Hereditary retinoblastoma iPSC model reveals aberrant spliceosome function driving bone malignancies

Abstract

The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSC–derived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3a–regulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.

Keywords: hereditary retinoblastoma; iPSCs; osteosarcoma; pRB; spliceosomal genes.

Fig. 1.

Generation of a premalignant RB patient–derived iPSC platform. (A) The RB family tree includes two RB patients (mother: RB-M; daughter: RB-D) with a heterozygous pRB(D511fs) mutation and one unaffected relative (father: WT-F). Arrow, proband. (B) SeV-4F (OCT4, SOX2, KLF4, and MYC)–reprogrammed RB and WT iPSCs highly express hESC pluripotency factors (NANOG and OCT4) and hESC surface markers (TRA-1-81 and SSEA4) and have high AP activity. (Scale bars, 100 µm.) (C) Schematic overview of correcting RB1 mutation by CRISPR-Cas9 nickase in the RB1 genomic locus (13q14.2). CRISPR-Cas9 sgRNA target sites are labeled in blue. RB1 exon 17 is labeled in pink. The c.1531-1532 ins A site is labeled in green and the affected GAT nucleotide encoding aspartic acid (D) is colored red. (D) Sanger sequencing indicates the RB1 c.1531-1532 ins A mutation is corrected in cRB iPSCs. (E) Western blot reveals comparable pRB protein levels in WT and cRB iPSCs and lower levels in RB iPSCs, indicating restoration of pRB protein upon correction of RB1 gene mutation. (F) AIG assay demonstrates in vitro tumorigenic ability for RB OBs but not WT and cRB OBs. Colonies larger than 50 µm after 1 mo of growth are considered positive. (Scale bars, 100 µm.) Representative photographs show positive colonies from RB-M2 and RB-D2 OBs. (G) Tumor xenograft experiments by subcutaneous transplantation in NU/NU mice demonstrate that RB but not WT or cRB OBs recapture in vivo cell proliferation ability. The numbers of xenografts are indicated. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Enrichment of RNA splicing and spliceosomal genes in RB iPSC-derived OBs. (A) Heatmap demonstrating a pairwise comparison of gene expression levels from WT, RB, and cRB RNA-seq during the time course of differentiation by Spearman’s correlation. The pairwise correlation coefficients range from 0.94 (blue) to 1 (orange). (B, Left) Enrichment of GO biological processes identified by EnrichmentMap analysis. Network visualization of enriched gene sets in RB OBs compared with WT and cRB OBs at day 24 indicates that GO biological processes involved in DNA replication and repair, cell cycle, RNA process, and bone development are enriched in RB OBs. Enriched gene sets in RB OBs are displayed in orange and enriched gene sets in WT and cRB OBs are shown in blue. FDR, false discovery rate. (B, Right) Enriched GO biological processes in RB vs. WT and cRB OBs are analyzed by GSEA and summarized by a heatmap. Nonsignificant GOs are shown in light gray. (C) qRT-PCR indicates that expression of spliceosomal genes is enriched in RB iPSC-derived OBs but not LFS iPSC-derived OBs compared with their corresponding WT controls. (D) qRT-PCR demonstrates that depletion of pRB in WT OBs leads to up-regulation of spliceosomal genes. (E) qRT-PCR indicates that H1-pRBKO hESC-derived OBs demonstrate increased spliceosomal gene expression compared with H1 hESC-derived OBs. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

pRB and E2F3a directly cotarget spliceosomal genes. (A, Top) Venn diagram depicts the overlap between pRB and E2F3a binding peaks in cRB OBs and defines pRB-specific (2,889), pRB/E2F3a–cotargeted (4,096), and E2F3a-specific (6,539) loci. (A, Bottom Left) Heatmaps depict pRB and E2F3a binding to the 5-kb genomic loci surrounding the identified ChIP-seq peaks, grouped by cluster. (A, Bottom Right) Composite plots show the average binding of pRB and E2F3a to the pRB-specific, pRB/E2F3a–cotargeted, and E2F3a-specific loci. (B) GO analyses of pRB-specific, pRB/E2F3a–cotargeted, and E2F3a-specific genomic regions. pRB/E2F3a–cotargeted genes are overrepresented for numerous RNA splicing pathway genes, including those involved in the processing of capped introns containing pre-mRNA, mRNA splicing, mRNA processing, and mRNA splicing minor pathway. (C) Increased expression of pRB/E2F3a–cotargeted spliceosomal genes in RB OBs compared with cRB OBs. (C, Upper) Heatmaps of ChIP-seq peak intensities in pRB/E2F3a–cotargeted spliceosomal gene regions (±5 kb from the TSS). Each box in the heatmap represents a 0.7-kb region. (C, Lower) Heatmap of spliceosomal gene expression in RB-D, RB-M, cRB-D, and cRB-M OBs is examined by RNA-seq. (D) Integrative Genomics Viewer snapshot of pRB and E2F3a occupancy over promoter regions of spliceosomal genes HNRNPD, HNRNPUL1, SF3A2, and SNRPA. (E) ChIP-qPCR validation of pRB and E2F3a binding peaks at identified spliceosomal genes. (E, Left) Schematic of amplicon locations of spliceosomal genes and upstream controls used for ChIP-qPCR validation. (E, Right) ChIP-qPCR at spliceosomal TSS peak sites and upstream controls to assess for pRB (Left), E2F3a (Right), or immunoglobulin G (IgG) enrichment (ChIP/input). ChIP-qPCR confirms specific enrichment of HNRNPD, HNRNPUL1, SF3A2, and SNRPA at peak regions in RB OBs. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

Spliceosome perturbation influences cell viability and RNA splicing of RB OBs. (A) Violin plots represent distributions of statistically significant ΔPSI values (P < 0.05) for different classes of RNA splicing events: A3, A5, AF, AL, MX, RI, and SE. Separate violins are displayed for each pairwise comparison of DMSO- or PB-treated cells, and the percentage of events is presented. ΔPSI values are shown for 1) PB-treated cRB OBs compared with DMSO-treated cRB OBs; 2) PB-treated RB OBs compared with DMSO-treated RB OBs; 3) DMSO-treated RB OBs compared with DMSO-treated cRB OBs; and 4) PB-treated RB OBs compared with PB-treated cRB OBs. (B) Inhibition of spliceosomal function by PB leads to significant increases of aberrant RIs in RB OBs compared with cRB OBs. (C) Visualization of RNA-seq read mappings at METTL2B, JAG1, ZC3HAV1, and BYSL in the DMSO- and PB-treated OBs. A sashimi plot displaying the major splice junctions is superimposed. Representative RT-PCR validation of RI events of METTL2B, JAG1, ZC3HAV1, and BYSL transcripts. Exons are shown in gray and introns are in red. Half-arrows indicate the locations of primers designed to validate RI events. (D) Inhibition of spliceosomal function by PB preferentially alters RNA splicing events on pRB- and pRB/E2F3a–targeted transcripts. (E) RB1 mutation–associated RI transcripts are more highly expressed than global transcripts in PB-treated RB OBs. FPKM, fragments per kilobase of exon per million mapped fragments. (F) Unique genes with perturbed splicing events in DMSO- or PB-treated cRB OBs vs. RB OBs are analyzed by BioPlanet 2019 and summarized by heatmaps. (G and H) Both PB and SD6 selectively inhibit the cell proliferation of RB OBs compared with cRB OBs. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

OS specimens commonly have low pRB, high E2F3a, and high REOSS gene expression, and spliceosome inhibitors selectively impair pRB-deficient OS proliferation. (A) HOS-pRBKO cells are more sensitive to HNRNPUL1, RBMX, SF3A2, or SNRPA knockdown–induced inhibitory effects than HOS-Ctrl cells. (B) Knockdown of BUD31, SRSF9, U2AF2, or XAB2 showed similar inhibitory effects between HOS-Ctrl and HOS-pRBKO cells. (C and D) Ectopic expression of HNRNPUL1, RBMX, SF3A2, or SNRPA promotes HOS cell growth and colony formation. (E) PB selectively suppresses cell growth of HOS-pRBKO compared with HOS-Ctrl. (F) Spliceosome inhibitor SD6 preferentially suppresses pRB-deficient OS growth in vivo. Nude mice inoculated with 143B-Ctrl and 143B-pRBKO cells are injected with DMSO or SD6 (3 mg/kg) for four cycles (3-d constitutive injection and 1-d break) and the tumor growth is measured twice weekly. The arrowhead indicates the day of initial DMSO or SD6 injection. (G) pRB expression is negatively associated with CHERP, HNRNPD, SNRPA, and RBMX expression in 74 primary human OS specimens. (G, Left) Two representative specimens. (G, Right) Percentages of specimens with low (−) or high (+) pRB expression in which CHERP, HNRNPD, SNRPA, and RBMX are or are not observed. (H) E2F3a expression is positively correlated with CHERP, HNRNPD, SNRPA, and RBMX expression in 74 primary human OS specimens. (H, Left) Two representative specimens. (H, Right) Percentages of specimens with low (−) or high (+) E2F3a expression in which CHERP, HNRNPD, SNRPA, and RBMX are or are not observed. Results are expressed as mean ± SEM. ns, nonsignificant; *P < 0.05, **P < 0.01, ***P < 0.001.