Abstract 1616: Dissecting the biology and therapeutic vulnerabilities of RB1-mutant osteosarcoma using hereditary retinoblastoma iPSCs

Abstract Patients with hereditary retinoblastoma (RB), an inherited autosomal dominant cancer disordercaused by germline mutations/deletions in the RB1 gene, have a >400 fold increased incidenceof osteosarcoma (OS), suggesting a strong mechanistic link between RB1 loss andosteosarcomagenesis. Although mice offer many advantages when conducting cancer research,unlike humans, Rb1 knockout mice do not develop OS, suggesting the urgent requirement foralternative disease models to understand how RB1 mutation leads to osteosarcomagenesis.Here, we generated patient-derived induced pluripotent stem cells (iPSCs) by reprogramming RBand healthy control fibroblasts and then created isogenic controls by correcting RB1 mutationusing CRISPR/Cas9. Performing in vitro soft-agar assay and in vivo xenografts, we foundpotential tumorigenic ability in RB osteoblasts (OBs) but not control OBs. In order to gain insightsinto RB1 loss associated osteosarcomagenesis, we compared global transcripts among RB andcontrol OBs. GSEA analysis indicated that OS associated genes are specifically enriched in RBOBs in comparison with control OBs, demonstrating that RB OBs acquire OS characteristics inthe absence of additional gene alterations. GO analysis revealed that genes involved in the mitoticcell cycle are enriched in RB OBs. Although RB1 is known to controlling G1/S related geneexpression by inhibiting E2F transcription factors, it is still nebulous if RB1 plays a role inregulating mitotic genes.To comprehensively identify preferential binding of RB1 to specific promoters in OBs, weconducted RB1 ChIP-seq to map genome-wide RB1-binding sites in iPSC-derived OBs. SinceE2F3a, a RB1 repressed transcription factor, is enriched expressed in OBs, we also performedE2F3a ChIP-seq to define whether these RB1-repressed targets are E2F3a-activated targets.Using the nonbiased de novo motif search algorithm DME, we further identified the most highlyenriched RB1-binding motif in all conditions and cross-compare with the E2F3a motifs. Weconfirmed that RB1 and E2F3a co-occupy the mitotic regulator promoters by ChIP-PCR. IHCstudies of clinical OS specimens supported the clinical correlation between RB1/E2F3a andmitotic regulators. High mitotic regulator genes expression is correlated with poor prognosis. Ourresults demonstrated that the mitotic regulators are co-regulated by both RB1 and E2F3a andthey play roles in osteosarcomagenesis.Taken together, our findings demonstrated multiple OS-related phenotypes in human RB iPSC-derived OBs and RB1/E2F3a-regulating mitotic regulators can be a therapeutic vulnerability inRB1-mutant OS. Citation Format: Dandan Zhu, Jian Tu, Zijun Huo, An Xu, Mo-Fan Huang, Ying Liu, Yi-Hung Chen, Ruiying Zhao, Dung-Fang Lee. Dissecting the biology and therapeutic vulnerabilities of RB1-mutant osteosarcoma using hereditary retinoblastoma iPSCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1616.