ATR inhibition enables complete tumour regression in ALK-driven NB mouse models

High-risk neuroblastoma (NB) often involves MYCN amplification as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes like MYCN and ALK result in increased replication stress in cancer cells, offering therapeutically exploitable options. We have pursued phosphoproteomic analyses highlighting ATR activity in ALK-driven NB cells, identifying the BAY1895344 ATR inhibitor as a potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterize NB cell and tumour responses to ATR inhibition, identifying key components of the DNA damage response as ATR targets in NB cells. ATR inhibition also produces robust responses in mouse models. Remarkably, a 2-week combined ATR/ALK inhibition protocol leads to complete tumor regression in two independent genetically modified mouse NB models. These results suggest that NB patients, particularly in high-risk groups with oncogene-induced replication stress, may benefit from ATR inhibition as therapeutic intervention.

D 0 The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement D 0 A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly D 0 The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section. 0 D A description of all covariates tested D 0 A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons D 0 A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) D D For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable. 0 D For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings 0 D For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes D D Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection Data analysis All data were generated from cell line and mice experiments as described in the manuscript. The source code and processed data used for the bioinformatics analysis are available via Github https://github.com/CCGGlab/ATR and https://doi.org/10.5281/zenodo.5594083.
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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our QQ_1ig,_ Data and code availability. Sample size for each experiment is indicated in the figure legend. RNA-Seq, Proteomics and Phosphor samples were performed with at least an n=3 (biological repeats). Detailed histological analyses of mice was performed with n=4 or more. RNA-Seq from mouse tumours was performed with at least n=3 biological samples. Mouse tumour treatment was carried out with at least n=3. Other experiments, proliferation, viability, cell assays, were performed with n>3 or more, with the exception of the proteomics/phosphor samples that were performed as either biological duplicates/triplicates or quadriplets as specified. No statistical sample size calculation was performed to predetermine sample sizes. Sample size was chosen based on a balance between cost and replicate power for RNA-Seq, Proteomics and Phospohr samples. Sample sizes were based on previous experience with the experiments performed. All available mice were used to provide sufficient statistical power in the mouse experiments.
Animals suffering from unrelated illnesses were removed in accordance with ethical regulations. These animals were excluded from analyses.
Cell experiments, including proliferation, viability, cell assays, were repeated >3 times. All experimental findings were successfully reproduced in multiple independent experiments. The number of independent experiments is indicated in the Figure legends.
Animals were followed over time in a non-biased manner and treatment initiated on tumour detection. The order of tumour development determined the animals treatment with either vehicle or drug, irrespective of age and sex. Therefore, animals were randomly assigned to vehicle or drug treatment groups. For experiments involving cell lines samples were randomly allocated into treatment groups.
Mouse treatment was performed on mice presenting with tumors following strict ethical procedures in a non-blinded manner. Tumor imaging was performed by ultrasound in a blinded manner. Quantification of IHC analysis of mouse tumors was performed in a blinded manner. For in vitro proliferation experiments, investigators were blinded for data acquisition and analysis. For routine cellular molecular biology experiments, investigators were not blinded as handling of cells, preparation of reagents and treatments required accurately recorded information.Proteomics/ phosphoproteomics was performed at the University of Gothenburg Core facility in a blinded manner and resulting data decoded by the respective investigators and bioinformatitions. NB cell lines CLB-BAR, CLB-GE, CLB-GAR, SK-N-AS, IMR32, NB-1, SKSYSY, Kelly and SK-N-BE(2) were employed in this study (genetic details are provided in Fig 51). CLB-BAR, CLB-GE and CLB-GAR were obtained from The Centre Leon Berard, France under MTA. All other cell lines are from ATCC.

Eukaryotic cell lines
Cell lines have been authenticated by STR profiling in the last 24 months.
Cell lines are regularly tested for mycoplasma contamination. All cell lines used in this study tested negative for mycoplasma contamination.
No commonly misidentified cell lines were used in the study.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals

Wild animals
Field-collected samples Ethics oversight