Dissecting the Biology and Therapeutic Vulnerabilities of RB1-Mutant Osteosarcoma Using RB iPSCs
Abstract
RB1 mutations are found in more than 30 of clinical osteosarcoma specimens, highlighting the crucial role of RB1 in preventing bone malignancy. Patients with hereditary retinoblastoma (RB), an inherited autosomal dominant cancer disorder caused by germline mutations/deletions in the RB1 tumor suppressor gene, have a >400-fold increased incidence of osteosarcoma, suggesting a strong mechanistic link between RB1 loss and osteosarcomagenesis. Our purpose are 1) To elucidate how loss of RB1 contributes to the upregulated FOXM1 transcriptional program; 2) To evaluate the therapeutic potential of FOXM1 inhibitors for RB1-mutant osteosarcoma treatment. We have reprogrammed RB patients fibroblasts to iPSCs. Then, we further applied CRISPR/Cas9 technology to target the RB1 genomic locus to correct the RB1 mutation in RB iPSCs and created corrected RB iPSCs (cRB) to serve as isogenic controls. We subsequently differentiated wild type, RB, and cRB iPSCs to OBs. Then we conduct ChIP-seq using an anti-RB1 antibody to map genome-wide RB1-binding sites among these iPSC-derived OBs. We found E2F3a but not E2F1 or E2F2 expression is enriched in RB and cRB OBs. RB1 and E2F3a ChIP-seq and ATAC-seq in RB and cRB OBs revealed that both RB1 and E2F3 bind the FOXM1 promoter and that RB OBs contain more open chromatin at FOXM1 promoter region than cRB OBs. Also, our studies of E2F3a and FOXM1 expression in the TCGA SARC database revealed the positive correlation between E2F3a and FOXM1 and high FOXM1 expression is correlated with poor prognosis. Meanwhile, our in vitro competition assay showed that RB OBs display increased sensitivity to Thiostrepton, suggesting that pharmacological inhibition of FOXM1 has potential in treating RB1-mutant cells.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jun 01, 2021
- Accession Number
- AD1142155
Entities
People
- Dandan Zhu
Organizations
- University of Texas Health Science Center at Houston