John Inge Johnsen

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

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

Figure S9. Immunofluorescence staining of hemizygous TH-MYCN tumors and matched ex vivo tumoroids. Related to Figure 4.

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

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

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

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

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

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