MDL-800, an allosteric activator of SIRT6, suppresses proliferation and enhances EGFR-TKIs therapy in non-small cell lung cancer

Article abstract of DOI:10.1038/s41401-020-0442-2

Sirtuin 6 (SIRT6), a member of the sirtuin family, is a nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylase that is involved in various physiological and pathological processes. SIRT6 is generally downregulated and linked to tumorige

Full text of DOI:10.1038/s41401-020-0442-2

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ARTICLE
MDL-800, an allosteric activator of SIRT6, suppresses
proliferation and enhances EGFR-TKIs therapy in non-small
cell lung cancer
1
1
1
2
1,3,4
Jia-lin Shang , Shao-bo Ning , Ying-yi Chen , Tian-xiang Chen and Jian Zhang
+
Sirtuin 6 (SIRT6), a member of the sirtuin family, is a nicotinamide adenine dinucleotide (NAD )-dependent deacetylase that is involved
in various physiological and pathological processes. SIRT6 is generally downregulated and linked to tumorigenesis in non-small cell lung
carcinoma (NSCLC), thus regarded as a promising therapeutic target of NSCLC. In this study, we investigated whether MDL-800, an
allosteric activator of SIRT6, exerted antiproliferation effect against NSCLC cells in vitro and in vivo. We showed that MDL-800 increased
SIRT6 deacetylase activity with an EC50 value of 11.0 ± 0.3 μM; MDL-800 (10–50 μM) induced dose-dependent deacetylation of histone
H3 in 12 NSCLC cell lines. Treatment with MDL-800 dose dependently inhibited the proliferation of 12 NSCLC cell lines with IC50 values
ranging from 21.5 to 34.5 μM. The antiproliferation effect of MDL-800 was significantly diminished by SIRT6 knockout. Treatment with
MDL-800 induced remarkable cell cycle arrest at the G /G phase in NSCLC HCC827 and PC9 cells. Furthermore, MDL-800 (25, 50 μM)
0
1
enhanced the antiproliferation of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in osimertinib-resistant
HCC827 and PC9 cells as well as in patient-derived primary tumor cells, and suppressed mitogen-activated protein kinase (MAPK)
−1
−1
pathway. In HCC827 cell-derived xenograft nude mice, intraperitoneal administration of MDL-800 (80 mg · kg · d , for 14 days)
markedly suppressed the tumor growth, accompanied by enhanced SIRT6-dependent histone H3 deacetylation and decreased p-MEK
and p-ERK in tumor tissues. Our results provide the pharmacological evidence for future clinical investigation of MDL-800 as a promising
lead compound for NSCLC treatment alone or in combination with EGFR-TKIs.
Keywords: non-small cell lung cancer; SIRT6 activator; MDL-800; deacetylation; EGFR-TKIs; MAPK; ERK
Acta Pharmacologica Sinica (2020) 0:1–12; https://doi.org/10.1038/s41401-020-0442-2
INTRODUCTION
sirtuins will be helpful in cancer therapy by themselves or in
combination with different therapies [6, 8].
Lung cancer is the leading cause of cancer-related morbidity and
mortality worldwide [1, 2]. Small cell lung carcinoma (SCLC) and
non-SCLC (NSCLC) are the two major types of lung cancer. NSCLC
accounts for ~85% of all lung cancer cases and is relatively
refractory to conventional therapies (surgery, radiation, che-
motherapy, and other targeted therapies, including epidermal
growth factor receptor tyrosine kinase inhibitors [EGFR-TKIs]) with
a low 5-year overall survival rate of ~15% [3–5]. Thus, there is a
pressing need to identify novel targets and develop effective
therapeutic agents for NSCLC treatment. Mammalian sirtuins
SIRT6 is a member of the sirtuin family, which is responsible
for many physiological and pathological processes [9]. Although
the function of SIRT6 in cancers is cell context dependent and
SIRT6 plays an oncogenic role in some tumors [10–14], the
downregulation of SIRT6 has been frequently observed in many
cancer types, indicating that SIRT6 functions as a tumor
suppressor [15–20]. Emerging studies indicate that the mRNA
and protein levels of SIRT6 are generally decreased in NSCLC
tissues and cell lines and are closely related to tumor
progression and poor prognosis of patients [21, 22]. Over-
expression of SIRT6 can suppress NSCLC cell proliferation by
inhibiting Twist1 and induce a radiosensitization effect in NSCLC
(
(
SIRT1–7) are conserved nicotinamide adenine dinucleotide
NAD )-dependent class III deacetylases that play central roles in
+
various biological events, including metabolism, genome stability,
longevity, and inflammation. Recently, sirtuins have gained
increasing attention as promising targets for the treatment of
age-related diseases, including cancers, type 2 diabetes, inflam-
matory disorders, and Alzheimer’s disease [6, 7]. Several chemical
modulators of sirtuins that currently exist regulate NAD+ levels
but lack selectivity. Specific and potent activators or inhibitors of
0 1
cells, leading to the induction of G /G cell cycle arrest and
apoptosis [22, 23], which is also associated with an improved
survival rate among NSCLC patients [24]. Thus, SIRT6 can serve
as a promising therapeutic target for NSCLC.
SIRT6 contains a large hydrophobic channel that harbors a
+
substrate with an acetylated lysine and cofactor NAD , where the
1
2
State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; Shanghai Lung Cancer Center, Shanghai
3
Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030, China; Medicinal Bioinformatics Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200025,
China and School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
4
Correspondence: Tian-xiang Chen (zjutxchen@163.com) or Jian Zhang (jian.zhang@sjtu.edu.cn)
These authors contributed equally: Jia-lin Shang, Shao-bo Ning
Received: 22 March 2020 Accepted: 12 May 2020
©
CPS and SIMM 2020

A SIRT6 activator exhibits potent anti-NSCLC efficacy
JL Shang et al.
2
acyl group is removed from the lysine using NAD+ through SIRT6
deacetylation [25]. In the genomic context, SIRT6 can deacetylate
histone H3 on acetylated K9, K18, and K56 (H3K9Ac, H3K18Ac, and
H3K56Ac) [26–28], leading to the transcription inhibition of
oncogenic genes, including c-Myc [15], Hif-1α [29], and c-Jun [30],
thus implicating SIRT6 in tumorigenesis [15, 17–20, 31]. These
findings lead to the hypothesis that pharmacological activation of
SIRT6 deacetylation may exert an anticancer effect against NSCLC.
Due to the clinical relevance of SIRT6 activation in cancers, many
efforts have been directed toward the development of activators of
SIRT6. Lamin A and fatty acids have been reported as endogenous
activators of SIRT6 to increase deacetylation [32, 33]. To our
knowledge, several synthetic small molecules and natural poly-
phenols have been identified to activate the deacetylation of SIRT6,
which has been detected only in vitro [34–36]. Moreover, few potent
SIRT6 activators have been reported to elucidate the anticancer
efficacy of SIRT6 activation [37, 38]. Using AlloSite [39], we
discovered a cellularly active SIRT6 allosteric activator, MDL-800,
with high potency and selectivity [37]. It is not yet clear whether a
small-molecule SIRT6 activator could be efficient for NSCLC.
Iron powder (14.0 g, 250 mmol) was added to crude compound
3 (38.3 g, 85.88 mmol) dissolved in acetic acid at room tempera-
ture. Then, the reaction was stirred under the same conditions for
12 h. The system was filtered, and the solvent was evaporated
under reduced pressure. The solid was dissolved with 400 mL
3
ethyl acetate and washed with saturated aqueous NaHCO brine
and dried over sodium sulfate. The organic layer was evaporated
in vacuo and subsequently purified by column chromatography
on silica gel (PE:EA = 2:1) to afford 4 (27.1 g, two steps 66%) as a
white solid.
Cl
O
Cl
O
2
H N
H
N
O
H
O
N
O
+
Cl
S
H
S
Cl
S
O
O
N
Cl
O
S
O
O
F
O
O
F
Br
Br
4
5
MDL-800
To a solution of methyl 5-amino-2-(N-(5-bromo-4-fluoro-2-
methylphenyl)sulfamoyl)benzoate (4, 27.1 g, 65.2 mmol) in 50
mL pyridine, 3,5-dichlorobenzene-1-sulfonyl chloride (5, 19.07 g,
78.2 mmol) was added at 0 °C, and the reaction was stirred at the
same temperature for ~1 h. Then, the reaction was moved to
room temperature and stirred for another 6 h. The reaction was
cooled to 0 °C, and the pH was adjusted to 3–4 with 2 N
hydrochloric acid. The precipitate formed was filtered and
subsequently purified by column chromatography on silica gel
(PE:EA = 3:1) to afford MDL-800 (32.52 g, 80%) as a white solid.
Here, we focused on an allosteric activator of SIRT6, MDL-800, as
a pharmacological tool to investigate whether it would show anti-
NSCLC activity. We found that MDL-800 effectively inhibited
NSCLC cell proliferation by activating SIRT6 deacetylation and
0 1
retarding the SIRT6-mediated G /G cell cycle. Notably, MDL-800
could enhance the antiproliferation of EGFR-TKIs in NSCLC
osimertinib-resistant (OR) cells and patient-derived primary tumor
cells and inhibit the mitogen-activated protein kinase (MAPK)
pathway. Finally, MDL-800 showed favorable in vivo pharmacoki-
netic profiles and antitumor efficacy against the NSCLC xenograft
model. Overall, we demonstrate that the activation of SIRT6 can be
a promising therapy in NSCLC with clinical application prospects.
1
H NMR (400 MHz, DMSO-d6) peaks were as follows: δ 11.36 (s,
1H), 9.46 (s, 1H), 8.01 (s, 1H), 7.82–7.81 (d, J = 4.0 Hz, 2H),
7.60–7.58 (d, J = 8.0 Hz, 1H), 7.43–7.41 (m, 1H), 7.28–7.16 (m, 3H),
1
3
3.74 (s, 1H), and 1.82 (s, 3H). C NMR (100 MHz, DMSO-d6) peaks
were as follows: 167.12, 158.34, 158.91, 142.06, 141.64, 137.89
(J = 8), 135.87, 133.83, 133.81, 132.60, 132.56, 132.12 (J = 3),
1
5
C
31.30, 125.81, 120.53, 138.78, 138.62, 138.39, 104.78 (J = 21),
3.64, and 17.32. HRMS (ESI+) m/z calculated for
+
MATERIALS AND METHODS
21
H
16BrCl
2
FN
2
O
6
S
2
(M + H) is 623.8994 and experimentally
Cloning, expression, and purification of wild-type (WT) SIRT6
According to previously described methods [37], WT full-length
human SIRT6 was inserted into the pET28a-LIC vector (Addgene
plasmid no. 26094). The plasmid was transformed into Escherichia
coli BL21(DE3) cells. Protein was purified using a nickel column and
gel filtration. Purified protein was dialyzed into the assay buffer (50
determined as 624.9072.
Cl
Cl
O
H
O
N
O
N
S
O
Cl
S
Cl
H
O
O
O
O
N
N
O
S
S
O
O
O
F
F
Br
Br
mM Tris-HCl [pH 8.0], 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl
2
)
MDL-800
MDL-800 NG
and used in all in vitro assays performed in this study.
A solution of MDL-800 (100 mg, 0.16 mmol) in DMF was stirred
at 0 °C. NaH (19 mg, 60%, 0.48 mmol) was added at the same
temperature, and the reaction was stirred for another 1 h under
the same conditions. Then, MeI (68 mg, 0.48 mmol) was added,
and the reaction was moved to room temperature and stirred for
another 4 h. Then, 50 mL ethyl acetate was added to the reaction,
Synthesis of target compounds
O
2
O N
O
O
H
2
H N
2
O N
O
Cl
N
O
+
S
F
O
S
Br
O
O
F
Br
and the mixture was washed with saturated aqueous NaHCO
3
1
2
3
brine and dried over sodium sulfate. The organic layer was
evaporated in vacuo and subsequently purified by column
chromatography on silica gel (PE: EA = 3:1) to afford MDL-
To a solution of 5-bromo-4-fluoro-2-methylaniline (1, 20 g, 98.5
mmol) in 50 mL pyridine was added methyl 2-(chlorosulfonyl)-5-
nitrobenzoate (2, 33.0 g, 118.2 mmol) at 0 °C, and the reaction was
continuously stirred at 0 °C for 1 h. Then, the reaction was moved to
room temperature and stirred for another 8 h. The reaction was
cooled to 0 °C, and the pH was adjusted to 3–4 with 1 N
hydrochloric acid. The precipitate formed was filtered, washed with
water, and dried to yield crude compound, which was directly used
in the next step without being purified.
1
800NG (68 mg, 63%) as a white solid. H NMR (500 MHz, DMSO-
d6) peaks were as follows: δ 8.09 (s, 1H), 7.68–7.53 (m, 5H),
7.40–7.38 (d, J = 10 Hz, 1H), 7.20–7.19 (d, J = 5 Hz, 1H), 3.66 (s, 3H),
13
3.27–3.19 (d, 6H), and 2.11 (s, 3H). C NMR (125 MHz, DMSO-d6)
peaks were as follows: 166.90, 159.14, 157.18, 144.98, 141.14 (J =
9), 138.82, 136.69, 135.94, 134.31, 134.21, 134.05, 133.65, 130.80,
128.11, 126.31, 125.35, 119.00 (J = 10), 53.35, 39.45, 38.04,
and 17.75. LRMS (ESI+) 652.9. HRMS (ESI+) m/z calculated
+
O
O
for
C
23
H20BrCl
2
FN
2
O
6
S
2
(M + H)
is
651.9307
and
O
2
N
2
H N
O
H
O
H
experimentally determined as 652.9383. (The HPLC UV spectral
purity of MDL-800NG was >95%.) The synthesis and spectral data
of MDL-800 were the same as previously reported [37]. The
spectral data for MDL-800NG are depicted in Supplementary
Information Fig. S1.
N
O
N
O
S
S
O
O
F
F
Br
Br
3
4
Acta Pharmacologica Sinica (2020) 0:1 – 12

A SIRT6 activator exhibits potent anti-NSCLC efficacy
JL Shang et al.
3
Scheme 1 The synthetic procedures of MDL-800 and MDL-800NG. Reagents: a pyridine, room temperature; b Fe, CH
room temperature; d NaH, MeI, DMF, 0 °C to room temperature.
3
COOH, 50 °C; c pyridine,
Fluor de Lys (FDL) assay
Academy of Sciences. The NCI-H358, NCI-H1299, NCI-H1650,
NCI-H23, NCI-H1792, NCI-H520, NCI-H460, NCI-H1975, HCC827,
and PC9 cell lines were maintained in RPMI-1640 medium
supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitro-
gen). Calu-1 cells were maintained in McCoy’s 5A modified
medium supplemented with 10% (v/v) FBS. A549 cells were
maintained in F-12K medium supplemented with 10% (v/v) FBS.
All cell lines were cultured according to the standard cell
culturing protocols of the American Type Culture Collection and
WT SIRT6 protein (5 μM) was incubated in a 50-μL reaction mixture
+
(
2.5 mM NAD , 75 μM RHKK-Ac-AMC, compounds/DMSO, and assay
buffer) at 37 °C for 2 h; the reaction was stopped by 40 mM
nicotinamide, and the mixture was incubated with 6 mg/mL trypsin
for 30 min at 25 °C. The fluorescence intensity was measured with a
microplate reader (Synergy H4 Hybrid Reader, BioTek) at excitation
and emission wavelengths of 360 and 460 nm, respectively. EC50
values were calculated by fitting the data points with the
dose–response function in GraphPad Prism V7. Each experiment
was independently performed three times in technical triplicates.
2
were maintained in a humidified 37 °C incubator with 5% CO .
The cell lines were authenticated by short tandem repeat
profiling.
Clinical sample collection
Clinical patient samples were collected in the Shanghai Lung Cancer
Center, Shanghai Chest Hospital, Shanghai Jiao Tong University
School of Medicine. Individual NSCLC tissue was isolated from a 45-
year-old female patient diagnosed with pulmonary invasive
adenocarcinoma of the left lower lobe in 2019. The study was
approved by the Ethics Committee of Shanghai Chest Hospital,
Shanghai Jiao Tong University School of Medicine. Informed written
consent was obtained from the patient.
Establishment of OR NSCLC cells
OR HCC827 and NCI-H1975 cells were established and maintained
as described previously [40–42]. In short, 1 × 10 HCC827 or NCI-
6
H1975 cells/mL were seeded in a six-well plate and incubated in
RPMI-1640 medium containing osimertinib (TargetMol, China).
Cells were grown in culture medium containing increasing
concentrations of osimertinib for 72 h with a recovery period
between treatments. After 6 months of passage, the remaining
HCC827 or NCI-H1975 cells that grew in the presence of 1 or 4 μM
osimertinib, respectively, were considered OR cells, while the
parental NCI-H1975 and HCC827 cell lines were only maintained in
osimertinib-free medium.
Cell culture
All NSCLC cell lines were obtained from the Cell Resource Center
of the Shanghai Institute for Biological Sciences, Chinese
Acta Pharmacologica Sinica (2020) 0:1 – 12

A SIRT6 activator exhibits potent anti-NSCLC efficacy
JL Shang et al.
4
Fig. 1 MDL-800 activates histone H3 deacetylation of SIRT6 in NSCLC cells. a Chemical structure of MDL-800 and MDL-800NG.
b Concentration-dependent effects of MDL-800 and MDL-800NG on the activation of SIRT6 deacetylation were assessed by FDL assays with
the acetylated peptide RHKK-Ac-AMC (75 μM). The data are shown as the mean ± SD from three or four independent experiments (MDL-800:
EC50 = 11.0 ± 0.3 μM). c Dose-dependent deacetylation effects of MDL-800 in NSCLC cell lines. Western blots of SIRT6, H3K9Ac, H3K18Ac, and
H3K56Ac levels in various NSCLC cell lines treated with the indicated concentrations of MDL-800 for 48 h. Histone H3 was used as the internal
control, and β-actin was used as the loading control.
Isolation and culture of patient-derived primary NSCLC cells
Patient-derived primary NSCLC cells were isolated from a 45-year-
old female patient diagnosed with pulmonary invasive adenocar-
cinoma of the left lower lobe in 2019. The tissues were cut into
cultured in RPMI-1640 supplemented with 10% FBS,100 U/mL
penicillin, and 100 g/mL streptomycin (Invitrogen, USA) in a
humidified atmosphere (37 °C, 5% CO ) and were used within two
2
or three passages.
3
small blocks of ~1 mm and washed extensively in PBS. Then, the
small pieces were digested with 1 mg/mL collagenase I (Solarbio,
China) for 3 h at 37 °C. Digested tissues were centrifuged at 400 ×
g for 5 min, and the pellet was washed in PBS and passed through
a 70 μm sterile cell strainer (Corning, USA).The isolated cells were
Cell viability
Cell viability was evaluated using a Cell Counting Kit-8 (CCK-8)
(Dojindo), following the manufacturer’s instructions. Briefly, NSCLC
cells were seeded (n = 3 wells/group) in 96-well plates at a density
Acta Pharmacologica Sinica (2020) 0:1 – 12

A SIRT6 activator exhibits potent anti-NSCLC efficacy
JL Shang et al.
5
into the lentiCRISPR v2 vector (Addgene plasmid no. 52961). The
lentivirus was generated by transfecting the lentiCRISPR v2 vector
containing sgRNA, psPAX2, and pMD2.G into HEK293T cells using
Neofect™ DNA transfection reagent, following the manufacturer’s
protocol. The viral supernatant was collected 48 h after transfec-
tion and used to infect the indicated cells with 8 μg/mL polybrene
(
Sigma-Aldrich). Then, the virally infected cells were selected in
medium containing puromycin (Sigma-Aldrich). Total protein was
isolated from single cell-derived colonies and assessed by Western
blotting.
Cell cycle analysis
Cells were seeded in six-well plates. After 24 h of seeding, cells
were treated with DMSO, MDL-800, or MDL-800NG for 48 h. Then,
both floating and adherent cells were collected and fixed with
7
5% ethanol at 4 °C overnight. Cells were incubated with RNase I
(
(
50 μg/mL) in 1× PBS at 37 °C for 30 min and stained with PI
propidium iodide) (50 μg/mL) for 15 min. Cells were assessed by
flow cytometry and FACS DIVA software, v6.2 (BD Biosciences). The
cell cycle distribution was analyzed using FlowJo 7.6.1 software.
Fig. 2 MDL-800 activates histone H3 deacetylation of SIRT6 in
NSCLC cells. a Western blots of SIRT6, H3K9Ac, H3K18Ac, and
H3K56Ac levels in HCC827 and PC9 cells treated with the indicated
concentrations of MDL-800NG for 48 h. b Western blots of SIRT6,
H3K9Ac, H3K18Ac, and H3K56Ac levels in vector or SIRT6-KO
HCC827 and PC9 cells treated with the indicated concentrations of
MDL-800 for 48 h. Histone H3 was used as the internal control, and
β-actin was used as the loading control.
RNA extraction and RT-qPCR
Total RNA was extracted from NSCLC cells using TRIzol reagent
(
Invitrogen), according to the manufacturer’s instructions. Then, 1
μg of total RNA was reverse transcribed to cDNA using a FastKing
RT Kit (Tiangen). The cDNA was amplified in triplicate using SYBR
Green Master Mix (Roche) in an ABI 7900 Thermal Cycler (Applied
Biosystems), following the manufacturer’s instructions. Mean cycle
threshold (Ct) values were used for the expression analysis. The
−
ΔΔCt
mRNA levels of target genes were calculated by the 2
method. The expression data were normalized to β-actin
expression levels and presented as the mean ± SEM of three
independent experiments. The RT-qPCR primer sequences are
listed in Supplementary Information Table S5.
3
of 5 × 10 cells per well. After attaching overnight, NSCLC cells
were treated with MDL-800 at various concentrations. Cells treated
with DMSO were used as positive controls. After 48 h of treatment,
CCK-8 (10 μL/well) was added and incubated for 1 h at 37 °C. To
measure the number of viable cells, the absorbance of each well
was detected at 450 nm using a microplate reader (Synergy H4
Hybrid Reader, BioTek). The relative viability of each group is
presented as the percentage change relative to the positive
control group. Each data point is the mean ± SD of two or three
independent experiments.
Combination drug treatment
Gefitinib, afatinib, and osimertinib were purchased from Target-
Mol. For cell viability assays, all drugs were dissolved in DMSO,
stored at −20 °C, and protected from light. The maximum final
DMSO concentration was less than 0.25%. Cells were treated with
MDL-800 at concentrations ranging from 25 to 50 μM with
gefitinib, afatinib, or osimertinib at the indicated concentrations.
Then, cell viability assays were conducted using the CCK-8
method. Combination effects were analyzed by the Chou–Talalay
combination index (CI) method [44]. The CI and the fraction of
cells affected values were generated using CompuSyn Version
1.0 software. CI values of <1, =1, and >1 indicate synergism,
additivity, and antagonism between the drugs, respectively.
Colony formation
Cells at a density of 300 cells/well were plated into six-well plates
and cultured in RPMI-1640 medium. Medium containing different
concentrations of MDL-800 or MDL-800NG (25 or 50 μM) and
DMSO (control) was replaced every 2 days. After 10 days, colonies
were fixed with methanol and stained with crystal violet
(
Beyotime) for 30 min. Finally, plates were washed gently with
water, images were captured with a Nikon Ti-S microscope, and
Pharmacokinetic studies in mice
colony numbers were quantified by ImageJ V4.
Pharmacokinetic studies were performed by BioDuro Inc.,
Shanghai, China, according to the standard protocol. Six-week-
old male C57BL/6J mice were grouped randomly (n = 5 mice per
group) and administered MDL-800 by a single intravenous (IV)
bolus at a dose of 45 mg/kg, intraperitoneal (IP) injection at a dose
of 65 mg/kg, or oral gavage (PO) at a dose of 90 mg/kg. Three
administration formulations were prepared in the vehicle with 5%
DMSO, 30% PEG-400, and 65% saline, and the pH was adjusted to
7.0–8.0. After treatment, mice were sacrificed, and plasma samples
were harvested at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h.
The drug concentration in plasma was analyzed by LC-MS/MS.
Pharmacokinetic parameters were calculated using Analyst Soft-
ware 1.6.3 from mean plasma concentration-time profiles. The
area under the curve (AUC) was calculated from the time versus
concentration data by the linear trapezoidal rule. The IP/PO
bioavailability was calculated as the ratio of AUC for MDL-800 from
IP/PO and IV dosages. The calculation was normalized to
relative doses.
Immunoblotting and antibodies
5
5
Cells (2 × 10 –3 × 10 cells) were seeded in six-well plates and
treated with DMSO, MDL-800, or MDL-800NG. After 48 h of
treatment, both floating and adherent cells were lysed with 1×
SDS buffer and separated by SDS-PAGE, followed by incubation
with specific primary antibodies overnight at 4 °C and then with
HRP-conjugated anti-rabbit IgG (#7074, Cell Signaling Technology)
or anti-mouse IgG (#7076, Cell Signaling Technology). An
Immobilon Western Chemiluminescent HRP Substrate Kit (Milli-
pore) was used for detection. Information regarding the primary
antibodies is shown in Supplementary Information Table S4.
Generation of CRISPR-Cas9 knockout cell lines
SIRT6-KO NSCLC cell lines were generated following a previous
protocol [43]. In brief, sgRNA targeting exon 1, CACTTG
CCCTTGTCCGCGTACGG (with the PAM underlined), was cloned
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A SIRT6 activator exhibits potent anti-NSCLC efficacy
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6
Fig. 3 MDL-800 extensively inhibits NSCLC cell proliferation. a Dose response of the viability of various NSCLC cell lines exposed to MDL-
8
00 for 48 h. Cell viability was determined by the CCK-8 assay. The viability of the drug treatment groups was normalized to the viability of the
corresponding DMSO-treated controls. The points indicate the mean ± SD of three independent experiments. b Dose response of the viability
of HCC827 and PC9 cells exposed to MDL-800 or MDL-800NG for 48 h. Relative viability was normalized to that of the DMSO controls. Error bar,
SEM of three replicates. c Dose response of the viability of vector or SIRT6-KO HCC827 and PC9 cells exposed to MDL-800. Error bar, SEM of
three replicates. d The colony formation of HCC827 and PC9 cells treated with DMSO, MDL-800, or MDL-800NG for 10 days was analyzed. The
cell colonies were stained with crystal violet and counted. The statistical analysis of the colony formation assay is shown (mean ± SEM).
e Representative images of the colony formation of HCC827 and PC9 cells treated with DMSO, MDL-800, or MDL-800NG. All P values were
determined by a two-tailed unpaired Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001).
Human NSCLC cell-derived xenograft (CDX) studies
of the mice were monitored every other day. After treatment or if
meeting the humane endpoint criteria, the mice were sacrificed
The HCC827 CDX model was established by following federal and
institutional guidelines approved by the Institutional Animal Care
and Use Committee at Shanghai Jiao Tong University School of
by CO asphyxiation. Tumors were dissected, photographed, and
2
weighed. Tumor tissues were collected and fixed in 10% neutral
6
Medicine. To establish the CDX, HCC827 cells (1 × 10 ) suspended
buffered formalin or snap frozen in liquid N
for the following analyses.
2
and stored at −80 °C
in 100 μL of 1× PBS were injected subcutaneously into the flanks
of 6-week-old male BALB/c nude mice. Mice were monitored daily,
and caliper measurements began when tumors became visible.
The tumor volumes were measured every other day using calipers.
Histological and immunohistochemical (IHC) analyses
Xenograft tumor tissues were fixed in 4% paraformaldehyde for
24 h, embedded in paraffin, and sectioned with a semiautomated
rotary microtome (Leica, RM2235). Morphology was evaluated by
hematoxylin and eosin (H&E) staining. The protein levels were
analyzed by immunohistochemistry using standard protocols
with specific primary antibodies (anti-H3K9Ac, 1:100 dilution,
ab32129, Abcam; anti-p-ERK, 1:400 dilution, 4370, Cell Signaling
Technology; anti-ERK, 1:500 dilution, 4696, Cell Signaling
Then, tumor volumes were calculated using the following formula:
2
(
L × W )/2, where L and W refer to the length and width of tumors,
3
respectively. When the mean tumor volume was ~100 mm , mice
were randomly grouped (n = 5), and treatment was initiated. Mice
received an IP injection of the vehicle alone (5% DMSO, 30% PEG-
4
00, and 65% saline, pH 7.0–8.0) or designated doses of MDL-800
every other day for 14 days. The tumor volumes and body weights
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A SIRT6 activator exhibits potent anti-NSCLC efficacy
JL Shang et al.
7
Fig. 4 MDL-800 induces cell cycle arrest in NSCLC cells. a Cell cycle distribution of HCC827 and PC9 cells treated with DMSO, 25 µM MDL-
8
00, or MDL-800NG for 48 h, and vector or SIRT6-KO HCC827 and PC9 cells exposed to DMSO and 25 µM MDL-800 for 48 h were measured by
PI staining. The data are presented as the mean ± SD of three independent experiments (**P < 0.01, two-way ANOVA). b RT-qPCR analyses
showing the mRNA levels of candidate genes in HCC827 and PC9 cells treated with DMSO, 25 µM MDL-800, or MDL-800NG for 48 h. The data
are normalized to β-actin levels and presented as the mean ± SEM of three independent experiments. P values were determined by a two-
tailed unpaired Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001, compared with the DMSO-treated group).
Technology; and anti-Ki-67, 1:200 dilution, ab15580, Abcam). All
assays were followed by staining with an HRP-conjugated
secondary antibody (anti-mouse, KIT-9702; anti-rabbit, KIT-9707,
Maixin Biotechnology), and samples were then counterstained
with H&E and mounted. Images were captured with a Nikon Ti-S
microscope.
11.0 ± 0.3 μM, while MDL-800NG did not activate SIRT6 deacetylase
activity even up to 200 μM (Fig. 1b). Next, we evaluated the cellular
histone H3 deacetylase activity of SIRT6 after MDL-800 treatment in
12 various NSCLC cell lines. As shown by Western blots in Fig. 1c,
MDL-800 could induce dose-dependent deacetylation of SIRT6-
targeted histone H3 in NSCLC cell lines. In the sensitive HCC827 and
PC9 cell lines, MDL-800 markedly enhanced histone H3 deacetyla-
tion of SIRT6 in a dose-dependent manner, but there was little effect
observed in cells treated with the negative control MDL-800NG
(Fig. 2a). To further investigate the target engagement of MDL-800
in NSCLC cells, we generated SIRT6-knockout (SIRT6-KO) HCC827
and PC9 cell lines using the CRISPR/Cas9 system. As expected, the
activating deacetylation effect of MDL-800 was not appreciable in
SIRT6-KO NSCLC cells compared with control vector cells (Fig. 2b),
indicating that MDL-800 activates SIRT6-specific histone H3
deacetylation in NSCLC cells.
Statistical analysis
All data were analyzed using Microsoft Office Excel 2016 and
GraphPad Prism V7. The graphs and error bars show the mean ±
SD of independent biological experiments unless stated other-
wise. Statistical analysis was performed using two-tailed Student’s
t tests or ANOVA unless stated otherwise. P < 0.05 was considered
statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001). The
investigators were not blinded to each experiment.
RESULTS
MDL-800 extensively inhibits NSCLC cell proliferation
MDL-800 activates histone H3 deacetylation of SIRT6 in NSCLC
cells
SIRT6 has been considered a promising therapeutic target in NSCLC
Increasing studies have demonstrated that SIRT6 can suppress
tumorigenesis by reducing the levels of H3K9Ac and H3K56Ac
[15, 17–20, 31]. To explore whether this association has a
pharmacological effect on NSCLC tumorigenesis, we assessed
the antiproliferation of MDL-800 in 12 different NSCLC cell lines.
We found that MDL-800 significantly reduced the proliferation of
various NSCLC cell lines in a dose-dependent manner (Fig. 3a),
with half-maximum inhibitory concentration (IC50) values at 48 h
ranging from 21.5 to 34.5 μM (Supplementary Information Table
S1), whereas MDL-800NG did not significantly affect the prolifera-
tion of NSCLC cells up to 80 μM (Fig. 3b). Similarly, SIRT6-KO
HCC827 and PC9 cells became more resistant to MDL-800 than the
vector control groups (Fig. 3c), supporting that MDL-800 inhibits
the proliferation of NSCLC cells by activating SIRT6 specifically. We
[
21, 22]. Having a potent and specific SIRT6 activator may provide a
unique opportunity to explore whether SIRT6 activation can be
useful as an anti-NSCLC strategy. We have identified the cellularly
active allosteric activator of SIRT6, MDL-800 [37]. To facilitate further
investigation of MDL-800 in NSCLC cells, we synthesized the
corresponding MDL-800NG as an inactivating control of MDL-800.
MDL-800NG differs from MDL-800 by only two groups (the two H
atoms in MDL-800 are changed to methyl groups in MDL-800NG)
(
Scheme 1 and Fig. 1a). FDL assays with an acetylated peptide
(RHKK-Ac-AMC) [37] showed that MDL-800 activated SIRT6 deace-
tylation with a half-maximal effective concentration (EC50) value of
Acta Pharmacologica Sinica (2020) 0:1 – 12

A SIRT6 activator exhibits potent anti-NSCLC efficacy
JL Shang et al.
8
Fig. 5 MDL-800 enhances the antitumor activity of EGFR-TKIs and suppresses the MAPK pathway in NSCLC cells. Cell viability was
assessed following 48 h of exposure to the indicated concentrations of MDL-800 alone or combined with gefitinib, afatinib, osimertinib in a
HCC827 and PC9 cells, b HCC827OR and NCI-H1975OR cells, and c NSCLC patient-derived primary cells. CI values for the various combinations
were calculated using CompuSyn Version 1.0 software. Synergism, additivity, and antagonism are defined as CI < 1, =1, and >1, respectively.
The data are presented as the mean ± SEM of three independent experiments.
also detected the long-term effect of MDL-800 on NSCLC cells by
colony formation assays. The results showed that the colony
numbers in the MDL-800-treated group were markedly decreased
but MDL-800NG had little impact on the colony formation (Fig. 3d,
e), further indicating that MDL-800 inhibits NSCLC cell
proliferation.
with previous results, we found that MDL-800 remarkably arrested
the cell cycle at the G /G phase of both NSCLC cell lines (HCC827
and PC9) and had a corresponding reduction in cells in G /M and
S phases, while MDL-800NG did not significantly affect the cell
cycle distribution at 25 µM (Fig. 4a and Supplementary Informa-
tion Table S2). Similarly, MDL-800 exerted no observable effect on
SIRT6-KO HCC827 and PC9 cells compared with control cells
(Fig. 4a and Supplementary Information Table S2), suggesting that
MDL activators induce a general impairment of cell cycle function
in cancers. Moreover, we assessed a panel of SIRT6-mediated
0
1
2
MDL-800 induces SIRT6-mediated cell cycle arrest in NSCLC cells
Many studies have implicated the crucial role of SIRT6 in the
regulation of cell cycle progression [16, 28, 31, 45, 46]. Consistent
Acta Pharmacologica Sinica (2020) 0:1 – 12

A SIRT6 activator exhibits potent anti-NSCLC efficacy
JL Shang et al.
9
Fig. 6 Western blots of the levels of SIRT6, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, H3K9Ac, and H3K18Ac in HCC827 and PC9 cells treated
with DMSO and 25 or 50 µM MDL-800 (left) or DMSO and 25 µM MDL-800 or MDL-800NG (right) for 48 h. Histone H3 was the internal
control; β-actin was the loading control.
genes related to tumor cell cycle progression (CDC25C, PCNA,
CCNA2, and CDC2) [45] and glucose metabolism (PKM2, GLUT1,
and LDHA) [29, 47]. As expected, MDL-800 potently abated the
transcription of these genes in HCC827 and PC9 cells compared
with that in the MDL-800NG-treated groups (Fig. 4b), indicating
that MDL-800 treatment results in SIRT6-driven G /G cell cycle
0 1
arrest and glucose metabolism repression in NSCLC cells.
and an EGFR-TKI is a potential clinical strategy for NSCLC
treatment.
Many studies have demonstrated that drug resistance to EGFR-
TKIs converges on the constitutive activation of the MAPK
pathway, albeit through different mechanisms [4, 5, 41, 48]. We
hypothesized that MDL-800 could suppress the feedback of MAPK
pathway activation, consequently enhancing the activity of EGFR-
TKIs in NSCLC. As depicted in Fig. 6, MDL-800 caused a reduction
in the phosphorylation of MEK and ERK (p-MEK and p-ERK,
respectively) in a dose-dependent manner in HCC827 and PC9
cells, while MDL-800NG did not significantly inactivate these
downstream effectors. In addition, MDL-800 also increased the
antiproliferation of U0126 (a specific MAPK inhibitor) in HCC827
and PC9 cells (Supplementary Information Fig. S2). These results
indicated that the pharmacological activation of SIRT6 can
overcome signaling redundancies and increase the sensitivity of
EGFR-TKIs.
MDL-800 enhances the antiproliferation of EGFR-TKIs and
suppresses the MAPK pathway
Targeted therapies for refractory NSCLC remain a major clinical
challenge, especially for first-line EGFR-TKI therapy [3–5]. The MDL-
800-sensitive HCC827, NCI-H1975, and PC9 cell lines are human
EGFR-mutant NSCLC cells. The EGFR mutations are as follows:
L858R in HCC827 cells, Del E746_A750 in PC9 cells, and L858R/
T790M in NCI-H1975 cells. First, we used HCC827 and PC9 cells to
determine whether an SIRT6 activator is effective against NSCLC
cell proliferation alone or combined with an EGFR-TKI to maximize
the anticancer efficacy. As expected, CCK-8 assay results showed
that MDL-800 plus each of the three generations of EGFR-TKIs
MDL-800 shows potent in vivo antitumor efficacy in NSCLC
Our cell-based assays revealed broad and robust antiproliferation
of MDL-800 in NSCLC. To assess the utility of MDL-800 in NSCLC
animal experiments, we characterized the pharmacokinetic
properties of MDL-800. MDL-800 was administered at the
indicated doses via IV bolus, oral gavage (PO), or IP injection in
C57BL/6J mice (n = 5 mice per group). Pharmacokinetics studies
showed that MDL-800 has rapid absorption and short half-life in
mouse plasma (Supplementary Information Table S3). In addition,
MDL-800 displayed relatively favorable bioavailability by IP
injection (F% = 73.6%).
To further evaluate the pharmacological effect of MDL-800
in vivo, we generated NSCLC CDXs. In the HCC827 CDX model,
MDL-800 treatment effectively suppressed tumor growth at 80
mg/kg, but no obvious weight loss was observed (Fig. 7a–d),
indicating potent in vivo antitumor activity of MDL-800. Western
blot analyses of xenograft tumor tissues showed enhanced SIRT6-
dependent histone H3 deacetylation and decreased phosphoryla-
tion of MEK and ERK (p-MEK and p-ERK) in the groups treated with
MDL-800 (Fig. 7e). Moreover, IHC staining showed that NSCLC
tumor regression was associated with reduced proliferation (Ki-67)
and H3K9Ac levels in xenografts after MDL-800 treatment, as well
as decreased levels of p-ERK (Fig. 7f). These results revealed the
potent antitumor efficacy of MDL-800-induced SIRT6 activation in
NSCLC in vivo.
(
gefitinib, afatinib, and osimertinib) led to a more robust reduction
in cell viability than either single agent alone in HCC827 and PC9
cells. The CI analyses via the Chou–Talalay method [44] revealed a
synergistic effect of MDL-800 and each of three EGFR-TKIs in
NSCLC cells, in which all the CIs were below 1 (Fig. 5a). These
results demonstrated that MDL-800 sensitizes EGFR-mutant NSCLC
cells to EGFR-TKIs.
To further explore whether MDL-800 could recover the drug
sensitivity of OR NSCLC cells, HCC827OR and NCI-H1975OR NSCLC
cells were established and cultured as described previously [40–
42]. We used MDL-800 alone and up to 50 μM or osimertinib alone
and up to 5 μM, and these causes no more than 50% inhibition in
HCC827OR and NCI-H1975OR cells, respectively (Fig. 5b). The
results showed that HCC827OR cells were less sensitive to MDL-
8
00 or osimertinib treatment than their parental HCC827 cells. This
may be due to the strong drug resistance effect of long-term and
sustained osimertinib treatment. However, the combination of
MDL-800 and osimertinib suppressed the proliferation of
HCC827OR and NCI-H1975OR cells more significantly than any
single treatment, suggesting that MDL-800 effectively restored the
osimertinib sensitivity of HCC827OR and NCI-H1975OR cells
(
8
Fig. 5b). This result implied that the activation of SIRT6 by MDL-
00 may contribute to improving acquired osimertinib resistance
in OR NSCLC cells. To further investigate the clinical potential of
this combination treatment, we also isolated primary tumor cells
from one NSCLC patient specimen. The CCK-8 assay and CI
analysis confirmed the synergetic effect in NSCLC patient-derived
primary cells cotreated with MDL-800 and osimertinib (Fig. 5c).
Our data demonstrated that the combination of an SIRT6 activator
DISCUSSION
NSCLC is a leading cause of cancer-related death worldwide but
has limited therapeutic targets. SIRT6 is a crucial epigenetic
regulator of gatekeeping physiological and pathological functions,
Acta Pharmacologica Sinica (2020) 0:1 – 12

A SIRT6 activator exhibits potent anti-NSCLC efficacy
JL Shang et al.
1
0
Fig. 7 MDL-800 displays robust antitumor efficacy in NSCLC cell-derived xenografts. a Representative image of HCC827 xenograft tumors
dissected from nude mice treated intraperitoneally with vehicle or 80 mg/kg MDL-800 every other day for 14 days. b Volume measurements of
representative xenograft tumors in different groups. The tumor volumes are shown as the mean ± SEM of n = 5 mice per group. P values were
calculated by a two-tailed Student’s paired t test. c Tumor weights in different groups of mice. The data are presented as the mean ± SD of n =
5
mice per group. P values were determined by a two-tailed Student’s unpaired t test. d Body weights in different groups of mice (mean ± SD
of n = 5 mice per group). e Western blots of SIRT6, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, H3K9Ac, H3K18Ac, and H3K56Ac levels in
representative xenograft tumors from each group. Histone H3 was the internal control, and β-actin was the loading control. f Representative
tissue sections from representative xenograft tumors with H&E, H3K9Ac, Ki-67, ERK1/2, and p-ERK1/2 staining after treatment with vehicle or
MDL-800. Representative images (×40 magnification) are shown. Scale bars, 100 μm.
including tumorigenesis, via SIRT6-mediated histone H3 deacetyla-
tion [15, 17–20, 31]. Although the role of SIRT6 in cancers is cell
context dependent and SIRT6 plays an oncogenic role in certain
cancers, including skin cancer, squamous cell carcinoma, prostate
cancer, and acute myeloid leukemia [10–14], SIRT6 mainly acts as
NSCLC [21, 22], suggesting that SIRT6 could serve as a promising
epigenetic target for NSCLC. However, the anticancer efficacy of
SIRT6 activation in NSCLC has not been fully explored in terms of the
lack of cellularly active SIRT6 activators. In this study, we
demonstrated that our allosteric activator of SIRT6, MDL-800,
showed favorable in vitro and in vivo anti-NSCLC activity by
enhancing SIRT6 deacetylation, indicating that pharmacological
activation of SIRT6 can be a promising therapy against NSCLC.
a
tumor suppressor in multiple cancers, including NSCLC
15–20, 22, 23]. Many studies have demonstrated that the down-
regulation of SIRT6 is closely associated with a poor prognosis in
[
Acta Pharmacologica Sinica (2020) 0:1 – 12

A SIRT6 activator exhibits potent anti-NSCLC efficacy
JL Shang et al.
1
1
Recent advances in targeted therapies have improved the
paradigm for NSCLC treatment [3, 4]. Given that EGFR is one of the
most frequently mutated genes in NSCLC and is responsible for
carcinogenesis, relapse, and metastasis, EGFR has been high-
lighted as a key target for NSCLC therapy [49]. EGFR-TKIs, from the
first-generation gefitinib to the second-generation afatinib and
the third-generation osimertinib, have shown profound clinical
efficacy and thus have been approved for the treatment of
advanced-stage EGFR-mutant NSCLC [3, 50–53]. However, pro-
longed treatment with EGFR-TKIs often causes acquired drug
resistance that limits the duration of their clinical benefit [3–5, 54].
Thus, it is urgent to discover potential strategies for increasing
sensitivity to EGFR-TKIs. Many studies have elucidated mechan-
isms for acquired resistance to EGFR-TKIs. For example, the
emergence of T790M and C797S, which are gatekeeper mutations,
accompanied by EGFR amplification, is detected in ~50% of EGFR-
mutant NSCLC, as well as mutational activation of downstream
kinases (BRAF or PIK3CA) and amplification of related receptor
tyrosine kinases (MET, ALK, or HER2) [3–5]. Mutations in EGFR can
lead to constitutive activation of downstream signaling cascades,
including the MAPK pathway, which regulates tumor cell
proliferation and has been frequently observed to be dysregulated
in NSCLC [5, 41, 48, 55, 56]. The activated MAPK pathway is
believed to be a major contributor to the tumorigenesis of NSCLC
and reduces the antitumor effect of EGFR-TKIs [5, 41, 48].
preclinical rationale for developing a promising strategy that
combines SIRT6 activators and EGFR-TKIs to defeat NSCLC.
ACKNOWLEDGEMENTS
This research is sponsored by the National Natural Science Foundation of China
(81925034, 91753117, 81721004 to JZ and 81901423 to YYC), Innovation Program of
Shanghai Municipal Education Commission (2019-01-07-00-01-E00036 to JZ),
Shanghai Science and Technology Innovation (19431901600 to JZ), Shanghai Health
and Family Planning System Excellent Subject Leader and Excellent Young Medical
Talents Training Program (2018BR12 to JZ), and Shanghai Health and Family Planning
Commission (20184Y0268 to YYC).
AUTHOR CONTRIBUTIONS
JZ conceived the project and supervised the project. JZ, JLS, and TXC analyzed the
data and wrote the manuscript. JZ, JLS, TXC, and SBN performed the biological
experiments, and YYC carried out the synthesis, purification, and characterization of
the compounds. All authors discussed the results and approved the final manuscript.
ADDITIONAL INFORMATION
The online version of this article (https://doi.org/10.1038/s41401-020-0442-2)
contains supplementary material, which is available to authorized users.
Competing interests: The authors declare no competing interests.
Notably, epigenetic alterations can be acquired during initial
EGFR-TKI treatment to induce drug tolerance. Histone deacetylase
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