ACY-1215 is efficacious as an anti-cancer drug in primary UM and metastatic UM cell lines. (A): Schematic diagram on the treatment regime followed. Days post treatment (dpt). (B): Representative image of clonogenic assay plates for Mel270 (top panel) and OMM2.5 cells (bottom panel) treated with 0.5% DMSO; 1, 5, 10, 20 or 50 μM ACY-1215 or 20 μM Dacarbazine for 96 h. (C,D): A dose-dependent, significant decrease in the surviving number of OMM2.5 colonies was observed, indicative of reduction in cell viability upon ACY-1215 treatment in comparison to 0.5% DMSO treatment. (E): A dose-dependent, significant decrease in OMM2.5 cell viability was observed following 96 h of ACY-1215 treatment in comparison to 0.5% DMSO treatment. (F): IC₅₀ value of ACY-1215 in OMM2.5 cell viability assays. One-way ANOVA with Dunnett’s Test for Multiple Comparisons statistical analysis was performed; error bars represent mean ± SEM, *** p value of 0.001, **** p value of 0.0001 (N = 3–4).

ACY-1215 demonstrates anti-cancer effects in vivo in zebrafish OMM2.5 xenografts. (A): Schematic depicting the workflow for assessing ACY-1215 effects in vivo. (B): Top panel shows representative images of Dil-labeled OMM2.5 cells transplanted into the perivitelline space of 2-day-old zebrafish larvae. Bottom panels present representative images of the distribution of OMM2.5 Dil-labeled cells in xenografts 3 days post treatment (dpt), with 0.5% DMSO (n = 30), 20 μM ACY-1215 (n = 31) or 20 μM Dacarbazine (n = 27). Days post fertilization (dpf). (C): At 3 dpt, OMM2.5 Dil-labeled cells have disseminated (white arrowhead) to the caudal vein plexus of the zebrafish larvae. (D): ACY-1215 treatment for 3 days resulted in a significant (****, p = 0.0001) reduction in normalized primary xenograft fluorescence on average in comparison to larvae treated with 0.5% DMSO or 20 μM Dacarbazine. (E): There was no observed difference in the average number of disseminated cells between vehicle control-treated or drug-treated groups after 3 days. Statistical analysis was performed using one-way ANOVA with Dunnett’s Test for Multiple Comparisons, and error bars present mean ± SEM.

Expression and activity of HDAC6 in UM/MUM cells. (A,A’): HDAC6 is expressed in Mel270, OMM2.5, Mel285 and ARPE19 cells (N = 3). (B,B’): 20 μM ACY-1215 treatment significantly increased acetylated α-tubulin expression levels at 4 h post treatment (hpt) (**, p = 0.0013) and 24 hpt (***, p = 0.0002) compared to 0.5% DMSO treatment. Student’s Unpaired T test statistical analysis was performed, and data are presented as mean ± SEM. Representative blots for each protein probed and densitometry analysis presented, plus raw blots are provided in Supplementary Figures S3 and S7. (C): Kaplan–Meier survival curves assessing correlation between expression of HDAC6 and overall survival (OS) or progression-free survival (PFS) in UM patients. Median values were used as cut-off for high (red) and low (blue) expression levels, with Log-rank p-values (categorical variable) and Cox p-values (continuous variable) calculated (n = 80).

Proteome profiling of ACY-1215-treated cells to uncover mechanism of action. (A): ACY-1215 treatment regime for proteome profiling of OMM2.5 cells. Hours post treatment (hpt). (B): Heat map showing all significant differentially expressed proteins at 24 h post 20 μM ACY-1215 treatment in OMM2.5 cells. A total of 4423 proteins were identified in MS, with 150 downregulated (blue) and 202 upregulated (red) proteins (N = 4). (C,D): Enriched protein pathway analysis for GO term: biological processes for down and upregulated proteins, given a fold change cut off of +/− ≥ 1.2, p ≤ 0.05 displayed as pie charts. *, p < 0.05 and **, p < 0.01 denotes GO-term significance.

Significantly altered proteins identified by proteomic profiling following ACY-1215 treatment in OMM2.5 cells for 24 h. (A): Table highlighting the top 10 most down and upregulated proteins at 24 h post treatment (hpt) with 20 μM ACY-1215. (B): List of proteins involved in the MITF signaling pathway that were downregulated upon 20 μM ACY-1215 treatment for 24 h.

Western blot validation of proteomics data in OMM2.5 cells. (A,A’): There was no change in MITF expression levels after 4 h of 20 μM ACY-1215 treatment, while treatment for 24 h with 20 μM ACY-1215 led to a significant (**, p = 0.002) reduction in MITF expression levels. (B,B’): Relative expression levels of p-ERK to total ERK remained unchanged after 20 μM ACY-1215 treatment for 4 h. At 24 h post treatment with 20 μM ACY-1215, relative expression levels of p-ERK to total ERK were significantly (****, p < 0.0001) downregulated when compared to the 0.5% DMSO treatment. (C,C’): Expression of cleaved PARP was significantly (*, p = 0.049) upregulated after 24 h treatment with 20 μM ACY-1215 in comparison to 0.5% DMSO-treated OMM2.5 cells. Representative blots for each protein probed and densitometry analysis presented, raw blots are provided in Supplementary Figure S7. β-actin, GAPDH or α-tubulin were used as loading controls. Student’s Unpaired T test statistical analysis was performed, and data presented as mean ± SEM.

Cell cycle progression is arrested in the S phase by ACY-1215 in OMM2.5 cells. (A): Schematic illustrating treatment protocol undertaken. Hours post treatment (hpt). (B): Flow cytometry data analysis plot legend. (C,E): 4 h of treatment with 10, 20 or 50 μM ACY-1215, or 50 μM Etoposide or 20 μM Dacarbazine did not alter the cell cycle profile. (D,F): A significant (****, p = 0.0001) reduction in the percentage of cells in G₁ phase and a significant (****, p = 0.0001) increase in the percentage of cells in the S phase were observed after 24 hpt with 10, 20 or 50 μM ACY-1215 or 50 μM Etoposide, in comparison to vehicle control. No changes in the cell cycle phases were observed following 20 μM Dacarbazine treatment. No alterations to G₂ phase were observed in all treatment groups. Statistical analysis was performed by two-way ANOVA with Dunnett’s Test for Multiple Comparisons and data represented as mean ± SEM (N = 4).

ACY-1215 activates the apoptotic pathway in OMM2.5 cells. (A): Diagram portraying treatment regime. Hours post treatment (hpt). (A’,B): Plots representing gating of cell singlets into different stages of apoptosis. (A’’,B’): Representative plots depicting OMM2.5 treated with 0.5% DMSO; 10, 20 or 50 μM ACY-1215; 50 μM Etoposide or 20 μM Dacarbazine, at 24 and 96 hpt, respectively. (A’’’,B’’): Representative micrographs of OMM2.5 cells at 24 and 96 h post treatment. (C): A significant reduction in the percentage of live cells and a significant increase in the percentage of early apoptotic cells was detected following 20 μM (**, p = 0.005 and *, p = 0.017, respectively) and 50 μM (****, p < 0.0001) ACY-1215 treatment compared to 0.5% DMSO treatment. (D): Live cell populations were significantly (****, p < 0.0001) reduced, and cell populations in late apoptotic stage and apoptotic stage were significantly (****, p < 0.0001) increased upon 50 μM Etoposide and all concentrations of ACY-1215 tested. The 10 μM ACY-1215 treatment resulted in a significant increase in the early apoptotic cell population compared to 0.5% DMSO. The 20 μM Dacarbazine treatment was comparable to vehicle control plots. Statistical analysis by two-way ANOVA, followed by Tukey’s Multiple Comparisons test, with error bars shown as mean ± SEM (N = 3).

Inhibition of MITF pathway reduces OMM2.5 cell proliferation in vitro. (A): Schematic diagram of treatment regime. Days post treatment (dpt). (B): Chemical structure of ML329, a small molecule MITF pathway inhibitor. (C): Representative image of clonogenic assay plates for OMM2.5 cells treated with 0.5% DMSO, 0.05–50 μM ML329, 20 μM Dacarbazine or 20 μM ACY-1215 for 96 h. (D): A dose-dependent, significant reduction in the number of OMM2.5 colonies was observed in ML329 treatment groups compared to the 0.5% DMSO treatment group. One-way ANOVA with Dunnett’s Test for Multiple Comparisons statistical analysis was performed; error bars represent mean ± SEM, **, p = 0.005, ****, p = 0.0001 (N = 3–4). (E): Kaplan–Meier survival curves demonstrating no correlation between expression of MITF and overall survival (OS) or progression-free survival (PFS) in UM patients. Median values were used as cut-off for high (red) and low (blue) expression levels, with Log-rank p-values (categorical variable) and Cox p-values (continuous variable) calculated (n = 80).

ML329 demonstrates anti-UM properties in zebrafish OMM2.5 xenografts. (A): Top panel shows OMM2.5 Dil-labeled cells xenografted into the perivitelline space of 2-day-old zebrafish larvae. Bottom panel presents zebrafish larvae 3 days post treatment (dpt) with 0.5% DMSO (n = 29) or 1.25 μM ML329 (n = 29). Days post fertilization (dpf). (B). Representative image of OMM2.5 Dil-labeled cells disseminated (white arrowhead) to the caudal vein plexus of zebrafish larvae at 3 dpt. (C): A significant (***, p = 0.0006) regression of the average normalized primary xenograft fluorescence of OMM2.5 Dil-labeled cells was observed when treated with 1.25 μM ML329. (D): No difference was detected in the average number of disseminated OMM2.5 Dil cells following treatment with 1.25 μM ML329 compared to vehicle control. Student’s T test was used for statistical analysis, with error bars presenting mean ± SEM.