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

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

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

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

The KIT/c-KIT proto-oncogene is frequently over-expressed in Merkel cell carcinoma (MCC), an aggressive skin cancer commonly caused by Merkel cell polyomavirus (MCPyV). Here, we demonstrated that truncated MCPyV-encoded large T-antigen (LT) suppressed macroautophagy/autophagy by stabilizing and sequestering KIT in the paranuclear compartment via binding VPS39. KIT engaged with phosphorylated BECN1, thereby enhancing its association with BCL2 while diminishing its interaction with the PIK3C3 complex. This process ultimately resulted in the suppression of autophagy. Depletion of KIT triggered both autophagy and apoptosis, and decreased LT expression. Conversely, blocking autophagy in KIT-depleted cells restored LT levels and rescued apoptosis. Additionally, stimulating autophagy efficiently increased cell death and inhibited tumor growth of MCC xenografts in mice. These insights into the interplay between MCPyV LT and autophagy regulation reveal important mechanisms by which viral oncoproteins are essential for MCC cell viability. Thus, autophagy-inducing agents represent a therapeutic strategy in advanced MCPyV-associated MCC.Abbreviation: 3-MA, 3-methyladenine; AL, autolysosome; AP, autophagosome; Baf-A1, bafilomycin A1; BARA, β-α repeated autophagy specific domain; BH3, BCL2 homology 3 domain; CCD, coiled-coil domain; CHX, cycloheximide; Co-IP, co-immunoprecipitation; CQ, chloroquine; CTR, control; DAPI, 4',6-diamidino-2-phenylindole; EBSS, Earle's balanced salt solution; ECD, evolutionarily conserved domain; EEE, three-tyrosine phosphomimetic mutations Y229E Y233E Y352E; ER, endoplasmic reticulum; FFF, three-tyrosine non-phosphomimetic mutations; FFPE, formalin-fixed paraffin-embedded; FL, full-length; GIST, gastrointestinal stromal tumor; IB, immunoblotting; IHC, immunohistochemistry; KIT-HEK293, KIT stably expressing HEK293 cells; KRT20/CK20, keratin 20; LT, large T-antigen; LT339, MCPyV truncated LT antigen; LTco, codon-optimized MCPyV LT antigen; MCC, Merkel cell carcinoma; MCPyV-, MCPyV-negative; MCPyV, Merkel cell polyomavirus; MCPyV+, MCPyV-positive; PARP1, poly(ADP-ribose) polymerase 1; PCI, pan-caspase inhibitor; PI, propidium iodide; PtdIns3K, class III phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; RB1, RB transcriptional corepressor 1; RTKs, receptor tyrosine kinases; KITLG/SCF, KIT ligand; sT, small T-antigen; sTco, codon-optimized MCPyV sT antigen; T-B, Tat-BECN1; T-S, Tat-scrambled; TEM, transmission electron microscopy.

High-risk neuroblastoma frequently exhibits segmental gain of chromosome 17q, including the locus of PPM1D, which encodes the phosphatase WIP1, a regulator of p53 activity, DNA repair, and apoptosis. High expression of PPM1D is correlated to poor prognosis, and genetic or pharmacologic inhibition of WIP1 suppresses neuroblastoma growth. Here, we show that combining drugs that target WIP1 and H3K27 demethylation induces synergistic cytotoxicity in neuroblastoma. We screened 527 different compounds together with inhibitors of WIP1 and identified a strong cytotoxic synergism between the WIP1 inhibitor SL-176 and GSK-J4, a specific inhibitor of the H3K27 demethylase JMJD3. Viability assays in neuroblastoma cell lines and treatment of tumor spheroids confirmed the synergistic effect of combining SL-176 with GSK-J4. Immunoblot experiments demonstrated a marked effect on WIP1 downstream targets and apoptosis markers, while qPCR showed a synergistic upregulation of p53 downstream targets PUMA and p21. RNA sequencing revealed a vast number of differentially expressed genes, suggesting a pervasive effect of this drug combination on transcription, with enrichment of pathways involved in DNA damage response. Finally, this drug combination was confirmed to reduce tumor growth in zebrafish xenograft experiments. In conclusion, the combination of the WIP1 inhibitor SL-176 and the epigenetic modifier GSK-J4 induces synergistic cytotoxicity in neuroblastoma cells by potentiating p53 downstream effects.