Supplementary,Figure,1 Supplementary Figure 1

A B

A2058 (BRAF*) IMR90 3 +700% 5 +2400% % change vs. day 0 % change vs. day 0 ! DT$$$V$$$DTV ! DT$$E$$DTE 4 +1500% p!ERK 2 +300% p!ERK 3 +700% ERK ERK 2 +300% H3K9Ac 1 +100% H3K9Ac (day3 vs. day 0) (day 3 vs. day ) 1 +100% H3 H3 0 0 0 0 DMSO DT V DT+V DMSO DT ENT DT+ENT log2 fold change in cell number log2 fold change in cell number

C D E Hs695THs695T Hs695T 2.040 2.0 Hs695T DMSODMSO NIH3T3 DMSO 293T 1.5 1.5 2.0 MEKi+BRAFi DTDT 5 +3100% 30 4 +1500% % change vs. day 0 DMSO % change vs. day 0 1.0 1.0 HDACi E E 4 +1500% 1.5 0.5 MEKi+BRAFi+HDACi DT 3 +700% 0.520 DTEDTE 3 +700% 0.0 1.0 E 20 40 60 0.080 2 +300% log2 fold growth -0.5 10 20 40 60 80 2 +300% hours of treatment 0.5 DTE log2 fold growth -0.5 -1.0 hours of treatment 1 +100%

1 +100% (day3 vs. day 0)

(day3 vs. day 0) -1.00 0.0 0 0 0 20 2040 4060 8060 80 0 0 DMSO DT ENT DT+ENT Caspase 3/7 positive cells (%) DMSO DT ENT DT+ENT log2 fold growth -0.5hours of treatment log2 fold change in cell number hours of treatment log2 fold change in cell number -1.0

F G A2058'(high'MITF) SKMEL5'(high'MITF) Resistant Sensitive High'MITFMITF Expression 4.5 4.0 3.5 3.0

2.5 DMSO MEKi VOR ENT MEKi+VOR MEKi+ENT DMSO MEKi VOR ENT MEKi+VOR MEKi+ENT 2.0 1.5 MITF MITF 1.0 0.5 Low'MITF Vinculin Vinculin

0.4

0.3 0.10 RPMI7951'(low'MITF) Hs695T'(low'MITF) 0.08 Resistant Sensitive 0.06 0.04 Relative MITF expression (MITF/Actin) 0.02 0.00 DMSO MEKi VOR ENT MEKi+VOR MEKi+ENT DMSO MEKi VOR ENT MEKi+VOR MEKi+ENT

A375 A2058 MEWO A101D Hs695T SKMEL5 SKMEL2 LOXIMVI MITF MITF UACC257 MALME3MRPMI7951

Vinculin Vinculin sensitive resistant

H Hs695T ! E$$$$$$DTE

HDAC1

HDAC2

HDAC3

GAPDH

Supplementary,Figure,1 Supplementary Figure 1. A subset of melanoma cell lines and control cell lines do not respond to BRAF/MEK/HDAC inhibitor therapy. Therapeutic responses are unrelated to MITF status or expression changes. (A-D) melanoma cells (A2058), transformed human embryonic kidney cells (HEK293T) and fibroblasts (IMR90 and

NIH3T3) were treated with DMSO, 100nM dabrafenib and 10nM trametinib (DT), 2μM vorinostat (V), 1µM entinostat (E) and the combination of MEK/BRAF and HDAC inhibitors.

Cells were manually counted prior to the addition of compounds and 3 days after treatment. Graphs represent log2 transformation of the fold change in cell number at day

3 versus day 0. Right axis shows percent change in cell number relative to day 0.

Immunoblots show levels of phosphorylated ERK (p-ERK), total ERK, histone H3 acetylation at lysine 9 (H3K9Ac) and total H3 after 48 hours of indicated treatment. (E)

Real-time quantification of apoptotic cell death using the live cell imager IncuCyte Zoom.

Red (nuclear restricted NucLight Red fluorescent protein for quantifiying live cells) and green (Apoptosis-3/7 Green reagent for quantifying apoptotic cells) fluorescent objects were monitored in the Incucyte ZOOM acquiring images every 2 hours (for 72 hours) following treatment with vehicle (DMSO) or drugs (MEK inhibitor, BRAF inhibitor, HDAC inhibitor) and then quantified with the IncuCyte integrated analysis software. (F) quantification of MITF protein levels of cell lines sensitive and resistant to MAPK/HDAC inhibition, as shown in Fig. 1A and Fig. 3D. (G) Western blots depicting MITF protein levels in different melanoma cell lines (high/low baseline MITF levels and sensitive/resistant to

MAPK/HDAC inhibition) after 48hrs of treatment with vehicle (Veh), MEK inhibitor (MEKi) and/or HDAC inhibitors (vorinostat, VOR; entinostat, ENT). Vinculin serves as a loading control. (H) Western blots depicting HDAC1, HDAC2 and HDAC3 protein levels after 48hrs of treatment with vehicle (-), HDAC inhibitor (entinostat, E) and combined

MEK/BRAF/HDAC inhibitor (DTE). GAPDH serves as a loading control. A MELJUSO YUDOSO YUGASP WM3670 Hs852T

MGMT

α"tubulin

Supplementary,Figure,2 Supplementary Figure 2. Differential expression of MGMT in NRAS-mutant cell lines. (A) Western blot depicting baseline MGMT protein expression levels in NRAS- mutant human melanoma cell lines shown in Fig. 2E. A Hs695T SKMEL2

H2O2 H2O2

DMSO DMSO DT T E E DTE TE 100 102 104 100 102 104 H2DCFDA H2DCFDA (ROS) (ROS)

B Hs695T Hs695T PARP3 2 PARP3 +300% 2 +300% % change vs. day 0 % change vs. day 0 2.5 1 2.5 +100% 1 +100% 0 DMSO DMSO 2.0 -1 0 DT 2.00 0 DT -2 -1 -50% E -3 -50% E 1.5 1.5 -4 -2 -75% DTE -75% (day3 vs. day 0) (day3 vs. day 0) DTE -5 1.0 1.0 -6 -88% -3 -88% DMSO Vit C

log2 fold change in cell number 0.5 log2 fold change in cell number 0.5 DMSO 0.0 NAC (1mM) 0.0 NAC (2.5mM) G1 S G2

G1 S G2 relative mRNA expression (vs. DMSO) relative mRNA expression (vs. DMSO)

C SKMEL2 PARP3 3 +300% % change vs. day 0 2.5 2 +100% DMSO

1 2.0 0 DT E 0 1.5 -50% DTE -1 -75% (day3 vs. day 0) 1.0 -2 -88% 0.5 log2 fold change in cell number 0.0 DMSO G1 S G2 NAC (2.5mM) relative mRNA expression (vs. DMSO) D A2058 RPMI7951 4 +1500%

4 +1500% % change vs. day 0 % change vs. day 0 DMSO DMSO DT DT 3 +700% 3 +700% VOR VOR DT+VOR 2 +300% DT+VOR 2 +300%

1 +100% 1 +100% (day 3 vs. day 0) (day 3 vs. day 0)

0 0 0 0 DMSO BSO (200µM) DMSO BSO (200µM) log2 fold change in cell number log2 fold change in cell number

E Baseline((ROS)Data 1 400 Sensitive Resistant 300

200

100

0 Sensitive Resistant Mean fluorescence intensity (DCFDA) Supplementary,Figure,3 Supplementary Figure 3. Therapeutic effects are not mediated by ROS. (A) Sensitive cell lines (Hs695T and SKMEL2) treated with DMSO, 100nM dabrafenib (D) and/or 10nM trametinib (T), 1µM entinostat (E) and the combination of MEK/BRAF and HDAC inhibitors for 24hrs (or 1mM H2O2 for 4hrs as a positive control) were stained with dichlorofluorescein diacetate (DCFDA), a dye that measures ROS. Graphs indicate relative mean DCFDA fluorescence intensity. ROS levels slightly decrease in Hs695T cells in response to DTE whereas they increase in SKMEL2 cells, due to the effects of T. (B,C) Sensitive melanoma cells (Hs695T and SKMEL2) were treated with DMSO, 100nM dabrafenib (D) and/or 10nM trametinib (T), 1µM entinostat (E) or the specified drug combinations, alone and in combination with the ROS scavenger NAC and the anti-oxidant Vitamin C. Graphs represent log2 transformation of the fold change in cell number at day 3 versus day 0.

Right axis shows percent change in cell number relative to day 0. Negative values represent cell death and a decrease in cell number. NAC is toxic in Hs695T cells. Vitamin

C does not suppress, but rather potentiates the therapeutic response. NAC is tolerated in

SKMEL2 cells and has no effect on the therapeutic response. (D) Resistant melanoma cells (A2058 and RPMI7951) were treated with DMSO, 100nM dabrafenib and 10nM trametinib (DT), 1µM vorinostat (VOR) or the specified drug combinations, alone and in combination with the ROS inducer buthionine sulfoximine (BSO, 200µM). Graphs represent log2 transformation of the fold change in cell number at day 3 versus day 0.

Right axis shows percent change in cell number relative to day 0. Negative values represent cell death and a decrease in cell number. BSO does not make resistant cells become sensitive to this combination. (E) Baseline ROS levels for 3 sensitive and 3 resistant melanoma cell lines as measured by DCFDA staining, demonstrating that ROS levels do not correlate with sensitivity. A Sensitive(((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((Resistant Hs695T RPMI7951

!H2AX Rad51 Merge !H2AX Rad51 Merge siLuc siLuc siBRCA2 siBRCA2

SKMEL2 A2058 !H2AX Rad51 Merge !H2AX Rad51 Merge siLuc siLuc siBRCA2 siBRCA2

B MGMT+ MGMT7

LIG4 ATM ATR RBBP8 PMS1 REV1 EPC2 SMC5 UHRF1BP1L RAD50 POLK MSH3 WRN XRCC4 RRM2B POLI TDP2 ESCO1 DCLRE1A PALB2 2.0 GTF2H1 RIF1 RECQL SLK 1.6 GTF2H3 SHPRH SMG1 ERCC4 1.2 RAD18 POLG2 HELQ 0.8 HEATR1 SMC6 REV3L VCPIP1 0.4 UBE2V2 MNAT1 FANCM PSMC6 0.0 SMC3 CUL4A RFC3 BRCA2 -0.4 RAD51AP1 ESCO2 BRIP1 FIGNL1 -0.8 RAD17 ERCC8 RAD52 SUPT16H -1.2 TP53BP1 PARP4 SMC1A ANKRD17 -1.6 CLSPN RNF168 NONO -2.0 XPC SHFM1 RBX1 CETN2 RAD9A CIB1 MGMT PNKP MPG PML XAB2 RAD23A MUTYH SMUG1 NSMCE1 MAD2L2 ERCC1 POLL UBE2V1

h h h h h h h h h h h h h h h h h h h h h h h h h Supplementary,Figure,4 w w w w w w w w w w w w w w w w w w w w w w w w w g g g g g g g g g g g g g g g g g g g g g g g g g i i i i i i i i i i i i i i i i i i i i i i i i i o o o o o o o o o o o o o o o o o o o o o o o o o l l l l l l l l l l l l l l l l l l l l l l l l l h h h h h h h h h h h h h h h h h h h h h h h h h

BRB-ArrayTools 4/25/2017 3:38:35 PM Supplementary Figure 4. (A) Melanoma cells, transfected with control (siLuc) or pooled siRNAs targeting BRCA2 were irradiated with 10Gy. Cells were fixed 5 hours later and analyzed by immunofluorescence for RAD51 and gamma H2AX. Insets show magnification of merged gamma H2AX and RAD51 signals in sensitive and resistant cell lines, respectively. (B) Heatmap of only non-cell cycle regulated DNA repair genes in

MGMT+ versus MGMT- melanomas. Heatmap of differentially expressed DNA repair genes not regulated by the cell cycle in (MGMT+) versus (MGMT-) melanomas at significance level p=0.001. “High” expression (MGMT+) was defined as the top 10% of tumors in the TCGA SKCM provisional dataset stratified by MGMT mRNA expression, whereas “low” (MGMT-) was defined as the bottom 10%. Gene list obtained from [23]. A siControl siPARP3 siControl siXRCC4 siControl siXRCC6 siControl siXRCC5 siControl siPNKP XRCC5 PNKP PARP3 XRCC4 XRCC6

GAPDH GAPDH GAPDH GAPDH GAPDH

ELK1 ELK3 1.4 1.4 1.2 1.2 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 DMSO DT ENT DT+ENT DMSO DT ENT DT+ENT relative mRNA expression (vs. DMSO) relative mRNA expression (vs. DMSO)

Supplementary,Figure,5 Supplementary Figure 5. (A) Immunoblots confirming siRNA target suppression for experiment shown in Figure 6D. (B) mRNA levels of ELK1 and ELK3 in sensitive cells after 24hrs of treatment with vehicle (Veh), 100nM dabrafenib and 10nM trametinib (DT) and/or 1µM entinostat (ENT) as determined by quantitative PCR.