JERUSALEM, ISRAEL, 7 April 2026 – The tumor begins before birth. Somewhere in the developing fetus, neural crest cells that should have matured into adrenal tissue or sympathetic ganglia take a wrong turn, and a child is born harboring a malignancy that may not declare itself for months. Neuroblastoma accounts for roughly 28 percent of all cancers diagnosed in infants across Europe and the United States. In its gentlest form it regresses on its own, a fire that puts itself out. In its cruelest, it metastasizes with a velocity that makes oncologists reach for words they would rather not use in front of parents. High-risk neuroblastoma carries a five-year survival rate of approximately 40 percent. That number has barely moved in a generation.
Now, a study published in the peer-reviewed journal Brain Medicine (Genomic Press) offers something that has been in short supply: a mechanistic explanation for how this cancer sustains itself, and a way to cut the wire.
The Messenger That Turned Mercenary
Nitric oxide is among the most ancient signaling molecules in biology. It dilates blood vessels. It carries messages between neurons. At physiological concentrations it is indispensable, a quiet civil servant. But at elevated concentrations it 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. Prof. Haitham Amal and colleagues at the Hebrew University of Jerusalem 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.
Two Roads to the Same Silence
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 logic was deliberate: if a drug and a genetic tool produce the same result, you are looking at biology, not pharmacological noise.
They produced the same result.
BA-101 reduced NADPH-diaphorase activity, the standard readout of NOS function, by 35 to 40 percent. Genetic silencing cut it by 45 to 50 percent. Nitrite levels, a stable proxy for nitric oxide production, fell 65 to 70 percent with BA-101 and 55 to 60 percent 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.
A Signaling Cascade Collapses from the Top
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.
Then the dominoes. 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 was the most telling detail: 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 reengage, like releasing a foot from an accelerator that someone had wired to the floor.
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.
The Mirror Experiment
Good science asks the question in reverse. If blocking nitric oxide suppresses mTOR signaling, then flooding the cell with nitric oxide should amplify it. The researchers exposed SH-SY5Y cells to SNAP, a nitric oxide donor, at 200 μM for 24 hours.
Every needle swung the other way. 3-nitrotyrosine rose (p < 0.05). TSC2 fell (p < 0.01). Phosphorylation of AKT, mTOR, and RPS6 all increased (p < 0.05 for each). The converse experiment produced the converse result, which is the kind of symmetry that separates a finding from a fluke.
From the Dish to the Animal
Cell culture can tell you a great deal. It cannot tell you whether a tumor inside a living body will respond the same way. The team established xenograft neuroblastoma by injecting SH-SY5Y cells subcutaneously into the flanks of six-week-old NOD-SCID mice, waited for palpable tumors, then administered BA-101 intraperitoneally at 80 mg/kg/day for 22 days. Control animals received vehicle alone. Six mice per group.
The control tumors 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 (p < 0.001 for both). Body weight did not differ significantly between groups, suggesting that the compound was tolerated without gross systemic toxicity. The in vivo data did what in vivo data must do: they confirmed that the mechanism observed in the dish operates in a whole organism.
“The magnitude of the in vivo suppression caught our attention,” said Prof. Haitham Amal , the study’s corresponding author, who holds appointments at the Institute for Drug Research, School of Pharmacy, Faculty of Medicine, 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.”
“What convinced me was the concordance between the pharmacological and genetic approaches,” said Dr. Shashank Kumar Ojha , first author of the study and a researcher at the Institute for Drug Research, Hebrew University of Jerusalem. “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.”
What Remains Unknown
The authors are forthright about limitations. The in vitro work relies 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.
These are honest caveats, and they matter. But they do not diminish the central finding. The nNOS–mTOR axis is real, it is druggable, and in mice it responds to intervention with a force that demands further study.
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 a different geometry of attack: 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.
There is a long distance between a mouse flank and a child’s bedside. The researchers know this better than anyone. But a door that was previously invisible has now been located, measured, and shown to open. What walks through it next will depend on the studies that follow.
The peer-reviewed research article in Brain Medicine titled “Targeting nNOS suppresses AKT–TSC–mTOR signaling and inhibits neuroblastoma growth,” is freely available via Open Access, starting on 7 April 2026 in Brain Medicine at the following hyperlink: https://doi.org/10.61373/bm026a.0027 .
The full reference for citation purposes is: Ojha SK, Tripathi MK, Khaliulin I, Choudhary V, Kartawy M, Amal H. Targeting nNOS suppresses AKT–TSC–mTOR signaling and inhibits neuroblastoma growth. Brain Medicine 2026. DOI: https://doi.org/10.61373/bm026a.0027 . Epub 2026 Apr 7.
About Brain Medicine: Brain Medicine (ISSN: 2997-2639, online and 2997-2647, print) is a peer-reviewed medical research journal published by Genomic Press, New York. Brain Medicine is a new home for the cross-disciplinary pathway from innovation in fundamental neuroscience to translational initiatives in brain medicine. The journal’s scope includes the underlying science, causes, outcomes, treatments, and societal impact of brain disorders, across all clinical disciplines and their interface.
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Brain Medicine
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Targeting nNOS suppresses AKT–TSC–mTOR signaling and inhibits neuroblastoma growth
7-Apr-2026
Haitham Amal is the Chief Scientific Officer (CSO) of Point6 Bio and NeuroNOS Ltd. NeuroNOS Ltd., which partly funded this work, has obtained a license for the patent applications related to the BA-101 molecule filed by Yissum (The Hebrew University Technology Transfer Company). No funding from Point6 Bio was received for this study. All other authors declare no competing interests.