TOP 15 networks significantly affected by upmodulated 1 2 3 4 -log (p-value) 1. Tyrosine metabolism; 2. Development_regulation of telomere: length and cellular immortalization; 3. Cytoskeleton remodeling; 4. Cell adhesion_integrin-mediated cell adhesion and migration; 5. Cytoskeleton remodeling_integrein outside-in signaling; 6. Cytoskeleton remodeling_regulation of actin cytoskeleton by Rh GTPases; 7. Apoptosis and survival_p53-dependent apoptosis; 8. Role of tissue factor in independent of coagulation protease signaling; 9. Transport_clathrin-coated vesicle cycle; 10. Sgnal transduction_AKT signaling; 11. Development_TGF-beta-dependent induction of EMT via MAPK; 12. Development_IGF-1 receptor signaling; 13. Development_melanocyte development and pigmentation; 14. Development_WNT_signaling pathway; 15. Development_FGFR signaling pathway;

Supplemental Figure 1: ontology network analysis of uveal melanoma cells following MEKi treatment (24h) and ABPP. Figure shows top 15 most significantly altered pathways. B) YAP ( MP Supplemental bosentan

YAP A) Mean Fluorescence intensity 100 ET-3 Control . EDNRBi + ET-3 EDNRBi 20 40 80 60 41 0 B) DAPI and and ** 92.1 ns , 92.1 Nuclear 80 C) ns OMM  Quantification Mel270 M) YAP ** ns Figure or 1 ns cells ET ET Control - 3 - ns 92.1 3 DAPI Cytoplasmic ns 2 + . : ns A) bosentan EDNRBi+ET EDNRBi Mel270 of ET Mel270 ns Cells nuclear/cytoplasmic - ns 3 YAP ns - 3 increases were ( 100

Fluorescence intensity C) 100 20 40 80 60 0 nM treated DAPI MP41 ** and MP41 ns nuclear Nuclear * 80 with YAP YAP MM28 ns  M, ns either staining ns accumulation respectively) MP41 * ET Control DAPI - Cytoplasmic ns 3 ET from ns - OMM1 3 EDNRBi+ET EDNRBi MM28 ns ( 100 A) ns . YAP Cells ns . nM, - 3 of were 1 YAP hr ), fixed in EDNRB 92 and . 1 stained , antagonist Mel 270 for , 2 92.1 Mel270

ns ns ns ns

1 ns ns luciferase luciferase activity

- ns Relative Relative YAP

0 Control ET-1 EDNRBi EDNRBi+ET-1 Supplemental Figure 3: ET-1 does not induce YAP reporter activity in 92.1 or Mel270 cells. Cells were treated with either ET-1 (100nM, 1 hr), EDNRB antagonist (bosentan, 80µM) or ET-1 + bosentan (100nM and 80µM, respectively). Cell 92.1 Mel270 MP41 OMM1 Viability (%) MEKi (nM) 100

0 10 0 10 0 10 0 10 0 DOT1Li (µM) 1 5 50

0 EZH2i (µM) 5 10 0

0 LSD1i (µM) 5 10

0 DNTMi (nM) 20 40

0 HATi (µM) 5 10

0 HDACi (nM) 10 20

Supplemental Figure 4: HDAC inhibitors increase the cytotoxic effects of MEK inhibition. Data show heatmaps showing the inhibition of the growth of uveal melanoma cell lines (92.1, MP41, Mel270 and OMM1) treated with of a MEK inhibitor (trametinib, 10nM) in combination with inhibitors of DOT1 (EPZ5676), EZH2 (Tazemetostat), LSD1 (GSK 2879552), DNMT (decitabine), HAT (anacardic acid) and HDAC (panobinostat). Cells were treated with vehicle, epigenetic inhibitors (0-10 µM), MEK inhibitor (trametinib, 10nM) or the combination for 72 h before being subjected to the MTT assay. A) Deacetylated products

Control MEKi 10nM MEKi 25nM 0.6 ***

0.4 **

* * (ng)

0.2 Deacetylated productsDeacetylated 0.0 92.1 Mel270

B) HDAC activity

Control MEKi 10nM MEKi 25nM 1.0 *** 0.8

0.6 * ** *

0.4

(ng/min/mg) Concentration Concentration 0.2

0.0 92.1 Mel270

Supplemental Figure 5: MEK inhibition leads to an increase in global acetylation in uveal melanoma cells. 92.1 and Mel270 cells were treated for 72 h with trametinib (10 and 25 nM) before being assayed for total protein acetylation and HDAC activity. Control MEKi HDACi MEKi+HDACi 20

15

10

ns ns ns ns ns Annexin Annexin V (%) ns ns ns ns 5

0

Mel-025 Mel-026 Mel-028

Supplemental Figure 6: MEKi, HDACi and the MEKi-HDACi combination do not affect the survival of primary uveal melanocytes. 3 uveal melanocyte lines (MEL-025, MEL- 206, MEL-028) were treated with vehicle, MEKi (trametinib,10nM), HDACi (panobinostat, 10nM) or the combination for 72 h. Apoptosis was measured by FITC-Annexin-V binding and flow cytometry. 40 92.1 MP41 *** Mel270 OMM1

*** ***

30

*** /mL)

pg * * 3 ( 3 ns ns - ns 20 ns ns * ns ns ns ns ns *

ns ns Endothelin

10 ns ns ns ns

0

Supplemental Figure 7: HDACi suppresses MEKi-induced release of ET-3 from uveal melanoma cells. Uveal melanoma cell lines were treated with vehicle, MEKi (trametinib,10nM), HDACi (panobinostat, 10nM) or a combination of the two drugs for 24 or 72 h. ET-3 concentrations were measured by ELISA. A) B) Fold change PIK3R3 DUSP3 Enrichment plot: PRKCB HALLMARK_PI3K_AKT_MTOR_SIGNALING MAPK10 RIT1 IRAK4 0.0 CDK2 PHLPP1 -0.5 PPP2R1B

RICTOR (ES) -0.10 MYD88 3 PLA2G12A -0.15 Enrichment Enrichment score TIAM1 PITX2 -0.20 GSK3B FGF17 CREB1 PIK3R1 positively correlated negatively correlated CAB39L 5.0 NCK1 2.5 MAP2K6 0.0 -2.5 PLCG1

-5.0 PPP1CA (PreRanked) -7.5 AP2M1

Ranked Ranked list metric 0 2500 5,000 7,500 10,000 12,500 SQSTM1 Rank in Ordered Dataset RIPK1 ITPR2 Enrichment profile Hits PIK3CA PTEN MAPK8 ACACA 2 MAPKAP1 CDKN1B TSC2 THEM4 CFL1 ACTR2 MAP3K7 ACTR3 RAF1 PRKAG1 C) PIKFYVE 92.1 Mel270 AKT1 RALB MAPK1 CALR CSNK2B DDIT3 RAC1 SMAD2

M.W. HDACi

M.W. HDACi

MEKi

MEKi

Control

MEKi+ HDACi Control MEKi+ HDACi MTOR 1 54 PTEN 54 PTEN UBE2D3 AKT2 116 Vinculin 116 Vinculin PTENP1 NFKBIB CDK4 PPP2CA MAPK9 GSK3A RPTOR PRKAR2A UBE2N ATF1 PDK1 MAP2K3 ARF1 ECSIT EIF4E AKT3 AKT1S1 MEKi vs Control HDACi vs Control MEKi+HDACi vs MEKi Supplemental Figure 8: Negative modulation of PI3K-AKT pathway by combinatory treatment with MEKi+HDACi. A) GSEA analysis of RNA-Seq experiments of the comparison between MEKi-treated only and MEKi+HDACi-treated uveal melanoma cells (24h) shows a decrease in PI3K-AKT-mTOR pathway activity. B) Heatmap shows the comparison between MEKi vs. Control, HDACi vs. Control and MEKi+HDACi vs. MEKi only. C) MEKi+HDACi combination increases expression of PTEN in uveal melanoma cells by Western Blot. line combination MEKi of the Supplemental palpable 92 and (Trametinib, . A) 1 Tumor weight (g) 0.0 0.5 1.0 1.5 B) uveal 92.1 MP tumors for Control 41 melanoma Figure 31 cell days 1 of mg/kg MEKi * line 92 9 . : Data . . 1 The xenograft HDACi po or ns shows MP daily), MEK MEKi MEKi HDACi+ 41 *** - HDACi final cells, HDACi model mean B) mice combination . Tumor weight (g) ( 0.0 0.1 0.2 0.3 0.4 0.5 After Panobinostat MP41 tumor were Control subcutaneous treated weights MEKi delivers * , 20 with from mg/kg, HDACi * inoculation vehicle each durable MEKi MEKi HDACi+ IP, *** group 3 (Control X responses and week) A) formation 92 group), or . 1 cell the in Control MEKi HDACi MEKi + HDACi

Supplemental Figure 10: Macroscopic findings of liver tumor formation after inoculation of MP41 by tail vein injection. Pictures were taken after 7 weeks (4 weeks until tumors were visible on MRI and then 3 weeks of treatment with vehicle (Control group), MEKi (Trametinib, 1mg/kg po daily), HDACi (Panobinostat, 20mg/kg, IP, 3X week) or the combination. Multiple foci of liver metastases are indicated (yellow arrows). Figure 2C

pAKT

AKT

GAPDH

Figure 3D

ROR2 ROR1

GAPDH

IGF-1R

Figure 3E 92.1

pAKT (Thr308)

AKT

GAPDH

Supplemental Figure 11: Uncropped blots Figure 3F 92.1 Mel270

IGF-1R

GAPDH

ROR1

ROR2

Figure 3G

pAKT (Thr308)

AKT

GAPDH

Supplemental Figure 11: Uncropped blots Figure 4F

92.1 Mel270

CTGF

AREG

GAPDH

Figure 4I

92.1 Mel270

OMM1 MP41 YAP YAP

GAPDH

GAPDH

Supplemental Figure 11: Uncropped blots Figure 6G 92.1 Mel270

BCL-2

Caspase3 Cleaved Caspase3

Parp Cleaved Parp

Vinculin

Figure 7A Mel270 MP41

pAKT (Thr308)

AKT

GAPDH

pAKT 92.1 (Thr308) AKT

GAPDH

Supplemental Figure 11: Uncropped blots 92.1 Figure 1D MP41 Figure 1D Control 10nM 25nM Control 10nM 25nM

24h 24h

72h 72h

Figure 1D Mel270 OMM1 Figure 1D Control 10nM 25nM Control 10nM 25nM

24h 24h

72h 72h

Figure 2D Control MEKi PI3Ki MEKi+PI3Ki 92.1

Mel270

MP41

Supplemental Figure 12: Flow cytometry plots Figure 3H

siControl siROR1/2 siIGF-1R Control MEKi Control MEKi Control MEKi 92.1

Mel270

siControl Figure 4J siYAP Control MEKi Control MEKi 92.1

Mel270

MP41

OMM1

Supplemental Figure 12: Flow cytometry plots Figure 6F Control MEKi HDACi MEKi+HDACi 92.1

MP41

Mel270

Supplemental Figure 12: Flow cytometry plots