ICG-001 Exerts Potent Anticancer Activity Against Uveal Melanoma Cells

Abstract

Purpose: Uveal melanoma (UM) is uniformly refractory to all available systemic chemotherapies, thus creating an urgent need for novel therapeutics. In this study, we investigated the sensitivity of UM cells to ICG-001, a small molecule reported to suppress the Wnt/β-catenin-mediated transcriptional program.

Methods: We used a panel of UM cell lines to examine the effects of ICG-001 on cellular proliferation, migration, and gene expression. In vivo efficacy of ICG-001 was evaluated in a UM xenograft model.

Results: ICG-001 exerted strong antiproliferative activity against UM cells, leading to cell cycle arrest, apoptosis, and inhibition of migration. Global gene expression profiling revealed strong suppression of genes associated with cell cycle proliferation, DNA replication, and G1/S transition. Gene set enrichment analysis revealed that ICG-001 suppressed Wnt, mTOR, and MAPK signaling. Strikingly, ICG-001 suppressed the expression of genes associated with UM aggressiveness, including CDH1, CITED1, EMP1, EMP3, SDCBP, and SPARC. Notably, the transcriptomic footprint of ICG-001, when applied to a UM patient dataset, was associated with better clinical outcome. Lastly, ICG-001 exerted anticancer activity against a UM tumor xenograft in mice.

Conclusions: Using in vitro and in vivo experiments, we demonstrate that ICG-001 has strong anticancer activity against UM cells and suppresses transcriptional programs critical for the cancer cell. Our results suggest that ICG-001 holds promise and should be examined further as a novel therapeutic agent for UM.

Figure 1

ICG-001 suppresses proliferation and induces growth arrest in UM cells. (A) ICG-001 suppresses the growth of a panel of UM cells. MTT assay was performed after 96 hours of ICG-001 treatment. Results shown are average ± SEM. (B) ICG-001 induces cell cycle arrest. Mel270 and Mel202 UM cells were treated with ICG-001 for 24 and 72 hours and stained with propidium iodide. Cell cycle distribution is shown as bar graphs. Experiments were repeated three times with similar results; shown here is one representative experiment performed with at least three technical replicates. Results shown are average ± SD. Representative histograms are shown in Supplementary Figure S1.

Figure 2

ICG-001 suppresses cell cycle gene expression in UM cells. Gene expression profiling was performed with the Illumina HumanHT-12 v4 Expression BeadChip array using RNA harvested from Mel202 cells treated with 3 μM of ICG-001 for 48 hours. Gene set enrichment analyses against MSigDB pathways reveal that ICG-001 potent suppresses cell cycle proliferation, DNA replication, G1/S transition cell cycle, cell cycle checkpoints genes, and transcriptional targets of E2F.

Figure 3

ICG-001 inhibits the expression of genes involved in the mTOR pathway, Wnt pathway, and stemness. (A, B) GSEA shows negative enrichment of transcriptional targets of mTORC1 and Wnt signaling pathways (MSigDB curated geneset). (C, D) GSEA of a WNT5A siRNA signature, derived from a prostate cancer cell line (LNCaP-abl), compared against the ICG-001 signature from Mel202 cells shows strong concordance. (E) Normalized enrichment scores from stemness gene sets (from MSigDB) compared against the ICG-001 transcriptional footprint. Strong inhibition of embryonic stem cell (ESC)-associated transcriptional programs is observed upon ICG-001 treatment of UM cells. Gene sets with a nominal P value <0.05 and FDR < 0.25 were defined as significantly enriched.

Figure 4

Strong concordance of gene signature of ICG-001 in UM with signatures of ICG-001 and p300 inhibitor in other cancers. Gene set enrichment analysis shows high concordance between the ICG-001 signature from UM and the transcriptomic footprints of ICG-001 (A–D) as well as with the signature of C646 (E–H), a small molecule inhibitor of p300, derived from the HCT116 (colon cancer) and PANC1 (pancreatic cancer) cell lines (GSE64038).

Figure 5

Strong concordance between the gene signatures of ICG-001, BET inhibitor JQ1, and siEP300. Comparison of publically available signatures from UM cell lines treated with JQ1, an inhibitor of BET family of bromodomain proteins, shows strong concordance with our signature of ICG-001 in UM cells. Similar concordance also is observed with the EP300 siRNA (siEP300) signature from C4-2B prostate cancer cell line. Gene sets with a nominal P value <0.05 and FDR < 0.25 were defined as significantly enriched.

Figure 6

ICG-001 suppresses UM cell migration in vitro and the expression of genes associated in vivo with UM aggressiveness. (A) ICG-001 suppresses the expression of genes that are associated with focal adhesion in UM (left), as well as SPP1 (osteopontin) and epithelial genes that are associated with UM aggressiveness in Class 2 UM (right). (B) Normalized enrichment scores for pathways associated with kinase signaling (ERK/MAPK, MEK, and HGF signaling) when compared against the transcriptomic footprint of ICG-001 in UM. (C) In vitro migration of UM cells was assessed by measuring their ability to heal a scratch wound in a cell monolayer. ICG-001 potently suppressed UM cell migration in the Mel270 (primary) and OMM2.5 (metastatic) UM cell lines. Results shown are average ± SD. Representative images are shown in Supplementary Figure S5.

Figure 7

ICG-001 suppresses the expression of genes associated with metastasis in vivo and its transcriptomic footprint is associated with increased patient overall survival. (A–D) GSEA of various UM clinical datasets against the transcriptomic footprint of ICG-001 in UM demonstrates that ICG-001 potently suppresses gene expression profiles associated with UM metastasis. (E) The ICG-001 gene expression profile, applied to a UM patient dataset (TCGA) shows a statistically significant association with clinical outcomes. UM patient samples exhibiting low ICG-001 signature (lower 20% of patients) had inferior overall survival.

Figure 8

ICG-001 suppresses tumor growth in a murine model of UM. A Mel270 UM xenograft model was used to evaluate the anticancer activity of ICG-001 in vivo. Treatment with ICG-001 substantially reduced growth of UM xenografts compared to the vehicle controls. Shown in (A) are the average tumor volumes of mice treated with ICG-001 or vehicle (average±SEM; *P value <0.05). After 5 weeks of treatment, several mice (those with the largest tumors) in the vehicle cohort (but not in the ICG-001 cohort) had to be removed from the study due to tumor diameter exceeding the study threshold, thus making moot any further direct comparison of volumes between the two cohorts. ICG-001 delayed the xenografts from reaching the study threshold of 1 cm diameter, thus prolonging animal survival on the study (Kaplan-Meier survival plot is shown in [B]).