Supplementary Fig. S1

a

A375 WM-3912 WM-293A Time (h) 0 0.5 2 8 24 48 0 0.5 2 8 24 48 0 0.5 2 8 24 48 BRAF + MEKi

P-ERK1/2 37

ERK1/2 37 BRAF (V600E) + NRAS (WT) BRAF (N581Y) + NRAS (WT) BRAF (V600D) + NRAS (WT)

WM-4205 WM-3936 WM-4231 Time (h) 0 0.5 2 8 24 48 0 0.5 2 8 24 48 0 0.5 2 8 24 48 BRAF + MEKi

P-ERK1/2 37

ERK1/2 37 BRAF (V600_K601delinsE) + NRAS (WT) BRAF (V600E) + NRAS (Q61K) BRAF (V624F) + NRAS (Q61R)

WM-3629 WM-3670 WM-4262 Time (h) 0 0.5 2 8 24 48 0 0.5 2 8 24 48 0 0.5 2 8 24 48 BRAF + MEKi

P-ERK1/2 37

ERK1/2 37 BRAF (D549G het) + NRAS (G12D hom) BRAF (G469E) + NRAS (G12D) BRAF (V600E) + NRAS (G13R)

WM-1963 WM-3960 Time (h) 0 0.5 2 8 24 48 0 0.5 2 8 24 48 BRAF + MEKi P-ERK1/2 37

ERK1/2 37 BRAF (N581S) + NRAS (A146T) BRAF (V600E) + NRAS (Q61K)

b c

BRAFi - + - + Sensitive to B+Mi Resistant to B+Mi MEKi - - + +

BRAF (V600E) A375 + NRAS (WT)

BRAF (D549G het) WM-3629 + NRAS (G12D hom)

BRAF (V600E) WM-3936 + NRAS (Q61K)

BRAF (V624F) WM-4231 + NRAS (Q61R)

Supplementary Figure S1. Combined BRAF and MEK inhibition mediates ERK reactivation associated resistance in BRAF mutant and BRAF/NRAS mutant melanoma cells. A, Immunoblots of indicated cell lines treated with PLX4720 (3µM) + trametinib (100nM) show BRAFi + MEKi-induced ERK reactivation in mutant melanoma cells. B, Cells were treated with BRAFi + MEKi for 48h, and MTT assay was performed. Graph shows BRAFi (1nM-10µM) + MEKi (10nM) treatment IC50 (µM) on indicated cell lines. (B+Mi: BRAFi + MEKi). Error bar indicates ± s.d. c, Cells were treated with BRAFi (1µM) + MEKi (10nM) and colony assay was performed. Micrographs demonstrate the effect of BRAFi + MEKi on clonogenic survival. Supplementary Fig. S2 A WM-4231 (BRAF/NRAS) (0.5h) (PLA+PDI) (PLA+PDI)

PLA PDI-ER Merge PLA PDI-ER Merge

NRAS 1.5 1.5 ** -3 ** P = 1.1 x 10 -3 1.0 1.0 P = 1.4 x 10

CI 0.5 CI 0.5 0 0 - + - + BRAFi + MEKi BRAF GRP78-NRAS GRP78-BRAF GRP78-PLA 1.5 1.5 ** MEK ** -3 -3 1.0 P = 1.5 x 10 1.0 P = 1.7 x 10 CI CI 0.5 0.5 0 0 - + - + BRAFi + MEKi ERK GRP78-MEK GRP78-ERK

Untreated BRAFi + MEKi C B WM-3918 (BRAF/NRAS-WT) (0.5h) WM-3918 (BRAF/NRAS-WT) (0.5h) (PLA+PDI) (PLA+PDI) PLA PDI-ER Merge PLA PDI-ER Merge

A375(BRAF-V600E) WM-3282(BRAF-V600K HOM)WM-4231(BRAF-V624F/NRAS-Q61R)WM-3912(BRAF-N581Y) WM-3936(BRAF-V600E/NRAS-Q61K)WM-3629(BRAF-D549G/NRAS-G12D)WM-3918(BRAF/NRASWT)WM-1366(BRAF WT/NRAS-Q61L)WM-4262(BRAF-V600E/NRAS-G13R) BRAFi + MEKi - + - + - + - + - + - + - + - + - + NRAS 25 NRAS

BRAF 75 BRAF

50 MEK GRP78-PLA ERK

37 MEK

75 GRP78

50 Calreticulin ERK

50 Tubulin Untreated BRAFi + MEKi ER fraction (0.5h) D E 0.5h ER Cyto ER Cyto BRAFi + MEKi - + - + BRAFi - + - + - + - + MEKi - - + + - - + + 75 CRAF 25 NRAS

75 ARAF 75 BRAF

50 Calreticulin 37 ERK1/2

50 MEK1/2 50 Tubulin

50 A375 Calreticulin

F 50 Tubulin WM-1366 (NRAS-Q61L/BRAF WT) (0.5h) A375 MEKi - + - + NRAS MEK GRP78-PLA GRP78-PLA ERK BRAF

Supplementary Figure S2. Combined BRAF and MEK inhibition induces ER translocation of MPKs. A, WM-4231 cells were treated with BRAFi + MEKi and processed for PLA (GRP78 and MPKs). Micrographs show BRAFi + MEKi-induced GRP78 and MPKs interaction (red) and colocalization (yellow; arrowhead) with ER (PDI; green) in BRAF/NRAS mutant cells. Representative immunofluorescence images from n=3 fields of view per condition. Scale bar, 10µm. Graph: quantification of colocalization index (CI) from three independent experiments in biological replicates. B, Indicated mutant melanoma cell lines were treated with BRAFi + MEKi for 0.5h, and ER fractionations were performed. Immunoblots show ER translocation of MPKs. C, WM-3918 (BRAF/NRAS WT) cells were treated with BRAFi + MEKi, and processed for PLA (GRP78 and MPKs). Micrographs show BRAFi + MEKi did not induce GRP78-MPKs interaction (red) and colocalization (yellow) with ER (PDI; green) in WT cells. Representative immunofluorescence images from n=3 fields of view per condition. Scale bar, 10µm. D, Cells were treated with BRAFi + MEKi for 0.5h and ER/cytosolic fractions were prepared. Immunoblots show BRAFi + MEKi inhibitor did not induce ARAF and CRAF translocation to the ER. E, A375 cells were treated with BRAFi and MEKi either alone or in combination for 0.5h and subcellular fractions were performed. Immunoblots show BRAF and MEK inhibitor induces ER translocation of MAPK. F, WM-1366 cells (NRASQ61A/BRAFWT) were treated with MEKi for 0.5h, and processed for PLA (GRP78 and MPKs). Micrographs show MEKi induces GRP78-MPKs interaction (red) and colocalization (yellow) with ER (PDI; green) in NRAS mutant cells. Representative immunofluorescence images from n=3 fields of view per condition. Scale bar, 10µm. Supplementary Fig. S3

A B C

A375 (0.5h) WM-4231 (0.5h) Cyto shNT + + -- + + -- GRP78 WT GRP78 KDELΔ shNT + + -- + + -- shGRP78 (#2) + +-- + +-- shGRP78 (#1) + +-- + +-- BRAFi + MEKi - + - + - + - + 25 NRAS BRAFi + MEKi - + - + - + - + 25 25 NRAS NRAS BRAF 75 BRAF BRAF 75 MEK 75 50 50 MEK 50 MEK 37 ERK ERK ERK 37 Tubulin 37 50 Calreticulin 50 Calreticulin ATP6V1A 50 50 ATP6V1A 50 50 ATP6V1A 75 GRP78 75 GRP78 Tubulin 75 50 Calreticulin 50 Tubulin ER Cyto 0 0.5 2 4 0 0.5 2 4 Time (h) ER Cyto BRAFi + MEKi (A375)

D IP-GRP78 INPUT GRP78 WT GRP78 KDELΔ GRP78 WT GRP78 KDELΔ ER Cyto ER Cyto ER Cyto ER Cyto 25 25 IB: N-ras IB: N-ras

IB: B-raf IB: B-raf 75 75

IB: ERK IB: ERK 37 37

IB: GRP78 IB: GRP78 75 75 Time (h) 0 0.5 2 4 0 0.5 2 4 0 0.5 2 4 0 0.5 2 4 Time (h) 0 0.5 2 4 0 0.5 2 4 0 0.5 2 4 0 0.5 2 4 BRAFi + MEKi BRAFi + MEKi

E

BRAFi + MEKi (A375) BRAFi + MEKi (WM-4231)

Time (h) 0 0.5 8 24 0 0.5 8 24 0 0.5 8 24 0 0.5 8 24 JNK 50

50 Calreticulin

50 Tubulin

Cytosolic ER Cytosolic ER

Supplementary Figure S3. . GRP78 mediates ER translocation of MPKs. A, GRP78 (construct #1) -silenced WM-4231 cells were treated with BRAFi + MEKi, and ER/cytosolic fractions were prepared. Immunoblots demonstrate GRP78 knockdown disrupts the BRAFi + MEKi-induced MPKs translocation to ER. B, GRP78 (construct #2)-silenced A375 cells were treated with BRAFi + MEKi, and ER/cytosolic fractions were prepared. Immunoblots demonstrate GRP78 knockdown disrupts the BRAFi + MEKi-induced MPKs translocation to ER. C, A375-GRP78-KDELΔ mutant cells were treated with BRAFi + MEKi, and ER/cytosolic fractions were performed. Immunoblots of the cytoplasmic fraction shown here demonstrates cytoplasmic retention of MPKs in GRP78-KDELΔ mutant cells D, A375-GRP78-KDELΔ mutant cells were treated with BRAFi + MEKi, and ER/cytosolic fractions were performed for immunoprecipitation with GRP78. Immunoblots show GRP78-KDELΔ mutant cells did not show translocation of GRP78-MPKs complex into the ER. Input controls are shown in right. E, A375 and WM-4231 cells were treated with BRAFi + MEKi, and ER/cytosolic fractions were prepared. Immunoblots show BRAFi + MEKi treatment did not induce JNK (Jun N- terminal ) translocation to the ER. Supplementary Fig. S4 INPUT A B IP: GRP78 BRAFi + MEKi BRAFi + MEKi KSR1-IP INPUT Time (h) 0 0.5 2 4 8 24 BRAFi + MEKi (A375) Time (h) 0 0.5 2 4 8 24 IB:KSR2 (A375) 0.5 h 24 h 0.5 h 24 h IB:KSR2 (A375) 75 - + - + 75 - + - + IB:KSR2 (WM-4231) IB: GRP78 IB:KSR2 (WM-4231) 75 75 75 IB:KSR2 (WM-3918) 75 100 IB: KSR1 IB:KSR2 (WM-3918) 75

75 IB:GRP78 (A375) C GRP78-IP INPUT 75 IB:GRP78 (WM-4231) (0.5h) (0.5h) A375 i-shKSR2 # 2 A375 i-shKSR2 # 2 75 IB:GRP78 (WM-3918) DOX - - + + DOX - - + + BRAFi + MEKi - + - + BRAFi + MEKi - + - + 25 ER (0.5h) 25 IB: NRAS D IB: NRAS A375 i-shKSR2 # 2 IB: BRAF DOX - - + + IB: BRAF BRAFi + MEKi - + - + 75 75 25 IB: MEK NRAS 50 IB: MEK 50 BRAF IB: ERK IB: ERK 75 37 37 50 IB: GRP78 IB: GRP78 MEK 75 75

KSR2 37 ERK 75 β-actin 50 Calreticulin 37 ATP6V1A 50

50 Tubulin E (WM-4231) F Control si RNA + + - - (A375) BRAFi + MEKi - A375 (0.5 h) KSR2 si RNA - - + + Control si RNA + + - - - + BRAFi + MEKi - + - + KSR2 si RNA - - + + BRAFi + MEKi - + - + KSR2 75 KSR2 β-actin 75 37 β-actin

(24h) 37 Control siRNA (24h) KSR2 siRNA

G BRAFi + MEKi (0.5h) GRP78-IP IF: GRP78 (green) and ERK (red) INPUT BRAFi + MEKi - + - + - + 150 - + BRAFi + MEKi (0.5h) 100 GRP78/ 100 25 150 NRAS GRP78/ BRAF GRP78 75 75 NRAS BRAF 50 85 100 BRAF GRP78 37 75 50 MEK 25 NRAS 50 37 50 MEK 20 37 25 anti-NRAS ab anti-BRAF ab anti-MEK ab ERK 37 100 (0.5h) GRP78-IP KSR2 BRAFi + MEKi - + - + - + 100 GRP78 100 100 75 BRAF/KSR2 BRAF/KSR2 GRP78 GRP78- 75 75 75 ERK GRP78 50 MEK 50 MEK 50 ERK 37 ERK ERK 37 37 25 25 NRAS 25 20 20 anti-ERK ab anti-KSR2 ab anti-GRP78 ab Supplementary Figure S4. KSR2 mediates GRP78 mediated ER translocation of MPKs. A, Cells were treated with BRAFi + MEKi (0.5h), and cell lysates were prepared for KSR1 immunoprecipitation (IP). BRAFi + MEKi does not induced an interaction between KSR1 and GRP78. B, Cells were treated with BRAFi + MEKi, and cell lysates were prepared for GRP78-IP. IB shows BRAFi + MEKi -induced KSR2-GRP78 interaction in BRAF (A375) and BRAF/NRAS (WM-4231) mutant melanoma cells but not in WT (WM-3918) melanoma cells. Input controls are shown in left panel. C, A375 i-shKSR2 (construct #2) cells were treated with BRAFi + MEKi. Whole cell lysates were prepared for GRP78-IP in the absence/presence of dox (2µg for 48h). Immunoblots show BRAFi + MEKi -induced GRP78-MPKs interaction was abrogated in the absence of KSR2. Input controls are shown in right. D, A375 i-shKSR2 (construct #2) cells were treated with BRAFi + MEKi. Cells were harvested for ER and cytosolic fractionations. Immunoblots show BRAFi + MEKi-induced ER translocation of MPKs was inhibited in KSR2 silenced cells. E, (Efficacy of KSR2 knockdown in Fig. 2e). WM4231 were transfected with pooled siRNA against scrambled (control) or KSR2, immunoblotting was performed. F, A375 cells were transfected with pooled siRNA against scrambled (control) or KSR2, immunoblotting was performed (left panel). KSR2-silenced cells were treated with BRAFi + MEKi, and processed for immunofluorescence (IF) with GRP78 (green) and ERK (red). Micrographs show KSR2 inhibits BRAFi + MEKi-induced GRP78-ERK interaction (yellow). Representative immunofluorescence images from n=3 fields of view per condition. Scale bar, 10µM. G, A375 cells were treated with BRAFi + MEKi, and cell lysates were prepared for GRP78-IP. IP Samples were prepared for native page and IB shows BRAFi + MEKi treatment induce complex formation/interaction of GRP78-KSR2-MPKs. Arrow head indicates GRP78 complex with MPKs and KSR2. Input controls are shown in right. Supplementary Fig. S5

A B C KSR2-IP IP:KSR2 BRAFi + MEKi (A375) Cyto PMF BRAFi + MEKi (A375) 0.5 h 24 h Time (h) 0 0.5 2 4 8 24 - + - + 25 25 IB: RAB5 IB: RAB7

A375 WM-4231 A375 WM-4231 IB: KSR2 - + - + - + - + BRAFi + MEKi IB: KSR2 75 - 75 KSR2 INPUT 75 INPUT BRAFi + MEKi (A375) eIF2α BRAFi + MEKi (A375) 37 0.5 h 24 h Time (h) 0 0.5 2 4 8 24 - + - + ATP6V1A 50 IB: KSR2 25 IB: RAB7 0.5 h 75

25 IB: RAB5 IB: KSR2 75

D (A375) E BRAFi + MEKi RAB5 -IP INPUT Time (h) 0 0.5 2 8 24 48 (0.5 h) (0.5 h) 25 DOX - - + + RAB5 DOX - - + + BRAFi + MEKi - + - + BRAFi + MEKi - + - + 25 25 25 IB: NRAS IB: NRAS RAB7 IB:BRAF IB:BRAF 75 β-actin 75 37 IB: MEK 50 IB: MEK 50 (WM-4231) BRAFi + MEKi IB: ERK IB: ERK 37 37 Time (h) 0 0.5 2 8 24 48 75 IB: GRP78 IB: GRP78 25 RAB5 75

25 IB: RAB5 25 IB: RAB5 25 RAB7 A375 i-shKSR2 # 1 A375 i-shKSR2 # 1

β-actin 37 (I-IP) (II-IP) (III-IP) F BRAFi + MEKi BRAFi + MEKi BRAFi + MEKi GRP78-IP Beads only KSR2-IP Beads only ERK-IP Beads only GRP78-IP KSR2-IP ERK-IP

Time (h) 0 0.25 0.5 0 0.25 0.5 0 0.25 0.5 0 0.25 0.5 (I-IP) (II-IP) (III-IP) 0 0.25 0.5 0 0.25 0.5 ERK ERK ERK 37 37 37 GRP78 KSR2 ERK 75 75 37

25 RAB5 25 RAB5 25 RAB5

Post I-IP Post II-IP INPUT INPUT INPUT supernatatnt supernatatnt 75 GRP78 KSR2 ERK 75 37

ERK ERK 75 GRP78 37 37 25 RAB5 25 RAB5 25 RAB5

Supplementary Figure S5.. KSR2 localizes on RAB5+ early endosomes and promotes ER translocation of GRP78-MPKs complex. A, Cells were treated with BRAFi + MEKi for 0.5h, and subcellular fractionations were performed. Immunoblots show BRAFi + MEKi-induced KSR2 expression in cytosol. T-eIF2α and ATP6V1A were used as cytosol (cyto) and plasma membrane fraction (PMF) markers respectively. B, A375 cells were treated with BRAFi + MEKi, and whole cell lysates were prepared for KSR2 immunoprecipitation (IP). Immunoblots (IB) show BRAFi + MEKi treatment induces KSR2 and RAB5 interaction. Input controls are shown in the bottom panel. C, A375 cells were treated with BRAFi + MEKi, and whole cell lysates were prepared for KSR2-IP. IB demonstrates BRAFi + MEKi-treatment did not induce KSR2 and RAB7 interaction in A375 cells treated for 0.5h and 24h. D, Cells were treated with BRAFi + MEKi and whole cell lysates were prepared. Immunoblots show BRAFi + MEKi did not induce RAB5 and RAB7 expression. E, A375 i-shKSR2 cells were treated with BRAFi + MEKi for 0.5h, and whole cell lysates were prepared for RAB5-IP. IB shows KSR2 knockdown inhibits the interaction of RAB5 and MPKs-GRP78 complex. Input controls are shown in right. F, A375 cells were treated with BRAFi + MEKi in a time dependent manner and lysates were prepared for three sequential IPs. For first IP, GRP78 was immunoprecipitated, and then of KSR2 in the post-IP lysates depleted of GRP78. A third ERK2 IP was subsequently carried out on the resulting post-KSR2 IP lysates. Supplementary Fig. S6 A WM-4231 (24h) B BRAFi + MEKi (WM-4231) Control si RNA + + - - 00.5 4 8 24 00.5 4 8 24 00.5 4 8 24 Time (h) SEC61 si RNA - - + + 25 0h NRAS BRAFi + MEKi - + - + 0.5h 3 4h 8h 37 SEC61 BRAF 75 2 24h

50 Fold change

MEK ERK expression 1 Actin 0 37 ERK 37 ER Cytosol Nucleus C A375 (8h) NT gRNA + + - - 50 Calreticulin SEC61 gRNA - - + + 100 BRAFi + MEKi - + - + HSP90

ERK2 HDAC 37 50 ER Cytosolic Nucleus HDAC 50

100 HSP90

Nuclear fraction F BRAFi + MEKi (WM-4231) D E BRAFi + MEKi 4 A375P 0 0.5 2 8 24 48 0 0.5 2 8 24 48 NT-gRNA-untreated 0 0.5 2 4 8 24 Time (h) NT-gRNA-BRAFi+MEKi P-ERK1/2 3 37 SEC61-KO-untreated 50 P-MEK 1/2 SEC61-KO-BRAFi+MEKi ERK1/2 37 2 SEC61-RES-BRAFi+MEKi 50 MEK 1/2 β-actin Fold change Fold 1 37 PERK-WT PERK-K618A β-actin 4 0 37 PERK-WT ** 3 PERK-K618A ELK1 DUSP6 SPRY2 2 -3 P = 6.8 X 10 H 1

G ERK/actin expression

0

- A375- WM-4231 BRAFi + MEKi 0h 2h 8h BRAFi + MEKi 0.5h 24h 48h Time (h) 0 0.5 2 4 8 24 0 0.5 2 4 8 24 - + + - + + PERKi PERKi + + + + + + ------+ + - - ++ ERK2 P-ERK1/2 37 37 HDAC 50 ERK1/2 100 37 HSP90 Nuclear fraction (8h) K I BRAFi + MEKi (A375) J BRAFi + MEKi PERK-WT PERK ΔC Time (h) 0 0.5 2 8 24 48 0 0.5 2 8 24 48 0 0.5 2 4 8 24 Time (h) 150 37 P-eIF2α P-IRE1α CYTO ER CYTO ER 100 25 eIF2α NRAS 37 β-actin PERK WT 37 P-ERK1/2 BRAF 75 37 150 P-IRE1α ERK1/2 50 MEK 37 100

β-actin C Δ PERK β-actin ERK 37 37 37 PERK-WT PERK ΔC ATP6V0A1 L M 50 A375 WM-4231 Control-siRNA + + - - PERK WT + + - - + + - - MEK-siRNA - - + + 50 Calreticulin PERK ΔC - - + + - - + + BRAFi+ MEKi - + - + BRAFi + MEKi - + - + - + - + 37 SEC61 37 P-ERK1/2 ERK2 37 ERK1/2 PERK 37 HDAC 100 50 50 MEK1/2 Actin 100 HSP90 37 β-actin Nuclear fraction (8h) 37 Supplementary Figure S6. BRAFi + MEKi induces ERK translocation from ER to cytosol to nucleus. A, Efficacy of SEC61 knockdown in Fig. 4d. WM4231 were transfected with pooled siRNA against scrambled (control) or SEC61, immunoblotting was performed. B, Cellular fractionations were performed in WM-4231 cells treated with BRAFi + MEKi. Immunoblots demonstrate ERK translocation from cytoplasm to ER to cytoplasm to nucleus during treatment. Calreticulin, HSP90, and HDAC were used as ER, cytosolic and nuclear markers respectively. Graph: quantification of fold change of ERK expression in ER, cytosol, and the nucleus from three independent experiments with biological replicates. C, NTgRNA and SEC61gRNA transfected cells were treated with BRAFi + MEKi, and nuclear fractions were prepared. Immunoblots show BRAFi + MEKi-induced nuclear ERK2 expression was blocked in SEC61 KO cells. D. NTgRNA, SEC61gRNA and SEC61 overexpressed cells were treated with BRAFi + MEKi for 8h, and RNA was isolated. Synthesized cDNA were analyzed for ERK downstream target by qPCR using sets of primers. Bar graph shows inhibited expression of DUSP6, SPRY2 and ELK1 expression in SEC61KO cells during BRAFi+MEKi inhibition.E, A375 cells were treated with BRAFi + MEKi and whole cell lysates were prepared. Immunoblots show dephosphorylation of MEK during drug treatment. F, PERK-WT and PERKK618A (cytoplasmic kinase domain dead) cells were treated with BRAFi + MEKi and whole cell lysates were prepared. Immunoblots show PERK cytoplasmic kinase domain activity regulates ERK reactivation during treatment. Graph: Quantification of fold change in ERK expression. Data shown as mean ± s.d. (n=3). G, Cells were pre-treated with PERK inhibitor (GSK2606414; 1µM) for 1h. Thereafter, BRAFi + MEKi were added for different time points and whole cell lysates were prepared. Immunoblots show PERK inhibitor significantly blunted BRAFi + MEKi induced ERK reactivation. H, Cells were pre-treated with PERK inhibitor for 1h. Thereafter, BRAFi + MEKi were added for 8h and nuclear fractions were prepared. Immunoblots display PERK inhibition decreased nuclear ERK2 expression. I, PERK-WT and PERKΔC cells were treated with BRAFi + MEKi, and whole cell lysates were prepared. Inhibition of p-eIF2α expression demonstrates the transfection efficacy of PERK inhibition. Immunoblots show the PERK c-terminal mutation had no effect on ERK reactivation. J, PERK-WT and PERKΔC cells were treated with BRAFi + MEKi, and whole cell lysates were prepared. Immunoblots show increased levels of IRE1α in PERKΔC cells. K, PERK-WT and PERK∆C expressed cells were processed for ER/cytosolic fractionation. Immunoblots demonstrate PERK constitutive activation did not induce MPKs translocation inside the ER. L, PERK-WT and PERKΔC cells were treated with BRAFi + MEKi for 8h, and nuclear fractionations were prepared. Immunoblots show PERKΔC cells did not affect BRAFi + MEKi induced ERK2 nuclear expression. M, A375 cells were transfected with pool of siControl or siMEK and treated with BRAFi + MEKi. Whole cell lysates were prepared at 8h. Immunoblots show MEK independent ERK phosphorylation during drug treatment. Supplementary Fig. S7

A B C ATF4-IP A375 (8h) A375 WM-4231 A375 WM-4231 ERK2 ATF4 Merge BRAFi + MEKi - + + + - - - + + + - - ERK WT BRAFi + MEKi - + - + - - + - + - - - + - + - ERK2-KD - - +- +- - - +- +- IB: ERK2 -+ 37 50 p-ATF4

INPUT ERK- WT 50 ATF4

50 IB: ATF4 100 HSP90 IB: ERK2 37 - HDAC Nuclear Co-IP (8h) BRAF + MEKi (8h) 50 Nuclear fraction (8h) ERK2-KD +

Nuclear colocalization 0.8 *** Control 0.6 BRAFi + MEKi

0.4 -4 0.2 P = 1.8 X 10 Nuclear CI 0 BRAFi + MEKi +- +- ERK2 WT + + - - ERK2-KD -- + + D A375 WM-4231 ATF4-WT-Control 3 3 ATF4-WT-Control -2 -3 -2 -3 -3 -2 ATF4-WT-BRAFi + MEKi ATF4-WT-BRAFi + MEKi 2 ATF4-S245A-Control 2 ATF4-S245A-Control ATF4-S245A-BRAFi + MEKi P = 2.1 X 10 P = 1.9 X 10 ATF4-S245A-BRAFi + MEKi P = 1.7 X 10 P = 6.8 X 10 1 P = 1.8 X 10 * P = 1.91 X 10 ** * * 1 ** * Relative expression Relative expression

0 0 ASNS CHOP ATF3 ASNS CHOP ATF3

E

MTHFD2 ASNS PSAT1 PHGDH 1.0 1.0 1.0 1.0 ** ** 0.8 0.8 0.8 0.8 * * 0.6 0.6 -3 0.6 0.6 P = 4.2 X 10 0.4 -3 0.4 0.4 -2 0.4 -2 ChIP % Input P = 1.4 X 10 P = 1.6 X 10 % Input P = 1.2 X 10 % Input 0.2 % Input 0.2 0.2 0.2 0.0 0.0 0.0 0.0 ATF4-WT + + + + - - - - ATF4-WT + + + + - - - - ATF4-WT + + + + - - - - ATF4-WT + + + + - - - - ATF4-S245A - - - - + + + + ATF4-S245A - - - - + + + + ATF4-S245A - - - - + + + + ATF4-S245A - - - - + + + + BRAFi + MEKi - + - + - + - + BRAFi + MEKi - + - + - + - + BRAFi + MEKi - + - + - + - + BRAFi + MEKi - + - + - + - +

IgG-IP IgG-IP IgG-IP IgG-IP IgG-IP IgG-IP IgG-IP IgG-IP ATF4-IP ATF4-IP ATF4-IP ATF4-IP ATF4-IP ATF4-IP ATF4-IP ATF4-IP

Supplementary Data Figure S7. ERK2 interacts with ATF4 and induce its phosphorylation to promote ATF4 function. A, A375 and WM-4231 cells were treated with BRAFi + MEKi for 8h, and nuclear extracts were prepared -4231 cells were treated for ATF4 immunoprecipitation (IP). Immunoblots show BRAFi + MEKi ATF4 induces ATF4 and ERK2 interaction in the nucleus. B, ERK2-WT-GFPERK an2 d ERK2-KD-GFP (green) A375 cells were treated with-GFP BRAFi (green) + A375 MEKi,cells an d processed for ATF4 (red) immunostaining. Micrographs showimmuno drugstaining. induce d significant ERK2 and ATF4 nuclear colocalization (yellow). Nuclear colocalization index (CI) was calculated as mean ±d s.d. as mean (n=3). ± s.d.C, ERK2-W(n=3). c, T and ERK2-KD cells were treated with BRAFi + MEKi for 8h,, and and nuclear nuclea r -induced ERK2 reactivation extracts were prepared. Immunoblots show BRAFi S245A + MEKi-induced ERK2 expressing A375 and reactivation is required for phosphorylation of ATF4. D, ATF4- S245A , and RNA was isolated for WT/ATF4 expressing A375 and WM-4231 cells-induced were ATF4 treated targeted wit h BRAFi + MEKi for 4h, and RNA was isolated fore ,cDNAATF4- WTsynthesis. and ATF4 GraphsS245A : relative expression of BRAFi + MEKi-induced .C ATF4hromatin targeted was isolated s measured by qPCR. E, ATF4-WT and ATF4S245A expressing A375 cells were treated with BRAFi + MEKi for 4h. Chromatin was isolated the an d immunoprecipitated for IgG and ATF4. Purified DNA Ki fragments-induced ATF4 wer e (b, d, e) analyzed for ATF4 binding partners by qPCR using setsssed of primers. using two Graph:-tailed t- % input shows that the mutation in the phosphorylation site (S245A) decreases BRAFi + MEKi-induced ATF4 binding with promoters of target genes MTHF2, ASNS, PSAT1, and PHGDH. Data shown as mean ± s.d. *P<0.05; n=3. Supplementary Fig. S8

A A375 D G shControl + - - A375 A375 GRP78 WT - - + KSR2 WT - + SEC61 WT - + shGRP78 + + + shKSR2 + + SEC61 gRNA + + - - - BRAFi + MEKi BRAFi + MEKi BRAFi + MEKi + + +

150 -3 P = 1.4 x 10 150 -3 150 -3 ** P = 1.3 x 10 P = 2.1 x 10 ** ** 100 100 100 50 50

% Colony formation 50 % Colony formation % Colony formation 0 sh Control+ + - - -- 0 0 SEC 61 gRNA + + + + sh GRP78 -- + + + + sh KSR2 + + + + BRAFi + MEKi - + - + BRAFi + MEKi- + - + - + BRAFi + MEKi- + - + KSR2 WT - - + + SEC61 WT - - + + GRP78 WT -- - - + +

B E H

-2 P = 2.2 x 10 P = 3.2 x 10 * 1.0 -2 1.0 1.0 P = 4.7 x 10 * 0.8 0.8 0.8 * -2 0.6 0.6 0.6 O.D.

O.D. 0.4 O.D. 0.4 0.4 0.2 0.2 0.2 0.0 0.0 0.0 Control siRNA + + - - Control siRNA + + - - Control siRNA + + - - SEC61 siRNA - - + + GRP78 siRNA - - + + KSR2 siRNA - - + + BRAFi + MEKi - + - + BRAFi + MEKi - + - + BRAFi + MEKi - + - +

C F I A375 (24h) A375 (24h) Control siRNA + + - - A375 (24h) Control siRNA + + - - GRP78 siRNA - - + + Control siRNA + + - - SEC61 siRNA - - + + BRAFi + MEKi - + - + KSR2 siRNA - - + + BRAFi + MEKi - + - + BRAFi + MEKi - + - + 50 50 50 Clv-Caspase 3 Clv-Caspase 3 Clv-Caspase 3

100 100 Clv-PARP Clv-PARP 100 Clv-PARP 50 SEC61 GRP78 KSR2 75 75 Actin Actin Actin 37 37 37

Supplementary Data Figure S8. ER translocation of MPKs promotes resistance to BRAFi + MEKi. A. GRP78-silenced cells were transfected with GRP78-WT plasmid and colony formation assay was performed. Micrographs and bar graph (below panel) show GRP78-WT over expression rescue the cell survival in GRP78 silenced cells. B, GRP78-silenced cells were treated with BRAFi + MEKi and MTT assay was performed. Bar graph demonstrates GRP78 inhibition significantly reduced the viability of cells during drug treatment. C, GRP78-silenced cells were treated with BRAFi + MEKi and whole cell lysates were prepared. Immunoblots show GRP78 inhibition induces apoptotic expression in the presence of drugs. D, KSR2-silenced cells were transfected with KSR2-WT plasmid and colony formation assay was performed. Micrographs and bar graph (below panel) show KSR2-WT over expression rescue the cell survival in KSR2 silenced cells. E, KSR2-silenced cells were treated with BRAFi + MEKi and MTT assay was performed. Bar graph demonstrates KSR2 inhibition significantly reduced the viability of cells. F, KSR2-silenced cells were treated with BRAFi + MEKi and whole cell lysates were prepared. Immunoblots show KSR2 inhibition induces apoptotic proteins expression in the presence of drugs. G, SEC61KO cells were transfected with SEC61-WT plasmid and colony formation assay was performed. Micrographs and bar graph (below panel) show SEC61-WT over expression rescue the cell viability in SEC61KO cells. H, SEC61-silenced cells were treated with BRAFi + MEKi and MTT assay was performed. Bar graph demonstrates SEC61 inhibition significantly reduced the viability of cells. I, SEC61-silenced cells were treated with BRAFi + MEKi and whole cell lysates were prepared. Immunoblots show SEC61 inhibition induces apoptotic proteins expression in the presence of drugs. Data shown as mean ± s.d.*P<0.05. Supplementary Fig. S9 A B

A375 A375 (24h) PERKi - + BRAFi + MEKi - + - + PERKi - - + + 50 Clv-Caspase 3 -

100 Clv-PARP

150 BRAFi + MEKi PERK + Actin 37

-3 150 P = 3.7 x 10 ** 100

50 % Colony formation 0 BRAFi + MEKi - + - + PERKi - - + + C D

A375 WM-4231 ATF4-WT ATF4-S245A ATF4-WT + + - - + + - - BRAFi + MEKi - + - + ATF4-S245A - - + + - - + + BRAFi + MEKi - + - + - + - + 100 cl-PARP WM-3936

50 cl-Cas3

37 Actin WM-3629 (24h) n.s. n.s. 4 -1 -1 P = 3.7 X 10 P = 2.3 X 10 3 ATF4-WT ATF4-WT+ BRAFi + MEKi 2 ATF4-S245A 1 ATF4-S245A+ BRAFi + MEKi Relative expression 0 WM-4205 cl-PARP cl-Cas3 A375

3 *** -4 P = 5.6 X 10 *** -4 P = 2.4 X 10 ATF4-WT 2 ATF4-WT+ BRAFi + MEKi WM-3960 1 ATF4-S245A

Relative expression ATF4-S245A+ BRAFi + MEKi 0 cl-PARP cl-Cas3 WM-4231 E Dab + Tra Dab + Tra GRP78-IP INPUT GRP78-IP INPUT Time (h) 0 2 4 8 24 48 0 2 4 8 24 48 Time (h) 0 0.5 0 0.5 0 0.5 0 0.5 50 P-ATF4

KSR2 75 50 ATF4

75 GRP78 β-actin 37 WM-293A WM-3912 WM-293A WM-3912 BRAF (V600E) + NRAS (WT) BRAF (N581Yt) + NRAS (WT) BRAF (V600E) + NRAS (WT) BRAF (N581Y) + NRAS (WT) F Dab + Tra Dab + Tra GRP78-IP INPUT GRP78-IP INPUT Time (h) 0 2 4 8 24 48 0 2 4 8 24 48 Time (h) 0 0.5 0 0.5 0 0.5 0 0.5 50 P-ATF4 KSR2 75 50 ATF4 GRP78 75 β-actin WM-3936 WM-3629 37 BRAF (V600E) + NRAS (Q61K) BRAF (D 549 G het) + NRAS (G12D hom) WM-3936 WM-3629 BRAF (V600E) + NRAS (Q61K) BRAF (D 549 G het) + NRAS (G12D hom)

Supplementary Data Figure S9. ATF4 phosphorylation promotes resistance to BRAFi + MEKi. A, A375 cells were treated with BRAFi + MEKi in presence or absence of PERK inhibitor (PERKi), and colony formation assay was performed. Micrographs show a significant reduction in colony formation in the presence of PERKi during drug treatment. B, A375 cells were treated with BRAFi + MEKi in presence or absence of PERKi for 24h, and whole cell lysates were prepared. Immunoblots showed significant cleavage of PARP and caspase-3 in the presence of PERKi during drug treatment. C, ATF4-WT and ATF4S245A cells were treated with BRAFi + MEKi for 24h, and whole cell lysates were prepared. Immunoblots showed significant cleavage of PARP and caspase-3 in ATF4S245A cells during drug treatment. Graph: quantifications of PARP and caspase-3 expression. D, ATF4S245A and ATF4-WT cells were treated with BRAFi + MEKi, and colony formation assay was performed. Micrographs show a significant reduction in colony formation in ATF4S245A cells as compared to ATF4-WT cells during BRAFi + MEKi treatment. E, Sensitive cells were treated with Dab (1µM) + Tra (100nM) for 0.5h and processed for GRP78-IP. Immunoblots show (left panel) drug induced an interaction between GRP78 and KSR2 in melanoma cells. Right panel - cells were treated with Dab (1µM) + Tra (100nM) in a time dependent manner and whole cell lysates were prepared. Immunoblots show drug induced phosphorylation of ATF4. F, Resistant cells were treated with Dab (1µM) + Tra (100nM) for 0.5h and processed for GRP78-IP. Immunoblots show (left panel) drug induced an interaction between GRP78 and KSR2 in melanoma cells. Right panel- cells were treated with Dab (1µM) + Tra (100nM) in a time dependent manner and whole cell lysates were prepared. Immunoblots show drug induced phosphorylation of ATF4. Data shown as mean ± s.d. *P<0.05; n=3. Supplementary Fig. S10 A B PDX Tumor tissue(22 days) Nu Cyto Control + + + - - - + + + - - - Pre-treatment Post-treatment BRAFi + MEKi - - - + + + - - - + + + 50 P-ATF4

50 ATF4 100 HSP90

HDAC 50 1 2 3 1 2 3 1 2 3 1 2 3

C D WCL GRP78-IP INPUT Control chow + + - - Control chow + - Control chow + - BRAFi + MEKi chow - - + + BRAFi + MEKi chow - + BRAFi + MEKi chow - + GRP78 25 25 75 NRAS NRAS 100 KSR2 BRAF BRAF 75 75 P-ERK1/2 37 50 MEK 50 MEK

ERK1/2 37 ERK ERK 20 37 37 LC3I 100 15 LC3II 100 KSR2 KSR2 75 P62 75 GRP78 75 GRP78 β-actin 37 A375 -xenograft Tumor tissue C1 C2 T1 T2 A375 -xenograft Tumor tissue E F

Nuclear fraction ER Cyto Control chow + + - - BRAFi + MEKi chow - + - + BRAFi + MEKi chow - - + + 25 NRAS ERK2 37

BRAF 50 75 P-ATF4

50 MEK 50 ATF4

ERK HDAC 37 50 100 HSP90 50 Calreticulin C1 C2 T1 T2

50 Tubulin A375 -xenograft Tumor tissue A375 -xenograft Tumor tissue

Supplementary Figure S10. Combined BRAF and MEK inhibitor showed resistance in vivo. A, CT scan performed at pre and post- treatment on a patient with a BRAFV600E amplified melanoma who failed to respond to dabrafenib, trametinib, and hydroxychloroquine. Scale bar, 85mm. Blue (tumor outlines). B, Nuclear and cytosolic fractions of PDX-4552 tumor lysates were prepared after 22 days of BRAFi + MEKi and vehicle/control chow treatment. Immunoblots show BRAFi + MEKi treatment induced ATF4 expression and autophagy induction. C, NSG mice bearing subcutaneous A375 xenografts were treated with vehicle/control chow and BRAFi + MEKi chow for 24h. Tumors were harvested and cell lysates were prepared. Immunoblots show induction of GRP78, KSR2, and autophagy. C1 and C2 lanes indicate tumor lysates from two different mice treated with vehicle chow. T1 and T2 lanes indicate tumor lysates of two different mice treated with BRAFi + MEKi chow. D, Harvested A375 xenograft tumor lysates were prepared for GRP78-immunoprecipitation (IP). Immunoblots show interaction between GRP78 and MPKs at 24h of BRAFi + MEKi chow treatment. E, Xenograft tumors were prepared for ER/cytosolic fractionations. Immunoblots show that BRAFi + MEKi treatment induces MPKs translocation to the ER at 24h of BRAFi + MEKi chow treatment. F, A375 mice xenograft tumors were prepared for nuclear fractionations. Immunoblots show ERK2 and ATF4 expression in the nucleus at 22 days of BRAFi + MEKi chow treatment. Supplementary Fig. S11

A B GRP78-IP INPUT BRAFi + MEKi (0.5h) BRAFi + MEKi (0.5h) ER Cyto - + - + BRAFi + MEKi - + - + 25 25 IB:NRAS IB:NRAS 25 NRAS

IB:BRAF IB:BRAF 75 75 BRAF 75

50 IB:MEK 50 IB:MEK ERK 37 IB:ERK IB:ERK 37 37 50 Calreticulin 100 100 IB:KSR2 IB:KSR2 50 Tubulin IB:GRP78 IB:GRP78 75 75 4522 4522 4522

C D

Nuclear fraction (8h) Control chow + + - - BRAFi + MEKi chow - - + + ERK2 25 n.s. 50 P-ATF4 30 -1 P = 5.6 X 10

50 ATF4 20

HDAC 50 10 100 HSP90 Mice weight (gm) 0 1 22 Days of treatment ATF4 WT ATF4 S245A E BRAFi + MEKi F - + Control + + + + + + + + + + + + + + + + + + + + BRAFi + MEKi - - - - - + + + ++ - - - - - + + + + + ATF4-WT 20 cl-Cas3 4522 0.00 0.00 0.00 0.01 0.23 0.98 0.12 0.32 0.36 0.23 1.23 0.98 0.92 0.89 0.76 1.45 1.26 1.34 1.38 1.29 ATF4-S245A 20 LC3I 15 LC3II

** 0.56 30.4 1.27 2.98 2.27 2.02 2.12 2.32 2.24 2.36 0.10 0.10 1.12 1.33 1.42 0.00 0.00 0.00 0.00 0.00 150 -3 ATF4 WT-Control P = 4.1X 10 ATF4 WT-BRAFi + MEKi 75 100 ATF4 S245A- Control P62 ATF4 S245A-BRAFi + MEKi 50 olony

C 50

ormation 00.2 0.32 0.33 0.31 0.02 0.01 0.15 0.02 0.21 0.00 1.32 0.98 0.23 0.81 0.75 1.67 1.32 1.23 1.42 1.44 % F 0 - + - + β-actin WT S245A 37 4552 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

PDX Tumor tissue (22 days) Supplementary Figure S111. BRAF and MEK inhibition mediates resistance via ATF4 phosphorylation in vivo. A, Primary cell line (4552) was established from PDX-4552 mice tumor, were treated with BRAFi + MEKi for 0.5h. Whole cell lysates (WCL) were prepared for GRP78- immunoprecipitation (IP). Immunoblots (IB) show drug-induced GRP78 interaction with MPKs. B, 4552 cells were treated with BRAFi + MEKi for 0.5h and ER/cytosolic fractionations were performed. Immunoblots show that BRAFi + MEKi treatment induces MPKs translocation to the ER in ex vivo cells. C, 4552 cells were treated with BRAFi + MEKi for 8h and nuclear/cytosolic fractionations were prepared. Immunoblots show ERK2 and ATF4 expression in the nucleus. D, Graph: ATF4-WT and ATF4S245A xenograft mice weight (gm) during the vehicle chow and BRAFi and MEKi chow treatment. Data shown as mean ± s.d.*P<0.05. E, 4552 cells were transfected with ATF4-WT and ATF4S245A constructs. Colony formation assay was performed in BRAFi + MEKi treated cells. Graph: quantification of the % colonies formation in BRAFi + MEKi treated ATF4-WT and ATF4S245A cells. F, Whole cell lysates prepared from tumor tissue obtained from ATF4-WT and ATF4S245A-4552 xenograft mice. Immunoblots show apoptosis induction and autophagy inhibition in ATF4S245A tumor.