I2) induces apoptosis and differentiation in various cancer cells. I2 can activate peroxisome proliferator-activated receptors type gamma (PPARγ), and its pathways are associated with its oxidant/antioxidant capacity. This work aimed to compare the effect of I2 supplementation in progenitor and CSC populations with low (MCF-7 and S-K-NAS) and high invasiveness (MDA-MB231 and SK-N-BE2) in mammary and neuroblastoma (NB) cell lines. Results showed that the CSC population enriched by the spheroid culture overexpressed stem messengers CD44, SOX2, and NMYC and exhibited the highest mitochondrial metabolism (membrane mitochondrial potential and O2-). The presence of I2 increases PPARγ expression and induces apoptosis through the Bax/Bcl2 index in all populations but silences NMYC expression and reduces mitochondrial metabolism in Stem NB. I2 also enhances the expression of nuclear erythroid factor 2 (Nrf2) in all populations, but the target antioxidant superoxide dismutase 2 (SOD2) is only elevated in progenitor cells. In contrast, the mitophagy inductors PTEN-induced putative kinase 1 (Pink1) and microtubule-associated protein1 light chain3 alpha (LC3) were overexpressed in Stem populations. I2-preselected SK-N-BE2 populations exhibited minor implantation and invasion capacities in the in vivo zebrafish model. These data indicate that I2 interferes with viability, implantation, and invasion capacity in all cell lines, but the molecular mechanisms vary depending on the progenitor or Stem condition.

Sequencing of neuroblastoma (NB) tumors has revealed genetic alterations in genes involved in DNA damage response (DDR) pathways. However, roles for specific alterations of DDR genes in pediatric solid tumors remain poorly understood. To address this, mutations in the DDR pathway including Brca2, Atm, and Palb2 were incorporated into an established zebrafish MYCN transgenic model (Tg(dbh:EGFP-MYCN)). These mutations enhance NB formation and metastasis and result in upregulation of cell-cycle checkpoint and DNA damage repair signatures, revealing molecular vulnerabilities in DDR-deficient NB. DDR gene knockdown in zebrafish and human NB cells increases sensitivity to the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib, and this effect is enhanced by inhibition of the ataxia telangiectasia and rad3-related (ATR) kinase. This work provides in vivo evidence demonstrating that alterations in certain DDR-pathway genes promote aggressive NB and supports combination PARP + ATR inhibitor therapy for NB patients with tumors harboring specific genetic alterations in DDR.

Targeting oncogenes and their interactive partners is an effective approach to developing novel targeted therapies for cancer and other chronic diseases. We and others have long suggested the MDM2 oncogene being an excellent target for cancer therapy, based on its p53-dependent and -independent oncogenic activities in a variety of cancers. The MYC family proteins are transcription factors that also regulate diverse biological functions. Dysregulation of MYC, such as amplification of MYCN, is associated with tumorigenesis, especially for neuroblastoma. Although the general survival rate of neuroblastoma patients has significantly improved over the past few decades, high-risk neuroblastoma still presents a poor prognosis. Therefore, innovative and more potent therapeutic strategies are needed to eradicate these aggressive neoplasms. This review focuses on the oncogenic properties of MYCN and its molecular regulation and summarizes the major therapeutic strategies being developed based on preclinical findings. We also highlight the potential benefits of targeting both the MYCN and MDM2 oncogenes, providing preclinical evidence of the efficacy and safety of this approach. In conclusion, the development of effective small molecules that inhibit both MYCN and MDM2 represents a promising new strategy for the treatment of neuroblastoma and other cancers.

Neuroblastoma (NB) is a predominantly pediatric cancer with greater than 90% of cases arising in children under the age of five. More than half of patients have metastases detected at diagnosis, and high-risk disease is associated with five-year survival rates of only 50-60 %. Standard therapy involves highly toxic chemotherapy, surgery, radiation, and immunotherapy, and less toxic, more specific targeted therapies are urgently needed. Genomic studies have identified common driver aberrations in high-risk NB, such as MYCN amplification. In addition, a proportion of high-risk patients harbor amplification or activating mutations in anaplastic lymphoma kinase (ALK), and co-occurrence of ALK mutations and MYCN amplification have been associated with aggressive disease. In this study, we analyzed the efficacy of a Phase Ia-cleared, orally bioavailable dual ALK and focal adhesion kinase (FAK) inhibitor, ESK440, in multiple preclinical NB models. ESK440 potently inhibited proliferation of NB cell lines, with increased sensitivity in cell lines harboring ALK aberrations. ALK, FAK, and downstream target activation were rapidly decreased upon ESK440 treatment, and this was associated with impaired cellular migration and invasion. Importantly, ESK440 treatment also decreased MYCN levels. NB cell line and patient-derived xenograft studies showed significant reduction in tumor growth in ESK440-treated mice with no signs of toxicity. In certain NB models, ESK440 showed comparable or enhanced efficacy to lorlatinib, another clinical ALK inhibitor, and a lorlatinib-resistant cell line (COG-N-561 LR) retained sensitivity to ESK440. These preclinical results indicate that ESK440 is a promising targeted agent for ALK-driven NB and support future clinical studies to evaluate its efficacy in NB patients.

c-MYC is an important driver of high-risk neuroblastoma. A lack of c-MYC-driven genetically engineered mouse models (GEMM) has hampered the ability to better understand mechanisms of neuroblastoma oncogenesis and to develop effective therapies. In this study, we showed that conditional c-MYC induction via Cre recombinase driven by a tyrosine hydroxylase promoter led to a preponderance of PDX1+ somatostatinoma, a type of pancreatic neuroendocrine tumor. However, c-MYC activation via an improved Cre recombinase driven by a dopamine β-hydroxylase promoter resulted in neuroblastoma development. The c-MYC murine neuroblastoma tumors recapitulated the pathologic and genetic features of human neuroblastoma and responded to anti-GD2 immunotherapy and difluoromethylornithine, an FDA-approved inhibitor targeting the MYC transcriptional target ODC1. Thus, c-MYC overexpression results in different but related tumor types depending on the targeted cell. The GEMMs represent valuable tools for testing immunotherapies and targeted therapies for these diseases. Significance: The development of c-MYC-driven genetically engineered neuroblastoma and somatostatinoma mouse models provides useful tools for understanding the tumor cell origin and investigating treatment strategies.

Neuroblastomas are pediatric tumors arising from undifferentiated cells of neural crest origin with stem cell-like characteristics. Dysregulation of Wnt/β-catenin signaling has been shown to be linked to the development of various tumors. Activated Wnt signaling results in β-catenin accumulation in the nucleus to support pro-neoplastic traits. DKK1, a secreted glycoprotein, is an inhibitor of Wnt signaling, and the addition of DKKI to the culture medium has been used to suppress the Wnt pathway. This study aimed to analyze the role of Dickopff-1 as a potential differentiating agent for the neuroblastoma cell line SH-SY5Y and neurospheres derived from it. The treatment of SH-5Y5Y derived neurospheres by DKK1 resulted in their disintegration and reduced proliferation markers like Ki67, PCNA. DKK1 treatment to the neurospheres also resulted in the loss of cancer stem cell markers like CD133, KIT and pluripotency markers like SOX2, OCT4, NANOG. DKK1 treatment caused reduction in mRNA expression of β-catenin and TCF genes like TCF4, TCF12. When the SH-SY5Y cancer cells were grown under differentiating conditions, DKKI caused neuronal differentiation by itself, and in synergy with retinoic acid. This was verified by the expression of markers like MAPT, DCX, GAP43, ENO2 and also with changes in neurite length. We concluded that Wnt inhibition, as exemplified by DKK1 treatment, is therefore a possible differentiating condition and also suppresses the proliferative and cancer stemness related properties of SH-SY5Y neuroblastoma cells.

Most childhood cancers possess distinct clinicopathological profiles from those seen in adulthood, reflecting their divergent mechanisms of carcinogenesis. Rather than depending on the decades-long, stepwise accumulation of changes within a mature cell that defines adult carcinomas, many pediatric malignancies emerge rapidly as the consequence of random errors during development. These errors-whether they be genetic, epigenetic, or microenvironmental-characteristically block maturation, resulting in phenotypically primitive neoplasms. Only an event that falls within a narrow set of spatiotemporal parameters will forge a malignant clone; if it occurs too soon then the event might be lethal, or negatively selected against, while if it is too late or in an incorrectly primed precursor cell then the necessary intracellular conditions for transformation will not be met. The precise characterization of these changes, through the study of normal tissues and tumors from patients and model systems, will be essential if we are to develop new strategies to diagnose, treat, and perhaps even prevent childhood cancer.

Neuroblastoma is the most common pediatric extracranial solid tumor and is derived from trunk neural crest cells (tNCC) and its progenitor sympathoadrenal (SA) cells. While human pluripotent stem cell (PSC) models of neuroblastoma have been described, the PSC were differentiated using protocols that made neural crest cells, but not specifically the trunk subtype. Here, we compared four recent protocols to differentiate pluripotent stem cells (PSC) toward SA cells and examined their efficiency at generating SA cells along with earlier cell states (neuromesodermal progenitors [NMP], tNCC), as well as generating MYCN-driven tumors. Interestingly, the protocols that created cells with the highest level of NMP markers did not produce cells with the highest tNCC or SA cell markers. We identified a protocol that consistently produced cells with the highest level of SA markers using two PSC lines of different genders. This protocol also generated tumors with the highest level of PHOX2B, a marker of neuroblastoma. Transcriptionally, however, each protocol generates tumors that resemble neuroblastoma. Two of the protocols repeatedly produced adrenergic neuroblastoma whereas the other two protocols were ambiguous. Thus, we identified a protocol that reliably generates adrenergic neuroblastoma.

Super-enhancers (SEs) typically govern the expression of critical oncogenes and play a fundamental role in the initiation and progression of cancer. Focusing on genes that are abnormally regulated by SE in cancer may be a new strategy for understanding pathogenesis. In the context of this investigation, we have identified a previously unreported SE-driven gene IRF2BP2 in neuroblastoma (NB).

The expression and prognostic value of IRF2BP2 were detected in public databases and clinical samples. The effect of IRF2BP2 on NB cell growth and apoptosis was evaluated through in vivo and in vitro functional loss experiments. The molecular mechanism of IRF2BP2 was investigated by the study of chromatin regulatory regions and transcriptome sequencing.

The sustained high expression of IRF2BP2 results from the activation of a novel SE established by NB master transcription factors MYCN, MEIS2, and HAND2, and they form a new complex that regulates the gene network associated with the proliferation of NB cell populations. We also observed a significant enrichment of the AP-1 family at the binding sites of IRF2BP2. Remarkably, within NB cells, AP-1 plays a pivotal role in shaping the chromatin accessibility landscape, thereby exposing the binding site for IRF2BP2. This orchestrated action enables AP-1 and IRF2BP2 to collaboratively stimulate the expression of the NB susceptibility gene ALK, thereby upholding the highly proliferative phenotype characteristic of NB.

Our findings indicate that SE-driven IRF2BP2 can bind to AP-1 to maintain the survival of tumor cells via regulating chromatin accessibility of the NB susceptibility gene ALK.

Neural crest cells (NCC) are multipotent migratory stem cells that originate from the neural tube during early vertebrate embryogenesis. NCCs give rise to a variety of cell types within the developing organism, including neurons and glia of the sympathetic nervous system. It has been suggested that failure in correct NCC differentiation leads to several diseases, including neuroblastoma (NB). During normal NCC development, MYCN is transiently expressed to promote NCC migration, and its downregulation precedes neuronal differentiation. Overexpression of MYCN has been linked to high-risk and aggressive NB progression. For this reason, understanding the effect overexpression of this oncogene has on the development of NCC-derived sympathoadrenal progenitors (SAP), which later give rise to sympathetic nerves, will help elucidate the developmental mechanisms that may prime the onset of NB. Here, we found that overexpressing human EGFP-MYCN within SAP lineage cells in zebrafish led to the transient formation of an abnormal SAP population, which displayed expanded and elevated expression of NCC markers while paradoxically also co-expressing SAP and neuronal differentiation markers. The aberrant NCC signature was corroborated with in vivo time-lapse confocal imaging in zebrafish larvae, which revealed transient expansion of sox10 reporter expression in MYCN overexpressing SAPs during the early stages of SAP development. In these aberrant MYCN overexpressing SAP cells, we also found evidence of dampened BMP signaling activity, indicating that BMP signaling disruption occurs following elevated MYCN expression. Furthermore, we discovered that pharmacological inhibition of BMP signaling was sufficient to create an aberrant NCC gene signature in SAP cells, phenocopying MYCN overexpression. Together, our results suggest that MYCN overexpression in SAPs disrupts their differentiation by eliciting abnormal NCC gene expression programs, and dampening BMP signaling response, having developmental implications for the priming of NB in vivo.

The olfactory epithelium undergoes neuronal regeneration from basal stem cells and is susceptible to olfactory neuroblastoma (ONB), a rare tumor of unclear origins. Employing alterations in Rb1/Trp53/Myc (RPM), we establish a genetically engineered mouse model of high-grade metastatic ONB exhibiting a NEUROD1+ immature neuronal phenotype. We demonstrate that globose basal cells (GBCs) are a permissive cell of origin for ONB and that ONBs exhibit cell fate heterogeneity that mimics normal GBC developmental trajectories. ASCL1 loss in RPM ONB leads to emergence of non-neuronal histopathologies, including a POU2F3+ microvillar-like state. Similar to small-cell lung cancer (SCLC), mouse and human ONBs exhibit mutually exclusive NEUROD1 and POU2F3-like states, an immune-cold tumor microenvironment, intratumoral cell fate heterogeneity comprising neuronal and non-neuronal lineages, and cell fate plasticity-evidenced by barcode-based lineage tracing and single-cell transcriptomics. Collectively, our findings highlight conserved similarities between ONB and neuroendocrine tumors with significant implications for ONB classification and treatment.

Lysophosphatidic acid (LPA) is a well-documented pro-oncogenic factor in different cancers, but relatively little is known on its biological activity in neuroblastoma. The LPA effects and the participation of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK) in LPA mitogenic signaling were studied in human neuroblastoma cell lines. We used light microscopy and [3H]-thymidine incorporation to determine cell proliferation, Western blot to study intracellular signaling, and pharmacological and molecular tools to examine the role of ALK. We found that LPA stimulated the growth of human neuroblastoma cells, as indicated by the enhanced cell number, clonogenic activity, and DNA synthesis. These effects were curtailed by the selective ALK inhibitors NPV-TAE684 and alectinib. In a panel of human neuroblastoma cell lines harboring different ALK genomic status, the ALK inhibitors suppressed LPA-induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), which are major regulators of cell proliferation. ALK depletion by siRNA treatment attenuated LPA-induced ERK1/2 activation. LPA enhanced ALK phosphorylation and potentiated ALK activation by the ALK ligand FAM150B. LPA enhanced the inhibitory phosphorylation of the tumor suppressor FoxO3a, and this response was impaired by the ALK inhibitors. These results indicate that LPA stimulates mitogenesis of human neuroblastoma cells through a crosstalk with ALK.

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC50 of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.

The proximal convoluted tubule (PCT) of the kidney is a crucial functional segment responsible for reabsorption, secretion, and the maintenance of electrolyte and water balance within the renal tubule. However, there is a lack of a well-defined endogenous transgenic line for studying PCT morphogenesis. By analyzing single-cell transcriptome data from the adult zebrafish kidney, we have identified the expression of odd-skipped-related 2 (osr2, which encodes an odd-skipped zinc-finger transcription factor) in the PCT. To gain insight into the role of osr2 in PCT morphogenesis, we have generated a transgenic zebrafish line Tg(osr2:EGFP), expressing enhanced green fluorescent protein (EGFP). The EGFP expression pattern closely mirrors that of endogenous Osr2, faithfully recapitulating its native expression profile. During kidney development, we can use EGFP to track PCT development, which is also preserved in adult zebrafish. Additionally, osr2:EGFP-labeled zebrafish PCT fragments displayed short lengths with infrequent overlap, rendering them conducive for nephrons counting. The generation of Tg(osr2:EGFP) transgenic line is accompanied by simultaneous disruption of osr2 activity. Importantly, our findings demonstrate that osr2 inactivation had no discernible impact on the development and regeneration of Tg(osr2:EGFP) zebrafish nephrons. Overall, the establishment of this transgenic zebrafish line offers a valuable tool for both genetic and chemical analysis of PCT.

Neuroblastoma is the most common extracranial solid tumor of childhood. While MYCN and mutant anaplastic lymphoma kinase (ALKF1174L) cooperate in tumorigenesis, how ALK contributes to tumor formation remains unclear. Here, we used a human stem cell-based model of neuroblastoma. Mis-expression of ALKF1174L and MYCN resulted in shorter latency compared to MYCN alone. MYCN tumors resembled adrenergic, while ALK/MYCN tumors resembled mesenchymal, neuroblastoma. Transcriptomic analysis revealed enrichment in focal adhesion signaling, particularly the extracellular matrix genes POSTN and FN1 in ALK/MYCN tumors. Patients with ALK-mutant tumors similarly demonstrated elevated levels of POSTN and FN1. Knockdown of POSTN, but not FN1, delayed adhesion and suppressed proliferation of ALK/MYCN tumors. Furthermore, loss of POSTN reduced ALK-dependent activation of WNT signaling. Reciprocally, inhibition of the WNT pathway reduced expression of POSTN and growth of ALK/MYCN tumor cells. Thus, ALK drives neuroblastoma in part through a feedforward loop between POSTN and WNT signaling.

Solid tumors, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. We found that chemokine-like factor (CKLF) is highly expressed by various solid tumor cells and transcriptionally up-regulated by MYCN. Using the MYCN-driven high-risk neuroblastoma as a model system, we demonstrated that as early as the premalignant stage, tumor cells secrete CKLF to attract CCR4-expressing CD4+ cells, inducing immunosuppression and tumor aggression. Genetic depletion of CD4+ T regulatory cells abolishes the immunorestrictive and protumorigenic effects of CKLF. Our work supports that disrupting CKLF-mediated cross-talk between tumor and CD4+ suppressor cells represents a promising immunotherapeutic approach to battling MYCN-driven tumors.

Dysregulated pre-mRNA splicing and metabolism are two hallmarks of MYC-driven cancers. Pharmacological inhibition of both processes has been extensively investigated as potential therapeutic avenues in preclinical and clinical studies. However, how pre-mRNA splicing and metabolism are orchestrated in response to oncogenic stress and therapies is poorly understood. Here, we demonstrate that jumonji domain containing 6, arginine demethylase, and lysine hydroxylase, JMJD6, acts as a hub connecting splicing and metabolism in MYC-driven human neuroblastoma. JMJD6 cooperates with MYC in cellular transformation of murine neural crest cells by physically interacting with RNA binding proteins involved in pre-mRNA splicing and protein homeostasis. Notably, JMJD6 controls the alternative splicing of two isoforms of glutaminase (GLS), namely kidney-type glutaminase (KGA) and glutaminase C (GAC), which are rate-limiting enzymes of glutaminolysis in the central carbon metabolism in neuroblastoma. Further, we show that JMJD6 is correlated with the anti-cancer activity of indisulam, a 'molecular glue' that degrades splicing factor RBM39, which complexes with JMJD6. The indisulam-mediated cancer cell killing is at least partly dependent on the glutamine-related metabolic pathway mediated by JMJD6. Our findings reveal a cancer-promoting metabolic program is associated with alternative pre-mRNA splicing through JMJD6, providing a rationale to target JMJD6 as a therapeutic avenue for treating MYC-driven cancers.

The MYC proto-oncogenes (c-MYC, MYCN , MYCL ) are among the most deregulated oncogenic drivers in human malignancies including high-risk neuroblastoma, 50% of which are MYCN -amplified. Genetically engineered mouse models (GEMMs) based on the MYCN transgene have greatly expanded the understanding of neuroblastoma biology and are powerful tools for testing new therapies. However, a lack of c-MYC-driven GEMMs has hampered the ability to better understand mechanisms of neuroblastoma oncogenesis and therapy development given that c-MYC is also an important driver of many high-risk neuroblastomas. In this study, we report two transgenic murine neuroendocrine models driven by conditional c-MYC induction in tyrosine hydroxylase (Th) and dopamine β-hydroxylase (Dbh)-expressing cells. c-MYC induction in Th-expressing cells leads to a preponderance of Pdx1 + somatostatinomas, a type of pancreatic neuroendocrine tumor (PNET), resembling human somatostatinoma with highly expressed gene signatures of δ cells and potassium channels. In contrast, c-MYC induction in Dbh-expressing cells leads to onset of neuroblastomas, showing a better transforming capacity than MYCN in a comparable C57BL/6 genetic background. The c-MYC murine neuroblastoma tumors recapitulate the pathologic and genetic features of human neuroblastoma, express GD2, and respond to anti-GD2 immunotherapy. This model also responds to DFMO, an FDA-approved inhibitor targeting ODC1, which is a known MYC transcriptional target. Thus, establishing c-MYC-overexpressing GEMMs resulted in different but related tumor types depending on the targeted cell and provide useful tools for testing immunotherapies and targeted therapies for these diseases.

Studies defining normal and disrupted human neural crest cell development have been challenging given its early timing and intricacy of development. Consequently, insight into the early disruptive events causing a neural crest related disease such as pediatric cancer neuroblastoma is limited. To overcome this problem, we developed an in vitro differentiation model to recapitulate the normal in vivo developmental process of the sympathoadrenal lineage which gives rise to neuroblastoma. We used human in vitro pluripotent stem cells and single-cell RNA sequencing to recapitulate the molecular events during sympathoadrenal development. We provide a detailed map of dynamically regulated transcriptomes during sympathoblast formation and illustrate the power of this model to study early events of the development of human neuroblastoma, identifying a distinct subpopulation of cell marked by SOX2 expression in developing sympathoblast obtained from patient derived iPSC cells harboring a germline activating mutation in the anaplastic lymphoma kinase (ALK) gene.

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signaling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-d ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-d combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify an ability of ATR inhibition to promote NB differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.

Survival rates among patients with high-risk neuroblastoma remain low and novel therapies for recurrent neuroblastomas are required. ALK is commonly mutated in primary and relapsed neuroblastoma tumors and ALK tyrosine kinase inhibitors (TKI) are promising treatments for ALK-driven neuroblastoma; however, innate or adaptive resistance to single-agent ALK-TKIs remain a clinical challenge. Recently, SHP2 inhibitors have been shown to overcome ALK-TKI resistance in lung tumors harboring ALK rearrangements. Here, we have assessed the efficacy of the SHP2 inhibitor TNO155 alone and in combination with the ALK-TKIs crizotinib, ceritinib, or lorlatinib for the treatment of ALK-driven neuroblastoma using in vitro and in vivo models. In comparison to wild-type, ALK-mutant neuroblastoma cell lines were more sensitive to SHP2 inhibition with TNO155. Moreover, treatment with TNO155 and ALK-TKIs synergistically reduced cell growth and promoted inactivation of ALK and MAPK signaling in ALK-mutant neuroblastoma cells. ALK-mutant cells engrafted into larval zebrafish and treated with single agents or dual SHP2/ALK inhibitors showed reduced growth and invasion. In murine ALK-mutant xenografts, tumor growth was likewise reduced or delayed, and survival was prolonged upon combinatorial treatment of TNO155 and lorlatinib. Finally, we show that lorlatinib-resistant ALK-F1174L neuroblastoma cells harbor additional RAS-MAPK pathway alterations and can be resensitized to lorlatinib when combined with TNO155 in vitro and in vivo. Our results report the first evaluation of TNO155 in neuroblastoma and suggest that combinatorial inhibition of ALK and SHP2 could be a novel approach to treating ALK-driven neuroblastoma, potentially including the increasingly common tumors that have developed resistance to ALK-TKIs.

These findings highlight the translatability between zebrafish and murine models, provide evidence of aberrant RAS-MAPK signaling as an adaptive mechanism of resistance to lorlatinib, and demonstrate the clinical potential for SHP2/ALK inhibitor combinations for the treatment of ALK-mutant neuroblastoma, including those with acquired tolerance or potentially resistance to ALK-TKIs.

Selection of the best tumor antigen is critical for the therapeutic success of chimeric antigen receptor (CAR) T cells in hematologic malignancies and solid tumors. The anaplastic lymphoma kinase (ALK) receptor is expressed by most neuroblastomas while virtually absent in most normal tissues. ALK is an oncogenic driver in neuroblastoma and ALK inhibitors show promising clinical activity. Here, we describe the development of ALK.CAR-T cells that show potent efficacy in monotherapy against neuroblastoma with high ALK expression without toxicity. For neuroblastoma with low ALK expression, combination with ALK inhibitors specifically potentiates ALK.CAR-T cells but not GD2.CAR-T cells. Mechanistically, ALK inhibitors impair tumor growth and upregulate the expression of ALK, thereby facilitating the activity of ALK.CAR-T cells against neuroblastoma. Thus, while neither ALK inhibitors nor ALK.CAR-T cells will likely be sufficient as monotherapy in neuroblastoma with low ALK density, their combination specifically enhances therapeutic efficacy.

Dysregulated pre-mRNA splicing and metabolism are two hallmarks of MYC-driven cancers. Pharmacological inhibition of both processes has been extensively investigated as potential therapeutic avenues in preclinical and clinical studies. However, how pre-mRNA splicing and metabolism are orchestrated in response to oncogenic stress and therapies is poorly understood. Here, we demonstrate that Jumonji Domain Containing 6, Arginine Demethylase and Lysine Hydroxylase, JMJD6, acts as a hub connecting splicing and metabolism in MYC-driven neuroblastoma. JMJD6 cooperates with MYC in cellular transformation by physically interacting with RNA binding proteins involved in pre-mRNA splicing and protein homeostasis. Notably, JMJD6 controls the alternative splicing of two isoforms of glutaminase (GLS), namely kidney-type glutaminase (KGA) and glutaminase C (GAC), which are rate-limiting enzymes of glutaminolysis in the central carbon metabolism in neuroblastoma. Further, we show that JMJD6 is correlated with the anti-cancer activity of indisulam, a "molecular glue" that degrades splicing factor RBM39, which complexes with JMJD6. The indisulam-mediated cancer cell killing is at least partly dependent on the glutamine-related metabolic pathway mediated by JMJD6. Our findings reveal a cancer-promoting metabolic program is associated with alternative pre-mRNA splicing through JMJD6, providing a rationale to target JMJD6 as a therapeutic avenue for treating MYC-driven cancers.

This review commemorates the 10-year anniversary of the discovery of physiological ligands Augα (Augmentor α; ALKAL2; Fam150b) and Augβ (Augmentor β; ALKAL1; Fam150a) for anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK), previously considered orphan receptors. This manuscript provides an in-depth review of the biophysical and cellular properties of ALK family receptors and their roles in cancer, metabolism, pain, ophthalmology, pigmentation, central nervous system (CNS) function, and reproduction. ALK and LTK receptors are implicated in the development of numerous cancers, and targeted inhibition of their signaling pathways can offer therapeutic benefits. Additionally, ALK family receptors are involved in regulating body weight and metabolism, modulating pain signaling, and contributing to eye development and pigmentation. In the CNS, these receptors play a role in synapse modulation, neurogenesis, and various psychiatric pathologies. Lastly, ALK expression is linked to reproductive functions, with potential implications for patients undergoing ALK inhibitor therapy. Further research is needed to better understand the complex interactions of ALK family receptors and Aug ligands and to repurpose targeted therapy for a wide range of human diseases.

Oncogene MYCN is closely related with malignant progression and poor prognosis of neuroblastoma (NB). Recently, long non-coding RNAs (lncRNAs) have been recognized as crucial regulators in various cancers. However, whether lncRNAs contribute to the overexpression of MYCN in NB is unclear.

Microarray analysis were applied to analyze the differentially expressed lncRNAs between MYCN-amplified and MYCN-non-amplified NB cell lines. Bioinformatic analyses were utilized to identify lncRNAs nearby MYCN locus. qRT-PCR was used to detect the expression level of lncRNA AC142119.1 in NB cell lines and tissues. Gain- and loss-of-function assays were conducted to investigate the biological effect of AC142119.1 in NB. Fluorescence in situ hybridization, RNA pull-down, RNA immunoprecipitation, mass spectrometry, RNA electrophoretic mobility shift, chromatin immunoprecipitation and chromatin isolation by RNA purification assays were performed to validate the interaction between AC142119.1 and WDR5 protein as well as MYCN promoter.

AC142119.1 was significantly elevated in NB tissues with MYCN amplification, advanced INSS stage and high risk, and associated with poor survival of NB patients. Moreover, enforced expression of AC142119.1 reinforced the proliferation of NB cells in vitro and in vivo. Additionally, AC142119.1 specifically recruited WDR5 protein to interact with MYCN promoter, further initiating the transcription of MYCN and accelerating NB progression.

We identified a novel lncRNA AC142119.1, which promoted the progression of NB through epigenetically initiating the transcription of MYCN via interacting with both WDR5 protein and the promoter of MYCN, indicating that AC142119.1 might be a potential diagnostic biomarker and therapeutic target for NB.

Deregulation of the MYC family of transcription factors c-MYC (encoded by MYC), MYCN, and MYCL is prevalent in most human cancers, with an impact on tumor initiation and progression, as well as response to therapy. In neuroblastoma (NB), amplification of the MYCN oncogene and over-expression of MYC characterize approximately 40% and 10% of all high-risk NB cases, respectively. However, the mechanism and stage of neural crest development in which MYCN and c-MYC contribute to the onset and/or progression of NB are not yet fully understood. Here, we hypothesized that subtle differences in the expression of MYCN and/or c-MYC targets could more accurately stratify NB patients in different risk groups rather than using the expression of either MYC gene alone. We employed an integrative approach using the transcriptome of 498 NB patients from the SEQC cohort and previously defined c-MYC and MYCN target genes to model a multigene transcriptional risk score. Our findings demonstrate that defined sets of c-MYC and MYCN targets with significant prognostic value, effectively stratify NB patients into different groups with varying overall survival probabilities. In particular, patients exhibiting a high-risk signature score present unfavorable clinical parameters, including increased clinical risk, higher INSS stage, MYCN amplification, and disease progression. Notably, target genes with prognostic value differ between c-MYC and MYCN, exhibiting distinct expression patterns in the developing sympathoadrenal system. Genes associated with poor outcomes are mainly found in sympathoblasts rather than in chromaffin cells during the sympathoadrenal development.

Neuroblastoma is among the most common childhood cancers. Neuroblastoma in advanced stages is one of the most intractable pediatric cancers, notwithstanding the recent therapeutic advances. ALK mutations are among the leading cause of hereditary neuroblastoma and account for more than 14% of the somatically acquired alterations. ALK kinase activity is currently one of the main targets for pharmacological strategies. However, evidence from ALK fusion-positive lung cancer studies has shown that resistance to ALK inhibition arises during the therapy, causing a relapse within several years. IgLONs are membrane-bound proteins involved in cell-to-cell adhesion. The expression of the IgLON family results altered in different cancers. We found that the IgLON member Negr1 is downregulated in neuroblastoma. The ectopic overexpression of Negr1 impairs neuroblastoma growth in vitro and in vivo. Negr1 exists as a GPI-anchored membrane-bound protein and as a soluble protein released upon metalloprotease cleavage. We generated and characterized a panel of Negr1-derived peptides. The treatment with Negr1 protein and derived peptides induce ALK downregulation and halt neuroblastoma progression in vitro and in vivo.

Neuroblastoma is a paediatric malignancy of incredibly complex aetiology. Oncogenic protein kinase signalling in neuroblastoma has conventionally focussed on transduction through the well-characterised PI3K/Akt and MAPK pathways, in which the latter has been implicated in treatment resistance. The discovery of the receptor tyrosine kinase ALK as a target of genetic alterations in cases of familial and sporadic neuroblastoma, was a breakthrough in the understanding of the complex genetic heterogeneity of neuroblastoma. However, despite progress in the development of small-molecule inhibitors of ALK, treatment resistance frequently arises and appears to be a feature of the disease. Moreover, since the identification of ALK, several additional protein kinases, including the PIM and Aurora kinases, have emerged not only as drivers of the disease phenotype, but also as promising druggable targets. This is particularly the case for Aurora-A, given its intimate engagement with MYCN, a driver oncogene of aggressive neuroblastoma previously considered 'undruggable.'

Aided by significant advances in structural biology and a broader understanding of the mechanisms of protein kinase function and regulation, we comprehensively outline the role of protein kinase signalling, emphasising ALK, PIM and Aurora in neuroblastoma, their respective metabolic outputs, and broader implications for targeted therapies.

Despite massively divergent regulatory mechanisms, ALK, PIM and Aurora kinases all obtain significant roles in cellular glycolytic and mitochondrial metabolism and neuroblastoma progression, and in several instances are implicated in treatment resistance. While metabolism of neuroblastoma tends to display hallmarks of the glycolytic "Warburg effect," aggressive, in particular MYCN-amplified tumours, retain functional mitochondrial metabolism, allowing for survival and proliferation under nutrient stress. Future strategies employing specific kinase inhibitors as part of the treatment regimen should consider combinatorial attempts at interfering with tumour metabolism, either through metabolic pathway inhibitors, or by dietary means, with a view to abolish metabolic flexibility that endows cancerous cells with a survival advantage.

Cancer is a leading cause of death worldwide, resulting in ∼10 million deaths in 2020. Major oncogenic effectors are the Myc proto-oncogene family, which consists of three members including c-Myc, N-Myc, and L-Myc. As a pertinent example of the role of the Myc family in tumorigenesis, amplification of MYCN in childhood neuroblastoma strongly correlates with poor patient prognosis. Complexes between Myc oncoproteins and their partners such as hypoxia-inducible factor-1α and Myc-associated protein X (MAX) result in proliferation arrest and pro-proliferative effects, respectively. Interactions with other proteins are also important for N-Myc activity. For instance, the enhancer of zest homolog 2 (EZH2) binds directly to N-Myc to stabilize it by acting as a competitor against the ubiquitin ligase, SCFFBXW7, which prevents proteasomal degradation. Heat shock protein 90 may also be involved in N-Myc stabilization since it binds to EZH2 and prevents its degradation. N-Myc downstream-regulated gene 1 (NDRG1) is downregulated by N-Myc and participates in the regulation of cellular proliferation via associating with other proteins, such as glycogen synthase kinase-3β and low-density lipoprotein receptor-related protein 6. These molecular interactions provide a better understanding of the biologic roles of N-Myc and NDRG1, which can be potentially used as therapeutic targets. In addition to directly targeting these proteins, disrupting their key interactions may also be a promising strategy for anti-cancer drug development. This review examines the interactions between the Myc proteins and other molecules, with a special focus on the relationship between N-Myc and NDRG1 and possible therapeutic interventions. SIGNIFICANCE STATEMENT: Neuroblastoma is one of the most common childhood solid tumors, with a dismal five-year survival rate. This problem makes it imperative to discover new and more effective therapeutics. The molecular interactions between major oncogenic drivers of the Myc family and other key proteins; for example, the metastasis suppressor, NDRG1, may potentially be used as targets for anti-neuroblastoma drug development. In addition to directly targeting these proteins, disrupting their key molecular interactions may also be promising for drug discovery.

Glioblastoma multiforme (GB) and high-risk neuroblastoma (NB) are known to have poor therapeutic outcomes. As for most cancers, chemotherapy and radiotherapy are the current mainstay treatments for GB and NB. However, the known limitations of systemic toxicity, drug resistance, poor targeted delivery, and inability to access the blood-brain barrier (BBB), make these treatments less satisfactory. Other treatment options have been investigated in many studies in the literature, especially nutraceutical and naturopathic products, most of which have also been reported to be poorly effective against these cancer types. This necessitates the development of treatment strategies with the potential to cross the BBB and specifically target cancer cells. Compounds that target the endopeptidase, matrix metalloproteinase 2 (MMP-2), have been reported to offer therapeutic insights for GB and NB since MMP-2 is known to be over-expressed in these cancers and plays significant roles in such physiological processes as angiogenesis, metastasis, and cellular invasion. Chlorotoxin (CTX) is a promising 36-amino acid peptide isolated from the venom of the deathstalker scorpion, Leiurus quinquestriatus, demonstrating high selectivity and binding affinity to a broad-spectrum of cancers, especially GB and NB through specific molecular targets, including MMP-2. The favorable characteristics of nanoparticles (NPs) such as their small sizes, large surface area for active targeting, BBB permeability, etc. make CTX-functionalized NPs (CTX-NPs) promising diagnostic and therapeutic applications for addressing the many challenges associated with these cancers. CTX-NPs may function by improving diffusion through the BBB, enabling increased localization of chemotherapeutic and genotherapeutic drugs to diseased cells specifically, enhancing imaging modalities such as magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), optical imaging techniques, image-guided surgery, as well as improving the sensitization of radio-resistant cells to radiotherapy treatment. This review discusses the characteristics of GB and NB cancers, related treatment challenges as well as the potential of CTX and its functionalized NP formulations as targeting systems for diagnostic, therapeutic, and theranostic purposes. It also provides insights into the potential mechanisms through which CTX crosses the BBB to bind cancer cells and provides suggestions for the development and application of novel CTX-based formulations for the diagnosis and treatment of GB and NB in the future.

Oncogenic drivers like MYCN in neuroblastoma subsets continues to present a significant challenge owing to its strong correlation with high-risk metastatic disease and poor prognosis. However, only a limited number of MYCN-regulatory proteins associated with tumor initiation and progression have been elucidated. In this minireview, I summarize the recent progress in understanding the functional role of MYCN and its regulatory partners in neuroblastoma metastasis.