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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).
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