RB1 loss triggers dependence on ESRRG in retinoblastoma

Abstract

Retinoblastoma (Rb) is a deadly childhood eye cancer that is classically initiated by inactivation of the RB1 tumor suppressor. Clinical management continues to rely on nonspecific chemotherapeutic agents that are associated with treatment resistance and toxicity. Here, we analyzed 103 whole exomes, 20 whole transcriptomes, 5 single-cell transcriptomes, and 4 whole genomes from primary Rb tumors to identify previously unknown Rb dependencies. Several recurrent genomic aberrations implicate estrogen-related receptor gamma (ESRRG) in Rb pathogenesis. RB1 directly interacts with and inhibits ESRRG, and RB1 loss uncouples ESRRG from negative regulation. ESRRG regulates genes involved in retinogenesis and oxygen metabolism in Rb cells. ESRRG is preferentially expressed in hypoxic Rb cells in vivo. Depletion or inhibition of ESRRG causes marked Rb cell death, which is exacerbated in hypoxia. These findings reveal a previously unidentified dependency of Rb cells on ESRRG, and they implicate ESRRG as a potential therapeutic vulnerability in Rb.

Fig. 1.. Genomic landscape of 103 primary Rbs.

Oncoprint of 103 primary Rb samples analyzed by WES. Data include status of the most commonly mutated genes, types of mutations, International Intraocular Retinoblastoma Classification (IIRC) status, family history of Rb, gender, laterality, and chromosome copy number aberrations. LOH, loss of heterozygosity.

Fig. 2.. Recurrent genomic aberrations point to ESRRG as a common target for deregulation in Rb.

(A) Ingenuity Pathway Analysis of the most common mutations in 103 Rb samples. (B) RNA-seq analysis of mRNA expression for ESR/ESRR family members in 16 primary Rb tumors. (C) Uniform manifold approximation and projection (UMAP) plots of scRNA-seq data from five primary Rb tumor samples obtained at enucleation, sorted by tumor sample (left) and Seurat cluster (right). (D) Dot plot analysis of scRNA-seq data showing relative single-cell expression of ESRRA, ESRRB, and ESRRG by tumor sample (left) and Seurat cluster (right). (E) Violin plots of scRNA-seq data showing ESRRG expression by tumor sample (left) and Seurat cluster (right). CPM, counts per million.

Fig. 3.. ESRRG regulates genes involved in retinogenesis and oxygen metabolism in Rb.

(A) Integrated analysis of ESRRG ChIP-seq peaks and RNA-seq differentially expressed genes with or without shRNA-mediated knockdown of ESRRG (shESRRG). ChIP-seq was performed in RB006 and RB018 cells, and RNA-seq was performed in RB006, RB016, RB018, and wRB6 cells. The pie chart displays the percentage of peaks located within gene regions that are associated with differentially expressed genes (inner circle) or up-regulated and down-regulated genes (outer donut). The rectangular plot (top right) exhibits peak distance from the TSS. The bar plot shows the presence and co-occurrence of the most significantly enriched transcription factor binding motifs (FDR < 0.05) found within the ESRRG ChIP-seq peaks associated with up-regulated (blue) or down-regulated (red) genes after ESRRG knockdown. UTR, untranslated region. (B) ESRRG ChIP-seq track plots and corresponding RNA-seq data with (shESRRG) and without (shGFP) ESRRG knockdown at key retinal transcription factors involved in retinal development and differentiation. (C) Presence and co-occurrence of ERRE, NEUROD, LHX, and CRX/OTX2 binding motifs within ±3 kb from the TSS of all significantly up-regulated (darker colors) and down-regulated (lighter colors) genes (FDR < 0.05) following ESRRG knockdown. Individual genes are depicted as radii, and each donut represents the indicated binding motif.

Fig. 4.. RB1 interacts with and inhibits ESRRG.

(A) Protein maps showing location of RB1-binding VXXLYD motifs in ESRs (ESR1 and ESR2) and estrogen-related receptors (ESRRA, ESRRB, and ESRRG). AF, activation function domain; a.a., amino acids. (B) Western blot (WB) for endogenously expressed ESRRG or RB1 in RB1-WT SH-SY5Y neuroblastoma cells following immunoprecipitation for RB1, ESRRG or an immunoglobulin G (IgG) control. (C) Western blot for ectopically expressed hemagglutinin (HA)–tagged RB1 (HA-RB1), V5-tagged WT ESRRG (V5-ESRRG-WT), or ESRRG with mutated VXXLYD motif (V5-ESRRG-MUT) in RB1-null C33A cells following immunoprecipitation for HA (left). Western blot for ectopically expressed HA-RB1, V5-ESRRG-WT, or V5-ESRRG-MUT in RB1-null C33A cells following immunoprecipitation for V5 (right). IP, immunoprecipitation. (D) Normalized ERRE luciferase reporter activity with or without ectopic expression of V5-ESRRG-WT or V5-ESRRG-MUT as well as with (shRB1⁺) or without (shRB1⁻) doxycycline-induced RB1 knockdown in HEK293 cells (left, n = 12) or with (RB1) or without (EV2) RB1 re-expression in C33A cells (right, n = 12), respectively. Data are shown as means ± SEM. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student’s t test). (E) Chromatin immunoprecipitation for ESRRG followed by quantitative PCR in RB1-null C33A cells with and without exogenously expressed RB1 (n = 4) and in RB1-WT SH-SY5Y neuroblastoma cells with (RB1-KO) or without (RB1-WT) RB1-KO (n = 4). (F) Western blot in HEK293 cells following ectopic expression of V5-ESRRG-WT, shRNA directed against ESRRG (shESRRG), or controls (GFP and shGFP, respectively). (G) ESRRG ChIP-seq peaks at the CDKN1A locus and corresponding RNA-seq data with (shESRRG) and without (shGFP) ESRRG knockdown. DBD, DNA binding domain; EV, empty vector; LBD, ligand binding domain.

Fig. 5.. ESRRG is a hypoxic adaptation and survival factor in Rb.

(A) Normalized gene expression of ESRRG (top left, n = 3) and normalized ERRE luciferase activity (top right, n = 6) in HEK293 cells with (shRB1⁺) or without (shRB1⁻) doxycycline treatment to induce shRNA-mediated knockdown of RB1 at the indicated time points in hypoxia (1% O₂). Normalized gene expression of ESRRG (bottom left, n = 4) and normalized ERRE luciferase activity (bottom right, n = 3) in C33A cells with ectopically expressed RB1 (RB1) or EV control at the indicated time points in hypoxia (1% O₂). (B) Representative images of LIVE (green)/DEAD (red) cell viability assays of three recently established Rb cell lines (RB006, RB018, and RB020) stably expressing shRNA directed against ESRRG (shESRRG) or GFP control (shGFP) cultured in normoxia versus hypoxia (1% O₂) for 21 days. (C) Normalized LIVE/DEAD cell ratios averaged for all three Rb cell lines illustrated in (B) in normoxia versus hypoxia O₂ (1%). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student’s t test). (D) Normalized LIVE/DEAD cell ratios in wRB6 cells (left, n = 3) and RB020 cells (right, n = 3) treated with the indicated micromolar concentrations of the ESRRG inverse agonist GSK5182 for 7 days in normoxia versus hypoxia (1% O₂). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student’s t test). All data are shown as means ± SEM.

Fig. 6.. ESRRG expression in human Rbs.

(A) Hematoxylin and eosin staining (left) and multiplex fluorescence immunohistochemistry (middle and right) of Rb sample #2587-18. ESRRG (white), TUBB3 (red), 4′,6-diamidino-2-phenylindole (DAPI) (blue), and HIF1A (green). V, viable tumor cells concentrated in cuffs around blood vessels; N, necrotic tumor cells beyond ~100 μm of tumor blood vessels. Arrows demarcate regions enriched for ESRRG- and HIF1A-expressing cells in hypoxic zones between viable and necrotic tumor cells. (B and C) Similar multiplex fluorescence immunohistochemistry findings in Rb samples #2647-18 (left panels) and #1109189582 (right panels). ESRRG (white), TUBB3 (red), DAPI (blue), and HIF1A (green). (D) Vitreous tumor cell cluster or “seed” in Rb sample #2647-18 demonstrating strong diffuse staining for ESRRG and HIF1A. ESRRG (white), TUBB3 (red), DAPI (blue), and HIF1A (green). (E) Hematoxylin and eosin staining (left) and multiplex fluorescence immunohistochemistry (right) in Rb sample #29-15 showing invasion of the optic nerve by tumor cells (arrows). ESRRG (white), TUBB3 (red), DAPI (blue), and PECAM1 (green).