Supplemental Materials:
Anti-apoptotic BCL-2 family proteins as 5-Azacytidine sensitizing
targets and determinants of response in myeloid malignancies
Supplementary Figure 1A
BCL-XL siRNA Drug Dose Response Curves
Supplementary Figure 1B,C
B
TF-1
9
NS
25
XL
NS XL
48
NS
XL
78
NS
XL
time (h)
siRNA
BCL-XL
-Tubulin
C
THP-1
NS
9
25
XL
NS XL
48
NS
XL
78
NS
XL
time (h)
siRNA
BCL-XL
-Tubulin
Supplementary Figure 1. BCL-XL siRNA treatment of AML-derived cell lines. (A) BCL-XL siRNA drug dose response assays with 5-Azacytidine in
vitro. Cell lines are listed at the top of each response curve plotting % viability (relative cell number) against the log of the 5-Azacytidine molar
dose. Buffer treated wells received diluted transfection reagent but no siRNA, non-silencing wells received a siRNA with no known mRNA
targets. Leftward curve shifts indicate 5-Azacytidine sensitization. (B, C) siRNA knock-down time course. Extracts from TF-1 (B) and THP-1 (C)
treated with non-silencing (NS) and BCL-XL (XL) siRNA were prepared at 9, 25, 48 and 78 hours post-transfection and probed by western blotting.
Supplementary Figure 2A
5-Aza + ABT-737
5-Aza + ABT-199
Supplementary Figure 2A continued
5-Aza + ABT-737
5-Aza + ABT-199
Supplementary Figure 2A continued
5-Aza + ABT-737
5-Aza + ABT-199
5-Aza [µM]
4
39
EC16
3
8
9.3
5-Aza + ABT-737 - Algebraic estimate
12
5-Aza + ABT-199 - Algebraic estimate
1.0
1.2
3
4
2
6
11
10
3
8
0.6
2712
1
11
6
5
10
9
0.4
0.2
156
EC85
1
5
2
4
0.8
CI +/- 1.96 s.d.
78
EC46
7
ABT-199 [µM]
20
EC3
3.1
CI +/- 1.96 s.d.
ABT-1737 [nM]
1.0
5-Aza [µM]
Supplementary Figure 2B
ABT-737
MDS- L
ABT-199
8
7
0.9
6
12
1
11
5
9
10
0.6
0.3
9
0
0
0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
1.0
3.1
9.3
2.0
EC5
4
8
12
4.0
EC28
3
7
11
8.0
EC76
2
6
10
16.0
EC99
1
5
9
5-Aza [µM]
8
HL-60
5-Aza + ABT-737 - Algebraic estimate
12
5-Aza + ABT-199 - Algebraic estimate
1.0
78
EC59
3
156
EC74
2
313
EC90
1
7
6
11
10
1.0
4
0.8
0.8
0.6
3
2
0.4
1
8
7
6
12
5
9
10
11
0.2
5
ABT-199
ABT-199 [nM]
4
ABT-737
9.3
9
CI +/- 1.96 s.d.
39
EC33
3.1
CI +/- 1.96 s.d.
ABT-737 [nM]
1.0
5-Aza [µM]
0.2
0.4
0.6
Fractional Effect
0.8
3
0.6
2
8
7
6
5
10
11
12
9
0.4
0.2
0
0
1
4
0
1.0
0
0.2
0.4
0.6
0.8
Fractional Effect
1.0
1.0
3.1
9.3
40
EC43
4
8
12
80
EC61
3
7
11
170
EC73
2
6
10
330
EC79
1
5
9
5-Aza [µM]
8
ABT-737
9.3
20
EC30
4
39
EC59
3
78
EC72
2
6
10
156
EC82
1
5
9
ML-2
12
5-Aza + ABT-199 - Algebraic estimate
1.0
11
2.0
0.8
2
4
CI +/- 1.96 s.d.
7
ABT-199
5-Aza + ABT-737 - Algebraic estimate
0.6
0.4
8
0.2
7
36
12
215
9
11
10
0
1.5
6
3
4
1.0
87
5
1
11
9
12
10
0.5
0
0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
ABT-199 [µM]
3.1
CI +/- 1.96 s.d.
ABT-737 [nM]
1.0
5-Aza [µM]
1.0
3.1
9.3
2.0
EC15
4
8
12
4.0
EC26
3
7
11
8.0
EC69
2
6
10
16.0
EC99
1
5
9
Supplementary Figure 2B continued
9.3
27.8
10
EC5
5
10
15
20
EC15
4
39
EC50
3
78
EC61
2
156
EC76
1
ABT-737
THP-1
5-Aza + ABT-737 - Algebraic estimate
5-Aza + ABT-199 - Algebraic estimate
1.0
9
1.0
14
0.8
7
6
13
12
0.8
CI +/- 1.96 s.d.
8
ABT-199
0.6
5
4
0.4
10
3
2 1
9 15
8
14
76
13
12
11
0.2
0.6
0.4
5
4
3
1
2
10
0.2
0
11
ABT-199 [µM]
3.1
5-Aza [µM]
CI +/- 1.96 s.d.
ABT-737 [nM]
5-Aza [µM]
9
87
0
0
0.2
0.4
0.6
0.8
Fractional Effect
1.0
0
0.2
0.4
0.6
615
14
13
12
11
0.8
Fractional Effect
1.0
3.1
9.3
27.8
0.13
EC17
5
10
15
0.25
EC26
4
9
14
0.50
EC31
3
8
13
1.00
EC43
2
7
12
2.00
EC56
1
6
11
5-Aza [µM]
5-Aza [µM]
9.3
TF-1
ABT-199
27.8
5-Aza + ABT-199 - Algebraic estimate
5-Aza + ABT-737 - Algebraic estimate
5
10
15
1.0
4
0.8
20
EC43
2.0
8
4
39
EC86
3
78
EC98
2
9
8
14
13
3 712
2
0.6
6
11
1
0.4
5
10
9
12
8
1.5
4
12
3
0
0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
7
211
6
1.0
10
1
5
9
0.5
0.2
7
CI +/- 1.96 s.d.
10
EC16
0
0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
ABT-199 [µM]
3.1
CI +/- 1.96 s.d.
ABT-737 [nM]
ABT-737
3.1
9.3
27.8
2.0
EC6
4
8
12
4.0
EC32
3
7
11
8.0
EC80
2
6
10
16.0
EC99
1
5
9
5-Aza [µM]
9.3
27.8
4
8
12
M07e
5-Aza + ABT-199 - Algebraic estimate
1.0
1.0
0.8
3
39
EC97
2
78
EC99
1
7
6
11
10
CI +/- 1.96 s.d.
0.8
20
EC92
0.6
4
0.4
9
5
1
6
8
32
10
7
11
12
0.2
5
ABT-199
5-Aza + ABT-737 - Algebraic estimate
9
CI +/- 1.96 s.d.
10
EC81
3.1
ABT-737
ABT-199 [µM]
5-Aza [µM]
0.6
0.4
6
5 12
4
3 11
2110
18
8
97
17
13
16
15
14
0.2
0
0
0
0.2
0.4
0.6
Fractional Effect
0.8
0
1.0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
3.1
9.3
27.8
0.13
EC49
6
12
18
0.25
EC54
5
11
17
0.50
EC61
4
10
16
1.00
EC66
3
9
15
2.00
EC65
2
8
14
4.00
EC74
1
7
13
5-Aza [µM]
9.3
ABT-737
27.8
SET-2
ABT-199
5-Aza + ABT-737 - Algebraic estimate
65
EC15
3
130
EC30
2
9
1.2
1.0
3
37
7
8
9
1
5
4
8
7
0.9
2
0.6
1
6
5
4
0.3
0.8
CI +/- 1.96 s.d.
261
EC78
6
5-Aza + ABT-199 - Algebraic estimate
8
9
0.6
4
2
6
1
5
0.4
0.2
0
0
0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
ABT-199 [µM]
3.1
5-Aza [µM]
CI +/- 1.96 s.d.
ABT-737 [nM]
ABT-737 [nM]
Supplementary Figure 2B continued
3.1
9.3
27.8
4.0
EC18
3
7
11
8.0
EC58
2
6
10
16.0
EC99
1
5
9
Supplementary Figure 2C,D
C
% population with Cleaved Caspase 3
60
56.0
50
40
29.3
30
20
10
0
1.4
2.8
Untreated
5-Aza
ABT-737
Combined
D
8 Hours 24 Hours 48 Hours
% population with
cleaved caspase 3
80
62.7 65.3
60
35.9
40
20
17.0
14.8
5.2 4.3
24.9
41.9
40.5
22.8
5.5
0
Untreated
1.0 uM 5-Aza
500 nM ABT-737
Combined
Supplementary Figure 2. ABT-737 versus ABT-199 combination with 5-Azacytidine in vitro. (A) Seven to nine doses of 5-Azacytidine spanning
a broad range from 0 to 100% reduction in viability are shown in combination with six doses of ABT-737 (left panel), compared to nine doses of
5-Azacytidine spanning a broad range from 0 to 100% reduction in viability with six doses of ABT-199 (right panel). A leftward shift of the 5Azacytidine dose response curve with the addition of the corresponding ABT-737 or ABT-199 dose indicates 5-Azacytidine sensitization. (B)
Combination Index values (CI) versus fractional effect (FE) plots are shown side-by-side for ABT-737 (left panel) and ABT-199 combination with 5Azacytidine (right panel). CI values < 0.8 indicate synergy. The numbers in the tables correspond to the specific dose combinations shown on the
CI versus FE plots. (C, D) Combination treatment with 5-Azacytidine + ABT-737 resulted in increased levels of cleaved caspase 3 (CC3), compared
to either treatment alone. The y-axis shows the percentage of the cell population positive for CC3 signal by flow cytometry for the given
treatment on the x-axis. (C) TF-1 cells were pre-treated with ABT-737 for 24h followed by 5-Azacytidine treatment for an additional 48h prior to
fixation for CC3 analysis by flow cytometry. (D) HL-60 cells were treated with ABT-737 and 5-Azacytidine simultaneously for 8, 24 and 48h prior
to fixation for CC3 measurement.
Supplementary Figure 3
Supplementary Figure 3. MCL-1 siRNA + ABT-737 drug dose response in vitro. Cell lines are listed at the top of each response curve plotting %
viability (relative cell number) against the log of the ABT-737 molar dose. Buffer treated wells received diluted transfection reagent but no
siRNA, non-silencing wells received a siRNA with no known mRNA targets. The most effective MCL-1 siRNA sequence (s6) was selected from
previous siDDR experiments shown in Table 1A, B. Leftward curve shifts indicate ABT-737 sensitization by MCL-1 siRNA.
Supplementary Figure 4A
BCL-XL
MCL-1
BCL-2
Supplementary Figure 4B,C
B
BCL-XL mRNA Expression in AML
C
Valk, Oncomine
0. No value (19)
1. FAB Subtype M0 (6)
2. FAB Subtype M1 (63)
3. FAB Subtype M2 (66)
4. FAB Subtype M3 (19)
5. FAB Subtype M4 (53)
6. FAB Subtype M5 (64)
7. FAB Subtype M6 (3)
Metzeler2, Oncomine
0. No value (1)
1. FAB Subtype M0 (1)
2. FAB Subtype M1 (23)
3. FAB Subtype M2 (34)
4. FAB Subtype M4 (11)
5. FAB Subtype M5 (6)
6. FAB Subtype M6 (3)
Supplementary Figure 4. Oncomine BCL-XL mRNA expression in primary AML samples by FAB classification. (A) mRNA expression by AML FAB
classification of public datasets GEO accession numbers: GSE19429, GSE6891, GSE12417. (B) Oncomine, Valk mRNA expression. (C) Oncomine,
Metzeler2 mRNA expression. For B and C, the number of primary specimens for each AML FAB classification is shown in parentheses.
Supplementary Figure 5
p=0.0011
no response
p=0.0011
response
no response
p=0.022
response
no response
response
Supplementary Figure 5. BH3 profiling discriminates 5-Azacytidine response in vitro. Response to 5-Azacytidine (N=13 AML-derived cell lines)
is plotted against % priming as determined by BH3 profiling for the BH3 peptides indicated above each plot.
Supplementary Table 1
Clinicopathologic variables (5-Azacytidine treated patients)
Variable
Resistant (N=17)
Sensitive (N=5)
All (N=22)
Source
p.value
0.309
Bone Marrow
7 (41.2%)
4 (80%)
11 (50%)
PBMC
10 (58.8%)
1 (20%)
11 (50%)
71.7±5.5
73.2±3.3
72.1±5
Age
Gender
0.47
0.324
Female
Male
6 (35.3%)
11 (64.7%)
0 (0%)
5 (100%)
6 (27.3%)
16 (72.7%)
FAB
N/A
M0
1 (5.9%)
0 (0%)
1 (4.5%)
M1
1 (5.9%)
0 (0%)
1 (4.5%)
M2
6 (35.3%)
0 (0%)
6 (27.3%)
M4
8 (47.1%)
0 (0%)
8 (36.4%)
M7
1 (5.9%)
0 (0%)
1 (4.5%)
Cyto
0.076
Intermediate
9 (52.9%)
1 (20%)
10 (45.5%)
Favorable
1 (5.9%)
2 (40%)
3 (13.6%)
Unfavorable
7 (41.2%)
1 (20%)
8 (36.4%)
Performance
0.004
0
0 (0%)
2 (40%)
2 (9.1%)
1
2+
7 (41.2%)
10 (58.8%)
2 (40%)
0 (0%)
9 (40.9%)
10 (45.5%)
Supplementary Table 1. Clinicopathologic variables of clinical specimens used for BH3 profiling.