Cutting Edge: Inhibiting TBK1 by

Compound II Ameliorates Autoimmune

This information is current as
of January 5, 2022.
Disease in Mice
Maroof Hasan, Nicole Dobbs, Shaheen Khan, Michael A.
White, Edward K. Wakeland, Quan-Zhen Li and Nan Yan
J Immunol 2015; 195:4573-4577; Prepublished online 2
October 2015;
doi: 10.4049/jimmunol.1500162
http://www.jimmunol.org/content/195/10/4573

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Supplementary http://www.jimmunol.org/content/suppl/2015/10/02/jimmunol.150016
Material 2.DCSupplemental
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Cutting Edge
Cutting Edge: Inhibiting TBK1 by Compound II
Ameliorates Autoimmune Disease in Mice
Maroof Hasan,*,† Nicole Dobbs,*,† Shaheen Khan,‡ Michael A. White,x
Edward K. Wakeland,‡ Quan-Zhen Li,‡ and Nan Yan*,†
TANK-binding kinase 1 (TBK1) is a serine/threonine
protein kinase that plays a crucial role in innate immu-
nity. Enhanced TBK1 function is associated with auto-
immune diseases and cancer, implicating the potential
benefit of therapeutically targeting TBK1. In this article,
we examined a recently identified TBK1 inhibitor Com-
pound II on treating autoimmune diseases. We found
that Compound II is a potent and specific inhibitor of
TBK1-mediated IFN response. Compound II inhibited
polyinosinic-polycytidylic acid–induced immune activa-
tion in vitro and in vivo. Compound II treatment also
ameliorated autoimmune disease phenotypes of Trex12/2
mice, increased mouse survival, and dampened the IFN
gene signature in TREX1 mutant patient lymphoblasts. In
addition, we found that TBK1 gene expression is elevated
in systemic lupus erythematosus patient cells, and sys-
temic lupus erythematosus cells with high IFN signature
responded well to Compound II treatment. Together,
our findings provided critical experimental evidence
for inhibiting TBK1 with Compound II as an effec-
tive treatment for TREX1-associated autoimmune dis-
eases and potentially other interferonopathies. The
Journal of Immunology, 2015, 195: 4573–4577.
T inal vasculopathy with cerebral leukodystrophy (13). Trex12/2
he innate immunity is the first line of defense against
invading pathogens. Our cell encodes several pattern
recognition receptors that recognize specific compo-
nents of pathogens and activate appropriate immune responses
(1). Many innate immune signaling pathways converge to
a key protein TANK-binding kinase 1 (TBK1), which orches-
trates the induction of IFN and inflammatory genes that are
critical mediators of immune defense (2, 3). Human TBK1
haploinsufficiency is associated with herpes simplex encephalitis
(4), demonstrating its importance in antiviral response. Pattern
recognition receptors can also recognize self-ligands that accu-
*Department of Internal Medicine, University of Texas Southwestern Medical
Center, Dallas, TX 75390; †Department of Microbiology, University of Texas
Southwestern Medical Center, Dallas, TX 75390; ‡Department of Immunology,
University of Texas Southwestern Medical Center, Dallas, TX 75390; xDepartment
of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX
75390
ORCIDs: 0000-0003-4640-6949 (M.H.); 0000-0002-1472-8129 (S.K.); 0000-0002-
7107-0992 (E.K.W.).
Received for publication February 2, 2015. Accepted for publication September 8, 2015.
This work was supported by the National Institutes of Health (Grants AI098569 and
AR067135 to N.Y.) and the Alliance for Lupus Foundation (to N.Y.).
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500162
The Journal of
Immunology
mulate inappropriately, leading to autoimmune and inflam-
matory diseases through immune pathways that often depend
on TBK1 (5, 6). TBK1 is a member of the IkB kinase (IKK)
family and is ubiquitously expressed. In addition to its role in
innate immunity, TBK1 also plays an important role in onco-
genic transformation through direct regulation of Akt survival
signaling (7). Therefore, TBK1 represents an attractive thera-
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peutic target for autoimmune diseases and cancer with under-
lying hyperactive TBK1 signaling. The in vivo importance of
TBK1 in autoimmune disease models has been hampered by
the apparent lethality of Tbk12/2 mouse (8, 9). Small molec-
ular inhibitors of TBK1 (e.g., BX795) often lack specificity,
thereby limiting their application (5). We have recently iden-
tified a 6-aminopyrazolopyrimidine derivative (Compound II)
through a biochemical screen of small-molecule inhibitors of
TBK1 and demonstrated its effectiveness on limiting cancer cell
proliferation (7). In this study, we examined Compound II on
treating autoimmune diseases.
Trex1 deficiency causes autoimmune and inflammatory
disease phenotypes in mice (10, 11), and we recently deter-
mined that the underlying immune activation depends on
TBK1 (12). TREX1 mutations in humans are also associated
with a spectrum of autoimmune and inflammatory pheno-
types, including Aicardi–Goutières syndrome (AGS), familial
chilblain lupus, systemic lupus erythematosus (SLE), and ret-
or TREX1 mutant patient cells induce cell-intrinsic activation
of IFN-stimulated genes (ISGs) that are critically dependent
on TBK1 and a downstream transcriptional factor IRF3 (12).
Besides the TREX1 disease model, TBK1 and its associated
IFN signaling pathway have also been implicated in the
pathogenesis of complex SLE and other autoimmune diseases
collectively called interferonopathies (14, 15). No effective
treatment is available for these diseases. Thus, there is an ur-
gent need for developing novel therapeutics based on molec-
ular understandings of these diseases.
Address correspondence and reprint requests to Dr. Nan Yan, University of Texas
Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390.
E-mail address: nan.yan@utsouthwestern.edu
The online version of this article contains supplemental material.
Abbreviations used in this article: AGS, Aicardi–Goutières syndrome; AST, aspartate
aminotransferase; BMDM, bone marrow–derived macrophage; IKK, IkB kinase; ISG,
IFN-stimulated gene; poly(I:C), polyinosinic-polycytidylic acid; qRT-PCR, quantita-
tive RT-PCR; SLE, systemic lupus erythematosus; TBK1, TANK-binding kinase 1.
This article is distributed under The American Association of Immunologists, Inc.,
Reuse Terms and Conditions for Author Choice articles.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
4574 CUTTING EDGE: INHIBITING TBK1 AMELIORATES AUTOIMMUNE DISEASE
Materials and Methods
Cells and mice
Trex1+/2 mice and TREX1-R114H patient lymphoblasts were described
previously (12). SLE cell lines were obtained from Oklahoma Medical Re-
search Foundation. Leukocyte subsets microarray analysis of healthy control
and SLEs were described by Becker et al. (14). Cells were maintained in
DMEM or RPMI 1640 with 10% (v/v) heat-inactivated FCS, 2 mM
L-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate (complete
DMEM) with the addition of 100 U/ml penicillin, 100 mg/ml streptomycin
and cultured at 37˚C with 5% CO2. Bone marrow–derived macrophages
(BMDMs) were generated as described previously (12). Experiments in WT
mice with Compound II and polyinosinic-polycytidylic acid [poly(I:C)] were
done by i.p. injections of DMSO or Compound II (10 mg/kg) for 3 d fol-
lowed by one i.p. injection of poly(I:C) (300 mg in 100 ml vol of PBS) and
isolated peritoneal cells after 2 h. Experiments with Trex12/2 mice were
started when the mice were 4 wk old. Trex12/2 mice were treated with
DMSO or Compound II (i.p. injections 3 d/wk) for 7 wk. Stocks of DMSO
and Compound II were further diluted in PBS to obtain 100 ml total volume
per injection for each mouse. Mouse serum and tissues were harvested after
indicated weeks of treatment. Mouse tissues were fixed in 4% PFA followed
by standard H&E staining for histology analysis. Experiments involving
human and mouse materials were approved by the Institutional Animal Care
and Use Committee and Institutional Review Board of University of Texas
Southwestern Medical Center.
Reagents and Abs
TRI Reagent (Invitrogen) was used for RNA isolation. Compound II was
chemically synthesized (7). Abs used in this study include anti–phospho–
IRF-3 (Ser396; Cell Signaling), anti-HMGB1 (Abcam), anti-Tubulin (Sigma),
anti–phospho-TBK1 (Cell Signaling), and anti–Phospho–NF-kB (Cell Sig-
naling); secondary Abs (GE Healthcare) were used for immunoblot analysis
according to standard protocols.
Quantitative PCR array, microarray, and serum analysis
Quantitative PCR array analysis of immune gene profiles was performed as in
Hasan et al. (12) using custom-ordered PCR array plates containing primer
sets prealiquoted (Bio-Rad). Each primer set was validated by Bio-Rad and in-
house. Leukocyte subsets from healthy control and SLE subjects were ana-
lyzed by microarray (14). Mouse serum aspartate aminotransferase (AST)
level was measured by Vitros 250 at Mouse Metabolic and Phenotyping Core
(University of Texas Southwestern Medical Center). Autoantibody arrays
were performed as described previously (16).
Statistical methods
Data are presented as the mean 6 SEM. GraphPad Prism 6 was used for
statistical analysis. Statistical tests performed are indicated in the figure leg-
ends: *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.
Results and Discussion
Compound II is a potent and specific inhibitor of TBK1-mediated IFN
response
To evaluate the effect of Compound II in TBK1-mediated IFN
response, we first compared Compound II with another com-
monly used TBK1 inhibitor, BX795. We pretreated RAW264.7
(a mouse macrophage cell line) with either drug at a range of
concentration (1 nM to 10 mM) followed by poly(I:C) trans-
fection to induce IFN response. Both drugs inhibited poly(I:C)-
stimulated IRF3 activation (measured by Ifnb and Ifit1 mRNA
levels), but not NF-kB activation (measured by Tnfa mRNA
level), suggesting that Compound II specifically targets the
TBK1/IRF3 pathway (Fig. 1A, Supplemental Fig. 1A). Com-
pound II also appeared to be significantly more potent, with
a more gradual dose response and lower IC50 (20 nM) com-
pared with BX795 (IC50 of 1 mM). We also found that BX795
at high doses was toxic to RAW264.7 cells, whereas Compound
II did not appear to be toxic across the entire the dose range
(Supplemental Fig. 1B).
We also analyzed phosphorylation of TBK1 substrates to
gain more molecular insights on the mechanism of inhibition.
Poly(I:C) transfection activates both IRF3 and NF-kB path-
ways in RAW264.7 cells as indicated by robust phosphory-
lation of TBK1, IRF3, and NF-kB. Compound II completely
inhibited TBK1 autophosphorylation (17) and significantly
delayed IRF3 phosphorylation without affecting either total
protein level (Fig. 1B). In contrast, BX795 (at the same dose
as Compound II, 1 mM) only partially inhibited IRF3 phos-
phorylation and had no effect on TBK1 autophosphorylation.
Neither drug inhibited NF-kB phosphorylation. These data
suggest that Compound II is a potent and specific inhibitor of
TBK1-mediated IFN response.
Compound II inhibits poly(I:C)-induced immune activation in vitro
and in vivo
We next examined whether Compound II also inhibits im-
mune activation of TBK1 in BMDMs or mice stimulated
with poly(I:C) through the TLR pathway. Poly(I:C) activates
TLR3, which signals through TBK1 and IRF3 to activate IFN
and inflammatory cytokines. We pretreated BMDMs with
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DMSO or Compound II overnight, then stimulated with
poly(I:C) for 2 h and measured IRF3 phosphorylation as a
marker for TBK1 activation. Compound II dramatically re-
duced poly(I:C)-induced IRF3 phosphorylation (Fig. 2A). We
also treated wild type C57BL/6 mice with DMSO or Com-
pound II by i.p. injections for 3 d, followed by poly(I:C)
stimulation (i.p) for 2 h, and measured immune activation in
peritoneal macrophages. Compound II significantly reduced
poly(I:C)-induced immune gene activation and IRF3 phos-
phorylation compared with DMSO control (Fig. 2B, 2C).
These data demonstrate that Compound II is a potent in-
hibitor of immune activation of TBK1 in vitro and in vivo.
Compound II ameliorate autoimmune disease phenotypes of Trex12/2
mice and TREX1-mutant patient cells
We previously showed that Trex1 deficiency induces cell-
intrinsic immune activation that depends on TBK1 and
IRF3 (12). We then asked whether Compound II could
suppress the immune gene signature in Trex12/2 cells. We
treated wild type and Trex12/2 cells with DMSO or Com-
pound II and measured immune gene activation by quantita-
tive RT-PCR (qRT-PCR). Ifit1 mRNA expression is elevated
90-fold in Trex12/2 cells compared with wild type cells, and
Compound II reduced Ifit1 mRNA level in Trex12/2 cells in
a dose-dependent manner (Fig. 2D). Many other ISGs were
also elevated in Trex12/2 cells compared with wild type cells
(but not IFN or inflammatory genes), and the entire ISG sig-
nature was completely suppressed by Compound II (Fig. 2E).
TREX1 patient lymphoblasts carrying R114H mutation (asso-
ciated with both AGS and SLE) display a strong ISG signature
similar to that of Trex12/2 mouse cells (12). We next treated
TREX1-R114H patient lymphoblasts and a healthy control with
DMSO or Compound II and measured expression of two ISGs,
CXCL10 and RSAD2 (Supplemental Fig. 1C). Both ISGs were
significantly reduced after Compound II treatment, suggesting
that inhibiting TBK1 by Compound II could be therapeutically
useful for treating TREX1 patients with elevated ISG signature.
We next treated Trex12/2 mice with Compound II and
examined its effect on autoimmune disease phenotypes asso-
ciated with Trex1 deficiency. Trex12/2 mice develop auto-
antibodies, splenomegaly, and liver dysfunction, and the
average survival of these mice is 8–10 wk (10, 11). We first
The Journal of Immunology 4575

TBK1-mediated IFN signaling, or it may require an earlier and

higher dose treatment. Collectively, our data suggest that tar-
geting TBK1 by Compound II is an effective treatment at
ameliorating autoimmune disease phenotypes associated with
Trex1 deficiency.
Compound II reduces immune gene signature in SLE patient cells
To further explore the potential application of Compound II
in complex autoimmune diseases such as SLE, we first ana-
lyzed the expression of TBK1 and other kinases in the IkB
family (i.e., IKKa, IKKb, and IKKe) in SLE and healthy
control primary leukocyte subsets. Three leukocyte pop-
ulations, CD4+CD3+ (T cells), CD19+CD32 (B cells), and
CD33+CD32 (myeloid cells), were sorted from PBMCs and
analyzed by microarray (14). We found that TBK1 expression

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FIGURE 1. Compound II is a potent and specific inhibitor of TBK1-
mediated IFN response. (A) qRT-PCR analysis of Ifnb and Tnfa mRNA
expression. RAW264.7 cells were pretreated with Compound II (CmpII,
same throughout) or BX795 at indicated doses (nM) overnight and trans-
fected with 0.5 mg poly(I:C) for 2 h. (B) Immunoblot analysis of TBK1,
IRF3, and NF-kB phosphorylation. RAW267.4 cells were pretreated with
Compound II or BX795 overnight at 1 mM followed by poly(I:C) trans-
fection as in (A). Protein lysates were collected at indicated time post-
transfection (top) and analyzed by immunoblot with indicated Ab (right).
Data are representative of at least two independent experiments. Error bars
represent SEM. *p , 0.05.

treated Trex12/2 mice i.p. with DMSO or Compound II

(three injections per week, 10 mg/kg per injection) and found
that overall survival of Trex12/2 mice was significantly im-
proved after 7 wk of Compound II treatment compared with
DMSO controls (Fig. 3A). Compound II–treated Trex12/2
mice also have reduced AST level in the serum (indicating
improved liver function) and reduced number of splenocytes
and spleen size compared with DMSO-treated Trex12/2 mice
(Fig. 3B, 3C). We also analyzed autoantibodies in the serum
using an autoantigen microarray that contains 95 autoanti-
gens that are commonly found in a variety of autoimmune
diseases (16). We found that Trex12/2 mice produce a wide
array of autoantibodies compared with wild type littermates
and Compound II treatment significantly reduced most of the
autoantibodies in Trex12/2 mice (Fig. 3D, 3E). Other phe-
notypes of Trex12/2 mice, such as diminished hair and reduced
mobility, were also dramatically improved after Compound II FIGURE 2. Compound II inhibits TBK1-mediated IFN signaling in vitro
treatment (data not shown). Trex12/2 mice develop severe
and in vivo. (A) Immunoblot analysis of phospho-IRF3 (pIRF3) in wild type
BMDMs stimulated with poly(I:C) after pretreatment with DMSO or
systemic inflammation in multiple organs and succumb to Compound II (1 mM). HMGB1 is the loading control. (B) Immunoblot
disease mostly caused by inflammatory myocarditis (10, 11). analysis of phospho-IRF3 (pIRF3) in peritoneal cells. C57BL/6 mice were
We found that Compound II–treated Trex12/2 mouse heart pretreated with DMSO or Compound II (10 mg/kg per injection per day) for
showed reduced inflammation and pathology compared with 3 d followed by one i.p. injection of poly(I:C). Peritoneal cells were isolated
DMSO-treated or untreated Trex12/2 heart, although inflam- 2 h after poly(I:C) stimulation. (C) qRT-PCR analysis of indicated mRNAs
mation in the liver did not appear to improve at the point of in peritoneal cells from (B). Expression value of each gene was normalized
to housekeeping gene Gapdh (same throughout). Untreated value for each
our analysis (Supplemental Fig. 2). These histological findings gene was set to 1. (D and E) qRT-PCR analysis of Ifit1 (D) and other im-
are consistent with the partial rescue we have observed, and mune genes (E) in WT and Trex12/2 MEFs treated with DMSO or Com-
suggest that some of the tissue damage caused by the inborn pound II (CmpII). Data are representative of at least three independent
genetic defect may not be completely reversible by blocking experiments. Error bars represent SEM. ****p , 0.001.
4576 CUTTING EDGE: INHIBITING TBK1 AMELIORATES AUTOIMMUNE DISEASE
FIGURE 3. Compound II ameliorates autoimmune diseases of Trex12/2
mice. (A) Survival study of Trex12/2 mice treated with DMSO or CmpII
(n = 13, mice were treated with DMSO or CmpII, 10 mg/kg per injection,
three injections per week for 7 wk). (B and C) Serum AST levels (B) and
splenocyte counts (C) from WT or Trex12/2 mice treated with DMSO or
CmpII (n= 4). (D) Serum autoantibody array analysis of WT or Trex12/2
mice treated with DMSO or CmpII (n = 4). (E) Representative autoantibody
titer data from (D). Mantel–Cox log-rank test (A). Unpaired t test (B, C, and
E). Error bars represent SEM. *p , 0.05, **p , 0.01.
was consistently elevated in SLE patient cells compared with
healthy controls (Fig. 4A), and the elevation is significant in
CD4+ and CD33+ populations. Despite that IKK family
kinases often act in similar signaling pathways, we did not
observe elevated expression of other IKK genes (IKKA, IKKB,
and IKKE) in SLEs compared with healthy controls, sug-
gesting that TBK1 or the underlying signaling pathway(s)
may be uniquely associated with SLE (data not shown).
To examine whether Compound II is effective at reducing
IFN signature in SLE patient cells, we selected several SLE
lymphoblast cell lines with low, intermediate, and high ISG
signature. We did not observe elevated expression of IFN
genes likely due to immortalization of these cell lines. SLE
cells with intermediate and high ISG signature responded well
to Compound II treatment as indicated by reduced CXCL10
mRNA expression (Fig. 4B). Together, our results identify
TBK1 as a potential target in SLE and demonstrate that
inhibiting TBK1 by Compound II could be potentially useful
for treating SLEs with high IFN signature.
In conclusion, our data demonstrated that inhibiting
TBK1 by Compound II is an effective treatment option for
a variety of autoimmune diseases with elevated IFN sig-
nature. Elevated TBK1 expression or function has been
associated with many important human immune disorders
and cancer (5–7, 14, 18). Thus, TBK1 represents an at-
tractive therapeutic target, and a potent and specific in-
hibitor of TBK1 could have broad applications. Several
small-molecule inhibitors of TBK1 including Compound
II are in various stages of development and clinical trials
for treating cancer. These drugs are potentially useful for
repurposing to treat autoimmune diseases. Prolonged in-
hibition of TBK1 may increase the possibility of viral
infections; thus, such treatments should proceed with cau-
tion or be used in combination with other antiviral thera-
pies. Our findings provided important experimental
evidence and a rational for using TBK1 inhibitors such as
Downloaded from http://www.jimmunol.org/ by guest on January 5, 2022
Compound II for treating autoimmune diseases such as
AGS, SLE, and potentially other interferonopathies, many
of which are life threatening and currently lack effective
treatment.
FIGURE 4. TBK1 expression is elevated in SLE leukocytes, and Compound
II reduces IFN signature in SLEs. (A) TBK1 expression in healthy control and
SLE leukocyte subsets (CD4+, CD19+, or CD33+, as indicated on the y-axis).
Individual population was isolated from whole PBMCs. Gene expression was
measured by microarray. The p values are shown on the top of each panel. (B)
qRT-PCR analysis of CXCL10 in SLE lymphoblasts treated with DMSO or
Compound II (CmpII). Five SLE lymphoblast cell lines were selected based
on their low (SLE1, SLE2), intermediate (SLE3, SLE4), or high (SLE5) IFN
gene signature. Each cell line was treated with DMSO or CmpII for 24 h
followed by qRT-PCR analysis of CXCL10. Data are representative of at least
three independent experiments. Error bars represent SEM. ns, not significant.
*p , 0.05, **p , 0.01, unpaired t test.
The Journal of Immunology
Acknowledgments
We thank Rolf Brekken and members of the Yan Laboratory for helpful
discussions.
Disclosures
The authors have no financial conflicts of interest.
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Supplemental Figures

Inhibiting TBK1 by Compound II ameliorates autoimmune disease in

mice

Maroof Hasan1,2, Nicole Dobbs1,2, Shaheen Khan3, Michael A. White4, Edward

Wakeland3, Quan-Zhen Li3, & Nan Yan1,2,*
1
Departments of Internal Medicine, 2Departments of Microbiology, 3Departments of
Immunology, 4Departments of Cell Biology, University of Texas Southwestern Medical
Center, Dallas, Texas, USA.

Correspondence to: N.Y. (nan.yan@utsouthwestern.edu)

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Supplemental Figure 1. Compound II is more potent and less toxic in RAW264.7 cells
compared to BX795, and reduces IFN gene signature in TREX1R114H/R114H patient
lymphoblasts. (A) Quantitative RT-PCR analysis of Ifit1 mRNA expression (as in Figure
1). RAW264.7 cells were pre-treated with Compound II or BX795 at indicated doses
(bottom) overnight, and transfected with 0.5 μg poly(I:C) for 2 hours. (B) Microscopic
images of RAW264.7 cells after overnight treatment with Compound II or BX795 at dose
indicated on the top. Arrows indicate dying cells. (C) Quantitative RT-PCR analysis of
indicated genes in healthy control (HC) and TREX1R114H/R114H lymphoblasts treated with
DMSO or Cmp II. Healthy control treated with DMSO value was set to 1. ***P < 0.005.
**** P < 0.001. Data are representative of at least three independent experiments. Error
bars, SEM.

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Supplemental Figure 2. Histology analysis of WT and Trex1-/- mice with or without

treatment. Two representative H & E staining images of heart and liver from untreated
WT or Trex1-/- mice (8-week old) or Trex1-/- mice treated with DMSO or Compound II
for 4 weeks (8-week old, as in Figure 3). Reduced inflammation and tissue pathology was
observed in Trex1-/- mice heart after Compound II treatment, but not in the liver.

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