Eliana Destefanis

Supplementary Figures from S1 to S23 and Supplementary tables from S1 to S3

Neuroblastoma (NB) is an aggressive childhood tumor, with high-risk cases having a 5-year overall survival probability of approximately 50%. The multimodal therapeutic approach for NB includes treatment with the retinoid isotretinoin (13-cis retinoic acid; 13cRA), which is used in the post-consolidation phase as an antiproliferation and prodifferentiation agent to minimize residual disease and prevent relapse. Through small-molecule screening, we identified isorhamnetin (ISR) as a synergistic compound with 13cRA in inhibiting up to 80% of NB cell viability. The synergistic effect was accompanied by a marked increase in the expression of the adrenergic receptor α1B (ADRA1B) gene. Genetic knockout of ADRA1B or its specific blockade using α1/α1B adrenergic antagonists led to selective sensitization of MYCN-amplified NB cells to cell viability reduction and neural differentiation induced by 13cRA, thus mimicking ISR activity. Administration of doxazosin, a safe α1-antagonist used in pediatric patients, in combination with 13cRA in NB xenografted mice exerted marked control of tumor growth, whereas each drug alone was ineffective. Overall, this study identified the α1B adrenergic receptor as a pharmacologic target in NB, supporting the evaluation of adding α1-antagonists to the post-consolidation therapy of NB to more efficiently control residual disease.Significance:Targeting α-adrenergic receptors synergizes with isotretinoin to suppress growth and to promote differentiation of neuroblastoma, revealing a combinatorial approach for more effective management of the disease and prevention of relapse.

Figure S9. Colony formation assays of five cell lines treated with MEK1/2 inhibitor trametinib, PRMT5 inhibitor MRTX1719, or the combination across all drug concentrations are shown.

Figure S5. MTAP copy number and mRNA assessment of Cancer DepMap cell lines.

Figure S14. qRT-PCR of RAS target genes in RAS-MAPK active and RAS-MAPK wildtype cells.

Toxicity Study of drug combinations in vivo. Mouse body weight measurements.

Figure S16. Differential GSEA pathway analysis shows G2/M checkpoint is impacted by MTAC-PRMT5i and PRMT5 mRNA is repressed by treatment with either ERK1/2i or A/B/C-RAFi.

Figure S12. Differential GSEA by RNAseq reveals pathways modified by ERK1/2 inhibitor ulixertinib, MTAC-PRMT5i MRTX1719, or the combination.

Figure S15. Differential mRNA GSEA expression of A172 CDKN2A/MTAP null glioblastoma cells treated with MTAC-PRMT5i compared to control.

Figure S18. Protein level crosstalk between PRMT5 and RAS-MAPK signaling shows increased phospho-Fra1.