SKLB1028, a novel oral multikinase inhibitor of EGFR, FLT3 and Abl, displays
exceptional activity in models of FLT3-driven AML and considerable potency in
models of CML harboring Abl mutants
Zhi-Xing Cao, Jing-Jing Liu, Ren-Lin Zheng, Jiao Yang, Lei Zhong, Yong Xu,
Li-Jiao Wang, Chun-Hun Zhang, Bing-Lin Wang, Shuang Ma, Ze-Rong Wang,
Huan-Zhang Xie, Yu-Quan Wei, and Sheng-Yong Yang*
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West
China Medical School, Sichuan University, Chengdu, Sichuan 610041, China
Corresponding Author:
Dr. Sheng-Yong Yang
State Key Laboratory of Biotherapy and Cancer center
West China Hospital, Sichuan University
Chengdu, Sichuan, 610041
P. R. China
Tel: 86-28-85164063
Fax: 86-28-85164060
Email: yangsy@scu.edu.cn
Supplementary Material
Materials
For this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), dimethyl
sulfoxide (DMSO), PEG400, propidium iodide (PI) and Hoechst stain were purchased
from Sigma Chemical Co. (St. Louis, MO). The primary antibodies against FLT3,
STAT5/p-STAT5, p44/42 MAPK/p-p44/42 MAPK, caspase-3, β-actin, c-Abl and
p-c-Abl were purchased from Cell Signaling Technology (Beverly, MA). The
antibody against phosphotyrosine was from Millipore (Upstate Biotechnology, Lake
Placid, NY). Sorafenib, sunitinib, imatinib and SKLB1028 were synthesized at the
State Key Laboratory of Biotherapy, Sichuan University (Sichuan, China). All of the
chemicals used in this study were of analytical purity and culture grade.
Kinase inhibition assays
The in vitro kinase inhibition assays were performed according to the KinaseProfiler
assay protocols from Upstate Biotechnology (Millipore). The Ambit in vitro
KINOMEscan kinase binding assays (Ambit Biosciences) were used to determine the
binding affinity (Kd) of the compounds.
Cell culture
Unless specifically mentioned, the cell lines were obtained from the American Type
Culture Collection (Manassas, VA, USA). The cells were grown in RPMI 1640 or
DMEM culture medium containing 10% fetal bovine serum (v/v) in 5% CO2 at 37℃,
except for MV4-11 cells, which were cultured in IMDM culture medium.
Cell viability assays
Cell viability was determined using the MTT assay method. The leukemia cells were
seeded in a 96-well plate at 1-4×104 cells per well, and an equal volume of medium
containing increasing concentration of inhibitors was added to each well. At the end
of the incubation period (72 hours at 37℃), 20 μL of 5 mg/mL MTT reagent was
added per well for 2–4 hours of incubation, and 50 μL of 20% acidified SDS was
added to each well to lyse the cells. The other cell lines were seeded in 96-well plates
at a density of 2–5×103 cells/well for 24 hours, followed by replacement of the
medium with serial dilutions of inhibitors in culture medium. After a 72-hour
incubation period, the MTT reagent was added for 2–4 hours, and 100% DMSO was
used to dissolve the cells. Finally, the light absorption (OD) of the dissolved cells was
measured at 570 nm using a SpectraMAX M5 microplate spectrophotometer
(Molecular Devices). All experiments were performed in triplicate. The percentage of
viable cells was calculated and compared with that of the control cells treated with
DMSO (0.1%).
Cell cycle and apoptosis assays
A flow cytometric assay was used for cell cycle and apoptosis assays. Briefly, after
treatment with increasing concentrations of SKLB1028 for 20 hours, MV4-11 cells
were harvested and washed with PBS. Next, the cells were stained with hypotonic 50
μg/mL propidium iodide solution containing 0.1% sodium citrate and 0.1% Triton
X-100, and the DNA content was immediately analyzed using flow cytometry (ESP
Elite, Beckman Coulter, Fullerton, CA). Hoechst staining was used to analyze the
morphological change in the nucleus. After 20 hours of drug treatment, the cells were
fixed overnight with 70% ethanol, harvested by centrifugation, and suspended with 1
g/mL Hoechst solution. Next, the cell suspension was fixed on a slide using a
coverslip, and the morphology of the nucleus was analyzed under a fluorescence
microscope (Carl Zeiss MicroImaging, Inc).
The extent of apoptosis was analyzed by detecting the level of cleaved caspase-3.
Briefly, MV4-11 cells were treated with SKLB1028 at the selected concentrations for
20 hours, and the cells were subsequently lysed with RIPA buffer (10 mM Tris-HCl
(pH 7.8), 1% NP40, 0.15 M NaCl, 1 mM EDTA, 10 µM aprotinin, 1 mM NaF and 1
mM Na3VO4). The extracted samples were separated using SDS-PAGE and
electroblotted onto PVDF membranes (Millipore, Bedford, MA). Immunoblotting was
then performed with an anti-caspase-3 antibody, and β-actin was used as the control.
Immunoprecipitation and western blot assays
FLT3 autophosphorylation analysis was performed using immunoprecipitation and
immunoblotting. After treatment with a series of concentrations of SKLB1028 for 5
hours at 37°C, MV4-11 cells were harvested, washed in ice-cold PBS, and lysed with
RIPA buffer (10 mM Tris-HCl (pH 7.8), 1% NP40, 0.15 M NaCl, 1 mM EDTA, 10
µM aprotinin, 1 mM NaF and 1 mM Na3VO4). The samples were incubated overnight
at 4°C with the anti-FLT3 antibody, and the immune complexes were precipitated
with protein A agarose (Roche Applied Science) at 4°C. The precipitated samples
were subjected to immunoblot analysis to detect FLT3 phosphorylation by probing
with the anti-phosphotyrosine antibody (Upstate Biotechnology, Lake Placid, NY).
Total FLT3 was measured with an anti-FLT3 antibody after the PVDF membrane had
been incubated with stripping buffer. STAT5 and Erk1/2 phosphorylation was also
detected by immunoblot analysis. Briefly, after 20 hours of SKLB1028 treatment,
MV4-11 cells were harvested and lysed with RIPA buffer. The samples were
analyzed by immunoblotting as outlined above using anti-phospho-STAT5,
anti-STAT5, anti-p44/42 MAPK and anti-phospho-p44/42 MAPK antibodies.
In vivo models
MV4-11 and K562 cells were harvested during the exponential-growth phase, washed
twice with serum-free medium, and re-suspended at a concentration of 1×108/mL.
One hundred microliters of the cell suspension was injected subcutaneously into the
hind flank of each female NOD-SCID mouse (6–7 weeks old). For the MV4-11 model,
the tumors were allowed to grow to 300–500 mm3, at which point the mice were
randomized into 4 groups (6 mice for each group) and dosed orally with SKLB1028
(5, 10, or 20 mg/kg/d) or vehicle. For the K562 model, 35 or 70 mg/kg/d SKLB1028
and 70 mg/kg/d imatinib mesylate were dosed orally when the tumors were 150–300
mm3. The compounds were dissolved in 25% (v/v) PEG400 plus 5% DMSO and
administered orally at a dose of 5 mL/kg. Tumor growth was measured every 3 days
using Vernier calipers for the duration of the treatment. The volume was calculated as
follows: tumor volume = a×b2/2 (a, long diameter; b, short diameter).
In the bone-marrow engraftment model, NOD-SCID mice were pretreated
intraperitoneally with cyclophosphamide at a dose of 150 mg/kg once per day for 2
days. Following a 24-hour rest period, the mice were intravenously injected with
8×106 MV-4-11 cells per mouse via the tail vein. Three weeks after inoculation,
SKLB1028, sunitinib malate or vehicle was administered orally once per day for 30
days. Survival was determined by observation of the animals, and animals with
hind-limb paralysis and those that were moribund were counted as dead. For all in
vivo experiments, a paired-tail Student’s t-test was used to assess the differences
between the treated and control groups. P<0.05 was considered significant.
Histopathology and IHC
NOD-SCID mice bearing tumors or having undergone bone marrow engraftment were
treated with SKLB1028 at a dose of 20 mg/kg/d (P.O.). At the indicated time after
dosing, individual mice were sacrificed. The tumors or femoral bones were fixed with
formalin and embedded in paraffin (femoral bones were decalcified). Sections
measuring
4–8
μm
in
thickness
were
prepared
for
histological
and
immunohistochemical analysis. Proliferation was detected by immunostaining with
the Ki67 antibody (Thermo Fisher Scientific, Fremont, CA). Apoptosis was
determined by transferase-mediated dUTP nick-end labeling (TUNEL) staining
(Roche Applied Science). Finally, images were captured with an Olympus digital
camera attached to a light microscope.
Pharmacokinetic analysis of SKLB1028 in rats
Jugular vein blood was collected for the pharmacokinetic analysis. Briefly, a catheter
was surgically placed into the jugular vein for the collection of serial blood samples
from male SD rats (200–220 g). After fasting overnight, the animals were treated
orally with SKLB1028 at a single dose of 60 mg/kg/d; the vehicle used was 5%
DMSO and 25% PEG400 with the compound dissolved at a concentration of 12
mg/mL. Blood was collected in heparin-containing tubes at the appropriate times, and
the plasma was isolated by centrifugation. The plasma concentrations of SKLB1028
were determined using liquid–liquid extraction followed by high-performance liquid
chromatography
(HPLC)
with
tandem
mass
spectrometric
detection.
Noncompartmental pharmacokinetic parameters (AUC, Tmax, Cmax, and t1⁄2) were
obtained from the blood concentration time profiles using DAS software.
Sub-acute toxicity test
For a preliminary safety estimate of SKLB1028, we performed a sub-acute toxicity
test. Female and male SPF SD rats (7–8 weeks old) weighing 200 to 220 g were used
in this study. All experiments were performed at the Animal Center, State Key
Laboratory of Biotherapy, Sichuan University (Sichuan, China). For the sub-acute
toxicity test, the male and female rats (n = 5, respectively) were orally treated with
SKLB1028 at doses of 100 mg/kg/d and 60 mg/kg/d. The clinical symptoms of the
animals, including mortality, body weight, movement, and gross findings, were
observed once per day for 14 days. The rats were fasted overnight after the last
administration and were sacrificed for toxicological analysis. Finally, gross
histological examinations were performed after removal of the major organs. Blood
was analyzed for serum biochemistry and hematological analysis using a Hitachi 7200
Blood Chemistry Analyzer and a Nihon Kohden MEK-5216K Automatic Hematology
Analyzer.
Statistical analysis
All in vitro experiments were performed in triplicate, and the data are expressed as
average values. In vivo survival curves were estimated using the Kaplan-Meier
method, and the differences among various treatments were evaluated using the
log-rank test. Differences in the therapeutic effects were analyzed using an analysis of
variance (ANOVA). Finally, significance was determined using a two-tailed t-test,
and differences were considered to be statistically significant when P <0.05.
Chemical characterization of SKLB1028
SKLB1028
9-isopropyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)-N8-(pyridin-3-yl)-9H-purine2,8-diamine. Yield 65.6 %. HPLC>98.6%. 1H NMR(400 MHz, DMSO-d6)： δ 9.22(s,
1H), 9.05(s, 1H), 8.94(d, J=2.8Hz, 1H), 8.39(s, 1H), 8.34(d, J=8.4Hz, 1H), 8.20(m,
1H), 7.63(d, J=8.8Hz, 2H), 7.37(m, 1H), 6.88 (d, J=8.8Hz, 2H), 4.88(m, 1H), 3.05(m,
4H), 2.45(m, 4H), 2.22(s, 3H), 1.69(s, 3H), 1.68(s, 3H)ppm。HRMS (ESI) m/z [M-H]calcd for C24H29N9: 443.2546, found: 442.2538.
Table S1. Kinase inhibition profile of SKLB1028.
Kinase
IC50 (μM)
EGFR WT
EGFR L858R
FLT3
Abl
Abl-T315I
Fyn
Hck
KDR
PDGFRalpha
CSF1R
FGFR2
FGFR1
c-Kit (h)
CSK(h)
ALK
BTK
CDK1
CDK2
IGF1R
INSR
MET
ERK2
GSK3beta
SYK
ZAP70
JAK1
JAK2
JNK2
JNK3
MK2
PDK1
PIM2
PKA
PKB
ROCK2
S6K
0.031
0.004
0.055
0.081
0.071
0.214
0.487
0.330
0.360
1.040
0.980
1.200
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
Table S2. Anti-proliferative profile of SKLB1028 in various cell lines
Cell line
Tumor type
IC50 (µM)
MV4-11
K562
RS4-11
SU-DHL-6
TF-1
Karpas299
Jurkat
RPMI8226
human acute myeloid leukemia
human chronic myeloid leukemia
human acute lymphocytic leukemia
human B cell lymphoma
human erythroleukemia
human large cell lymphoma
human acute lymphocytic leukemia
human multiple myeloma
mouse hematopoietic cells that stably
expressed human FLT3-ITD
mouse hematopoietic cells
human normal liver cell line
Chinese hamster ovary cell line
0.002
0.190
0.790
1.250
1.430
2.760
2.990
>10
Ba/F3-FLT3-ITD
Ba/F3
LO2
CHO
0.01
>5
>10
>10
Affinities (Kd values) of SKLB1028 binding to various FLT3 and Abl mutants.
We examined the affinity of SKLB1028 binding to various FLT3 and Abl mutants,
and the results are provided in Table S3. The binding affinities (Kd values) of
SKLB1028 are 0.003 µM, 0.001 µM, 0.0012 µM, 0.0056 µM, 0.0038 µM, 0.036 µM,
and 0.58 µM for wt-FLT3 and the FLT3 D835H, D835Y, ITD, K663Q, N841I, and
R834Q mutants, respectively. The Kd values of SKLB1028 for binding to Abl F317I,
Q252H, T315I-nonphosphorylated, and T315I-phosphorylated are 0.480 µM, 0.0026
µM, 0.0024 µM, and 0.056 µM, respectively. These data further demonstrate the
potency of SKLB1028 against various FLT3 and Abl mutants.
Table S3. Binding affinities of SKLB1028 to various FLT3 and Abl mutations.
kinase
FLT3 (WT)
FLT3(D835H)
FLT3(D835Y)
FLT3(ITD)
FLT3(K663Q)
FLT3(N841I)
FLT3(R834Q)
Abl (F317I)-phosphorylated
Abl (Q252H)-phosphorylated
Abl (T315I)-nonphosphorylated
Abl (T315I)-phosphorylated
aKINOMEscan
Kd (nM)a
3
1
1.2
5.6
3.8
36
580
480
2.6
2.4
56
kinase binding assays (Ambit Biosciences) were
employed for determining the binding affinities (Kd).
Table S4. The final organ coefficients of different groups in the sub-acute toxicity
test.
Heart %
Liver %
Spleen %
Lung %
Kidney %
Vehicle♀
Vehicle ♂
SKLB1028
100 mg/kg♀
SKLB1028
100 mg/kg♂
SKLB1028
60 mg/kg♀
SKLB1028
60 mg/kg♂
0.43±0.09
0.44±0.03
0.39±0.02
0.40±0.02
0.42±0.04
0.44±0.03
3.00±0.22
3.07±0.18
2.98±0.13
2.87±0.12
3.29±0.19
2.97±0.12
0.26±0.02
0.23±0.04
0.23±0.05
0.22±0.06
0.24±0.02
0.21±0.03
0.42±0.09
0.39±0.03
0.43±0.03
0.37±0.03
0.44±0.03
0.41±0.04
0.37±0.03
0.40±0.02
0.37±0.03
0.36±0.03
0.36±0.04
0.38±0.02
Table S5. The final blood biochemical parameters of different groups in the
sub-acute toxicity test
ALB (g/L)
ALP (U/L)
ALT (U/L)
AST(U/L)
BUN (mM)
CHOI (mM)
CK (U/L)
CREA (uM)
TBIL (uM)
GLU (mM)
TP (g/L)
TG (mM)
Vehicle♀
Vehicle ♂
SKLB1028
100 mg/kg♀
SKLB1028
100 mg/kg♂
SKLB1028
60 mg/kg♀
SKLB1028
60 mg/kg♂
31.3±1.0
30.0±0.3
27.6±0.7
27.1±0.9
29.4±0.9
29.5±1.8
121.8±31.9
175.0±39.5
217.0±20.5
204.0±59.1
224.8±26.0
167.3±58.1
36.3±5.3
42.8±7.3
52.5±10.3
59.5±10.2
43.0±1.2
43.0±5.2
119.0±38.3
115.5±50.4
154.3±31.4
183.5±59.3
112.3±7.4
123.5±17.3
6.2±0.4
5.53±0.9
6.7±0.6
7.2±1.1
5.7±0.4
6.2±1.0
1.9±0.2
1.5±0.3
0.95±0.2
0.8±0.2
1.0±0.3
0.9±0.2
26.0±9.9
35.5±2.4
33.8±17.7
32.0±15.4
25.0±12.3
24.3±5.6
27.8±2.6
24.0±5.0
28.5±1.9
26.8±4.7
26.5±3.7
24.3±5.7
2.03±0.73
1.49±0.39
1.25±0.54
1.30±0.22
1.48±0.85
1.0±0.4
9.1±0.7
10.7±3.4
8.4±0.8
8.2±2.0
8.0±2.4
7.8±2.4
54.9±2.6
52.7±0.2
50.5±1.2
47.9±1.5
52.1±3.0
55.0±2.2
0.30±0.04
0.43±0.20
0.22±0.10
0.29±0.05
0.21±0.09
0.26±0.07
Table S6. The final hematological parameters of different groups in the sub-acute
toxicity test.
WBC
RBC
HGB
HCT
PLT
PCT
MCV
MCH
MCHC
RDW
MPV
PDW
Vehicle♀
Vehicle ♂
SKLB1028
100mg/kg♀
SKLB1028
100mg/kg♂
SKLB1028
60mg/kg♀
SKLB1028
60mg/kg♂
3.1±1.0
4.7±1.3
2.8±0.8
4.6±0.9
3.1±1.1
4.4±1.6
6.6±0.4
7.0±0.3
7.2±0.3
7.7±0.3
7.0±0.3
6.6±0.6
116.0±6.1
127.3±4.0
120.8±3.6
132.5±5.6
120.3±2.5
122.8±12.0
36.6±1.1
41.6±1
39.6±1.1
43.3±1.5
38.7±1.4
38.6±3.4
442±23.6
412±20.8
695±80.2
528±52.7
638±91.7
424±32.4
0.27±0.04
0.26±0.02
0.48±0.1
0.35±0.06
0.44±0.1
0.27±0.02
55.5±2.8
59.5±1.1
55.4±1.4
56.6±0.4
55.1±1.1
58.6±1.1
17.7±0.9
18.3±0.2
16.9±0.3
17.3±0.2
17.1±0.4
17.9±0.5
317.8±7.3
307.8±2.8
304.8±4.9
306.0±5.2
310.8±6.1
304.8±9.8
13.1±0.9
13.5±0.2
13.1±0.4
13.3±0.6
12.7±0.5
13.3±0.4
6.38±0.2
6.37±0.1
7.38±0.4
6. 73±0.3
7.32±0.5
6.67±0.2
12.2±0.3
12.7±0.7
13.1±0.6
12.7±0.4
13.3±0.3
12.5±0.3
Figure S1. G1 cell cycle arrest and apoptosis of MV4-11 cells is induced by
SKLB1028. MV4-11 cells were analyzed by FCM after treatment with the indicated
concentrations of SKLB1028 for 24 hours. (A) SKLB1028 treatment resulted in G1
cell cycle arrest and apoptosis in a dose-dependent manner. (B) A histogram analysis
revealed an increase in the percentage of G1 and sub-G1 apoptotic cells and a
reduction in the percentage of S phase cells.
Figure S2. Morphological changes in the nuclei of MV4-11 cells are induced by
SKLB1028. After a 20-hour treatment with SKLB1028, MV4-11 and K562 cells were
stained with Hoechst, and the microscopic appearance of cell nuclei was analyzed
using a fluorescence microscope (original magnification 400×). Apoptotic cells
containing condensed and fragmented fluorescent nuclei were observed in
SKLB1028-treated cells.
Figure S3. Evaluation of survival time upon treatment with SKLB1028.
NOD-SCID mice were injected with 8×106 MV4-11 cells after 2 days of
cyclophosphamide treatment. Treatment with SKLB1028 or sunitinib malate began on
day 21 after injection and continued for 30 days. Survival was determined by
observation, and animals were euthanized when they demonstrated poor health
conditions or hind-limb paralysis. Kaplan-Meier survival curves show that SKLB1028
resulted in a significantly longer survival time than sunitinib malate at a dose of 10
mg/kg/d（P<0.01, log-rank test）.
Figure S4. The influence of SKLB1028 on rat body weight in the sub-acute
toxicity test. After 14 days of SKLB1028 treatment at doses of 100 mg/kg/d and 60
mg/kg/d, the rats’ body weights were measured. No significant difference was
observed between the SKLB1028 and vehicle groups for both male (A) and female (B)
animals.
Figure S5. Pharmacokinetic characteristics of SKLB1028. The pharmacokinetics
of SKLB1028 were analyzed using SD rats (5 rats per group). The plasma of rats
treated orally with 60 mg/kg/d SKLB1028 was collected via jugular vein cannulation.
The SKLB1028 concentration in the plasma was determined using liquid–liquid
extraction followed by LC-MS detection, as described above.