Histone deacetylase inhibitors induce growth arrest and differentiation in uveal melanoma

Tumor samples

This study was approved by the Institutional Review Board of Washington University in St. Louis and adhered to the tenets of the Declaration of Helsinki. Primary UM samples were collected at the time of enucleation as previously described (12). Informed consent was obtained for each patient. All samples were confirmed to be uveal melanomas by pathologic evaluation. Tumor samples were snap-frozen for DNA and RNA isolation or collected in HAM’S F-12 medium for tissue culture. Primary UM cells were grown in serum-free MDMF medium on collagen-covered tissue culture plates in 5% CO₂ and 4% O₂ (13). Elimination of contaminating cells was confirmed by microscopic inspection and Melan-A staining. The multi-gene prognostic assay for assignment of tumors to class 1 or class 2 groups was performed as described elsewhere (4). To determine BAP1 status of primary tumors, genomic DNA was subjected to Sanger sequencing of all BAP1 exons as described elsewhere (14). The UM cell lines 92.1, OCM1A and Mel202 (kindly provided by Drs. M. Jager, J. Kan-Mitchell and B. Ksander, respectively) were grown in RPMI-1640 supplemented with 10% FBS, L-glutamine and antibiotics at 5% CO₂. These UM cell lines are all BAP1-wildtype with a gene expression profile similar to class 1 tumors.

Gene Set Enrichment Analysis (GSEA) and Connectivity Mapping

Gene expression profiles from fourteen class 1 and ten class 2 UMs were obtained on the Affymetrix U133A platform, as previously described (15). Curated gene sets enriched in class 1 UM tumors versus class 2 UM tumors were identified with GSEA (version 2.0.4; Broad Institute), using a significance threshold set at a nominal P-value < 0.01 (16), as we previously described (5). To identify compounds that could potentially induce differentiation of class 2 UM cells, we interrogated the Connectivity Map database (Broad Institute) (17) for compounds that caused gene expression changes that closely resembled the gene expression differences between normal uveal melanocytes versus class 2 UM cells, as well as class 1 UM cells versus class 2 UM cells. The 100 most differentially expressed genes were chosen using Student’s t-Test. This gene set was compared to those from the Connectivity Map database, and potential compounds were chosen using the following parameters (e.g., positive enrichment score with a P<0.05).

Chemosensitivity assays

The effects of four HDAC inhibitors, VPA (Sigma), TSA (Sigma), LBH-589 (Selleck) and SAHA (Selleck), were studied in UM cell lines and primary UM cells. Drug concentrations ranged from 0.5–2 mM for VPA, 50–250 nM for TSA, 5–60 nM for LBH-589 and 0.5–2.5 µM for SAHA. DMSO was added as vehicle in TSA, LBH-589 and SAHA untreated controls.

MTS assays were performed according to manufacturer’s instructions (CellTiter 96® AQueous Assay reagent; Promega). Bromodeoxyuridine (BrdU; 1:1000; Amersham Biosciences) incorporation assays were performed using an anti-BrdU-peroxidase-conjugated antibody according to the manufacturer’s instructions (Roche; dilution 1:75). Tetramethylbenzidine (TMB; Sigma) was added as substrate for signal detection and colorimetric changes were measured at 370 nm using a microplate spectrophotometer (SpectraMAX 190; Molecular Devices). Flow cytometry was performed after treatment of cells for 48h using a standard propidium iodide staining protocol as previously described (18) using a FACScan analyzer (BD Biosciences). The percentage of cells in each phase was determined with the VenturiOne software (Applied Cytometry). For clonogenic assays, flow cytometry (Moflo; Cytomation) was used to seed one viable cell per well in ultra-low attachment 96-well plates containing MDMF medium only or MDMF with drug. Cells were monitored with phase contrast microscope, and cell number was assessed for each well at 7 days. Cell morphology was assessed by phase contrast microscopy and morphologic changes were quantified by counting the number of dendritic arborizations per cell in primary UM cells or by determining the maximal cell length/width ratio (spindle morphology index) in UM cell lines using ImageJ. Higher values are consistent with increasing melanocytic differentiation.

Multi-gene prognostic assay and qPCR

Total RNA extracted with TRIzol (Invitrogen) was DNase-treated and reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNA was pre-amplified for 14 cycles with pooled primers according to the manufacturer’s protocol (Applied Biosystems). The multi-gene prognostic assay was performed with RNA from primary tumor cells treated with VPA, LBH-589 and SAHA, and molecular class was determined using Support Vector Machine (SVM), as described elsewhere (4). BAP1 mRNA levels were measured by qPCR as previously described (6).

BAP1 knockdown

BAP1 was depleted in the 92.1 UM cell line by using a lentiviral-based shRNA. Lentiviral pLKO.1 shRNA vectors for GFP (clonetechGfp-438s1c1) and BAP1 (NM_004656.2-321s1c1) (developed by the RNAi Consortium (TRC)) were purchased from the Children's Discovery Institute/Genome Sequencing Center at Washington University in St. Louis. Viral production and infections were carried out according to the The RNAi Consortium recommendations (Broad Institute). Lentiviruses encoded by the pLKO.1 shRNA vectors were packaged in 293FT cells (Invitrogen) after cotransfection of the shRNA plasmids with pCMV-dR8.2 dvpr and pCMV-VSV-G lentiviral plasmids (Addgene plasmids #8454 and #8455) using TransIT-LT1 (Mirus Bio) (19). 92.1 UM cell line was infected for 24 hours with lentiviral supernatants in the presence of 8 µg/ml polybrene. Puromycin (3 µg/ml) was added to the cells at 24 hours post-infection for selection.

Western blotting and immunofluorescence

Western blotting was performed as previously described (18) using anti-BAP1 (1:250; Santa Cruz), anti-alpha-tubulin (loading control, 1:1000; Sigma), anti-acetyl-histone H3Lys9 (1:200; Cell signaling), anti-histone H3 (1:200; Cell signaling), anti-ubiquityl-histone H2A (1:100; Millipore), and anti-histone H2A (1:200; Millipore) antibodies. Histone proteins for western blotting were extracted from cells according to previously published protocol (20). Western blotting densitometry was performed with the AlphaEaseFC software using the Spot Denso Analysis Tool. Intensity of each Ub-H2A and total H2A bands was measured using a rectangular spot and Ub-H2A was normalized to total H2A after background subtraction. Immunofluorescence was performed by plating 5×10³ 92.1 cells on Lab-Tek 8-well Permanox chamber slides (Nunc) and using anti-BAP1, anti-acetyl-histone H3Lys9 and anti-ubiquityl-histone H2A antibodies.

Animal studies

Animal experiments were approved by the Washington University in St. Louis Animal Studies Committee. 1×10⁶ 92.1 UM cells were resuspended in Cultrex (Trevigen) and injected subcutaneously into the flanks of NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ JAX® (NOD SCID gamma) mice (Jackson Laboratory). Mice were then given intraperitoneal injection of VPA (0.1 mg per g of body weight) every other day, beginning at day 7. Tumor volume was monitored once a week and the mice were euthanized after 42 days. Tumors were collected and volume measured using the ellipsoid volume formula (π xyz/6 mm³).

Statistical analysis

Except where otherwise noted, data were analyzed for statistical significance using MedCalc software, version 9.5.1.0.

In silico screening for compounds that reverse the effects of BAP1 loss

BAP1 loss in UM cells results in morphologic and transcriptomic changes consistent with a loss of melanocytic differentiation and a shift from class 1 to class 2 transcriptomic profile (6). Thus, we sought to identify therapeutic compounds that may reverse the effects of BAP1 loss by restoring a more differentiated, class 1-like transcriptomic profile. We used two complementary in silico approaches – GSEA and Connectivity Mapping – to compare genes that are differentially expressed between class 1 and class 2 UMs to curated gene sets associated with perturbation of cancer cells with therapeutic compounds. Using GSEA, the gene set that was most similar to the genes up-regulated in class 1 UMs (relative to class 2) was PEART_HISTONE_UP (Fig. 1), which consisted of genes up-regulated by the HDAC inhibitors SAHA and depsipeptide (21). We obtained similar results with the Connectivity Mapping, which identified three HDAC inhibitors (VPA, TSA and SAHA) among its top matches (Supplementary Table S1).

HDAC inhibition blocks proliferation of UM cells

Initially we chose VPA to test the effects of HDAC inhibition in UM cells. As expected, VPA caused a dose-dependent increase in histone H3 acetylation (Supplementary Fig. S1). In all three UM cell lines (92.1, OCM1A and Mel202), VPA inhibited proliferation but did not significantly reduce the fraction of viable cells, induced a G1 cell cycle arrest and markedly reduced the clonogenicity of UM cells (Fig. 2). The spindle morphology index increased after treatment with the HDAC inhibitors (Supplementary Fig. S2). Similar changes were seen with TSA and LBH-589, except that these agents significantly reduced the fraction of viable cells and increased the proportion of cells undergoing apoptosis (Fig. 2), consistent with increased cytotoxicity.

VPA inhibits UM tumor growth in vivo

Since VPA induced cell cycle arrest rather than cell death, we predicted that it may have therapeutic potential in an adjuvant setting to block the growth of micrometastatic disease. To model this situation, we established small subcutaneous flank tumors in NOD SCID gamma mice and tested whether VPA could prevent the growth of these tumors. Treatment with VPA or control was initiated when the tumors became barely palpable (usually about 7 days after injection). VPA markedly reduced the growth and final volume of these tumor deposits compared to control (Fig. 3).

BAP1 loss sensitizes UM cells to HDAC inhibition

BAP1 is the catalytic subunit of the PR-DUB complex that deubiquitinates histone H2A (22). As expected, RNAi-mediated knock down of BAP1 in UM cells (Fig. 4A) induced a marked increase in H2A ubiquitination (Fig. 4B, upper panel). Since a recent report showed that HDAC inhibitors decrease histone H2A ubiquitination through transcriptional repression of the PRC1 component BMI1 (23), we wished to determine whether HDAC inhibition may reverse this H2A hyperubiquitination in BAP1-deficient UM cells. Indeed, VPA markedly reduced the levels of histone H2A ubiquitination in BAP1-depleted cells (Fig. 4B, lower panel and Fig. 4C upper panel), while total histone H2A level remained unchanged (Fig. 4C, lower panel). We showed in Fig. 2A that VPA inhibited proliferation of BAP1-wildtype UM cell lines, but it did not reduce the fraction of viable cells. However, when we stably depleted BAP1 in UM cells using lentiviral shRNA, VPA significantly reduced the fraction of viable cells compared to control cells (Fig. 4D), indicating that BAP1 loss sensitized the UM cells to HDAC inhibition.

We then investigated whether HDAC inhibition could reverse the phenotypic consequences of BAP1 loss: loss of melanocytic differentiation and acquisition of the class 2 gene expression profile (6). Since UM cell lines undergo genetic and epigenetic artifacts in long term culture, for these experiments we used primary UM cells from five class 2 tumors (MM137, MM138, MM151, MM161 and MM162) and one class 1 tumor (MM131) which were obtained immediately after surgical resection and placed in short term culture. While only four of the class 2 samples contained detectable BAP1 mutations, all five showed low BAP1 RNA levels compared to the class 1 tumors (Supplementary Table S2 and Supplementary Fig. S3). In cells treated with VPA for 3 and 7 days, cell bodies became enlarged and flattened, and there was a marked increased in the number of dendritic arborizations, consistent with melanocytic differentiation (Fig. 5A–B). Untreated or VPA-treated cells were subjected to a multi-gene expression profile that classifies UMs as class 1 or class 2 (4). The results of this assay were reported as a SVM discriminant score; the more negative the score, the more class 1 the expression profile, the more positive the more class 2. VPA shifted all of the class 2 UM cells towards a class 1 profile, and it even shifted the class 1 UM cells towards a more class 1 profile (Fig. 5C and Supplementary Fig. S4). This effect was dose-dependent, and similar results were observed with LBH-589 (Supplementary Figs. S4 and S5C). The genes most affected by VPA were HTR2B (down-regulated by VPA) and LMCD1 (up-regulated by VPA) (Supplementary Fig. S5), both strong indicators of metastasizing UMs (4). A fourth HDAC inhibitor, SAHA, induced effects similar to VPA in UM cell lines and primary UM cells (Supplementary Fig. S6).