Clonogenic Assay MediaA12 CXCL1

n

Figure S1

Conditioned Media

250 WTTbk1-/-

250 WTTbk1-/-

CB

CXCL10 CCL5

6

8

10

mRN

A e

xpre

ssio

n

WT

Tbk1-/-

M-CSF

n Pi

xel I

nten

sity

Pix

el In

tens

ity

100

150

200Tbk1 /

100

150

200

CXCL10 CCL5

0

2

4

WT Tbk1-/-

Rela

tive

Tbk1-/-

M-CSF

Mea

n

Mea

n

0

50

M-C

SFCC

L1 IL-6

XCL1

0CX

CL1

M-C

SFCC

L2CX

CL2

CCL5

XCL1

2M

P-1

0

50

M-C

SFCC

L1IL

-6XC

L10

CXCL

1M

-CSF

CCL2

CXCL

2CC

L5XC

L12

MP-

1

D

GM CX C M C CX TIGM CX C M C CX TI

S f t ki lt ti i Tbk1 / MEF

EWT MEF bk /

Conditioned Media

CXCL10

CCL5

Clonogenic Assay Media

Summary of cytokine alterations in Tbk1-/- MEFs WT MEFs

Tbk1-/- MEFs

+CCL5 +CXCL10 +IL-6

+Tbk1-/- CM

Tbk1-/- MEFs Tbk1-/- MEFs Tbk1-/- MEFs

Tbk1-/- MEFs +WT CM

CCL1

CXCL1

CXCL2

M-CSF

+CCL5IL-6

+CCL5CXCL10

Tbk1-/- MEFs Tbk1-/- MEFs

CXCL2

CXCL12

IL-6

Figure S2A

600800

10001200 IC50>10μM IC50=452nM IC50=29nM

10Ex

pres

sion

D IFN-γ Stimulated RAW Macrophages

50

100

50

100

(%Co

ntro

l)

(%Co

ntro

l)

His-TBK1 + IKKε-Tide GST-IKKε + IKKε-Tide

ol)His-TBK1 + IKKε-Tide GST-IKKε + IKKε-Tide

ol)

0200400

Con

IFNγ

γ/D

MSO

MRT

0.01

MRT

0.05

/MRT

0.1

/MRT

0.5

g/M

RT1

g/M

RT5

/MRT

10CY

T0.0

1CY

T0.0

5/C

YT0.

1/C

YT0.

5N

g/CY

T1N

g/CY

T5g/

CYT1

0RU

X0.0

1RU

X0.0

5/R

UX0

.1/R

UX0

.5N

g/RU

X1N

g/RU

X5g/

RUX1

0

DM

SO0.

010.

05 0.1

0.5 1 5 10

0.01

0.05 0.1

0.5 1 5 10

0.01

0.05 0.1

0.5 1 5 10Rela

tive

CXC

L1

-IFN

γ+I

FNγ

-4 -3 -2 -1 0 -4 -3 -2 -1 0Log Concentration (μM)

RuxolitinibIC50=1.2 μM

Log Concentration (μM)

[AD

P]

[AD

P] RuxolitinibIC50>3 μM

50

100

C50(μM): 0.040 MRT67307

IC50=40 nM

4 2 0

50

100

yMRT67307

IC50=242 nM[AD

P] (%

Cont

ro

[AD

P] (%

Cont

ro

IFNγ

IFN

g/M

IFN

g/M

IFN

g/IF

Ng/

IFN

gIF

Ng

IFN

g/IF

Ng/

CIF

Ng/

CIF

Ng

IFN

gIF

NIF

NIF

Ng

IFN

g/R

IFN

g/R

IFN

g/IF

Ng/

IFN

IFN

IFN

gMRT67307 CYT387 Ruxolitinib

300350

pres

sion

IC50=114nM IC50=434nM IC50>10μM

LPS Stimulated RAW Macrophages

Concentration (μM)

E

-4 -2 -4 -2 0

050

100150200250300

ativ

e CX

CL10

exp

B

pSTAT1 (Y701)

DM

SO MRT67307 CYT387 Ruxolitinib

0.1 0.5 1.0IFN-γ 0.1 0.5 1.0(-) 0.1 0.5 1.0 (μM)

Log Concentration (μM)Log Concentration (μM)

n

C

0

RAW

RAW

-LPS

RAW

-LPS

/DM

SOLP

S/M

RT0.

01LP

S/M

RT0.

05LP

S/M

RT0.

1LP

S/M

RT0.

5LP

S/M

RT1

LPS/

MRT

5LP

S/M

RT10

LPS/

CYT0

.01

LPS/

CYT0

.05

LPS/

CYT0

.1LP

S/CY

T0.5

LPS/

CYT1

LPS/

CYT5

LPS/

CYT1

0LP

S/RU

XO0.

01LP

S/RU

XO0.

05LP

S/RU

XO0.

1LP

S/RU

XO0.

5LP

S/RU

XO1

LPS/

RUXO

5LP

S/RU

XO10

DM

SO0.

010.

05 0.1

0.5 1 5 10

0.01

0.05 0.1

0.5 1 5 10

0.01

0.05 0.1

0.5 1 5 10

MRT67307 CYT387 RuxolitinibConcentration (μM)

Rel

F

-LPS

+LPS

β-Actin

STAT1

IFN-γ Stimulated RAW Macrophages

IC-50 Values for TBK1/IKKε Inhibition

In vitro kinase Cell-based assays

TBK1 IKKεMRT67307 40 nM 242 nM

LPS-->IFNB1 LPS-->pIRF3228 nM <100 nM

2000400060008000

10000120001400016000 IC50>10 μM IC50=587nM IC50=20nM

eG

BP1

Expr

essi

o

CYT387 58 nM 42 nMRuxolitinib >3000 nM 1200 nM

201 nM 100 nM9,200 nM >1000 nM

02000

Con

IFNγ

IFNγ/

DM

SOIF

Ng/

MRT

0.01

IFN

g/M

RT0.

05IF

Ng/

MRT

0.1

IFN

g/M

RT0.

5IF

Ng/

MRT

1IF

Ng/

MRT

5IF

Ng/

MRT

10IF

Ng/

CYT0

.01

IFN

g/CY

T0.0

5IF

Ng/

CYT0

.1IF

Ng/

CYT0

.5IF

Ng/

CYT1

IFN

g/CY

T5IF

Ng/

CYT1

0IF

Ng/

RUX0

.01

IFN

g/RU

X0.0

5IF

Ng/

RUX0

.1IF

Ng/

RUX0

.5IF

Ng/

RUX1

IFN

g/RU

X5IF

Ng/

RUX1

0

DM

SO 0.01

0.05 0.1

0.5 1 5 10

0.01

0.05 0.1

0.5 1 5 10

0.01

0.05 0.1

0.5 1 5 10

MRT67307 CYT387 RuxolitinibConcentration (μM)

Rela

tive

-IFN

γ+I

FNγ

Figure S3A

ityKRAS Mutant KRAS WT B

0 8

1

1.2

shLacZshGFP pTBK1

(S172)

A54

9

HCC

44

H23

H17

92

H46

0

H20

09

H19

44

H28

H14

37

H15

68

H20

52

H52

2

Baseline (24 h Culture)

Rela

tive

viab

ili

0.2

0.4

0.6

0.8 shKRAS-1shTBK1-1shTBK1-2

TBK1

(S172)

pSTAT3(Y705)

STAT3

0

C D FE

β-Actin

12hEGF

A549 SpheroidsC

TBK1

pTBK1

0hIL-1β:

A549 cells

0.5h

1h 2h 3h 4h 5h 6h 7h 8h

1000

1500

2000ev

els

(pg/

ml)

IL-1β stimulation

DA549 cells

F

0 6

0.8

1

1.2 A549 cellsE

Via

bilit

y

24hβ-Actin

TBK1

1000

0

5000h

0.5h 1h 2h 4h 6h 8h

ml)

CCL5

Le

IL 1β stimulation

Time

0min

20m

in

40m

in

1h 2h 4h

pTBK1EGF: 0

0.2

0.4

0.6

Rela

tive

V

72h

100 μM0

250

500

750

0h 5h 1h 2h 4h 6h 8h

IL-6

Lev

els

(pg/

m IL-1β stimulation

β-Actin

TBK1

pTBK1

00.

5 1 2 4 6 8

Time

Figure S4 A H1819 Cells

on 12

14

16shLACZshIKKe-1shIKKe-2

DDMSO CYT387

A549 Cells

IC-50s for Growth Inhibition

A549 HCC44 H1437 H1568

C

εε

100

B Ruxolitinib

Fold

Pro

lifer

atio

4

6

8

10

MRT67307 Ruxolitinib

A549 HCC44 H1437 H1568

MRT67307 1.9 μM 2.1 μM >10 μM >10 μM

CYT387 1.7 μM 1.8 μM >10 μM 4.6 μM

Ruxolitinib >10 μM 9.8 μM >10 μM >10 μM

50A549HCC44H1437H1568

% C

ontr

olE

0

2

Day 0 Day 7

Dependent Independent Dependent Independent 0h 36h

G EGFGDC0941 Nearest NeighborsAZD6244 Nearest Neighbors F

-0.5 0.0 0.5 1.0Log (concentration/µM)

AZD6244 1CI-1040 0.7054GDC0941 0.5864VTX-11E 0.5517

Dependent Independent

*

A54

9H

CC

44H

23H

1792

H18

19H

1944

H20

09H

522

H14

37H

1568

H19

75H

2052

InhibitorPearson

Correlation (r)GDC0941 1MK-2206 0.6269AZD6244 0.5864LY294002 0.5365

*

InhibitorPearson

Correlation (r)

Dependent Independent

A54

9H

CC

44H

23H

1792

H18

19H

1944

H20

09H

522

H14

37H

1568

H19

75H

2052

MRT67307

Ruxolitinib

0h 36h

0h 36h

0 55BX-795 0.5398Flavopiridol 0.3988MRT-67307 0.3494JNK Inhibitor-1 0.335MK-2206 0.3186GDC0879 0.2012CYT-387 0.1894LY294002 0.02612AG490 -0.04855PLX 4720 0 05636

LY294002 0.5365BX-795 0.3627CI-1040 0.3375VTX-11E 0.3078MRT-67307 0.247IKK-2 (TPCA) 0.1611CYT-387 0.08016PLX-4720 -0.00775AG490 -0.01269BAY-11-7082 -0.07524Flavopiridol 0 1347

MRT67307+

Ruxolitinib

0h 36h

PLX-4720 -0.05636IKK-2 (TPCA) -0.1024Nifuroxazide -0.1491Imatinib -0.1754BAY-11-7082 -0.182

i

Relative Viability

Row Min Row Max

Flavopiridol -0.1347Nifuroxazide -0.311GDC0879 -0.3117JNK Inhibitor-1 -0.3403Imatinib -0.4293

i

Relative Viability

Row Min Row Max # D

ispe

rsed

Ce

lls/S

pher

oid

05

10152025

Figure S5 E MRT67307 CYT387 RuxolitinibDMSO

IL-1β0 15 30

IL-6

pSTAT3

IL-6 IL-6 IL-6IL-1βIL-1β0 15 30 0 15 30 0 15 30 0 15 30 0 15 30 0 15 30 0 15 30

IL-1βTime:(min)

200300400500600700

203040506070

evel

s (p

g/m

l)

293T cells

Leve

ls (p

g/m

l)AA549 cells

pTBK1

STAT3

TBK1

0100200

EGFP

TBK1

-WT

TBK1

-KD

IKBK

E-W

T

IKBK

E-KD

01020

EGFP

TBK1

-WT

TBK1

-KD

IKBK

E-W

T

IKBK

E-KD

IL-6

Le

CCL5

EGFP

TBK1

-WT

TBK1

-KD

IKKε

-WT

IKKε

-KD

EGFP

TBK1

-WT

TBK1

-KD

IKKε

-WT

IKKε

-KD

B MEF

ssio

n

2

2.5 MRT67307 CYT387 RuxolitinibDMSO

Fβ-Actin

0.6

0.8

1

1.2WTTbk1-/-

10

15

20WTTbk1-/-

CL5

expr

essi

on

L-6

expr

essi

on

B MEFs

lati

veIK

KεEx

pres

0.5

1

1.5

0

0.2

0.4

24h 48h 72h

0

5

24h 48h 72h

Rela

tive

CC

Rela

tive

IL

Time Time

Rel

IL-1β:

G300 MRT67307 CYT387 RuxolitinibDMSO

n

0

0h0.

5h 1h 2h 3h 4h 0h0.

5h 1h 2h 3h 4h 0h0.

5h 1h 2h 3h 4h 0h0.

5h 1h 2h 3h 4h

20

IKKε

β-Actin

12h 24h 36h 48h 60h 72h 84h 96hTbk1-/- MEFsC

D

100

150

200

250

veIL

-6 E

xpre

ssio

n

IL-6

exp

ress

ion

10

15

20

WT DMSOWT RuxoTbk1-/- DMSOTbk1-/- Ruxo

0

50

0h0.

5h 1h 2h 3h 4h 0h0.

5h 1h 2h 3h 4h 0h0.

5h 1h 2h 3h 4h 0h0.

5h 1h 2h 3h 4hIL-1β:

Rela

tiv

Rela

tive

0

5

24h 48h 72hTime

Figure S6 A

pSTAT3

0h 1h 2h 0h 1h 2h 0h 1h 2h 0h 1h 2h 0h 1h 2h 0h 1h 2h

A549 A549-C1 A549-C2CYT387 Ruxolitinib CYT387 Ruxolitinib CYT387 Ruxolitinib

B

)

293T Cells

) 18002000 300

β-Actin

STAT3

L-6

Leve

ls (p

g/m

l)

CL5

Leve

ls (p

g/m

l)

400600800

10001200140016001800

100

150

200

250

ILCC

0200400

0

50

EGFP

+DM

SO

-WT+

DM

SO

GFP

+CYT

387

WT+

CYT

387

-KD

+ D

MSO

KD+

CYT3

87

88C+

DM

SO

8C+

CYT3

87

92R+

DM

SO

2R+

CYT3

87

EGFP

+DM

SO

-WT+

DM

SO

GFP

+CYT

387

WT+

CYT

387

ε-KD

+ D

MSO

KD+

CYT3

87

88C+

DM

SO

88C+

CYT

387

92R+

DM

SO

92R+

CYT

387

A549-IKKε-Y88C Cells

C

EGFP IKKε-WT

DA549 Cells

E

IKKε

-

EG

IKKε

-W

IKKε

-

IKKε

-K

IKKε

Y8

IKKε

-Y88

IKKε

-G9

IKKε

-G92 E

IKKε

-

EG

IKKε

-W

IKKε

IKKε

-

IKKε

Y

IKKε

-Y8

IKKε

-G

IKKε

-G9

IKKε-KD IKKε-Y88C

TACTGC

Figure S7

DMSO CYT387

A C

pERK1/2

pMAPK Pathway Array (A549 Cells)

-5%

0%2 Wks 4 Wks

t (%

cha

nge)

B

pERK1/2 pERK1/20 5 10 20

MRT67307

40 60 0 5 10 20 40 60 min

CYT387

Ruxolitinib

pERK1/2

A549 Cells H23 Cells

-20%

-15%

-10%

Mea

n W

eigh

CYT387

CYT387+Docetaxel

AZD6244

pERK1/2β-Actin

ERK1/2

D E

pSTAT3

STAT3

0 20 40 60 minCYT387 AZD6244 CYT387+AZD6244

H23 Cells

pSTAT3

STAT3150

200

250

300

350

0 20 40 60 0 20 40 600 20 40 60 minCYT387 AZD6244 CYT387+AZD6244

0 20 40 60 0 20 40 60

vels

(pg/

ml)

D EA549 Cells

β-Actin

pERK1/2

ERK1/2

β-Actin

pERK1/2

ERK1/20

50

100

150

IL-6

Lev

β β Actin

F

1

1.2 A549 Cells1

1.2 H23 Cells1

1.2 H1792 Cells1

1.2 H1944 Cells

ty ty ity ty

250%

300%

e)

AZD6244

CYT387

KrasLSL-G12D/WT; p53Flox/FloxG

0.2

0.4

0.6

0.8

0.2

0.4

0.6

0.8

0.2

0.4

0.6

0.8

0.2

0.4

0.6

0.8

* ** ****Re

lati

ve V

iabi

li

Rela

tive

Via

bili

Rela

tive

Via

bili

Rela

tive

Via

bilit

50%

100%

150%

200%

Bur

den

(% C

hang

e CYT387

CYT387+AZD6244

0 0 0 0

-100%

-50%

0%0 1 2 3 4 5 6 7 8

Tum

or

Weeks