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Neuroblastoma is the most common tumor among children under a year of age, and while in its gentlest form neuroblastoma can regress on its own, it can also take an aggressive form, with high-risk neuroblastoma carrying a five-year survival rate of about 40%.

Researchers at The Hebrew University of Jerusalem have now discovered a mechanistic explanation for how neuroblastoma sustains itself and identified a potential approach to severing that mechanism, by inhibiting nitric oxide (NO) production to suppress mTOR signaling. The collective results from work in human neuroblastoma cells and experiments in a mouse xenograft model showed that inhibiting the enzyme neuronal nitric oxide synthase (nNOS) to inhibit NO production suppressed mTOR signaling and slowed tumor growth.

Professor Haitham Amal, PhD, head of The Laboratory of Neuromics, Cell Signaling, and Translational Medicine, is senior and co-corresponding author of the team’s published paper in Brain Medicine, titled “Targeting nNOS suppresses AKT–TSC–mTOR signaling and inhibits neuroblastoma growth.” In their paper the team concluded “Inhibition of nNOS suppresses mTOR signaling, reduces cellular malignancy, and attenuates tumor growth in vivo, identifying the nNOS-mTOR axis as a promising therapeutic target in neuroblastoma.”

Neuroblastoma accounts for roughly 28% of all cancers diagnosed in infants across Europe and the United States. “Neuroblastoma (NB) refers to a spectrum of neuroblastic tumors that originate from the neural crest cells during fetal development,” the authors wrote. “Neuroblastoma is predominantly a pediatric malignancy, with approximately 97% of cases occurring in children.”

NBs can range from spontaneous regression to maturation to an aggressive, deadly metastatic disease. And as the investigators noted, “Despite major advances in multimodal therapy, high-risk neuroblastoma remains associated with poor prognosis, frequent relapse, and therapy resistance, underscoring the need for a better understanding of the signaling pathways that regulate tumor cell survival, differentiation, and metabolic adaptation.”

Nitric oxide (NO) is an essential regulator of carcinogenesis in various tumors, including NB, the authors pointed out. “Nitric oxide (NO) is a ubiquitous free radical signaling molecule produced in multiple organs and tissues), such as those of the central and peripheral nervous systems.” But at elevated concentrations NO becomes reactive, generating nitrogen species that chemically modify proteins through a process called S-nitrosylation. That modification has been implicated in every stage of cancer progression.

The relationship between nitric oxide and tumors is not simple. Very high concentrations can damage DNA and trigger apoptosis. Lower, sustained levels appear to do the opposite, promoting survival and metastasis. Amal and colleagues had previously demonstrated that nitric oxide drives glioblastoma progression. The question that remained was whether the same enzyme, neuronal nitric oxide synthase, was performing a similar service for neuroblastoma, and if so, through which downstream pathway. The answer turned out to be mTOR.

The team attacked nNOS from two directions. They treated human SH-SY5Y neuroblastoma cells with BA-101, a selective pharmacological inhibitor, at 100 μM for 24 hours. Separately, they silenced the nNOS gene with small interfering RNA. The reasoning was that if a drug and a genetic tool produce the same result, you are looking at biology, not pharmacological noise.

The experiments produced the same result. BA-101 reduced NADPH-diaphorase activity, the standard readout of NOS function, by 35-40%. Genetic silencing cut it by 45-50%. Nitrite levels, a stable proxy for nitric oxide production, fell 65-70% with BA-101 and 55-60% with siRNA. Colony formation, the most direct measure of proliferative capacity, dropped significantly after both BA-101 treatment (p < 0.001) and nNOS silencing (p < 0.01). The cells were losing their ability to multiply.

What followed downstream was systematic. Protein tyrosine nitration, measured by 3-nitrotyrosine immunoreactivity, fell sharply after BA-101 treatment (p < 0.01) and nNOS silencing (p < 0.001). The chemical signature of nitrosative stress was fading.

The results then confirmed that AKT phosphorylation decreased (p < 0.01 with BA-101; p < 0.05 with siRNA), while total AKT remained unchanged. Phosphorylation of mTOR itself declined under both conditions (p < 0.01 each). The downstream mTORC1 substrate ribosomal protein S6 followed (p < 0.05 with BA-101; p < 0.01 with siRNA).

And here, the most telling detail, that TSC2, a master negative regulator of mTOR signaling, rose significantly under both treatments (p < 0.05). Removing the nitric oxide signal had allowed the cell’s own braking system to re-engage. In summary, the authors noted, “Pharmacological inhibition of nNOS with BA-101 (100 μM, 24 h) or genetic silencing of nNOS with siRNA caused upregulation of the key negative regulator TSC2 and decreased phosphorylation of AKT, mTOR, and RPS6, indicating suppression of mTOR pathway activity.”

Synaptophysin, a neuroendocrine tumor marker used to gauge the malignant identity of neuroblastoma cells, decreased significantly with BA-101 (p < 0.01) and nNOS knockdown (p < 0.05). The tumor cells were not merely growing more slowly. They were becoming, at a molecular level, less recognizably cancerous. In summary, the investigators noted, “Our results show that inhibition of NO production in the human NB cell line (SH-SY5Y cells), either by pharmacological intervention using the selective nNOS inhibitor BA-101 (41) or by genetic ablation using the specific siRNA, successfully suppressed NB malignancy.”

But if blocking nitric oxide suppresses mTOR signaling, then flooding the cell with nitric oxide should amplify it. The researchers tested this by exposing SH-SY5Y cells to SNAP, a nitric oxide donor, at 200 μM for 24 hours. This converse experiment produced the converse result. 3-nitrotyrosine rose (p < 0.05), and TSC2 fell (p < 0.01). Phosphorylation of AKT, mTOR, and RPS6 all increased (p < 0.05 for each).

The team then tested their findings in a xenograft mouse model of neuroblastoma, treated with BA-101. “Importantly, to extend these findings to an in vivo context, we further assessed the impact of pharmacological nNOS inhibition on tumor growth in a xenograft NB model,” they stated. The investigators found that while tumors in control animals grew to approximately 1.5 cm in their largest dimension, the treated tumors did not. Final tumor volume and weight were dramatically reduced in the BA-101 group. ‘Quantitative analysis revealed a dramatic decrease in the final tumor volume and weight in the BA-101-treated group (p < 0.001) compared with controls,” they noted.

Body weight did not differ significantly between groups, suggesting that the compound was tolerated without gross systemic toxicity. In summary, the authors wrote, “Our finding demonstrate that the pro-tumorigenic effects of nNOS in SH-SY5Y involve activation of themTOR signaling pathway.” Importantly, both genetic inhibition of nNOS using siRNA and pharmacological inhibition with BA-101 effectively suppressed mTOR pathway activation and reduced malignant properties of NB cells, highlighting the therapeutic relevance of targeting nNOS signaling.  “These findings indicate that pharmacological inhibition of nNOS effectively suppresses xenograft tumor progression, highlighting the critical role of nNOS-derived NO in promoting neuroblastoma growth in vivo.”

“The magnitude of the in vivo suppression caught our attention,” said Amal, the study’s corresponding author, who holds appointments at the Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, and the Rosamund Stone Zander and Hansjoerg Wyss Translational Neuroscience Center at Boston Children’s Hospital, Harvard Medical School. “We had demonstrated the role of nitric oxide in glioblastoma previously, but the consistency of the neuroblastoma results across every assay, from protein phosphorylation to colony formation to xenograft growth, points to nNOS as something more than a contributor. It appears to be a central driver of the signaling that sustains this tumor.”

Added first author Shashank Kumar Ojha, PhD, first author of the study and a researcher at the Institute for Drug Research, The Hebrew University of Jerusalem, added, “What convinced me was the concordance between the pharmacological and genetic approaches. When BA-101 and siRNA independently produce the same pattern of effects across NADPH-diaphorase activity, nitrosative stress markers, mTOR pathway phosphorylation, and clonogenic growth, you can be confident the biology is real. That reproducibility is what gives you a therapeutic hypothesis worth testing further.”

The authors acknowledged limitations to their study. The in vitro work relied on a single cell line, SH-SY5Y, which cannot capture the full genetic heterogeneity of neuroblastoma or the complexity of the tumor microenvironment. The chemical identity of BA-101 is currently undisclosed pending patent issuance, which means independent replication by other laboratories must wait. Whether nitrosative stress directly underlies its functional impairment, or whether an intermediary mechanism is involved, remains an open question that the authors explicitly flag for future investigation. “Future studies using patient-derived cells, organoids, or genetically engineered mouse models will be important to further validate and extend these observations,” they stated. Nevertheless, the authors suggest, the limitations do not diminish the central discovery of a druggable nNOS–mTOR axis.

mTOR inhibitors such as rapalogs and catalytic mTOR inhibitors have shown limited efficacy as monotherapies in neuroblastoma, undermined by feedback activation and resistance mechanisms. The present study suggests the potential for a different attack strategy. Rather than targeting mTOR at the lock, intervene upstream at the hand that turns the key. By reducing nitric oxide-dependent mTOR activation, nNOS inhibition may sidestep the compensatory pathways that have frustrated direct mTOR blockade. “Collectively, these results identify the nNOS-mTOR axis as a key driver of neuroblastoma progression and suggest that nNOS inhibition represents a promising strategy for NB treatment,” they concluded.

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