The review, led by Samruddhi Kaknale and Varnitha Manikantan of Symbiosis Medical College for Women in Pune, India, together with collaborators at Symbiosis School of Biological Sciences, Karunya Institute of Technology and Sciences, and Manipal Institute of Technology Bengaluru, systematically maps the pathobiology of infant neuroblastoma, the mechanics of CRISPR/Cas9 targeting, and the nanocarrier platforms that might deliver the editing machinery safely in vivo. The authors are careful to frame the work as an investigational hypothesis rather than an established clinical application, noting that direct evidence in infant neuroblastoma remains limited and that most findings come from general neuroblastoma models, non-cancer models, or adult cancers that should not be interpreted as proof of efficacy in infants.

At the heart of the therapeutic concept lie two molecular hallmarks: MYCN amplification and activating ALK mutations. MYCN is a potent transcription factor that drives proliferation, suppresses apoptosis, and propels tumor progression, and its amplification is an established adverse prognostic feature associated with advanced stage, rapid growth, and poor outcome. ALK mutations, meanwhile, produce constitutive kinase activity that fuels oncogenic signaling. Because these alterations define the biology of high-risk disease, they represent the most attractive targets for precision gene editing, offering a shift from broad cytotoxic chemotherapy toward highly selective molecular intervention.

The CRISPR/Cas9 system itself is elegantly simple in principle. It consists of the Cas9 nuclease and a single guide RNA, a synthetic chimeric RNA containing a scaffold for Cas9 binding and a 20-nucleotide spacer complementary to the target DNA. For the commonly used Streptococcus pyogenes Cas9, target recognition requires a 5′-NGG-3′ protospacer-adjacent motif. Once directed to its genomic address, Cas9 induces a targeted double-strand break that the cell repairs through endogenous mechanisms. The predominant pathway, non-homologous end joining, is error-prone and typically generates small insertions or deletions that frameshift and inactivate the target gene, a powerful strategy for knocking out oncogenes such as MYCN or ALK. The less frequent homology-directed repair pathway uses a homologous DNA template and can, in principle, correct specific point mutations, such as activating ALK variants, with meticulous precision.

Recent advances have refined this toolkit further. Base editors harness a catalytically impaired Cas9 fused to a deaminase to convert single nucleotides directly, for example C to T or A to G, without double-strand breaks. Prime editors combine nCas9 with reverse transcriptase and a prime editing guide RNA to direct templated insertions, deletions, and all twelve possible base-to-base conversions, offering search-and-replace capabilities that reduce the risk of indels and chromosomal rearrangements. The review notes that direct evidence for base or prime editing in neuroblastoma remains limited, marking these approaches as avenues for future investigation rather than validated options.

Preclinical evidence, however, is already compelling in cell lines and animal models. High-throughput CRISPR screens have identified genetic dependencies in neuroblastoma, including a reliance on the PRC2 components EZH2, EED, and SUZ12 in MYCN-amplified models. Direct knockout of MYCN and ALK in various neuroblastoma cell lines has produced significant reductions in cell proliferation, inhibition of tumor growth in vitro and in vivo, and induction of apoptosis. CRISPR interference targeting the MYCN promoter has effectively repressed MYCN expression and suppressed neuroblastoma cell growth, while knockout of uPAR, a gene linked to cellular invasion, has inhibited invasive phenotypes, suggesting the technology can also blunt metastasis by targeting genes involved in tumor cell motility.

Yet the review is unflinching about the obstacles. Delivering Cas9 and its guide RNA into a sufficient percentage of tumor cells in vivo remains the central challenge, complicated by the large size, negative charge, immunogenicity, and degradation susceptibility of CRISPR cargoes. The Cas9 protein, derived from bacteria, can elicit innate and adaptive immune responses that clear edited cells or render subsequent treatments unproductive, and preexisting adaptive immunity to Cas9 proteins has been identified in humans. Off-target editing persists despite improvements in guide RNA design and high-fidelity Cas9 variants, and achieving uniform editing efficiency across a heterogeneous solid tumor microenvironment, with poor penetration and variable cellular uptake, is extraordinarily difficult. For infants, whose expected lifespans stretch across decades, persistent Cas9 activity, potential germline exposure, chromosomal rearrangements, large deletions, structural variants, and long-term effects in normal tissues demand careful assessment and lifelong follow-up before any clinical translation.

Nanoparticle delivery systems emerge as the leading candidates to surmount these barriers. Lipid nanoparticles, the most clinically advanced platform and the technology behind successful mRNA vaccines, typically comprise ionizable lipids, helper lipids like cholesterol and phospholipids, and PEGylated lipids. Ionizable lipids are positively charged at acidic pH but neutral at physiological pH, facilitating nucleic acid complexing and endosomal escape after cellular uptake, while PEGylated lipids preserve systemic circulation by preventing rapid elimination by the reticuloendothelial system. Delivery of Cas9 mRNA with sgRNA allows transient protein expression, minimizing off-target effects and immune activation associated with prolonged Cas9 activity, whereas pre-formed Cas9 ribonucleoproteins offer reduced immunogenicity and faster editing onset but are more complicated to encapsulate due to their size.

Polymeric and inorganic nanocarriers expand the design space. Biodegradable polymers such as PLGA, PEG, chitosan, and dendrimers can encapsulate CRISPR components with adjustable degradation and sustained release, and PLGA’s capacity to safeguard sensitive payloads makes it widely used for surface functionalisation aimed at neuroblastoma cell targeting. Gold nanoparticles are biocompatible and easily functionalized for direct conjugation with Cas9 and sgRNA, with photothermal applications, while magnetic nanoparticles enable magnetic targeting and MRI tracking, and mesoporous silica nanoparticles provide high loading, controlled release, and rigid protection of CRISPR cargo. For the minority of patients with CNS involvement or relapse, blood-brain barrier penetration strategies, including receptor-mediated transcytosis via ligands binding the transferrin, insulin, or low-density lipoprotein receptors, cell-penetrating peptides like TAT and R8, and focused ultrasound combined with microbubbles to transiently and locally disrupt the barrier, are being explored, though the review cautions that enhanced permeability does not necessarily indicate efficient nanoparticle delivery to the brain and may increase the risk of unintended CNS exposure and neurotoxicity.

The authors conclude that robust preclinical validation in relevant in vitro and in vivo models, including organoids, patient-derived xenografts, and orthotopic systems that precisely mimic the human disease, is essential for determining efficacy, characterizing biodistribution, evaluating off-target effects, and establishing a preliminary safety profile. Ethical and regulatory considerations add further complexity, as infants lack capacity for informed consent, requiring parents or legal guardians to decide in the child’s best interests amid the potentially irreversible nature of genomic modification. Future work should prioritize more selective nanoparticles, gene-editing tools with high precision and minimal off-target effects, and internationally harmonised regulatory frameworks for paediatric applications. With sustained interdisciplinary collaboration, the convergence of nanoparticle delivery and CRISPR/Cas9 precision may ultimately enable safer and more effective therapies for infants with neuroblastoma, though the review emphasizes that current evidence remains predominantly preclinical and is not yet sufficient to establish the optimal nanoparticle or CRISPR technique for infant neuroblastoma.

Subject of Research: Nanoparticle-enabled CRISPR/Cas9 gene editing as a potential therapy for infant neuroblastoma

Article Title: Precision gene editing in infant neuroblastoma: a review of nanoparticle: enabled CRISPR/Cas9 therapies

Article References: Kaknale, S., Ranjan, A., Enoch, I. V. M. V., Vyas, A., & Manikantan, V. (2026). Precision gene editing in infant neuroblastoma: a review of nanoparticle: enabled CRISPR/Cas9 therapies. Molecular Biology Reports, 53(1), Article 1725. https://doi.org/10.1007/s11033-026-12916-2

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12916-2

Keywords: infant neuroblastoma, CRISPR/Cas9, nanoparticles, lipid nanoparticles, gene therapy, MYCN, ALK, drug delivery, paediatric oncology, base editing, prime editing, preclinical models

The story of ALK itself is one of the great sagas of precision oncology. First identified in 1994 as part of an NPM-ALK fusion in non-Hodgkin lymphoma, the gene later achieved fame when the EML4-ALK fusion was discovered in non-small-cell lung cancer in 2007, opening the door to a series of remarkably effective ALK inhibitors such as crizotinib and its successors. ALK can be activated in cancer through several distinct mechanisms: gene fusions that create constitutively active chimeric kinases, activating point mutations, copy-number gains and amplifications, and abnormal transcriptional regulation. Each of these mechanisms is now routinely tested for in clinical laboratories, and each carries a different therapeutic implication. The question raised by ALK ATI is whether this alternative transcript represents a fifth, previously underappreciated route to ALK-driven malignancy, or whether it is a biological curiosity that has been overinterpreted by clinicians hungry for new actionable targets.

The original discovery, reported by Wiesner and colleagues in Nature in 2015, was striking. The researchers found that a subset of melanomas expressed ALK protein without any detectable gene fusion, and traced this expression to an alternative promoter within intron 19. In experimental models, the truncated protein appeared to be phosphorylated, to activate oncogenic signalling pathways, to drive tumour formation, and, tantalisingly, to confer sensitivity to crizotinib. Because the kinase domain remained intact and the inhibitory drugs target that domain, the logic seemed sound: a tumour dependent on ALK ATI signalling might respond to ALK inhibitors even without a fusion. This raised the prospect of a tumour-agnostic biomarker, one that could extend ALK-directed therapy to patients with melanoma, sarcoma and other solid tumours whose cancers lacked canonical ALK rearrangements.

Subsequent laboratory work added layers of intrigue. One study showed that the ALK ATI isoform, in a kinase-activity-dependent manner, induces structural changes in chromatin within the nucleus, suggesting the truncated protein might remodel gene expression programs directly rather than simply relaying signals at the cell surface. Another report found that ALK ATI interacts with the c-Myc oncogene and promotes cancer stem-cell-like properties in sarcoma models, hinting at a role in tumour initiation, metastasis and treatment resistance. More recently, a 2025 study in ovarian high-grade serous carcinoma described a transcriptional variant of ALK that appeared to promote apoptosis, an unexpected and paradoxical finding that complicates any simple story of ALK ATI as a straightforward oncogene. Together, these results painted a picture of a versatile and context-dependent molecule whose effects vary dramatically depending on tumour type and cellular environment.

But the edifice began to crack under the weight of independent replication attempts. As the Spanish review documents in detail, subsequent experiments did not consistently reproduce the original claims of growth-factor-independent transformation or sensitivity to ALK inhibitors. A 2021 study using genomic and experimental evidence concluded that ALK ATI does not predict single-agent sensitivity to ALK inhibitors. In melanoma models expressing the alternative transcript, researchers generally observed low levels of ALK phosphorylation and no selective drug response, undermining the central premise that ALK ATI marks tumours that will benefit from ALK-directed therapy. The discrepancy between the initial excitement and the follow-up data illustrates a familiar pattern in translational cancer research: early findings from a limited set of models can generate hypotheses that broader and more rigorous testing fails to confirm.

The clinical literature on ALK ATI is equally fragmented. Studies across melanoma, sarcoma, ovarian carcinoma and histiocytic neoplasms have been retrospective, have employed heterogeneous assays, and have reported inconsistent prevalence figures and contradictory prognostic associations. In melanoma, where the phenomenon was first described, surveys of primary and metastatic cutaneous tumours confirmed that ALK expression through alternative transcriptional initiation occurs, but its correlation with outcomes has been disputed. In histiocytic neoplasms, a 2026 study reported that ALK ATI drives nuclear ALK expression even in the absence of ALK fusions, adding another layer of complexity to the interpretation of ALK staining in these rare tumours. Meanwhile, isolated case reports, such as a PRKCA-fused blue melanocytoma expressing a truncated ALK isoform, demonstrate that the biology of truncated ALK expression can intersect with other genetic events in unpredictable ways.

Perhaps the most consequential part of the review concerns diagnostics. Standard clinical tests for ALK are simply not designed to detect ALK ATI, and misreading them can lead to dangerous conclusions. ALK immunohistochemistry, which detects the protein regardless of how it is produced, cannot distinguish between expression driven by a fusion, by amplification, by ATI or by aberrant expression in inflammatory conditions such as malakoplakia. Break-apart fluorescence in situ hybridization, which detects physical disruption of the ALK locus, will be negative in ALK ATI because the gene is not rearranged. Confirmation that a tumour expresses ALK ATI requires an RNA-based assay specifically designed to demonstrate transcription initiating from intron 19 and continuing into ALK exons 20 through 29, together with the exclusion of a canonical ALK fusion. Without this molecular rigour, a positive ALK stain in a fusion-negative tumour tells the clinician almost nothing actionable.

The authors’ bottom line is deliberately cautious and carries immediate practical weight for oncologists and pathologists. Current evidence, they conclude, supports ALK ATI as a context-dependent biological phenomenon rather than a validated tumour-agnostic driver or predictive biomarker. In plain terms, finding the transcript in a tumour does not establish that the tumour depends on it, and it certainly does not establish that the patient will benefit from an ALK inhibitor. Accordingly, the review states that ALK-inhibitor treatment should not be selected solely on the basis of ALK ATI outside a clinical trial or a carefully documented individual assessment. This is a direct challenge to any practice of treating ALK-immunopositive, fusion-negative tumours with expensive and toxic targeted drugs on the assumption that the alternative transcript is driving them.

The broader lesson extends beyond a single transcript. Pan-cancer transcriptome analyses have revealed that regulation through alternative promoters is pervasive across the human genome, meaning that many cancer genes likely have truncated or altered isoforms awaiting discovery. Distinguishing passenger transcription from true oncogenic dependence will require the same combination of rigorous functional validation, standardised RNA-based diagnostics and prospective clinical trials that ultimately separated genuine ALK fusions from lookalike findings. For now, ALK ATI stands as a cautionary tale and an open scientific question: a kinase fragment that once promised to widen the reach of precision oncology, and that may yet prove important in specific tumour contexts, but that today remains, in the measured words of the Alicante team, unproven as a basis for treatment decisions.

Subject of Research: The role of the ALK ATI alternative transcript as a potential driver and biomarker in solid tumours

Article Title: ALK ATI in solid tumours: biological evidence, diagnostic challenges, and clinical relevance. a narrative review

Article References: Borregón, M., Algarra, M.-A., & Benítez-Fuentes, J.-D. (2026). ALK ATI in solid tumours: biological evidence, diagnostic challenges, and clinical relevance, a narrative review. Molecular Biology Reports, 53(1), Article 1657. https://doi.org/10.1007/s11033-026-12833-4

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12833-4

Keywords: ALK, ALK ATI, alternative transcription initiation, solid tumours, melanoma, sarcoma, ALK inhibitors, crizotinib, molecular diagnostics, precision oncology, biomarkers, kinase signalling

ALK-positive NSCLC arises when a chromosomal rearrangement fuses the ALK kinase gene to a partner gene, most commonly EML4, producing a constitutively active signaling protein that drives uncontrolled proliferation. Since the first ALK inhibitor, crizotinib, entered the clinic, a succession of increasingly potent tyrosine kinase inhibitors (TKIs) has transformed outcomes for these patients. The latest generation agent, lorlatinib, demonstrated durable benefit in the phase III CROWN study. Yet resistance remains an inevitability for many patients. ALK’s kinase domain is a mutable target, and sequential treatment with TKIs selects for compound mutations that no drug approved to date can fully suppress. Patients who progress on available TKIs have limited options, a gap the authors identify as the central motivation for an orthogonal therapeutic approach.

Targeted protein degradation offers such an approach. Rather than blocking an enzyme’s active site, degraders eliminate the target protein altogether, harnessing the ubiquitin-proteasome system that cells use for routine protein turnover. Two broad strategies exist. Proteolysis-targeting chimeras, or PROTACs, are large bifunctional molecules that physically tether a target protein to an E3 ubiquitin ligase. Molecular glue degraders, by contrast, are typically smaller, more drug-like compounds that bind the E3 ligase substrate receptor and remodel its surface to recognize a neosubstrate. Their smaller size confers potential advantages in oral bioavailability and tissue penetration, but discovering glues for a chosen target has historically been a matter of luck rather than design, because the interaction is not obvious from sequence alone.

The Triana team found TRI-611 through a deliberate engineering effort. Using a time-resolved fluorescence resonance energy transfer screen that reports when ALK and cereblon (CRBN), the substrate adaptor of the CRL4 ubiquitin ligase complex, are brought into proximity, the researchers identified an initial hit compound and then optimized it for potency, selectivity and drug-like properties. Structural and biophysical work, including cryo-electron microscopy of the ternary complex, revealed that TRI-611 operates through a distinctive mechanism. The glue promotes contact between the ALK kinase domain and CRBN through a degron interface positioned distal to the kinase active site. This arrangement matters for two reasons: the drug does not need to compete with ATP at a mutation-prone catalytic pocket, and its binding surface tolerates mutations that disable orthosteric inhibitors.

That structural feature translates directly into resistance coverage. In engineered cell lines and patient-derived models, TRI-611 degraded not only wild-type EML4-ALK but also versions carrying clinically important TKI-resistance mutations, including the gatekeeper mutation L1196M and the solvent-front mutation G1202R, individually and in combination. Biochemical assays demonstrated that the drug supported ubiquitin transfer onto the mutant kinase domain by the CRBN-containing ligase complex, and degradation potency correlated tightly with anti-proliferative activity across a panel of resistant lines. Proteomic profiling after TRI-611 treatment showed a remarkably clean footprint: among thousands of quantified proteins, ALK fusion proteins were selectively depleted, with minimal effects on known CRBN neosubstrates or other kinases. In an era when glue degraders are often criticized for promiscuous neosubstrate recruitment, that selectivity is a notable technical achievement.

The pharmacology was engineered with the brain in mind. ALK-positive NSCLC has a notorious tendency to metastasize to the central nervous system, and the blood-brain barrier excludes many otherwise effective drugs, making brain metastases a frequent site of progression. Guided by central nervous system multiparameter optimization principles, the team tuned the compound’s permeability and efflux properties. In mice, TRI-611 achieved brain-to-plasma exposure ratios comparable to lorlatinib, a benchmark for brain penetration, and unbound brain concentrations exceeded the levels needed for target degradation. Once-daily oral dosing produced regressions of subcutaneous xenografts derived from both cell lines and patients, including the DFCI-669 model and the MGH953-7 patient-derived model, the latter harboring TKI-resistance mutations. Critically, intracranial xenograft models, including one driven by the doubly resistant L1196M/G1202R mutant, also responded, with luminescence-based tumor measurements showing marked shrinkage in treated animals.

Perhaps the most clinically provocative finding concerns combination therapy. Because TRI-611 binds the kinase domain at a surface remote from the ATP pocket, it does not interfere with orthosteric TKIs, and the two modalities can be used together. In vitro, the combination of TRI-611 with alectinib or lorlatinib produced greater-than-additive killing of ALK-dependent cells, quantified by Bliss synergy analysis. In vivo, pairing the degrader with lorlatinib yielded synergistic and durable tumor regressions in subcutaneous and intracranial models, outperforming either agent alone. The logic is compelling: the TKI suppresses signaling immediately while the glue removes the protein irreversibly, and degrading the target may suppress the evolutionary escape routes that kinase inhibition alone leaves open. The combination also addresses a second resistance mechanism, MET amplification, which can bypass ALK entirely; pairing TRI-611 with a MET inhibitor such as capmatinib restored growth control in MET-overexpressing models.

The study is thorough on the mechanistic details that regulators and clinicians will want. Degradation required the proteasome, as shown by rescue experiments with bortezomib, and was enhanced by CRBN overexpression, confirming the ligase dependence of the effect. Treated tumors showed loss of phosphorylated STAT3, a downstream readout of ALK signaling, within hours of dosing, linking pharmacokinetics to pharmacodynamics. Structural biology pinned the molecular determinants: mutation of a key ALK residue in the degron interface, D1389W, abolished both degradation and the compound’s anti-proliferative effect, demonstrating that the observed binding mode is causally responsible for the biology. Cryo-EM maps and a deposited crystal structure provide a template that other groups can mine for the emerging rules of glue-induced neosubstrate recognition.

Caveats remain, as they always do at the preclinical-to-clinical boundary. The efficacy data come from xenograft and cell-line models rather than patients, and the long-term consequences of chronically eliminating ALK, which has physiological roles outside cancer, will need careful toxicological evaluation. The authors note that their work was conducted by employees of Triana Biomedicines, with a patent covering TRI-611 filed by the company, and the compound’s clinical profile will ultimately be decided in human trials. Still, the bar this molecule clears is high: potent, selective, orally bioavailable, brain-penetrant degradation of a validated oncogenic driver, active against the resistance mutations that defeat today’s best drugs, and compatible with rational combinations. If the clinical experience mirrors the preclinical one, TRI-611 could mark the moment molecular glue degraders graduated from the lab bench to a front-line role against gene-fusion-driven cancers, and the strategy of degrading rather than inhibiting oncogenic kinases may rapidly expand beyond ALK.

Subject of Research: A selective, brain-penetrant molecular glue degrader of ALK fusion proteins for ALK-positive non-small-cell lung cancer

Article Title: TRI-611, a selective, brain-penetrant molecular glue degrader of ALK

Article References: Conery, A. R., La, D. S., Alekseyenko, A. A., Marcoux, D., Bart, A. G., Harlow, M. L., Arsenault, P. R., Cantone, N. R., Casaubon, R. L., Constan, A., Kamadurai, H. B., Medikonda, A. P., Nunes, D. E., Szeto, H., Wigle, T. J., Yu, M., Zagulyaeva, A., Zarate, C. M., Highfield, L., … Palombella, V. J. (2026). TRI-611, a selective, brain-penetrant molecular glue degrader of ALK. Nature. https://doi.org/10.1038/s41586-026-10998-3

Image Credits: AI Generated

DOI: 10.1038/s41586-026-10998-3

Keywords: ALK, TRI-611, molecular glue degrader, non-small-cell lung cancer, targeted protein degradation, cereblon, EML4-ALK, TKI resistance, brain penetration, lorlatinib, oncogenic fusion, drug discovery