We report a child with an antenatally detected brain tumor that progressed over three years’ time despite surgery, chemo- and proton therapy. Retrospective whole-genome and transcriptome sequencing with methylation analysis of primary tumor tissue led to the molecular diagnosis infant-type hemispheric glioma, and identified a novel SNRNP70::ALK fusion, providing a therapeutic target for compassionate-use precision treatment with the ALK tyrosine kinase inhibitor lorlatinib. Functional studies confirmed the fusion protein to be expressed and active in the patient’s tumor. After two years of therapy, the child has sustained partial tumor regression on MRI and no new neurological symptoms. We conclude that comprehensive multi-omics analyses are required for correct molecular diagnosis in childhood CNS tumors and can radically impact patient outcome by identifying molecular targets for precision treatment.

Abstract There is growing recognition of how metabolic dependencies influence sensitivity or resistance to neuroblastoma (NB) therapies. In this regard, the impact of Anaplastic Lymphoma Kinase (ALK), a receptor tyrosine kinase which is mutated in approximately 10% of primary NB cases, and a clinical target in NB on cellular metabolism is unclear. In this study, we combined phosphoproteomics and BioID-mediated proximity interaction screening in neuroblastoma cell lines, identifying the sodium bicarbonate transporter SLC4A7, a member of the Solute Carrier (SLC) family, and the multifunctional protein Carbamoyl Phosphate Synthetase 2-Aspartate Transcarbamylase-Dihydroorotase (CAD) downstream of ALK signaling in NB cells. SLC4A7 and CAD are both important molecular components in the metabolism of pyrimidine nucleotides. ALK activation leads to phosphorylation of both SLC4A7 and CAD, resulting in increased sodium bicarbonate transport and pyrimidine biosynthesis that is blocked by the addition of ALK tyrosine kinase inhibitors (TKIs). Combined targeting of both ALK signaling and nucleotide synthesis leads to a more effective inhibition of NB tumor growth in both cell and genetically modified mouse NB models. Finally, we show that SLC4A7 and CAD are also targets in ALK-driven non-small cell lung cancer (NSCLC), revealing a novel therapeutic strategy that leverages metabolomic dependencies to target ALK-driven malignancies such as NB and NSCLC. Citation Format: Ruth Palmer, Wei-Yun Lai, Tzu-Po Chuang, Joel Johansson, Emre Can Tuysuz, Dan E. Lind, Alexander Schmidt, Michael N. Hall, Bengt Hallberg. Anaplastic lymphoma kinase harnesses the SLC4A7 bicarbonate transporter to intensify de novo nucleotide synthesis in neuroblastoma and lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4732.

High-risk neuroblastoma is characterized by MYCN amplification and high MYCN or MYC gene expression. These patients have a poor prognosis and there is an urgent need for more effective drugs. While strategies to develop inhibitors that directly target the MYC proteins have remained largely unsuccessful, recent preclinical studies have identified ATR, a key protein of the DNA damage response, as a promising alternative therapeutic target. Here we identified a strong RUVBL1 and RUVBL2 signature in transcriptomics data derived from different MYCN-driven mice tumors treated with ATR inhibitors. The RUVBL proteins form a complex with ATPase activity that has broad cellular functions and we demonstrate that pharmacological inhibition of this protein complex results in a strong reduction of MYC(N) signaling, cell cycle arrest, DNA damage and apoptosis. We confirmed the association with MYCN and identified the RUVBL genes as independent prognostic biomarkers in human primary neuroblastoma data.

Levels of oncogenic ALK mutations in tissue samples

One of the key features of high-risk neuroblastoma (NB) is MYCN -amplification. While MYCN is still regarded as therapeutically challenging despite intensive efforts to find targeting compounds, inhibitors against Anaplastic Lymphoma Kinase (ALK) are now being evaluated among ALK-driven NB patients with promising results. There is a pressing need to find alternative treatment regimens for ALK naïve MYCN -amplified patients, as current regimes are accompanied by significant mortality and morbidity. Here we show that genetically removing Alk in the Th-MYCN -driven engineered NB mouse model (GEMM) resulted in decreased tumor penetrance, and survival of Th-MYCN homozygote GEMMs, suggesting that Alk activity contributes to aggressiv e neuroblastoma development in this model. Given the high levels of replication stress induced in Th-MYCN tumors, we employed inhibitors of Ataxia Telangiectasia Rad3 related (ATR), combining ATR and ALK inhibition in a 14-day treatment regime, and comparing with ATR inhibitor monotreatment. Th-MYCN tumor bearing mice that received ATRi/ALKi combination treatment exhibited significantly increased survival compared to ATRi alone, that was sustained over time. Together, our data highlight a potentially effective treatment option for the currently challenging category of MYCN -amplified NB patients lacking ALK mutations. ### Competing Interest Statement The authors have declared no competing interest. Swedish Cancer Society, CAN21/1459, CAN21/1525 Swedish Research Council, 2023-02433, 021-01192 Barncancerfonden, PR2021-27, PR2022-0029 Knut and Alice Wallenberg Foundation, KAW 2015.0144 Assar Gabrielsson Foundation, FB23-71 Gothenburg Society of Medicine, GLS- 986836 Research Foundation - Flanders, FWO.OPR.2023.0063

Coverage with the NB-ALK sequencing panel

Sequencing results, ALK hotspot mutation sites

Solute Carrier Family 3, Member 2 (SLC3A2 or 4F2hc) is a multifunctional glycoprotein that mediates integrin-dependent signaling, acts as a trafficking chaperone for amino acid transporters, and is involved in polyamine transportation. We identified SLC3A2 as a potential Anaplastic Lymphoma Kinase (ALK) interacting partner in a BioID-proximity labeling screen in neuroblastoma (NB) cells. In this work we show that endogenous SLC3A2 and ALK interact in NB cells and that this SLC3A2:ALK interaction was abrogated upon treatment with the ALK inhibitor lorlatinib. We show here that loss of ALK activity leads to decreased SLC3A2 expression and reduced SLC3A2 protein stability in a panel of NB cell lines, while stimulation of ALK with ALKAL2 ligand resulted in increased SLC3A2 protein levels. We further identified MARCH11, an E3 ligase, as a regulator of SLC3A2 ubiquitination downstream of ALK. Further, knockdown of SLC3A2 resulted in inhibition of NB cell growth. To investigate the therapeutic potential of SLC3A2 targeting, we performed monotreatment of NB cells with AMXT-1501 (a polyamine transport inhibitor), which showed only moderate effects in NB cells. In contrast, a combination lorlatinib/AMXT-1501 treatment resulted in synergistic inhibition of cell growth in ALK-driven NB cell lines. Taken together, our results identify a novel role for the ALK receptor tyrosine kinase (RTK), working in concert with the MARCH11 E3 ligase, in regulating SLC3A2 protein stability and function in NB cells. The synergistic effect of combined ALK and polyamine transport inhibition shows that ALK/MARCH11/SLC3A2 regulation of amino acid transport is important for oncogenic growth and survival in NB cells.

Patients with anaplastic lymphoma kinase (ALK)-driven neuroblastoma may respond to tyrosine kinase inhibitors, but resistance to treatment occurs and methods currently used for detection of residual disease have limited sensitivity. Here, we present a national unselected cohort of five patients with relapsed or refractory ALK-driven neuroblastoma treated with lorlatinib as monotherapy and test the potential of targeted circulating tumor DNA (ctDNA) analysis as a guide for treatment decisions in these patients. We developed a sequencing panel for ultrasensitive detection of ALK mutations associated with neuroblastoma or resistance to tyrosine kinase inhibitors and used it for ctDNA analysis in 83 plasma samples collected longitudinally from the four patients who harbored somatic ALK mutations. All four patients with ALK p.R1275Q experienced major responses and were alive 35 to 61 months after starting lorlatinib. A fifth patient with ALK p.F1174L initially had a partial response but relapsed after 10 months of treatment. In all cases, ctDNA was detected at the start of lorlatinib single-agent treatment and declined gradually, correlating with clinical responses. In the two patients exhibiting relapse, ctDNA increased 9 and 3 months, respectively, before clinical detection of disease progression. In one patient harboring HRAS p.Q61L in the relapsed tumor, retrospective ctDNA analysis showed that the mutation appeared de novo after 8 months of lorlatinib treatment. We conclude that some patients with relapsed or refractory high-risk neuroblastoma show durable responses to lorlatinib as monotherapy, and targeted ctDNA analysis is effective for evaluation of treatment and early detection of relapse in ALK-driven neuroblastoma. SIGNIFICANCE:We present five patients with ALK-driven relapsed or refractory neuroblastoma treated with lorlatinib as monotherapy. All patients responded to treatment, and four of them were alive after 3 to 5 years of follow-up. We performed longitudinal ctDNA analysis with ultra-deep sequencing of the ALK tyrosine kinase domain. We conclude that ctDNA analysis may guide treatment decisions in ALK-driven neuroblastoma, also when the disease is undetectable using standard clinical methods.

Detection of relapse with ctDNA analysis 9 months prior to clinical presentation in a patient (patient 2) with ALK p.R1275Q-positive neuroblastoma treated with lorlatinib. A, MIBG scans. B, Levels of ctDNA. C, NSE. CS, Curie score; ND, not detected; TID, temozolomide, irinotecan and dinutuximab; URL, upper reference limit (16.3 µg/L).

ObjectiveTo assess the influence of F1174S mutation on kinase activity and drug sensitivity of the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) fusion (EML4-ALK) variants 1 and 3.MethodsWe constructed mammalian expression plasmids of both wildtype and F1174 mutant EML4-ALK variants 1 and 3, and then characterized them with cell models by performing immunoblotting, neurite outgrowth assay, focus formation assay as well as protein stability assay. Drug sensitivity to ALK tyrosine kinase inhibitors was also compared between wildtype and F1174 mutant EML4-ALK fusions. In addition, we characterized the effect of different F1174 kinase domain mutations in the context of EML4-ALK fusions.ResultsIn contrast to the oncogenic ALK-F1174S mutation that has been reported to be activating in the context of full-length ALK in neuroblastoma, EML4-ALK (F1174S) variant 1 exhibits impaired kinase activity leading to loss of oncogenicity. Furthermore, unlike the previously reported F1174C/L/V mutations, mutation of F1174 to S sensitizes EML4-ALK variants 3a and 3b to crizotinib.ConclusionThese findings highlight the complexity of drug selection when treating patients harboring resistance mutations and suggest that the F1174S mutation in EML4-ALK variant 1 is likely not a potent oncogenic driver. Additional oncogenic driver or other resistance mechanisms should be considered in the case of EML4-ALK variant 1 with F1174S mutation.

Anaplastic Lymphoma Kinase inhibitors (ALK TKIs) are approved for the treatment of ALK-positive non-small cell lung cancer (NSCLC) and are in clinical trial for ALK-aberrant high-risk neuroblastoma (NB) patients, particularly loratinib. However, resistance to ALK inhibitors can occur in patients, via the activation of bypass-signalling pathways, and there is a need to identify these mechanisms as well as drugs that inhibit them to design therapeutic approaches that prevent resistance, and to treat ALK TKI relapsed/refractory disease. Using genome-wide CRISPR-Cas9 overexpression screens, we identified and validated FGFR2 as a desensitizer to lorlatinib in aberrant ALK-expressing high-risk NB. FGFR2 and FGFR2-associated pathways are up-regulated in lorlatinib-resistant NB cells. Moreover, high-throughput screens using a library of 1,430 FDA approved drugs identified kinase inhibitors including those targeting FGFR2 as efficacious in reducing the survival of lorlatinib resistant NB cells. Hence, the FGFR pathway was investigated as a therapeutic target applying the pan-FGFR inhibitor erdafitinib or the multi-kinase inhibitor ponatinib, resulting in reduced survival of lorlatinib-resitant cells in comparison to their lorlatinib-sensitive counterparts. Moreover, both FGFR inhibitors act synergistically with lorlatinib in vitro and in vivo , using patient-derived xenografts (PDXs) and genetically engineered models (GEMM) of ALK-expressing NB. FGFR2 mRNA expression also correlate with a poorer prognosis for NB patients, regardless of sub-type, suggesting that a broader range of patients may benefit from FGFR inhibitors. Overall, our data suggests that FGFR2 potentially plays roles in lorlatinib resistance in NB and that combined pharmacological inhibition of ALK and FGFR constitutes a therapeutic approach to treat high-risk NB. ### Competing Interest Statement The authors declare no potential conflicts of interest although, one author, G.A.A.B. has received institutional consultancy fees from Roche, Takeda, Novartis, and Janssen outside of this research project.

Early detection of relapse and emergence of an HRAS p.Q61L mutation during lorlatinib treatment in a patient (patient 5) with ALK p.F1174L-positive neuroblastoma. A, MIBG scans at initial diagnosis, at the first relapse, after 4 months of lorlatinib (partial response), and at second relapse after 10.5 months of lorlatinib treatment. B, Allele frequency of HRAS p.Q61L in tumor tissue at different time points. C, Levels of ctDNA. D, Clinical tumor markers, values are normalized to the upper reference limit (URL) for each marker. CGA, chromogranin A; Lorla, lorlatinib; ND, not detected.

Lasting response to lorlatinib in a patient (patient 1) with relapsed neuroblastoma harboring ALK p.R1275Q. A, Chest computed tomography (top) and MIBG scans (bottom) at time of initial diagnosis, at relapse, and after 2 months of lorlatinib treatment. Red arrows denote tumors. B, Levels of ctDNA during and after lorlatinib treatment. ND, not detected. C, Chromogranin A. URL, upper reference limit (102 µg/L). NSE, urine VMA and urine HVA were within normal ranges at time of relapse and were therefore not further analyzed. HVA, homovanillic acid; VMA, vanillylmandelic acid.

Metabolic dysfunction-associated steatotic liver disease (MASLD) and its advanced subtype, metabolic dysfunction-associated steatohepatitis (MASH), have emerged as the most common chronic liver disease worldwide, yet there is no targeted pharmacotherapy presently available. This study aimed to investigate the possible in vivo function of STE20-type protein kinase MST4, which was earlier implicated in the regulation of hepatocellular lipotoxic milieu in vitro, in the control of the diet-induced impairment of systemic glucose and insulin homeostasis as well as MASLD susceptibility. Whole-body and liver-specific Mst4 knockout mice were generated by crossbreeding conditional Mst4fl/fl mice with mice expressing Cre recombinase under the Sox2 or Alb promoters, respectively. To replicate the environment in high-risk subjects, Mst4–/– mice and their wild-type littermates were fed a high-fat or a methionine–choline-deficient (MCD) diet. Different in vivo tests were conducted in obese mice to describe the whole-body metabolism. MASLD progression in the liver and lipotoxic damage to adipose tissue, kidney, and skeletal muscle were analyzed by histological and immunofluorescence analysis, biochemical assays, and protein and gene expression profiling. In parallel, intracellular fat storage and oxidative stress were assessed in primary mouse hepatocytes, where MST4 was silenced by small interfering RNA. We found that global MST4 depletion had no effect on body weight or composition, locomotor activity, whole-body glucose tolerance or insulin sensitivity in obese mice. Furthermore, we observed no alterations in lipotoxic injuries to the liver, adipose, kidney, or skeletal muscle tissue in high-fat diet-fed whole-body Mst4–/– vs. wild-type mice. Liver-specific Mst4–/– mice and wild-type littermates displayed a similar severity of MASLD when subjected to an MCD diet, as evidenced by equal levels of steatosis, inflammation, hepatic stellate cell activation, fibrosis, oxidative/ER stress, and apoptosis in the liver. In contrast, the in vitro silencing of MST4 effectively protected primary mouse hepatocytes against ectopic lipid accumulation and oxidative cell injury triggered by exposure to fatty acids. In summary, these results suggest that the genetic ablation of MST4 in mice does not mitigate the initiation or progression of MASLD and has no effect on systemic glucose or insulin homeostasis in the context of nutritional stress. The functional compensation for the genetic loss of MST4 by yet undefined mechanisms may contribute to the apparent discrepancy between in vivo and in vitro phenotypic consequences of MST4 silencing.

High-risk neuroblastoma is characterized by MYCN amplification and high MYCN or MYC gene expression. These patients have a poor prognosis and there is an urgent need for more effective drugs. While strategies to develop inhibitors that directly target the MYC proteins have remained largely unsuccessful, recent preclinical studies have identified ATR, a key protein of the DNA damage response, as a promising alternative therapeutic target. Here we identified a strong RUVBL1 and RUVBL2 signature in transcriptomics data derived from different MYCN-driven mice tumors treated with ATR inhibitors. The RUVBL proteins form a complex with ATPase activity that has broad cellular functions and we demonstrate that pharmacological inhibition of this protein complex results in a strong reduction of MYC signaling, cell cycle arrest, DNA damage and apoptosis. We confirmed the association with MYCN and identified the RUVBL genes as independent prognosticators in human primary neuroblastoma data.  ### Competing Interest Statement The authors have declared no competing interest.

Neuroblastoma (NB) is the most common cancer in infancy with an urgent need for more efficient targeted therapies. The development of novel (combinatorial) treatment strategies relies on extensive explorations of signaling perturbations in neuroblastoma cell lines, using RNA-Seq or other high throughput technologies (e.g. phosphoproteomics). This typically requires dedicated bioinformatics support, which is not always available. Additionally, while data from published studies are highly valuable and raw data (e.g. fastq files) are nowadays released in public repositories, data processing is time-consuming and again difficult without bioinformatics support. To facilitate NB research, more user-friendly and immediately accessible platforms are needed to explore newly generated as well as existing high throughput data. To make this possible, we developed an interactive data centralization and visualization web application, called CLEAN (the Cell Line Explorer web Application of Neuroblastoma data; https://ccgg.ugent.be/shiny/clean/). By focusing on the regulation of the DNA damage response, a therapeutic target of major interest in neuroblastoma, we demonstrate how CLEAN can be used to gain novel mechanistic insights and identify putative drug targets in neuroblastoma.

Background/Objectives: Neuroblastoma (NB) is a childhood cancer with heterogeneous characteristics, posing challenges to effective treatment. NBs express somatostatin receptors that facilitate the use of somatostatin analogs (SSTAs) as tumor-seeking agents for diagnosis and therapy. High-risk (HR) NBs often have gain-of-function mutations in the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). Despite intensive multimodal treatment, survival rates remain below 40% for children with HR-NB. The aim of this work was to investigate the combined effect of the SSTA 177Lu-octreotide with the ALK inhibitor lorlatinib. Methods: Mice bearing human HR-NB CLB-BAR tumors were treated with lorlatinib, 177Lu-octreotide, and a combination of these pharmaceuticals or saline (control). Tumor volume was monitored and tumor samples were evaluated for cleaved caspase-3 and expression of 84 human genes involved in apoptosis. Results: Combination treatment with 177Lu-octreotide and lorlatinib demonstrated synergistic antitumor effects. An increased number of cleaved caspase 3-positive cells was observed in tumors from mice treated with 177Lu-octreotide alone and in combination with lorlatinib. Modulation of Bcl-2 family gene expression was observed only in the presence of both 177Lu-octreotide and lorlatinib, with BID down-regulated and HRK up-regulated on days 2 and 7, respectively. Conclusions: The data suggest that ALK signaling pathway inhibition may contribute to radiosensitization in radionuclide therapy with 177Lu-octreotide and could improve treatment outcomes in patients with HR-NB.

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.

Long-term response to lorlatinib in a patient (patient 4) with refractory ALK p.R1275Q-positive neuroblastoma. A, MIBG or 68Ga-DOTATATE scans at the time of diagnosis, after induction of chemotherapy, and after 5 months of lorlatinib treatment. Red arrows denote tumors. B, Levels of ctDNA. C, Plasma methoxy noradrenaline. ND, not detected; URL, upper reference limit (0.7 nmol/L).