GAPDH
i
D
R
aj
5T4
(EPR5530)
H1
97
5
L
TU
M
62
M
D
2
A
43
5/
5T
4
M
DA
M
46
D
8
A3
61
D
YT
2
Ca
ov
3
TO
V1
12
D
A.
5T4 A1 Binding sites / Cell
Raji
H1975
TOV-112D
CaOV-3
MDA-361-DYT2
MDA-468
MDA435-5T4
TUM622
Supplementary Fig. S1
B.
5T4 Antibody Binding Capacity
250000
100000
50000
0
A.
Supplementary Fig. S2
Control
PF384 (50nM)
MDA-468, 8 Days
2.0
Impedance Index
MMAF-Ome
(0.06 nM)
MMAF-Ome
+ PF384
1.5
***
****
1.0
0.5
0.0
Control
B.
PF-384+
MMAF-OMe (0.8)
75
MMAF(0.8)
50
25
0
Spheroid Viability (% control)
Spheroid Viability (% control)
PF-384
100
100
80
60
**
***
PF-384
(25 nM)
MMAF-OMe
(0.8 nM)
40
20
0
0.1
1
10
100
1000
PF-384 (nM)
C.
Fold Induction (Caspase 3/7)
Fold Induction (Caspase 3/7)
6
*
***
2
0
MMAF-OMe
PF-384
MMAF-OMe
+PF-384
H-1975, 24h
MDA468, 24h
8
4
MMAF-OMe MMAF-OMe
+ PF-384
MDA-468, Spheroid Growth, 7 Days
MDA-468, Spheroid Growth, 7 Days
125
PF-384
MMAF-OMe
+PF-384
6
4
*
2
**
0
MMAF-OMe
PF384
MMAF-OMe
+PF384
A.
Supplementary Fig. S3
Mitotic Cells MDA468
Mitotic Cells MDA468
24 hr
15
20
24 hr
36 hr
% P-Histone H3
10
5
15
10
5
PF
C
38
nt
r
4
(1
5T
00
4n
A
M
D
)
PF
C
(1
5T 38
0
4- 4 (
g)
A 10
D
C 0nM
(1
0 )+
g
)
PF
Cn
38
tr
4
(1
00
5T
nM
4A
)
DC
PF
(1
5T 384

4g)
A (10
D
0
C nM
(1
)
g +
)
PF
C
38
nt
r
4
(1
00
5T
nM
4A
)
DC
PF
(1
5T 384

4g)
A (10
D
0
C nM
(1
)
g +
)
B.
C
38
nt
r
4
(1
5T
00
4n
A
M
)
P F DC
(1
5T 38
0
g
4- 4 (
A 10
)
D
C 0nM
(1
0 )+
g
)
0
0
PF
% P-Histone H3
36 hr
C.
Mitotic Cells H-1975, 24h
25
Cell Cycle MDA468, 36h
Cell Cycle MDA468, 24h
60
20
10
sub G1
sub G1
G1
G1
S
40
G2
% Cells
15
% Cells
% P--Histone H3
60
20
S
40
G2
20
5
0
(1
0
(0 0 nM
.1
nM )
+
)
M 384
M
A (1 0
F
0
(0
n
.5 M )
nM
)
PF384
(100nM)
5T4-ADC
(10g)
0
PF384/
5T4-ADC
Control
PF
84
M
M
A
-3
+
PF
Control
F
M
A
nM F
)
M
.5
(0
PF
00 384
nM
)
M
M
A
(0 F .1 O
nM m
) e
(1
C
nt
r
0
D.
H-1975
16h
-
10
50
-
-
-
-
-
5
50
24h
10 10 50
50
-
10
50
-
-
5
50
-
-
-
5
50
50
5
10 10 50
50
5T4-ADC, g/ml
5
50
PF-384, nM
50
5
P-Aurora A/B/C
Aurora B
Vinculin
PF384
(100nM)
5T4-ADC
(10g)
PF384/
5T4-ADC
Supplementary Fig. S4
A.
B.
P-AKT1
H-1975, PF-384
Pixel Density Fold (/ Cntrl)
P-AKT3
P-pan-AKT
P-S6K T389
P-S6K T421/S424
P-GSK3 S9
P-eNOS S1177
P-PRAS40 T246
H-1975, 5T4-ADC 16h
6 hr
16 hr
1.0
1.0
P-AKT1 S473
0.8
Pixel Density Fold (/ Cntrl)
1.2
P-AKT2
P-PRAS40 T246
0.6
P-GSK3 S9
0.4
0.2
0.0
P-AKT1
S473
C.
MDA-468, 24h
-
1
10
1
1
10
10
5T4-ADC, g/ml
-
5
50
-
-
5
50
5
50
PF-384, nM
P-AKT (S473)
P-AKT (T308)
AKT
P-AKT473
Relative Densitometric Index
-
1.00
P-AKT308
0.75
0.50
0.25
0.00
1
2
3
4
5
6
7
8
P-H3 (S28)
cPARP
GAPDH
Relative Densitometric Index
P-GSK (S21/9)
GSK
2
3
4
5
6
7
8
9
9
P-GSK
1.25
P-GSK
1.00
0.75
0.50
0.25
0.00
1
0.6
0.4
0.2
0.0
P-S6K
P-S6K P-GSK3 P-eNOS P-PRAS40
T389 T421/S424 S9
S1177
T246
-
0.8
1
2
3
4
5
6
7
8
9
P-AKT1
S473
P-PRAS40
T246
P-GSK3
S9
A.
Supplementary Fig. S5
B.
D.
MMAF-Ome (1 nM)
MMAF-OMe (0.5nM)
Downregulated
DAP3, RBM3, EEF2, EIF3C, EIF3CL, EIF5, EIF5A, HARS,
MRPL13, MRPL16, MRPS15, NACA, RPL23, EIF4A2,
EIF4A1
Upregulated
FAU, TUFM, RARS2, EEF1D, MRPL15, MRPL18,
MRPL24, MRPS16, PTRH2, RPL13, RPL14, RPL18,
RPL18A, RPL21P14, RPL22, RPL34, RPL35, RPL36A,
RPL36AL, RPS13, RPS14, RPS19, RPS27A, RPS3A
Downregulated
RPLP2 (S102, S105), EIF2S2 (S105, T111), RPLP0 (S304,
S307), EIF4ENIF1 (S74, S77), EIF4EBP1 (S65, T68, T70),
EIF5B (S134, S135, S137), EEF1B2 (S106), EEF1D
(S133), TARBP2 (S152), UPF1 (S1111, S1112), PUM1
(S124)
Upregulated
RSL1D1 (T423, S427), RPS6 (S235, S236, S240), eEF2
(T54, T57, T59), AKT (S124)
5T4ADC (1 g/ml)
Downregulated
TARBP2 (S152), EIF4G1 (S1596), EIF4EBP1 (S65, T69,
T70), EIF4ENIF1 (S74, S77), EIF2S2 (S105, T111), EIF5
(S389, S390), EIF5B (S134, S135, S137), RPLP2 (S102,
S105), EEF1B2 (S106), NGDN (S142, S143), RPLP0
(S304, S307), EEF1D (S133)
Upregulated
RPS6 (S235, S236, S240), EIF5B (S113, S107), eEF2
(T59)
MMAF-Ome (5nM)
Downregulated
DAP3, RBM3, EEF2, EIF5A, MRPL13, MRPL16, MRPL41,
MRPL43, PA2G4, RPS21, EIF4A1
Upregulated
FAU, RARS2, EIF3L, MRPL15, MRPL18, MRPL3, MRPL9,
MRPS7, RPL13, RPL13A, RPL18, RPL18A, RPL19,
RPL21P14, RPL24, RPL26, RPL27, RPL30, RPL31,
RPL34, RPL35, RPL36A, RPL36AL, RPS13, RPS14,
RPS15A, RPS16, RPS26, RPS27A, RPS3A, RPLP0,
RPLP1, RPLP2, RPL32
C.
E.
Total Proteomics
MMAF-OMe (0.5 nM)
Phosphoproteomics
MMAF-OMe (5 nM)
MMAF-OMe
Decreased
Decreased
Increased
Increased
5T4-ADC
5T
4-
5T
4-
C
on
C
PT on
tr
X
o
A
(1 l
D
0
C
n
(1
M
0
)
g
/
PT m
5T X- l)
1
4
A 0/
D
C
tr
o
A
D X (1 l
C
n
(1
M
0
g )
/
P m
5T TX l)
4 -1
A
D /
C
PT
% P-Histone H3
5T
5T
C
PT on
tr
X
ol
4(
A
D 10n
C
M
(1
)
g
PT /m
5T X- l)
1
4
A 0/
D
C
C
o
PT ntr
ol
4X
A
(
D
C 1nM
(1
g )
P /m
5T TX l)
4 -1
A
D /
C
% P-Histone H3
Supplementary Fig. S6
A.
B.
8
Mitotic Cells MDA-468, 24h
6
15
10
5
0
MDA-468
4
2
0
20
Mitotic Cells, MDA-468 24h
Control
5T4-ADC
MMAF-OMe
PTX
5T4-ADC/PTX
MMAF-Ome/PTX
Supplementary Table S1
Cell Lines
Type
Mutations
TUM622
NSCLC, AA37622 PDX-derived
K-RAS+/-
NCI-H1975
NSCLC, Adenocarcinoma
EGFR+/- (L858R/T790M), PIK3CA+/-(G118D), TP53-/-, CDKN2A-/-
Calu-6
NSCLC
TP53-/-, KRAS+/-, BRCA2+/-
NCI-H358
NSCLC, Bronchioloalveolar adenocarcinoma
KRAS+/-, FGFR1+/-
HCC2429
NSCLC
N.A.
MDA-MB-435-5T4
Melanoma, pleural effusion
BRAF+/-(V600E), TP53+/-, CDKN2A+/-
MDA-MB-468
Breast, Basal
HER2-/ER2-/PrgR-; PTEN-/-, RB1del-/-, SMAD4-/-, TP53-/-
MDAMB-361-DYT2
Breast, Luminal
HER2+/ER2+/PrgR-; 2XPIK3CA+/-, AKT1+/-, EGFR+/-
MDA-231
Breast, Basal
HER2+/ER-/PrgR-; K-RAS+/-, B-RAF+/-, NF2-/-. TP53-/-, CDKN2A-/-
CAOV-3
Ovarian, Adenocarcinoma
TP53-/-, EGFR+/-(R255Q), STK11-/-
TOV-112D
Ovarian, malignant adenocarcinoma; endometrioid
carcinoma
TP53-/-, HER2+
OV-90
Ovarian, Malignant Papillary Serous Adenocarcinoma
TP53-/-, SMAD4-/-. BRAF+/-, CDKN2A-/-
OVCAR-3
Ovarian, Adenocarcinoma
TP53-/-, PPP2R1A+/-
SKOV-3
Ovarian, Adenocarcinoma
PIK3CA+/-(H1047R), TP53-/-, NF1+/-, ATM+/-, APC+/-
HT-29
Colorectal adenocarcinoma
BRAF+/-(V600E), PIK3CA+/-(P449T), SMAD4-/-, TP53-/-, 2XAPC+/-
NCI-N87
Gastric carcinoma, Adenocarcinoma, derived from
metastatic site, liver
SMAD4-/-, TP53-/-, HER2+/-, IL2+/-, PCSK7+/-
Raji
NHL
MYC+/-, TP53+/-
Supplementary Table S2A
Total Proteomics, DAVID
Condition
Change
GO
Category
Count
P Value
MMAF-Ome (0.5 nM)
Decreased
GOTERM_BP_ALL
translation
14
MMAF-Ome (5 nM)
Decreased
GOTERM_BP_ALL
translation
10
1.1E-3
MMAF-Ome (0.5 nM)
Increased
GOTERM_BP_ALL
translation
24
6.3E-14
MMAF-Ome (5 nM)
Increased
GOTERM_BP_ALL
translation
34
2.5E-22
5.6E-5
Supplementary Table S2B
Phospho Proteomics, DAVID
Condition
MMAF-Ome
1 nM
Change
Decreased
GO
GOTERM_BP_ALL
Category
Count
P Value
ribonucleoprotein complex
biogenesis
9
8.7E-4
ribosome biogenesis
6
1.1E-2
Regulation of translation
6
1.8E-2
5T4-ADC
3 g/ml
Decreased
GOTERM_BP_ALL
Regulation of translation
10
8.3E-4
MMAF-Ome
1 nM
Increased
GOTERM_BP_ALL
RNA metabolic process
20
1.2E-4
5T4-ADC
3 g/ml
Regulation of translation
5
3.9E-2
Increased
GOTERM_BP_ALL
mRNA metabolic process
16
1.7E-6
Supplementary Table S3
Fixed Drug Ratios
CI Index at ED50
Aur101 : WYE-132
HT-29
N-87
1:2.7
0.18 (0.09)
0.04 (0.02)
1:8.3
0.39 (0.04)
0.35 (0.04)
1:25
0.43 (0.04)
0.53 (0.28)
1:75
0.58 (0.02)
0.52 (0.12)
1:225
0.87 (0.21)
0.49 (0.16)
1:675
0.57 (0.05)
0.41 (0.09)
1:2025
0.43 (0.07)
0.40 (0.13)