Supplemental Figure 1

A B C NSC EV NSC H3 WT NSC H3K27M

800 800 1500 Legend Scramble 600 600 x1000 ) x1000 ) x1000 ) ( ( ( 1000 t t t n n HRAS n 400 400 u u u o o o C C C

500 KRAS 200 200 el l el l el l C C C NRAS 0 0 0 0 48 96 144 0 48 96 144 0 48 96 144 Hours Hours Hours D *** E F 8 *** 1.5 1.5

*** ro l ro l 6 1.0 siRNA1 siRNA2 1.0 siRNA1 siRNA2 Con t Con t

o o t 4 * * t * * * * * * e * * * * * * e v i * * v i 0.5 * * 0.5 Fold Change 2 Fold Change Rela t Rela t

Relative to EV Scrambl e 0.0 0.0 0 Caspase Activity Fold Change

H-RASK-RASN-RASH-RASK-RASN-RAS H-RASK-RASN-RASH-RASK-RASN-RAS KRAS KRAS KRAS HRAS NRAS HRAS NRAS HRAS NRAS Scramble Scramble

Scramble Scramble Scramble NSC-EV NSC-H3 WT EV H3WT H3K27M

G H I 800 Control Con WT H3 H3K27MEZH2 inb 1.5 H3-WT Active RAS ro l 600 H3-K27M

1.0 siRNA1 siRNA2 Total RAS *** Con t

x1000 ) (

o EZH2 GSK343 t t * * * * * * H3K27me3 n e 400 u v i o 0.5 ***

H3K27M C

Fold Change W.C.L. el l Rela t p16 C 200 0.0 B-ACTIN H-RASK-RASN-RASH-RASK-RASN-RAS 0 Scramble NSC-H3 K27M 0 1 2 3 4 5 Days Supplemental Figure 2

A DIPG007 siRNA2 B NSC siRNA1 and 2 * * * * * 100 * * * * * * * * * * * 100

75 75 iabilit y iabilit y V 50

V 50 Cell Cell 25 25

0 0 ZAK ZAK OR3 OR3 MYC MYC ZAK OR3 MYC MOCK LIN28A LIN28A LIN28B LIN28B MAPK7 MAPK7 AURKA AURKA MOCK LIN28A LIN28B MAPK7 AURKA MAP2K3 MAP2K3 MAP2K5 MAP2K5 MAP3K2 MAP3K2 MAP3K7 MAP3K7 PDGFRA PDGFRA Scramble MAP2K3 MAP2K5 MAP3K2 MAP3K7 PDGFRA Scramble LAM T LAM T LAM T siRNA1 siRNA2

C NSC H3K27M siRNA1 and 2 D DIPG007 * * * * * * * * * * * * * * * * * * * * * * 100 1.2 1.0 75 0.8 iabilit y

V 50 0.6 0.4 Cell 25 Fold Change 0.2 Relative to Contro l 0 0.0 ZAK OR3 MYC ZAK ZAK OR3 OR3 MYC MYC MOCK LIN28A LIN28B MAPK7 AURKA LIN28A LIN28A LIN28B LIN28B MAPK7 MAPK7 AURKA AURKA MAP2K3 MAP2K5 MAP3K2 MAP3K7 PDGFRA MAP2K3 MAP2K3 MAP2K5 MAP2K5 MAP3K2 MAP3K2 MAP3K7 MAP3K7 PDGFRA PDGFRA Scramble LAM T LAM T LAM T siRNA2 siRNA1 siRNA2 Scramble siRNA siRNA1 E F NSC-EV NSC-H3K27M 1.2 1.2 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 Fold Change Fold Change 0.2 0.2 Relative to Contro l Relative to Contro l 0.0 0.0 ZAK ZAK OR3 OR3 MYC MYC LIN28A LIN28B MAPK7 LIN28A LIN28B AURKA MAPK7 AURKA MAP2K3 MAP2K5 MAP3K2 MAP3K7 MAP2K3 MAP2K5 MAP3K2 MAP3K7 PDGFRA PDGFRA LAM T LAM T Scramble siRNA siRNA1 siRNA2 Scramble siRNA siRNA1 siRNA2 Supplemental Figure 3 A ERK5 B ERK5 * * * 6 8 2 2 (TPM) og og L 6 4 ession ession L 4 2 RNA Exp r 2

RNA Seq Exp r 0

GTEx DIPG brain

Normal Brain Normal Brain 44 DIPG_Kool (n=12) DIPG_Paugh (n=37) H3.3 G34R/V C H3.1 K27M H3.3 K27M H3 WT

H3F3A (H3.3) 48%

HIST1H3B (H3.1) 10%

MAPK7 2.1%

PDGFRA 15%

NF1 9%

MAPK3 0%

MAPK1 0.3%

MAP2K1 0%

MAP2K2 0%

MAP2K5 0.3%

MAP3K2 0.3%

Genetic Alteration Missense Mutation (putative driver) Missense Mutation (unknown significance)

Truncating Mutation (unknown significance) Amplification Deep Deletion

Truncating Mutation No alterations Supplemental Figure 4 A

LGG GBM RNA-Seq ERK5 Log2 RSEM expression

B 3 * * *

2

1 Fold Change Relative to Contro l Cleaved Caspase 3/7 0

Dox Dox Dox Control Control Control DIPG-13p DIPG-IV SF8628 Supplemental Figure 5 A SF8628 B DIPG13p 100 100 1000 H3 WT C * H3 K27M 75 75 800 y y 600 * iabili t iabili t 50 50 V V

% % 400 25 25 200 0 0 EC 50 Values (TG02 nM) -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 0 Log Drug (uM) Log Drug (uM) NSC BIX02189 3.16 uM BIX02189 2.30 uM GBM 001 XMD8-92 4.51 uM XMD8-92 2.51 uM DIPG 007 SU-DIPG13 SU-DIPG IV NSC H3 WT SU-pcGBM2 NSC H3 K27M

ERK5-IN-1 512 nM ERK5-IN-1 500 nM CNMC-XD-760 TG02 70 nM TG02 80 nM

D E F SF8628 DIPG13p SF8628 DIPG 13p DMSO control * * * * 105 105 100

104 104 (% ) 103 103 en t 50

102 102 er c P 0 0 Annexin PB VL1-A Annexin PB VL1-A Annexin PB VL1-A 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0 ERK5-IN-1 PI GL1-A PI GL1-A

5 5 10 10 Con Con TG02 TG02

4 4 10 10 ERK5-IN-1 ERK5-IN-1

3 3 10 10 Viable Early apoptosis 102 102 Late apoptosis Necrosis 0 0

Annexin PB VL1-A Annexin PB VL1-A Annexin PB VL1-A G 2 3 4 5 2 3 4 5 DIPG13p Control 0 10 10 10 10 0 10 10 10 10 3 PI GL1-A PI GL1-A

) ERK5-IN-1

TG02 6 105 105 2 TG02

104 104

3 3 1 10 10 * * 2 * 102 10 Cell Count (x 10 ^ 0 * * * 0 0 Annexin PB VL1-A Annexin PB VL1-A Annexin PB VL1-A Annexin PB VL1-A 2 3 4 5 0 102 103 104 105 0 10 10 10 10 1 3 5 7 9 PI GL1-A PI GL1-A Day Supplemental Figure 6

siRNA con A B 8 ERK5 siRNA siRNA ConERK5 siRNAsiRNA ConERK5 siRNA * 6 pERK5

ERK5 4 B-ACTIN 2 SU-pcGBM2 CNMC-XD-760 Cell Count (x10^6 ) 0 2 4 6 8 10 Days CNMC-XD-760

C siRNA con D 8 ERK5 siRNA * EV ERK5-HAERK5-HA T733E 6 HA * B-ACTIN 4

2 Cell Count (x10^6 )

0 2 4 6 8 10 Days SU-pcGBM2

E F G * * * * * * 600 3000 1500 Control * * TG02 U F 1000 ERK5-IN-1

400 2000 R rar y 200 1000 500 rbi t A EC50 (nM ) TG02 0 0 Cleaved Caspase 3/7 0 EC50 (nM ) ERK5-IN-1 ector ector ector ector ector ERK5 ERK5 ERK5 ERK5 ERK5 T733E T733E T733E T733E T733E ERK5 ERK5 ERK5 ERK5 ERK5 Empty V Empty V Empty V Empty V Empty V Supplemental Figure 7

MYC C * A 1.5 B 800

600 1.0 * EV MYC MYC S62D 400 0.5 MYC

Fold Change 200 EC50 (nM ) TG02 Relative to Contro l B-ACTIN 0.0 0 DOX DOX MYC ector TG02 TG02 ehicle ehicle V V ERK5-IN-1 ERK5-IN-1 MYC S62D

DIPG-IV SF8628 Empty V D E F MYCN * 4000 1.5

* * * Control DOX ERK5-IN-1 TG02 3000 * 1.0 ERK5 pS731 pT733 2000 0.5 ERK5

1000 Fold Change

Relative to Contro l MYCN 0 0.0 EC50 (nM ) ERK5-IN-1 B-ACTIN DOX MYC ector TG02 ehicle V ERK5-IN-1 MYC S62D Empty V G H

VehicleVehicleERK5-IN-1ERK5-IN-1TG02 TG02 H3.3 WT H3.3 K27M H3.3 WT H3.3 K27M pERK5 S731/T733 ERK5 Cleaved PARP ASP 214 B-ACTIN

ERK5 - - + + Erk5 siRNA

1000 I H3.3 WT control siRNA

750 H3.3 WT Erk5 siRNA

* * H3.3 K27M control siRNA 500 * H3.3 K27M Erk5 siRNA Cell Count 250

0 1 3 5 7 9 Days