We have been working on neuroblastoma (NB), which is a childhood cancer arising from the neural crest that accounts for 15% of all pediatric tumour-related deaths, looking for new targets that might be therapeutically exploited . This is important as aggressive NB often responds to initial treatment but then relapses, representing a major clinical problem with poor prognosis and survival rates [2, 3]. Our groups at the University of Gothenburg have been working in close collaboration with the Van den Eynden group at Ghent University, combining wet-lab approaches with bioinformatics to better understand the underlying molecular mechanisms involved in NB initiation and progression. Part of this work has been centered on the identification of actionable therapeutic targets and pathways involved in this pathogenesis, and as part of this, we have performed a number of phosphoproteomics analysis of NB cells treated with anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) [4-6]. NB is rather a mutation poor, and chromosomal aberrations play an important role in diagnosis and prognosis, with the most common genetic anomalies being deletions of parts of chromosome arms 1p and 11q, 17q gain, triploidy, as well as MYCN and ALK amplification [7-9]. Although few genes are mutated, the ALK receptor tyrosine kinase has been implicated as an oncogenic driver in NB, where it can be activated by amplification and gain-of-function mutations [7, 8, 10]. While some reported clinical cases suggest that ALK TKI therapy can provide clinical response in NB  results from the phase I trials with ALK TKIs in NB suggest that, in contrast to other tumor types such as non–small cell lung cancer, single-agent therapy with ALK TKIs may not be effective in ALK-positive pediatric NB patients, underscoring the need to identify additional therapeutic targets [11, 12].
In our recent study, we followed up on an earlier comprehensive phosphoproteomics analysis  that identified ATR activity in NB cell lines. Digging into these phospho-proteomic analyses, we noted changes in several putative ATR target sites (S/TQ motifs). Because this raises the possibility that ALK activity in NB cells modulates ATR/ATM activity, we examined ATR and ATM in our phosphoproteomic dataset, identifying dephosphorylation of ATR in response to ALK inhibition. We also investigated ATR and pATR, pATM, pFOXM1, and pCHK1, concluding that our NB cell lines exhibit DNA damage response (DDR) pathway activity. To further explore ATR as a therapeutic target in NB, we tested the effect of both ATRi and siRNA targeting ATR on NB cell lines, showing that NB cell lines are highly sensitive to ATR inhibition, with the BAY 1895344  ATR kinase inhibitor being most potent. These experiments were complemented by RNA-Seq and proteomics analyses, including the first phosphoproteomic analysis of BAY 1895344, showing that ATR regulates S/G2 checkpoint and phosphorylation of key DDR components in NB cells.
Having shown that the ATR inhibitor, BAY 1895344 , is a potent inhibitor of NB cell growth, we further explored its effect in xenografts and two independent genetically engineered mouse models (GEMMs) of ALK-driven NB. We reasoned that any treatment of ALK-positive NB would require a combination with ALK TKIs, leading us to devise a 14 day combined ATR/ALK inhibition therapeutic protocol (3 days BAY 1895344 alone, 4 days lorlatinib alone, 3 days BAY 1895344/lorlatinib combination, 4 days lorlatinib alone). Remarkably, this short therapeutic intervention completely ablated tumours in all ALK-driven-NB GEMMs treated, and even more surprisingly, mice remained tumor free. This is in stark contrast to the treatment of these ALK-driven GEMMs with ALK TKIs alone, where tumours resume aggressive growth as soon as ALK inhibition is stopped . Given this striking response to the 14 days BAY 1895344/lorlatinib regime, we maintained all remaining mice over time without any therapeutic interventions monitoring regularly with ultrasound for tumour development Remarkably, except for one animal, all other mice have remained tumour free and still are tumour free (for more than a year from treatment). We also performed gene expression analysis of ALK-driven mouse tumors treated with BAY 1895344, leading to the identification of robust inflammatory signatures. This led us to test whether host immune cell involvement may assist in the rapid tumour loss observed in response to ATR inhibition. Histological analysis of BAY 1895344 treated tumors with anti-CD68 antibodies identified a remarkable infiltration of immune cells in response to treatment, in comparison with treated controls.
Our findings require further follow-up, particularly regarding the immune response to the combined ATR/ALK inhibition regime. Other important questions include whether there is a developmental role for ATR that makes ALK-driven NB in mice exquisitely sensitive to ATR inhibition? In addition, most importantly, can this be carried over to human pediatric NB patients? Our mouse ATRi/ALKi protocol lasted 14 days, with no obvious side effects, and after that, mice have been completely free from treatment and fully healthy. While there is not much information on the use of the BAY 1895344 ATR inhibitor in humans, a ‘first-in-human’ trial employing BAY 1895344 in adult cancer patients has recently been reported , providing more information on potential clinical use. Taken together, our findings have taken a simple hit in a phosphoproteomics analysis through to the development of an effective cure for ALK-driven NB in mice models. These results suggest that combined ALK and ATR inhibition may represent an effective therapeutic strategy in patients with ALK-positive NB, and highlight the need for further exploration.
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- Ladenstein, R., et al., Busulfan and melphalan versus carboplatin, etoposide, and melphalan as high-dose chemotherapy for high-risk neuroblastoma (HR-NBL1/SIOPEN): an international, randomised, multi-arm, open-label, phase 3 trial. Lancet Oncol, 2017. 18(4): p. 500-514.
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- Borenas, M., et al., ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation. EMBO J, 2021. 40(3): p. e105784.
- Guan, J., et al., Clinical response of the novel activating ALK-I1171T mutation in neuroblastoma to the ALK inhibitor ceritinib. Cold Spring Harb Mol Case Stud, 2018. 4(4).
- Van den Eynden, J., et al., Phosphoproteome and gene expression profiling of ALK inhibition in neuroblastoma cell lines reveals conserved oncogenic pathways. Sci Signal, 2018. 11(557).
- De Brouwer, S., et al., Meta-analysis of neuroblastomas reveals a skewed ALK mutation spectrum in tumors with MYCN amplification. Clin Cancer Res, 2010. 16(17): p. 4353-62.
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- Javanmardi, N., et al., Analysis of ALK, MYCN, and the ALK ligand ALKAL2 (FAM150B/AUGalpha) in neuroblastoma patient samples with chromosome arm 2p rearrangements. Genes Chromosomes Cancer, 2019.
- Fischer, M., et al., Ceritinib in paediatric patients with anaplastic lymphoma kinase-positive malignancies: an open-label, multicentre, phase 1, dose-escalation and dose-expansion study. Lancet Oncol, 2021.
- Mosse, Y.P., et al., Safety and activity of crizotinib for paediatric patients with refractory solid tumours or anaplastic large-cell lymphoma: a Children's Oncology Group phase 1 consortium study. Lancet Oncol, 2013. 14(6): p. 472-80.
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- Yap, T.A., et al., First-in-Human Trial of the Oral Ataxia Telangiectasia and RAD3-Related (ATR) Inhibitor BAY 1895344 in Patients with Advanced Solid Tumors. Cancer Discov, 2021. 11(1): p. 80-91.