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Figure legends
Figure 1. Expression of HGF in ATL cell lines and clinical samples
A, Cytokine and growth factor arrays containing 80 cytokines and growth factors were performed
in HUT102 (lymph node adult T-cell leukemia/lymphoma [LN-ATL]), MT-1, TL-Om1, and ATN-
1 (peripheral blood adult T-cell leukemia/lymphoma [PB-ATL]) cell lines. The four dots at the
top left, two dots on the bottom right, and two dots at the top middle represent positive and
negative controls for experiments, respectively. B, Amounts of HGF and CCL2 in cell culture
supernatants quantified by ELISA. Error bars indicate standard deviations (SD). **P < 0.01. C,
10
The mRNA expression levels of ligands (HGF and CCL2) and their corresponding membrane
11
receptors, c-Met and CCR2, in ATL cell lines (HUT102, MT-1, TL-Om1, and ATN-1), a human
12
T-cell leukemia virus type I (HTLV-1)–immortalized cell line (TL-Su), and normal CD4+ cells.
13
The y-axis indicates the relative mRNA levels of each gene normalized to that of GAPDH. Error
14
bars indicate SD. D, Hepatocyte growth factor (HGF) protein expression in two representative
15
patients with clinical ATL (Pt. 9 and Pt. 13 in Table 1). Hematoxylin eosin (HE) staining (upper
16
panels), immunohistochemical staining of CD4 (middle panels), and HGF (lower panels) were
17
performed in the peripheral blood, and lymph node or pharynx. Scale bars indicate 50 µm.
18
19
Figure 2. HGF promotes ATL cell proliferation and invasion
20
A, Cell proliferation analysis after HGF (100 ng/mL) or phosphate buffered saline (PBS)
21
treatment, three times every 24 h, in MT-1 and TL-Om1 cell lines (top). Viable cell numbers were
22
counted by trypan blue staining (bottom). Squares indicate cells treated with HGF, and circles
23
indicate control cells. The y-axis indicates cell number relative to control at 0 h. Error bars indicate
24
SD. **P < 0.01. B, Cell proliferation analysis in ATL cells, with and without HGF overexpression.
25
HGF protein expression in stable cell lines overexpressing HGF (OE) and vector control (Ctrl;
24
Totani H. et al.
top). Viable cell numbers were counted by trypan blue staining (bottom). Squares indicate HGF
overexpressing cells, and circles indicate control cells. The y-axis indicates the cell number
relative to control at 0 h. Error bars indicate SD. *P < 0.05, **P < 0.01. C, Depletion of HGF in
HUT102 by two independent short hairpin (sh)RNA sequences (shHGF #1 and shHGF #2, top
left). The protein expression levels of HGF and β-actin (loading control) were quantified and
calculated as HGF/β-actin ratios (top right). The bar graph shows relative cell numbers in sh-
control (shCtrl, black), shHGF #1 (right gray), and shHGF #2 (dark gray) at 24 and 48 h (bottom).
Error bars indicate SD. *P < 0.05, **P < 0.01. D, E, Cell invasion assay with HGF treatment (D)
or HGF overexpression (E). The number of infiltrating cells was counted after HGF treatment
10
(100 ng/mL) or overexpression (gray), or PBS treatment (black). The y-axis indicates cell number
11
after HGF treatment relative to that of control. Error bars indicate SD. **P < 0.01. Scale bars
12
indicate 100 μm. F, 6-week-NOG mice were intraperitoneally injected with 5×106 HGF-
13
overexpressing (TL-Om1-HGF-Venus) or control (TL-Om1-Venus; n = 8, each group) cells.
14
Tumor formation was confirmed after 50 days of transplantation. The appearance of mice (top),
15
laparotomy (middle), and spleen (bottom) in control (Ctrl) and HGF overexpression (OE) groups.
16
The arrowhead indicates the tumor. G, Weights of tumors, livers, and ascites, and weights and
17
area of spleens in mice (Ctrl and OE, n=8 and 8, respectively) shown in F. Error bars indicate SD.
18
**P < 0.01. n = 8. H, HE staining and immunohistochemistry with anti-human CD4 in livers and
19
spleens of mice in F. Scale bars indicate 100 µm.
20
21
Figure 3. Effects of HGF/c-Met signaling and its downstream pathway on ATL cell growth
22
A, HGF/c-Met signaling pathway in each ATL cell line. After 15 min HGF treatment (100 ng/mL),
23
cellular protein was examined by western blotting. The quantification of bands is shown as values
24
under the bands (left). Experiments were performed in triplicate and the data calculated is shown
25
Totani H. et al.
in bar graphs (right). The y-axis indicates a mean relative ratio of phosphorylated to total amount
of each protein expressed, with (gray) and without (black) HGF treatment. Error bars indicate SD.
**P < 0.01. N.S., not significant. B, HGF/c-Met signaling pathway in HGF-overexpressing TL-
Om1 cells. The quantification of bands is shown as values under the bands (left). Experiments
were performed in triplicate and data is shown in bar graphs (right). The y-axis indicates the mean
relative ratio of phosphorylated to total amount of each protein expressed in control (black) and
HGF-overexpressing cells (gray). Error bars indicate SD. *P < 0.05, **P < 0.01. N.S., not
significant. C, Effects of c-Met inhibitor (PHA-665752) on the downstream signal in HUT102
cells after 6 h of treatment. Values under the bands indicate the relative ratio of phosphorylated to
10
total amount of each protein expressed (left). The y-axis indicates the mean relative ratio of
11
phosphorylated to total amount of each protein expressed (right). Error bars indicate SD. **P <
12
0.01. D, The numbers of viable HUT102 cells treated with PBS (Ctrl, black), PHA-665752 (right
13
gray), and PHA-665752 plus HGF (100 ng/mL) for 72 h. The y-axis indicates cell number relative
14
to control. HGF was added every 24 h. Error bars indicate SD. *P < 0.05, **P < 0.01. E,
15
Phosphorylated Akt/Akt after treatment of PHA-665752, with or without HGF (100 ng/mL). The
16
values under the bands (left) and the bar graphs indicate the mean relative ratio of phosphorylated
17
to total amount of each protein expressed after treatment with PHA-665752, with or without HGF
18
(right). Error bars indicate SD. **P < 0.01.
19
20
Figure 4. Regulation of HGF expression by epigenetic mechanisms
21
A, Schema of HGF promoter (P) and enhancer (E) regions. B, Chromatin immunoprecipitation
22
(ChIP–PCR) analyses of BRD4 and H3K27Ac in the HGF promoter (P) and enhancer (E) regions.
23
HUT102 and TL-Om1 cell lines were treated with JQ1 (gray, 0.25 µM) or control
24
(dimethylsulfoxide, black). Error bars indicate SD. *P < 0.05, **P < 0.01. N.S., not significant.
26
Totani H. et al.
C, The expression level of HGF mRNA. HUT102 cells were treated with JQ1 at 0, 0.25, or 0.5
µM for 48 h. The y-axis indicates the relative ratio of HGF to GAPDH expression. Error bars
indicate SD. **P < 0.01. D, Effects of JQ1 on the HGF/c-Met signaling pathway after 72 h of
treatment. The values under the HGF bands indicate the relative ratio of HGF to β-actin
expression or relative expression ratio of the phosphorylated to total amount of each protein (left).
Experiments were conducted in triplicates and data are shown in bar graphs (right). Error bars
indicate SD. *P < 0.05, **P < 0.01. E, The viable cell number after treatment with JQ1 was
evaluated by trypan blue staining (top). The y-axis indicates cell number relative to control. Values
under the bands indicate the relative ratio of phosphorylated Akt to total amount of Akt expression
10
(bottom). Experiments were conducted in triplicate and the data calculated is shown in bar graphs
11
(right). Short and long indicate short and long exposure times for signal detection by ECL,
12
respectively. Error bars indicate SD. **P < 0.01. F, The number of viable cells after treatment
13
with 0.25 µM JQ1 or JQ1 plus caspase inhibitors for 24 h. Z-VAD-FMK, Z-DEVD-FMK, and Z-
14
LEHD-FMK were used as pan-caspase inhibitor, caspase-3 inhibitor, and caspase-9 inhibitor,
15
respectively (all at 10 μM). The y-axis indicates cell number relative to control. Error bars indicate
16
SD. * P < 0.05, **P < 0.01. G, Percentage of apoptotic cells after treatment with JQ1 or JQ1 plus
17
caspase inhibitors. The y-axis indicates percentage of apoptotic cells. Error bars indicate SD. **P
18
< 0.01. H, Treatment schema for JQ1. Six-week-old NOG mice were intraperitoneally injected
19
with 5×106 HUT102 cells. After seven days of transplantation, PBS or JQ1 (5 mg/kg) was
20
intraperitoneally injected five times a week for three weeks. The therapeutic effect was evaluated
21
30 days after transplantation (n = 6, each group). I, Mice appearances (top), laparotomy (middle),
22
and spleen (bottom) in control (Ctrl) and JQ1 treatment groups. Arrowheads indicate a tumor. J,
23
Effect of tumor reduction by JQ1. Error bars indicate SD. **P < 0.01. n = 6. K, HE staining (top),
24
immunohistochemical staining of human (h) CD4 (middle), and hCD25 (bottom) in ATL tumors.
27
Totani H. et al.
Scale bars indicate 50 µm. The right-hand panel shows the tumor area relative to the control group.
Error bars indicate SD. *P < 0.05. n = 6.
Figure 5. Serum HGF expression in clinical samples
A, Levels of HGF in sera from 51 patients with ATL (acute type: n = 38, lymphoma type: n = 13)
and from 10 non-leukemic, healthy patients. The median and mean HGF levels are indicated by
a solid horizontal line in the box and the “+”, respectively. The box ends denote upper and lower
quartiles. The whiskers represent minimum and maximum values. *P < 0.05. B, Serum HGF
levels of patients with ATL. Box-and-whisker plots represent patients with (n = 43) and without
10
(n = 8) non-PB lesions. *P < 0.05. C, Progression-free survival (PFS) and overall survival (OS)
11
of 26 ATL patients with non-PB lesions who received mogamulizumab treatment. The reference
12
date was defined as the starting date of mogamulizumab administration. Patients with ATL were
13
divided into two groups according to the mean value of HGF (1.80 ng/mL), low (n = 15) and high
14
(n = 11), respectively. Survival was calculated by the Kaplan–Meier method with log-rank and
15
Wilcoxon tests.
28
Totani H. et al.
Table 1. HGF expression in 15 ATL patients with non-PB lesions
PB lesion
non-PB lesion
Serum HGF
Case
Age
Sex
ATL type
ATL tumor
HGF
HGF
location
expression
expression
(ng/mL)
67
female
lymphoma
small intestine
NA
74
female
acute
lymph node
NA
65
male
lymphoma
lymph node
++
NA
NA
60
female
acute
lymph node
NA
60
female
lymphoma
lymph node
NA
NA
58
male
acute
lymph node
NA
0.82
59
female
lymphoma
lymph node
NA
0.71
71
female
acute
lymph node
NA
1.08
68
female
acute
lymph node
1.64
10
57
female
lymphoma
pharynx
NA
1.36
11
48
male
lymphoma
lymph node
NA
1.18
12
68
male
acute
tongue
++
6.03
13
68
female
acute
pharynx
NA
14
72
male
acute
tonsil
++
NA
2.24
15
76
female
acute
bone marrow
++
NA
-, no positive cells; +, less than 5% of positive cells; ++, more than 5% of positive cells; NA, not
available.
29
Figure 1
LN-ATL
PB-ATL
HUT102
CCL2
Concentration (ng/mL)
15
HGF
**
10
120
HGF
mRNA expression
(Target/GAPDH)
ATN-1
110
CCL2
**
100
90
80
TL-Om1
HGF
Concentration (pg/mL)
MT-1
c-Met
0.3
0.6
0.2
0.4
0.1
0.2
0.0
0.0
CCL2
0.5
0.20
0.4
0.15
0.3
0.10
0.2
0.05
0.1
0.00
0.0
Pt. 9
Lymph node
HE
CD4
HGF
CCR2
0.25
Pt.13
Peripheral blood
Pharynx
Peripheral blood
Figure 2
MT-1
24
48
72 (h)
**
10
**
**
24 48 72
Time (h)
TL-Om1
Ctrl
OE
**
**
24 48 72
Time (h)
**
**
0.0
Ctrl #1 #2
**
**
1.0
0.8
0.6
Ctrl
sh#1
sh#2
0.4
0.2
HGF (-)
MT-1
Ctrl
OE
**
**
HGF (+)
HGF/β-actin 1.00 0.33 0.20
**
1.2
**
24 48 72
Time (h)
HGF (-)
45 kDa
0.5
Ctrl
HGF
HGF (+)
β-actin
1.0
Relative cell number
83 kDa
HGF/β-actin
#2
**
**
Ctrl
OE
**
HGF
Relative cell number
24 48 72
Time (h)
shRNA
#1
45 kDa
Ctrl
β-actin
MT-1
Ctrl
HGF
**
10
83 kDa
TL-Om1
15
Ctrl
HGF
HGF
TL-Om1
Relative cell number
MT-1
15
Relative cell number
TL-Om1
Ctrl OE Ctrl OE
Relative cell number
HGF (100 ng/mL)
MT-1
TL-Om1
**
MT-1
TL-Om1
0.0
Ctrl
OE
Tumor
Weight (g)
**
Spleen
Ascites
Liver
**
**
0.3
0.2
0.1
**
**
60
0.08
Area (mm2)
48
Time (h)
Weight (g)
24
0.06
0.04
0.02
Ctrl
OE
0.0
Ctrl
OE
Ctrl
Liver
HE
hCD4
OE
Ctrl
OE
OE
Spleen
Liver
20
0.00
Ctrl
40
Spleen
Ctrl
OE
Figure 3
MT-1
TL-Om1
ATN-1
HGF
83 kDa
pc-Met
145 kDa
c-Met
170 kDa
140 kDa
pc-Met/c-Met 1.00 1.74 1.00 17.01 1.00 15.67 1.00 13.79
pAkt
60 kDa
Akt
60 kDa
pAkt/Akt 1.00 1.22 1.00 5.00
1.00 27.49 1.00
Erk1/2
1.00 3.36
1.00
Stat3
NA
1.00
NA
1.00
1.5
**
**
20
**
10
N.S.
1.0
0.5
0.0
kDa
kDa
kDa
kDa
86
79
86
79
kDa
kDa
kDa
kDa
40
**
30
20
10
**
**
**
NA
β-actin
HGF
83 kDa
pc-Met
145 kDa
c-Met
170 kDa
140 kDa
pc-Met/c-Met 1.00 55.02
pAkt
60 kDa
Akt
pAkt/Akt 1.00
60 kDa
6.20
44
42
44
42
pErk1/2
Erk1/2
pErk/Erk 1.00
kDa
kDa
kDa
kDa
1.80
β-actin
45 kDa
**
PHA-665752
60
40
20
10
Ctrl OE
10
(μM)
pc-Met
145 kDa
c-Met
170 kDa
140 kDa
pc-Met/c-Met 1.00
0.46
0.14
Akt
0.65
**
0.43
β-actin
**
0.0
0 0.1 1 10
60 kDa
0.68
**
0.5
1.0
60 kDa
pAkt/Akt 1.00
1.0
0.20
pAkt
Ctrl OE
0.1
pc-Met/c-Met
OE
80
pAkt/Akt
Ctrl
pAkt/Akt
TL-Om1
pc-Met/c-Met
45 kDa
pErk/Erk
44
42
44
42
2.13
pStat3
pStat3/Stat3 1.00 0.76 1.00
30
2.70
pErk1/2
pErk/Erk 1.00 1.22 1.00 1.34
HGF (-)
HGF (+)
pStat3/Stat3
pErk/Erk
pc-Met/c-Met
HUT102
HGF
pAkt/Akt
45 kDa
** ** **
0.5
0.0
0 0.1 1 10
PHA-665752 (µM)
N.S.
Ctrl OE
**
Relative cell number
1.2
**
**
**
**
HGF
1.0
0.8
0.6
PHA-665752
2.5
Ctrl
PHA-665752
PHA-665752 + HGF
0.2
0.0
2.5
5.0
PHA-665752 (µM)
1.0
pAkt
60 kDa
Akt
60 kDa
pAkt/Akt 1.00
0.4
(μM)
β-actin
0.49
pAkt/Akt
0.5
0.0
0.71
45 kDa
**
**
Figure 4
+100 (kb)
+50
TSS
-50
H3K27Ac
**
**
1.2
1.0
0.8
0.6
0.4
0.2
0.0
HUT102
0.25 0.5
83 kDa
HGF
pAkt
60 kDa
Akt
60 kDa
pAkt/Akt 1.00 0.37 0.22
pErk
44
42
44
42
Erk
pErk/Erk 1.00 0.48 0.54
kDa
kDa
kDa
kDa
pStat3
86 kDa
Stat3
86 kDa
79 kDa
pStat3/Stat3 1.00 0.048 0.004
β-actin
**
% of apoptosis cells
Relative cell number
1.5
1.0
0.5
Early apoptosis
40
Tumor
**
Weight (g)
0.0
1.0
20
**
1.0
**
0.5
0.0
JQ1 0 0.25 0.25 (µM)
HGF-OE +
0.5
0.0
**
**
** **
(short)
83 kDa
HGF
(long)
1.0
**
0.5
**
0.0
pAkt
60 kDa
Akt
60 kDa
β-actin
Total apoptosis
50
**
**
1.0
0.5
**
**
0.0
pAkt/Akt 1.00 0.54 0.80
**
10
45 kDa
**
40
10
1.5
0.0
Ctrl JQ1
Area (mm2)
Weight (g)
* *
0.5
TL-Om1
placebo or JQ1 (i.p.)
i.p.
analyses
30
0.5
**
14–18
7–11
Ctrl
Ctrl
JQ1
HE
Ctrl JQ1
hCD4
20
hCD25
2.5
2.0
1.5
1.0
0.5
0.08
0.06
0.04
0.02
0.00
Ctrl
21–25
JQ1
Ascites
**
40
Ctrl JQ1
20
60
Ctrl JQ1
**
0.0
10
80
**
Late apoptosis
Spleen
0.10
0.08
0.06
0.04
0.02
0.00
0.5
20
1.0
**
1.0
15
Ctrl JQ1
**
0.0
30
Liver
**
**
HUT102
1.5
1.0
45 kDa
**
0.5
pErk/Erk
pc-Met/c-Met 1.00 0.23 0.14
1.0
pStat3/Stat3
c-Met
170 kDa
140 kDa
HGF/β-actin
145 kDa
N.S. N.S.
TL-Om1
pc-Met/c-Met
pc-Met
HGF/β-actin 1.00 0.33 0.17
JQ1 (µM)
JQ1 (µM)
pAkt/Akt
Relative mRNA expression
0.5
N.S.
**
Relative cell number
1.0
N.S.
% input
% input
1.5
HGF
Ctrl
JQ1
pAkt/Akt
-100
2.0
Relative tumor area
BRD4
JQ1
30 (day)
Figure 5
N.S.
HGF (ng/mL)
HGF (ng/mL)
Low
High
1.0
0.5
Low
High
1.0
Overall Survival
Progression-Free Survival
0.5
P=0.036
P=0.011
0.0
0.0
500
1000
1500
Time (Days)
2000
2500
500
1000
1500
Time (Days)
2000
2500
Supplementary Figure S1
Ctrl
OE
Liver
Spleen
Liver
Spleen
HE
hCD25
HE
hCD4
Liver
Spleen
Supplementary Figure S2
MG132
Concanamycin A
0.5
pc-Met
145 kDa
c-Met
170 kDa
140 kDa
pc-Met/c-Met 1.00
1.84
1.46 1.94
2.15 2.07 2.28 2.52
2.02
2.34
β-actin
45 kDa
Met/β-actin 1.00
1.12
Time (h)
1.23 1.64
0.25
3.19 3.23 2.87 2.98
0.5
2.78
3.73
c-Met/β-actin
Time (h)
pc-Met/c-Met
MG132 - + - + + - + + - +
Concanamycin A - - + + - + + - + +
0 0.5
3 (h)
24
c-Met
170 kDa
140 kDa
β-actin
45 kDa
MT-1
ATN-1
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
c-Met/β-actin 1.00
1.01
0.84
0.54
0.57
0.52
c-Met
170 kDa
140 kDa
β-actin
45 kDa
ATN-1
c-Met/β-actin 1.00
0.94
0.96
0.42
0.36
0.40
c-Met/β-actin
MT-1
Supplementary Figure S3
+50
TSS
-50
-100 (kb)
H3K27Ac
H3K4me1
H3K4me3
BRD4
MED1
HGF
**
**
Relative cell number
1.2
**
1.0
0.8
0.6
0.4
0.2
0.0
0.25
0.25 (µM)
pAkt
60 kDa
Akt
60 kDa
pAkt/Akt 1.00
β-actin
0.17
0.53
45 kDa
pAkt/Akt
JQ1
HGF
1.0
0.5
0.0
Totani H. et al.
Supplementary Figure legends
Supplementary Fig. S1. Organ invasion by HGF-overexpressing ATL cells
A, Tumorigenesis and organ invasion by hepatocyte growth factor (HGF)-overexpressing TL-
Om1 cells. Hematoxylin and eosin (HE) staining and immunohistochemical staining of anti-
human (h) CD25 in the livers and spleens of mice in Fig. F. Scale bars indicate 100 µm. B, HE
staining and immunohistochemistry with anti-hCD4 in liver and spleen. Scale bars indicate 100
µm.
10
Supplementary Fig. S2. Internalization of c-Met protein after HGF stimulation
11
A, After treatment of HUT102 cells with 20 μM MG132, 100 nM concanamycin A, or both, for
12
0, 0.5, 1, and 3 h, expression levels of c-Met and c-Met phosphorylation were analyzed. The
13
values under the bands (left) and the bar graphs (right) indicate the relative ratio of c-Met
14
phosphorylation to c-Met (right upper), or that of c-Met to β-actin (right lower). B, Expression
15
changes of c-Met in MT-1 and ATN-1 cells after stimulation with HGF (100 ng/mL). Expression
16
levels of c-Met were quantified at 0, 0.25, 0.5, 1, 3, and 24 h after HGF stimulation. The values
17
under the bands (left) and the bar graphs (right) indicate the relative ratio of c-Met to β-actin.
18
19
Supplementary Fig. S3. Effects of JQ1 and HGF on HUT102 cell proliferation
20
A, Enrichment of histone markers (H3K27Ac, H3K4me1, and H3K4me3), BRD4, and MED1 in
21
the upstream of HGF gene in the monocytes and acute myeloid leukemia cells that express HGF
22
(ChIP-Atlas data). "P" and "E" indicate promoter and enhancer regions, respectively. B, Cell
23
growth and downstream signal changes by JQ1 treatment, with and without HGF stimulation in
24
HUT102 cells. The viable cell number was evaluated by trypan blue staining (top). The y-axis
Totani H. et al.
indicates the relative cell number to the untreated control (upper). The values under the bands
(lower left) and the bar graph (lower right) indicate the relative ratio of phosphorylated Akt/total
amount of Akt expression. Error bars indicate the standard deviation (SD). *P < 0.05, **P < 0.01.
Supplementary Table S1. Sequences of primers and shRNA
Primer sequences for qRT-PCR
Target gene
Primer sequence (5’ to 3’)
HGF
Forward: ACTGCAGACCAATGTGCTAATAGA
Reverse: TGCTATTGAAGGGGAACCAG
c-Met
Forward: TTACGGACCCAATCATGAGC
Reverse: ATAAGTCAACGCGCTGCAA
CCL2
Forward: AGCAAGTGTCCCAAAGAAGC
Reverse: GCTGCAGATTCTTGGGTTGT
CCR2
Forward: CTGAGACAAGCCACAAGCTG
Reverse: GACTTCTTCACCGCTCTCGT
GAPDH, TaqMan
Hs00266705_g1, Thermo Fisher Scientific
Primer sequences for plasmid construction
Target gene
Primer sequence (5’ to 3’)
HGF
Forward: TTGCTACAGGCATCGTGGTGTC
Reverse: GCGCCCACCCTTTCATGACTGTGGTACCTTATATGTTAAA
Primer sequences for ChIP-qPCR
Target gene
Primer sequence (5’ to 3’)
HGF-E
Forward: CAACTGCCCTTTGAGGAAAA
Reverse: GAGAAGCTGCAGAACTGTTGG
HGF-P
Forward: GTGCCTAAAAGAGCCAGTCG
Reverse: AGGGGGCTGGAAGAGAGTAA
shRNA targeting sequences
Target gene
Target sequence (5’ to 3’)
Luciferase
GTGCGTTGCTAGTACCAAC
HGF#1
GATTGATTTACCTAATTAT
HGF#2
GCAAAGACTACCCTAATCA
...
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