Neuroblastoma (NB) is a pediatric malignancy developing in the sympathoadrenal lineage of the neural crest, characterized by clinical heterogeneity ranging from spontaneous regression to poor outcomes. Activating mutations in the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) are frequently observed in both sporadic and familial NB, yet the functional role of ALK in tumor initiation is not fully understood. Using a patient-derived human induced pluripotent stem cell (iPSC) model of sympathoadrenal development, we show that upon sympathoadrenal lineage commitment, ALK R1275Q, the most common hotspot mutation found in familial NB, sustain a proliferative, immature Schwann cell precursor (SCP)-like cell state with elevated ALK signaling and increased susceptibility to MYCN-driven transformation. While ALK-mutant cells alone did not form tumors in vivo , they cooperated with MYCN to accelerate tumor initiation, suggesting that ALK R1275Q creates a permissive but insufficient state for transformation. These findings define an ALK-driven cell progenitor-like state that facilitates the initiation of NB during embryonal development.

Figure S13. Survival probability for neuroblastoma patients non-MYCN amplified tumors with tumoroid enriched gene expression programs. Related to Figure 5.

Figure S2. Trajectory analysis and joint alignment of TH-MYCN tumors and normal murine embryo trunk (E13.5). Related to Figure 2.

Figure S8. Homozygous and hemizygous ex vivo tumoroid cultures. Related to Figure 4.

Table S2 shows gene lists used for signature scores, corresponding to Fig. 2A and Fig. 5A.

Figure S4. Tumor hallmark gene expression patterns in human and mouse tumors.

Figure S5. Identification of malignant cells by inferCNV. Related to Figure 3.

Figure S3. TH-MYCN tumor microenvironment is composed of Schwannian and mesenchymal stroma, and a diverse immune-cell repertoire. Related to Figures 2 and 3.

ABSTRACT MYCN amplification drives replication stress in high-risk neuroblastoma, yet how MYCN-amplified tumour cells tolerate this stress to sustain proliferation remains poorly understood. Here we show that FBXL12, an SCF ubiquitin ligase substrate receptor that targets the Fanconi anaemia protein FANCD2 for degradation at replication forks, as well as the broader Fanconi anaemia and replication stress transcriptional program are elevated in high-risk and MYCN-amplified neuroblastoma. High FBXL12 expression independently predicts poor survival across neuroblastoma patient cohorts. FBXL12 loss stabilizes FANCD2 on chromatin, elevates ATR-dependent replication stress signalling and DNA damage during S phase, and impairs proliferation of MYCN-amplified neuroblastoma cells in vitro and in vivo. Mechanistically, MYCN directly engages the FBXL12-FANCD2 complex and antagonises FBXL12-mediated degradation of FANCD2 at replication forks, revealing that the oncogenic driver of replication stress also actively preserves the chromatin-bound FANCD2 pool required to tolerate it. Beyond S phase, FBXL12 loss disrupts FANCD2-dependent mitotic DNA synthesis and transmits unresolved replication intermediates into daughter cells. FBXL12-deficient cells consequently show transcriptional activation of MYC target gene, ATR, and mTOR signalling programs, and this pathway-concordant state confers differential sensitivity to ATR, and mTOR-targeting compounds, nominating candidate therapeutic strategies for this disease subset. Together, these findings define a MYCN-FBXL12-FANCD2 axis as a clinically relevant vulnerability in high-risk neuroblastoma. HIGHLIGHTS FBXL12 is upregulated in high-risk neuroblastoma and predicts poor survival FBXL12 loss traps FANCD2 on chromatin and drives S-phase DNA damage MYCN antagonises FBXL12-mediated degradation of FANCD2 at replication forks FBXL12 loss impairs mitotic DNA synthesis and damages daughter cells FBXL12-deficient cells show ATR/mTOR activation and drug sensitivity

Figure S1. Cell states and cell cycle phases are consistent across TH-MYCN samples and genotypes. Related to Figure 1.

Figure S10. Cryopreserved and thawed ex vivo tumoroid cultures from homozygous TH-MYCN. Related to Figure 4.

Figure S6. Identification of conserved and context-specific signaling pathways in tumors from NB patients and TH-MYCN mice.

Figure S14. Survival probability for neuroblastoma patients MYCN amplified tumors with tumoroid enriched gene expression programs. Related to Figure 5.

Table S1 shows characteristics of TH-MYCN mouse samples used for tumoroid cultures, scRNA-seq and immunostainings.

Figure S11. TH-MYCN tumoroids preserve transcriptomic and histological features of original tumors. Related to Figure 4.

Figure S7. Conserved signaling pathways, receptor-ligand interactions and survival analysis of NB patients.

Figure S12. Distinct ex vivo tumoroid enriched cluster embedding and expression patterns. Related to Figure 5.

Figure S15. Cross-species integration analysis of common cell clusters. Related to Figure 5.

Segmental gain of chromosome 17q is the most common genetic aberration in high-risk neuroblastoma, but its role in disease progression is poorly understood. This study aims to address the contribution of 17q gain to neuroblastoma malignancy. We analyzed the genetic and transcriptional landscape of 417 neuroblastoma patients across various risk groups and clinical stages using multi-omic approaches. Single-cell RNA/DNA sequencing and SNP arrays were combined to characterize genomic aberrations, while evolutionary trajectories were mapped to explore the accumulation of genetic changes in patients with neuroblastoma. Additionally, DNA and RNA sequencing were used to assess mutational burden and gene expression patterns. Our findings suggest that chromosome 17 gain is an early genetic event acquired during neuroblastoma development, correlating with the accumulation of additional chromosomal aberrations and poor prognosis. Increased segmental gains of chromosome 17q were observed during clonal evolution, relapse disease and metastasis. We identified PPM1D, a p53-inducible Ser/Thr phosphatase located on chr17q22.3, as a key player activated by segmental 17q-gain, gene-fusion, or gain-of-function somatic and germline mutations, further promoting neuroblastoma development/progression. Gain of chromosome 17 is an early driver of genetic instability in neuroblastoma, with PPM1D emerging as a potential candidate gene implicated in high-risk disease progression.