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(a) Histogram of pten expression from first database analyzed 7. Black bars
indicate samples considered to express low pten (22% of tumors); crosshatched
bars indicate high pten samples. (b) GBMs with decreased pten exhibit
increased activation of the Shh signaling pathway; robust z-scores for gli1 and
gli2 are higher in tumors expressing low pten compared with others
(Supplementary Figure 1a), (*) p = 0.005. (c) Copy number analysis of pten
status (Broad database, TCGA 8). Red line indicates cut-off used for loss of pten
Vehicle LDE 225 BKM 120 Combo
hBT1
12
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4hBT 112
(PTEN-Deficient)
Vehicle LDE 225 BKM 120(100 nM)
Combo
*
1.4
0.4
0.2
0
0.6
0.8
1
1.21.4
0.4
0.2
0
0.6
0.8
1
1.2
1.3
0.60.50.4
0.70.8
0.9
1.11
1.2
*BK
M 1
20(1
00 n
M)
Cy
BKM
120
(5
00 n
M)
Cy+
BKM
(1
00 n
M)
Cy+
BKM
(5
00 n
M)
BKM
120
(1
00 n
M)
LEQ
506
BKM
120
(5
00 n
M)
LEQ
+BKM
(1
00 n
M)
LEQ
+BKM
(5
00 n
M)
Vehi
cle
LDE
225
GD
C-0
941
(500
nM
)
LDE+
GD
C
(500
nM
)
GD
C-0
941
(100
nM
)
LDE+
GD
C
(100
nM
)
Vehi
cle
Rel
ativ
e vi
abilit
y
hBT 70 Alternative PI3K inhibitor hBT 70 Alternative Shh pathway inhibitors
Rel
ativ
e vi
abilit
y
caSupplementary Figure 1
b
e
Vehicle LDE 225
PTENParental
pten gli1 gli2
BKM 120 Combo
DAY 7DAY 14
DAY 21 (7d post-treatment) DAY 28 (14d post-treatment)
hBT112 Over Time
0.6
0.8
0
1.2
1.4
1.6
1.0
0.5
0
1.5
2.0
2.5
1.8
* *
* *
**
n.s.
f
d
g h
i
Relative PTEN Copy Number
Freq
uenc
y
hBT# PTEN Status by WB
PTEN Status by aCGH
EGFR (aCGH) EGFRvIII (IHC) IDH1 IDH2
DNA change TP53 Protein
Change Variant Class 112 Not Detected Normal copy Amplified WT WT g.7579548G>A p.P47S Missense Mutation
g.7579801G>C None 5'UTR 145 Not Detected Loss normal copy WT WT g.7579619G>T None Intron 188 Present Loss Amplified No WT WT g.7579619G>T None Intron 239 Present Normal copy Gain WT WT g.7579801G>C None 5'UTR
TP53 TP53
50 100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
0
5
10
15
Num
ber o
f GB
Ms
pten Expressionpten loss pten neutral
Vehi
cle
* * *
Fold
cha
nge
rela
tive
to v
ehic
le
0.2
-0.2
0.4
0.6
0.8
1.2
1.4
1
0Low pten High pten
**
Robu
st Z
-sco
re
gli 1gli 2
Nature Medicine: doi:10.1038/nm.3328
2
(–0.3 threshold represents minimum in copy number distribution). (d) hBT112
neurospheres were imaged and viability was quantified by Trypan Blue exclusion
(n = 3). Error bars: +/– SEM. (*) F = 12, DFn = 1, DFd = 20, p = 0.0025 by two-
way ANOVA factorial interaction. Scale bar = 1 mm. (e) Combination treatment
of alternative PI3K inhibitor GDC-0941 with LDE225 reduces cell viability in
hBT70 monolayer (n = 3). Error bars: +/– SEM. F = 2.72, DFn = 2, DFd = 12, p =
0.1065 by two-way ANOVA factorial interaction. (f) Combination treatment of
BKM120 with Smo-inhibitors, Cyclopamine, or LEQ506 reduces cell viability in
hBT70 neurospheres (n = 3). Error bars: +/– SEM. (*) F = 5.60, DFn = 2, DFd =
2, p = 0.0192 for Cyclopamine and (*) F = 4.99, DFn = 2, DFd = 12, p = 0.0265
for LEQ506 for interaction with BKM120, by two-way ANOVA factorial interaction.
(g) hBT112 neurospheres were assayed for viability (n = 3). Error bars: +/– SEM,
(*) p < 0.05 and (**) p < 0.01 by one-way ANOVA. (h) Table of mutation status of
hBT112, 145, 188 and 239 GBMs. Absence of PTEN protein is the only known
factor common to lines responsive to combination therapy. (i) Acute
overexpression of PTEN in hBT112 cells decreases expression of gli1 and gli2 (*,
p = .005 for gli1 and p = .00006 for gli2 by Student t-test, mRNA normalized to
gapdh), (n = 3). Results shown are normalized to no virus.
Nature Medicine: doi:10.1038/nm.3328
3
(a) hBT145 cells were treated with vehicle, LDE225 (1 µM), BKM (100 nM) or
combination therapy. Apoptotic index measured by activated caspase-3 is
increased with combination therapy (n = 3). Error bars: +/– SEM, (*) p < 0.01 by
chi-square analysis. (b) Combination treatment leads to a significant reduction of
cell size as measured by M-phase cells, normalized to cells treated with vehicle.
Data represent means from > 50 cells per condition (n = 3). Error bars: +/– SEM,
(*) p = 0.002 by t-test. (c) Hematoxylin/Eosin staining of hBT112 tumor
xenografts at end treatment shows decreased cell size and increased pyknotic
nuclei with combination therapy. Scale bar = 50 µM. (d) Combination therapy
leads to increased deaths shortly post-mitosis; a cell divides and both daughter
cells rapidly die (75% die within one hour after division and 90% die within two
hours after division). All mitoses were analyzed; the proportion wherein both
daughters rapidly die is shown. Data represent means from > 52 cells per
0.88
0.9
0.92
0.98
0.96
0.94
1
1.06
1.04
1.02
*
5
10
25
20
15
*
0
Mitotic Catastrophe
Per
cent
of m
itose
s
Rel
ativ
e ce
ll si
ze
Supplementary Figure 2
b
d
a
0
0.2
0.4
0.6
0.8
1
hBT1
45
Act
ivat
ed C
aspa
se3+
cel
ls (%
) *
c
H&
E
Vehicle BKM 120 ComboLDE 225
Vehicle BKM 120 ComboLDE 225
Vehicle BKM 120 ComboLDE 225
Vehicle BKM 120 ComboLDE 225
Nature Medicine: doi:10.1038/nm.3328
4
condition, from three independent real time imaging experiments, (*) p < 0.05 by
chi-square analysis.
(a) Immunoblots of phosphorylated and total AKT in hBT70 cells treated for 24
hours with LDE225 (1 µM), BKM120 (500 nM) or combination, Actin: loading
control (n = 3). (b) Real-Time RT PCR for gli1 mRNA normalized to gapdh (n =
2). (c) Immunoblots of phosphorylated and total S6K and S6 proteins from
hBT70. Actin: loading control. (d) Phosphorylated S6 decreases in hBT112 cells
after treatment with PF-4708671, as shown by integrated intensity of staining
over number of cells counted. The effect is similar to that seen with combination
therapy. Data represent means of > 2000 cells (n = 3). Error bars: +/– SEM, (*) p
= 0.017, (**) p = 0.0008 by z-test, compared to one. (e) Immunoblot of cyclinD1
gli1 hBT70
pAKT (Ser)
pAKT (Thr)
AKT
Actin
hBT 70 (PTEN-Deficient)Vehicle LDE 225 BKM 120 Combo
Actin
cyclinD1
Actin
cyclinD1
Vehicle LDE 225 BKM 120 Combo
a b
Supplementary Figure 3
0
0.2
0.4
0.6
0.8
1
1.2
pS6
S6
Actin
pS6 kinase
S6 kinase
Vehicle LDE 225 BKM 120 Combo
Vehicle LDE 225 BKM 120 Combo
BDNF
PION
TNFR S1B
KCNH2
RARRES2
ISG15
c
ed
hBT 70 (PTEN-Deficient)
hBT 70 (PTEN-Deficient)
hBT 112 (PTEN-Deficient)
Fold
cha
nge
rela
tive
to v
ehic
le
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Vehicle ) Combo
gni
niats f
o ytisnet
ni evitaleR
phospho-S6
* **
f
Vehicle LDE 225 BKM 120 Combo
Nature Medicine: doi:10.1038/nm.3328
5
from treated hBT70 and hBT112 cells. Actin: loading control. (f) hBT70 and
hBT112 cells were treated with vehicle, LDE225 (1 µM), BKM120 (500 nM), or
combination. mRNA expression was determined with Affymetrix Human Genome
U133A 2.0 chips. Results are shown as heat map generated with unsupervised
clustering. Data represent means of two independent experiments per line.
These genes are significantly altered in combination therapy in both GBM lines.
The full data set is in Table1.
Supplementary Table 1
Affymetrix data for hBT70 and hBT112 cells treated with vehicle control, LDE225
(1 µM), BKM120 (500 nM), or the combination of both for 5 d.
Nature Medicine: doi:10.1038/nm.3328