EXTENDED METHODS

Western Blot and Immunoprecipitation.

EBC buffer (50mM Tris-HCl pH8.0, 120mM NaCl, 0.5% NP40, 0.1mM EDTA and 10%

glycerol) supplemented with complete protease inhibitor and phosphoSTOP tablets (Roche

Applied Bioscience) was used to harvest whole cell lysates. Cell lysates concentration was

measured by Protein assay dye (Bio Rad). Equal amount of cell lysates was resolved by

SDS-PAGE. For immunoprecipitation, cells were lysed in EBC buffer supplemented with

protease inhibitor and phosphatase inhibitor. The lysates were clarified by centrifugation and

then incubated with primary antibodies or FLAG/HA antibody conjugated beads (HA beads,

Roche Applied Bioscience; FLAG M2 beads, Sigma) overnight at 4℃. For primary antibody

incubation, cell lysates were incubated further with protein G sepharose beads (Roche

Applied Bioscience) for 2 hours at 4℃. The bound complexes were washed with EBC buffer

for 3 times and were eluted by boiling in SDS loading buffer. Bound proteins were resolved

in SDS-PAGE followed by western blot analysis.

Overexpression vectors.

pcDNA3-FLAG-TBK1 was a kind gift from Prof. Dr. Michael Kracht (Universität Giessen)(1).

pcDNA4-HA-p62 was purchased from Addgene (#28027). Quick Change XL Site-Directed

Mutagenesis Kit (200516, Agilent Technologies) was used to generate TBK1 (K38A, P48A,

P678A) and p62 (S366A, S366D) mutants. For lentiviral based plasmids, TBK1 and p62

were cloned into pLenti6-FLAG or pLenti6-HA vectors respectively by standard molecular

cloning techniques.

Lentiviral sgRNA, shRNA vectors and siRNAs.

sgRNA sequences target TBK1 were described in ref(2). The guide RNAs were cloned into

pLentiCRISPR V2 -Puromycin. sgVHL, sgEglN1, sgHIF2α and sgARNT were described

previously(3-5). shTBK1 sequences were obtained from Broad Institute TRC shRNA library.

To generate inducible shTBK1 vectors, target sequences were cloned into pLKO-Tet-On

vector. shSTING was a kind gift from Dr. Samuel Bakhoum (MSKCC)(6). Ctrl and EglN1

siRNA were purchased from Dharmacon (ON-TARGETplus EglN1 siRNA). All sg, sh target

sequences are as follows:

sgTBK1-1: CATAAGCTTCCTTCGTCCAG

sgTBK1-2: TCCACGTTATGATTTAGACG

sgTBK1-3: GACAGCAGATTATCTCCAGG

sgVHL-1: CATACGGGCAGCACGACGCG

sgVHL-2: GCGATTGCAGAAGATGACCT

sgVHL-3: ACCGAGCGCAGCACGGGCCG

sgEglN1-1: CGGACAGCAGATCGGCGACG

sgEglN1-2: ATGCCGTGCTTGTTCATGCA

sgHIF2α-1: AATCTCCTCATGGTCGCA

sgHIF2α-2: TCATGAGGATGAAGTGCA

sgARNT-1: TTGGCAGTAGTCGCCGCCA

sgARNT-2: GTGGCATCTGCGGCCATGG

shTBK1 (3182): GCAGAACGTAGATTAGCTTAT

shTBK1 (3185): GCGGCAGAGTTAGGTGAAATT

shEglN1 (1044): ACGCCACTGTAACGGGAAGCT

shEglN1 (1045): TGGAGATGGAAGATGTGTGAC

Virus Production and Infection

293T cell line was used for lentiviral generation. Lentiviral infection was carried out similarly

as previously described(7). Briefly, post-transfection with lipofectamine 3000, viruses were

collected twice after 48 and 72 hours. After passing through 0.45μm filters, appropriate amount

of viruses was used to infect target cells in the presence of 8 μg/ml polybrene (Santa Cruz).

Subsequently, target cell lines were cultured in the presence of puromycin (2 μg/ml) or

blasticidin (10 μg/ml) depending on the vector.

Anchorage Independent 3-D soft agar Growth Assay.

Cells were plated at a density of 3000 cells/ml for UMRC2 and 15000 cells per ml for UMRC6

in complete medium with 0.4% agarose, onto bottom layers composed of medium with 1%

agarose followed by incubation at 4⁰C for 10 minutes. Afterwards, cells were moved to 37⁰C

incubator. For every 3 days, five drops of complete media were added onto the plate. After

two weeks for UMRC2 or four weeks for UMRC6 incubation, colonies were stained by 100

μg/ml iodonitrotetrazoliuim chloride (Sigma, I8377-1G) solution overnight in the incubator and

then foci number was counted.

Immunohistochemistry (IHC) Staining and Imaging Analysis.

The tissue slides described in the manuscript contained totally de-identified patient

information. The approval for usage of these slides were obtained from UNC-Chapel Hill

Institutional Review Board. IHC was carried out in the Bond Autostainer (Leica Microsystems

Inc.; Norwell MA). Briefly, slides were dewaxed in Bond Dewax solution (AR9222) and

hydrated in Bond Wash solution (AR9590). Antigen retrieval was performed for 20 min at

100ºC in Bond-Epitope Retrieval solution 1, pH-6.0 (AR9961). Slides were incubated with

primary antibody (TBK1, 1:100, 30min; pTBK1, 1:100, 1hr; p62, 1:2000, 30min; VHL, 1:200,

30min). Antibody detection was performed using the Bond Intense R detection system

(DS9263) with ImmPress HRP anti-rabbit IgG (MP-7451; Vector Laboratories; Burlingame,

CA). Stained slides were dehydrated and coverslipped. Positive and negative controls (no

primary antibody) were included during the run. Antibodies used in IHC: TBK1 (3504), pTBK1

(5483) and p62 (88588) were from Cell Signaling Technology, VHL (556347) was from BD

Biosciences.

For imaging analysis, stained slides were digitally scanned at 20x magnification using Aperio

ScanScope-XT (Aperio Technologies, Vista, CA) were uploaded to the Aperio

eSlideManager database (Leica Biosystems Inc; eSlideManager version 12.3.3.7075) at the

Translational Pathology Laboratory at UNC. TMA images were digitally segmented into

cores using TMALab (Aperio). Individual TMA cores were separately analyzed using the

Aperio Cytoplasmic v2 algorithm with slight adjustments for cell shape. The number and

percentage of cells with light (1+), medium (2+) and strong (3+) nuclei and/or cytoplasmic

staining was determined. H Scores were calculated using the following formula: 3 x

percentage of strongly staining cells + 2 x percentage of moderately staining cells +

percentage of weakly staining cells, giving a range of 0 to 300.

TBK1 PROTAC (UNC6587)

1. General Chemistry Procedures

All reagents and solvents were obtained from commercial suppliers and were used without

further purification unless otherwise stated. N-(3-((5-bromo-2-((4-

hydroxyphenyl)amino)pyrimidin-4-yl)amino)propyl)-N-methylcyclobutanecarboxamide (1)

was purchased from WuXi App Tec. and was synthesized according to reported literature

procedures (8). 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione was purchased from

CarboSynth. Reactions were carried out using conventional glassware and room temperature

was generally 22 °C. Reactions were carried out at elevated temperatures as noted using a

temperature regulated hot plater-stirrer. Thin layer chromatography was carried out using

Merck silica plates coated with fluorescent indicator and analyzed under 254 nm UV light.

Analytical Liquid Chromatography Mass Spectrometry (LCMS) data for all compounds were

acquired using an Agilent 6110 Series system with the UV detector set to 254 nm. Samples

were injected (<10 µL) onto an Agilent Eclipse Plus 4.6 × 50 mm, 1.8 µm, C18 column at room

temperature. Mobile phases A (H2O + 0.1% acetic acid) and B (MeOH + 0.1% acetic acid)

were used with a linear gradient from 10% to 100% B in 5.0 min, followed by a flush at 100%

B for another 2 minutes with a flow rate of 1.0 mL/min. Mass spectra (MS) data were acquired

in positive ion mode using an Agilent 6110 single quadrupole mass spectrometer with an

electrospray ionization (ESI) source. Normal phase column chromatography was performed

with a Teledyne Isco CombiFlash®Rf 200 using RediSep®Rf SILICA columns with the UV

detector set to 254 nm and 280 nm. Preparative High-Performance Liquid Chromatography

(HPLC) was performed using an Agilent Prep 1200 series with the UV detector set to 220 nm

and 254 nm. Samples were injected onto a Phenomenex Luna 75 x 30 mm (5 µm) C18 column

at room temperature.

2. Analysis of products

Analytical LCMS (at 254 nm) and NMR were used to establish the purity of targeted

compounds. 1H spectra were obtained on a Bruker AV 400 at 400 MHz. Chemical shifts are

reported in ppm and coupling constants are reported in Hz with MeOD-d4 referenced at 3.31

(1H). All compounds that were evaluated in biochemical and biophysical assays had >95%

purity.

3. Synthetic Schemes

Supplementary Scheme 1. Conditions for the synthesis of UNC6587. a) i) (1) (1.0 equiv),

tert-butyl 1-(tosyloxy)-3,6,9,12,15-pentaoxaoctadecan-18-oate (1.1 equiv), K2CO3 (3.0 equiv),

DMF; ii) 20% TFA/CH2Cl2; b) i) (2) (1.0 equiv), SOCl2, 60 °C; ii) 4-amino-2-(2,6-dioxopiperidin-

3-yl)isoindoline-1,3-dione (1.1 equiv), THF, 60 °C.

4. Chemistry Experimental

Abbreviations

Trifluoroacetic acid (TFA), dimethylformamide (DMF), tetrahydrofuran (THF).

1-(4-((5-bromo-4-((3-(N-methylcyclobutanecarboxamido)propyl)amino)pyrimidin-2-

yl)amino)phenoxy)-3,6,9,12,15-pentaoxaoctadecan-18-oic acid (2)

To a flask containing N-(3-((5-bromo-2-((4-hydroxyphenyl)amino)pyrimidin-4-

yl)amino)propyl)-N-methylcyclobutanecarboxamide (1) (50 mg, 115 μmol, 1.0 equiv) in DMF

(1.25 mL), was added K2CO3 (48 mg, 345 μmol, 3.0 equiv) and the reaction was heated to 60

°C for ~10 mins. To the reaction was added tert-butyl 1-(tosyloxy)-3,6,9,12,15-

pentaoxaoctadecan-18-oate (66 mg, 127 μmol, 1.1 equiv) and the reaction was left to stir at

60 °C for 16 hrs. The reaction was cooled, concentrated in vacuo, and neutralized with TFA

before column chromatography (0-10% MeOH in CH2Cl2) to yield the desired intermediate,

tert-butyl 1-(4-((5-bromo-4-((3-(N-methylcyclobutanecarboxamido)propyl)amino)pyrimidin-2-

yl)amino)phenoxy)-3,6,9,12,15-pentaoxaoctadecan-18-oate. Subsequent hydrolysis with 20%

TFA in CH2Cl2 and evaporation in vacuo yielded 2 as a clear oil which was used in the next

step without further purification (50 mg, 60%, over 2 steps).

1H NMR (MeOD-d4, 400 MHz): δ 7.87 (d, J = 12.9 Hz, 1H), 7.50 – 7.45 (m, 2H), 6.92 – 6.86

(m, 2H), 4.12 – 4.07 (m, 2H), 3.84 – 3.79 (m, 2H), 3.72 – 3.59 (m, 15H), 3.56 – 3.52 (m, 2H),

3.50 – 3.35 (m, 5H), 3.29 – 3.23 (m, 1H), 2.87 (ap d, 3H), 2.31 – 2.13 (m, 4H), 2.06 – 1.75 (m,

6H).

LCMS: expected mass for [M+H]+ (C32H48BrN5O9), 726.26 m/z; found, 726.20 m/z.

1-(4-((5-bromo-4-((3-(N-methylcyclobutanecarboxamido)propyl)amino)pyrimidin-2-

yl)amino)phenoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-3,6,9,12,15-

pentaoxaoctadecan-18-amide (UNC6587)

To a flask containing 1-(4-((5-bromo-4-((3-(N-

methylcyclobutanecarboxamido)propyl)amino)pyrimidin-2-yl)amino)phenoxy)-3,6,9,12,15-

pentaoxaoctadecan-18-oic acid (2) (50 mg, 69 μmol, 1.0 equiv) was added SOCl2 (1.0 mL,

13.7 mmol) and the reaction was heated at 60 °C for 1 hr. The reaction was cooled,

concentrated in vacuo, and THF (0.28 mL) and 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-

1,3-dione (21 mg, 76 μmol, 1.1 equiv) were added. The reaction was heated at 60 °C for 16

hrs, cooled to room temperature, concentrated in vacuo, and purified by preparative HPLC

(10-100% MeCN in H2O (0.1% TFA)) to yield the desired product as a yellow solid (UNC6587)

(4.2 mg, 6.2%, over two steps).

1H NMR (MeOD-d4, 400 MHz): δ 8.71 (dd, J = 0.6, 8.5 Hz, 1H), 7.93 (ap d, J = 16.0 Hz, 1H),

7.76 (dd, J = 7.4, 8.5 Hz, 1H), 7.56 (dd, J = 0.6, 7.3 Hz, 2H), 7.38 – 7.31 (m, 2H), 5.12 (dd, J

= 5.5, 12.4 Hz, 1H), 4.18 – 4.10 (m, 2H), 3.86 – 3.79 (m, 4H), 3.71 – 3.66 (m, 5H), 3.66 – 3.54

(m, 10H), 3.53 – 3.44 (m, 2H), 3.43 – 3.37 (m, 2H), 3.28 – 3.22 (m, 1H), 2.91 (s, 2H), 2.89 –

2.85 (m, 1H), 2.85 – 2.82 (m, 1H), 2.80 – 2.75 (m, 1H), 2.75 – 2.69 (m, 3H), 2.30 – 2.12 (m,

5H), 2.10 – 1.92 (m, 2H), 1.89 – 1.77 (m, 3H).

LCMS: expected mass for [M+H]+ (C45H57BrN8O12), 981.33 m/z; found, 981.10 m/z.

5. Spectral Analysis of compounds

1H NMR Analysis of 2

LCMS Analysis of 2

1H NMR Analysis of UNC6587

LCMS Analysis of UNC6587

References

1. Buss H, Dorrie A, Schmitz ML, Hoffmann E, Resch K, Kracht M. Constitutive and interleukin-1-inducible phosphorylation of p65 NF-{kappa}B at serine 536 is mediated by multiple protein kinases including I{kappa}B kinase (IKK)-{alpha}, IKK{beta}, IKK{epsilon}, TRAF family member-associated (TANK)-binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATA-binding protein-associated factor II31-mediated interleukin-8 transcription. The Journal of biological chemistry 2004;279(53):55633-43 doi 10.1074/jbc.M409825200.

2. Kiessling MK, Schuierer S, Stertz S, Beibel M, Bergling S, Knehr J, et al. Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening. BMC genomics 2016;17(1):723 doi 10.1186/s12864-016-3042-2.

3. Zhang J, Wu T, Simon J, Takada M, Saito R, Fan C, et al. VHL substrate transcription factor ZHX2 as an oncogenic driver in clear cell renal cell carcinoma. Science 2018;361(6399):290-5 doi 10.1126/science.aap8411.

4. Cho H, Du X, Rizzi JP, Liberzon E, Chakraborty AA, Gao W, et al. On-target efficacy of a HIF-2alpha antagonist in preclinical kidney cancer models. Nature 2016;539(7627):107-11 doi 10.1038/nature19795.

5. Chakraborty AA, Nakamura E, Qi J, Creech A, Jaffe JD, Paulk J, et al. HIF activation causes synthetic lethality between the VHL tumor suppressor and the EZH1 histone methyltransferase. Science translational medicine 2017;9(398) doi 10.1126/scitranslmed.aal5272.

6. Bakhoum SF, Ngo B, Laughney AM, Cavallo JA, Murphy CJ, Ly P, et al. Chromosomal instability drives metastasis through a cytosolic DNA response. Nature 2018;553(7689):467-72 doi 10.1038/nature25432.

7. Zhang Q, Gu J, Li L, Liu J, Luo B, Cheung HW, et al. Control of cyclin D1 and breast tumorigenesis by the EglN2 prolyl hydroxylase. Cancer cell 2009;16(5):413-24 doi 10.1016/j.ccr.2009.09.029.

8. Crew AP, Raina K, Dong H, Qian Y, Wang J, Vigil D, et al. Identification and Characterization of Von Hippel-Lindau-Recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1. Journal of medicinal chemistry 2018;61(2):583-98 doi 10.1021/acs.jmedchem.7b00635.

Supplementary Figure S1

293T

HA-VHL

HA

TBK1

pTBK1

Tubulin

＋

STING

pSTING

TBK1

sg

Ctr

l

sg

TB

K1

-2

Tubulin

Kinaseassay

LysisInput

UMRC2

UMRC2 lysis

Tubulin

DM

SO

DM

OG

DF

O

HIF2α

786-O

DM

SO

VHLEV

HA-VHL

TBK1

pTBK1

RCC4

1%O2－

Tubulin

TBK1

pTBK1

VHL

HA-VHL

HIF2α

＋ － ＋

EV

786-O UMRC2

ARNT

TBK1

pTBK1

Tubulin

sgARNTctrl sg1 sg2 ctrl sg1 sg2

Tubulin

TBK1

pTBK1

sg1 sg2

HIF2α

sgHIF2α

786-O

ctrl

A B C

D E

G H I

F

Tubulin

TBK1

pTBK1

HIF1α

DM

OG

DF

O

Caki-1

TBK1

pTBK1

Tubulin

HIF2α

VHL

FG

45

92

DF

O

FG

45

92

DF

O

FG

45

92

DF

O

RCC4VHL 786-OVHL UMRC2VHL

IKKε

pIKKε

Kinaseassay

Input

TBK1

STING

VHL

pTBK1

Tubulin

pSTING

UMRC2VHL

DM

OG

DF

O

HIF2α

J

RCC4 786-O UMRC2

Tubulin

VHL

IKKε

pIKKε

HIF2αVHLEV VHLEV VHLEV

IL-1

be

ta p

g/m

l

K

Supplementary Figure S1. VHL interacts with TBK1 and suppresses TBK1 activity.

A, Immunoblots of lysates from 293T cells transfected with HA-VHL as indicated.

B, Immunoblots of kinase assay samples using lysates from UMRC2 cells infected with

lentivirus encoding either sgCtrl or sgTBK1 as kinase and STING protein as substrate.

C, Immunoblots of lysates from RCC4, 786-O, UMRC2 and UMRC6 cells restored with

empty vector (EV) or VHL.

D, Quantification of IL-1β level by ELISA from conditional medium of indicated cell lines.

E-G, Immunoblots of lysates from indicated cells treated with hypoxia (1% O2), DMOG

(1mM), DFO (200μM) or FG4592 (200μM) overnight as indicated.

H-I, Immunoblots of lysates from 786-O or UMRC2 cells infected with lentivirus encoding

either Ctrl sgRNA, HIF2 α sgRNA or ARNT sgRNA.

J, Immunoblots of lysates from 786-OEV/VHL cells treated with DMOG (1mM) or DFO

(200μM) overnight as indicated.

K, Immunoblots of kinase assay samples using lysates from UMRC2 cells treated with

DMSO, DMOG (1mM) or DFO (200μM) as kinase and STING protein as substrate.

FLAG-VHL

FLAG-EglN3

GST-TBK1

FLAG-EglN2

＋

＋

＋

FLAG

GST

IP F

LA

G

GST

FLAG

＋

FLAG-EglN1

Tubulin

Inp

ut

＋ ＋ ＋＋＋

293T

＋

＋

HA-EglN1

DFO

Tubulin

TBK1

pTBK1

HA

＋

293T

scr 1044 1045

DMOG＋ ＋ ＋

Tubulin

TBK1

pTBK1

EglN1

HIF1α

shEglN1

293T

be

ad

s

HA-EglN1

GST-TBK1＋＋

0.3

% I

np

ut

Input: HA-EglN1

GST-TBK1

HA-VHL

GST pull-down

＋

Tubulin

TBK1

pTBK1

HA

＋ ＋ HA-EglN1

FG4592

Caki-1

HIF1α

＋

A B C

D E F

MEF

Tubulin

TBK1

pTBK1

EglN1

EglN2

EglN3

EglN1+/+ -/-

EglN2

+/+ -/-

+/+ -/-

Supplementary Figure S2

Supplementary Figure S2. EglN1 interacts with TBK1 and suppresses TBK1

activity.

A, Immunoblots of whole cell lysates (input) and immunoprecipitations (IP) from

293T cells transfected with indicated plasmids.

B, Immunoblots of lysates from wild type MEF cells and MEF cells with EglN1, EglN2

or EglN3 knock out respectively. Arrow head indicate EglN2 band.

C, Immunoblots of lysates from 293T cells infected with lentivirus encoding either

scramble shRNA (scr) or EglN1 shRNA and treated with DMOG (1mM) overnight as

indicated.

D-E, Immunoblots of lysates from Caki-1 and 293T cells transfected with indicated

plasmid and treated with FG4592 (200μM) or DFO (200μM) overnight.

F, Immunoblots of GST pull-down samples as indicated.

Kinase domain

Pro48A

B

Pro48

Positions within

proteins

Hydroxyl (P)p-value (t-test)

TBK1/TBK1+DMOG

678 HTMTPIYP(hydroxyl)SSNTLVEMTLGMK 0.0004 ∞48 VFNNISFLRP(hydroxyl)VDVQMREFEVLK 0.0394 1.623306675 HTMTP(hydroxyl)IYPSSNTLVEMTLGMK 0.1457 1.344338528 LSP(hydroxyl)GGSLADAWAHQEGTHP(0.105)K 0.4226 0182 ELEDDEQFVSLYGTEEYLHP(hydroxyl)DMYER 0.4591 1.246946544 LSPGGSLADAWAHQEGTHP(hydroxyl)K 0.5284 1.193486378 TTEENP(hydroxyl)IFVVSR 0.746 1.048734

Supplementary Figure S3

Pro678

C

TBK1IKKε

Pro48

D

KVFNNISFLRPVDVQMREFEVL KVFNNISFLRPVDVQMREFEVL KVFNSISFLRPVDVQMREFEVL

KVFNNLSFLRPADVQMREFEVL

KVFNNLSFLRPLDVQMREFEVL

Pro485040

H. sapines

M. musculus

G. gallus

X. laevis

D. rerio

E

Supplementary Figure S3. Identification of TBK1 Pro48 as a hydroxylation site.

A, Mass spectrometry analysis result suggested significant decreased hydroxylation

level on TBK1 Pro48 and Pro678.

B, MS/MS spectrum for identified hydroxylated TBK1 peptides at Pro48 and Pro678.

C, Crystal structure of the TBK1 dimer (PDB ID: 4IM0). The two subunits in the dimer

are colored pink and green respectively. Pro48 (highlighted) is located in the loop prior

to helix C in the kinase domain and largely exposed to solvent.

D, Amino acid sequence alignment between human IKKε and TBK1 performed by

Vector NTI suggested Pro48 only existed in TBK1.

E, Amino acid sequence alignment between TBK1 of several vertebrates performed by

Vector NTI suggested sequences surrounding Pro48 is highly conserved.

Supplementary Figure S4

A B

TubulinIP

TB

K1

EV VHL

TBK1

TBK1

VHL

Ub

UMRC2

HIF2α

Inp

ut

IP H

IF2

α

Tubulin

VHL

Ub

HIF2α

HIF2α

UMRC2

EV VHL＋ ＋ MG132

Inp

ut

Supplementary Figure S4. VHL doesn’t induce TBK1 ubiquitination. A-B, Immunoblots of whole cell lysates (input) and immunoprecipitations (IP) from UMRC2 cells.

A BCtrl sg1 Ctrl sg2 sg3sgTBK1

VHL

EV

RC

C4TBK1

VHL

Ctrl sg2 sg3 sgTBK1

HA-VHL

EV

Ctrl sg2 sg3

VHL

Ctrl sg1

EV

Ctrl sg1

HIF2α

RCC4Tubulin

Supplementary Figure S5

Supplementary Figure S5. Loss of TBK1 selectively suppresses VHL null ccRCC cell growth. A-B, Immunoblots of lysates and crystal violet staining of RCC4EV/VHL cells infected with lentivirus encoding either Ctrl sgRNA or TBK1 sgRNA. C-D, Immunoblots of lysates and crystal violet staining of HKC and 293T cells infected with lentivirus encoding either Ctrl sgRNA or TBK1 sgRNA. E, Representative crystal violet staining and 3-D soft agar growth pictures of UMRC2 cells infected with lentivirus encoding FLAG-TBK1 or FLAG-TBK1-K38A. F-G, Representative 3-D soft agar growth and quantification of UMRC2EV/VHL cells treated with 4μM CMPD1 (duplicate wells). H, Crystal violet staining of RCC4EV/VHL cells treated with BX-795 (1.4μM) or MRT-67307 (1.4μM). I-J, Representative 3-D soft agar growth and quantification of UMRC2 cells treated with 4μM CMPD1, 2uM PT2399 or combined CMPD1 and PT2399 (duplicate wells). Error bars represent SEM, ***P<0.001, n.s. denotes no significance.

vec TBK1-WT TBK1-K38A

UMRC2

C D E

UM

RC

2

CMPD1

EV

VHL

CMPD1DMSODMSO

1mm

F

EV

VHL

BX-795

RCC4

DMSO MRT-67307

RCC4

DMSO

Tubulin

TBK1

sgTBK1Ctrl sg1 sg2 sg3

HKC

293T

Tubulin

TBK1

sgTBK1Ctrl sg1 sg2 sg3

sg1 sg2 sg3CtrlsgTBK1

HKC

293T

G H

DMSO CMPD1 PT2399 CMPD1+PT2399

1mm

UM

RC

2

I J

DMSO

CMPD1

PT2399

CMPD1+

PT2399

0

200

400

600

800

1000 n.s.***

UMRC2Tubulin

TBK1

UNC6587/μM

IKKε

0 0.01 0.03 0.1 0.3 1 3 10 30

Tubulin

TBK1

UNC6587/μM0 0.01 0.03 0.1 0.3 1 3 10

UMRC6

IKKε

30

A

B C

D E F

Tubulin

TBK1

VHL

UNC6587

Cereblon

UMRC2

＋＋

EV VHL

HIF2α

IKKε

DMSO UNC65870.0

0.5

1.0

1.5 EVVHL

*

Fo

ld c

han

ge

Supplementary Figure S6

Supplementary Figure S6. Depletion of TBK1 by UNC6587 selectively suppresses VHL null ccRCC cell growth. A, Molecular structure of TBK1 PROTAC (UNC6587). B-C, Immunoblots of lysates from UMRC6 and UMRC2 cells treated with different dosage of TBK1 PROTAC (UNC6587) as indicated for 24hr. D-F, Immunoblots of lysates, representative 3-D soft agar growth and quantification of cell colony numbers (duplicate wells) of UMRC2EV/VHL cells treated with 3μM TBK1 PROTAC (UNC6587). Error bars represent SEM, *P<0.05.

DMSO UNC6587

EV

VHL

UM

RC

2

DMSO UNC6587

1mm

Supplementary Figure S7

Supplementary Figure S7. Representative VHL staining pictures of tumor and paired normal tissues from 10 pairs of ccRCC with normal VHL sequence.

1

2

3

4

6

7

8

9

105

Normal Tumor Normal Tumor

Tubulin

TBK1

STING

pTBK1

786-O

shSTINGCtrl sh1 sh2

UMRC6

Ctrl sh1 sh2

A B

sg2 sg3Ctrl

UMRC6 UMRC2

sgTBK1

pS396(IRF3)

IRF3

Tubulin

TBK1

sg1 sg2Ctrl

C

Supplementary Figure S8

Supplementary Figure S8. The function of TBK1 in ccRCC is independent of innate immune and Akt signaling. A-C, Immunoblots of lysates from UMRC6, UMRC2 and 786-O cells infected with lentivirus encoding indicated sgRNA or shRNA. C, experiments were performed in medium containing 1%FBS.

pT308(Akt)

pS473(Akt)

Akt1

Tubulin

TBK1

sgTBK1

UMRC2

sg1 sg2Ctrl

UMRC6

sg2 sg3Ctrl

p62

pS366(p62)

CMPD1 3h

r

0 15

m

1h

r

786-O Tubulin

3h

r

0 15

m

1h

r

UMRC2

GST-TBK1WT K38A

＋＋＋ HA-p62

GST

HA

Tubulin

293T

F

UMRC6

Ctrl sg2

＋＋

sgTBK1-2sg3

＋

Tubulin

TBK1

p62

LC3 I

NH4Cl

LC3 II

G

A

C

D E

B

TBK1

GST-p62

pS366

GST-p62

TBK1

＋＋

＋＋

In vitro kinase assay

EV VHL

BA1＋＋

TBK1

VHL

pTBK1

Tubulin

p62

HIF2α

UMRC6

Supplementary Figure S9

Supplementary Figure S9. TBK1 phosphorylates p62 on Ser366 and promotes p62 protein stability. A, Heatmap of the significantly changing (ANOVA p-value<0.05, log2 fold change ±0.5) phosphopeptides identified in the global quantitative phosphoproteomic analysis.B, Immunoblots of samples from in vitro kinase assay with cold ATP as indicated. C, MS/MS spectrum for identified phosphorylated p62 peptide at Ser366. D, Immunoblots of lysates from 786-O and UMRC2 cells treated with 1μM CMPD1 for indicated time points. E, Immunoblots of lysates from 293T cells transfected with indicated plasmids. F, Immunoblots of lysates from UMRC6 cells infected with lentivirus encoding either Ctrl or TBK1 sgRNA and then treated with NH4Cl (10mM) as indicated. G, Immunoblots of lysates from UMRC6EV/VHL cells treated with DMSO or 10nM BA1 overnight.

Normal

Tumor

0

10

20

30

40100

200

300

400 **n=45

Re

lati

ve

p6

2 le

ve

l

Normal

Tumor

0

5

10

15

2020406080 ***

n=45

Re

lati

ve

p6

2 le

vel

TMA1 TMA2

Supplementary Figure S10

A B

Supplementary Figure S10. p62 protein level increases in ccRCC tumor tissues. A-B, Quantification of relative p62 protein level of tumor and paired normal tissues

ccRCC TMA1 (A) and TMA2 (B). Error bars represent SEM, **P<0.01, ***P<0.001

(Wilcoxon matched pairs test).