H9_16h
A
C
B 5nM
P 3nM
B 5nM +P 3nM
p27
NOXA
HH3
RFC-1
α-Tubulin
P12 16h
P12_16h
B
C
B 5nM
P 2nM
P 3nM B+P 2nM
B+P 3nM
p27
NOXA
HH 3
HH-3
α-Tubulin
Supplementary Data A. (A) Changes in protein
expression following treatment with bortezomib
pralatrexate in the CTCL line,, H9.
and p
p27, NOXA, HH3 and RCF-1 expression in H9 cells
treated with bortezomib 5nM and/or pralatrexate
3nM after 16 hours of exposure was analyzed by
Western blot. α-Tubulin was used to normalized
protein loading in both cell lines.
The western blot shown represents the time point
at which the difference in protein expression were
maximized. (B) Changes in protein expression
following treatment with bortezomib and
pralatrexate in the T-ALL line , P12.
(B) p27, NOXA and HH3 expression in P12 cells
after exposure for 16 hours to bortezomib 5nM and
pralatrexate 2nM, 3nM and their combination was
analized by Western Blot.
α-Tubulin was used to normalized protein loading
in both cell lines.
The western blot shown represents the time point
att which
hi h the
th difference
diff
i protein
in
t i expression
i were
maximized.
250
Control
200
Tumor Volum
me (mm³)
Bortezomib
150
Pralatrexate
100
Bortezomib + Pralatreaxate (6 CR)
50
0
1
3
6
9
12
15
18
Days
Supplementary Data B. SCID Xenograft Beige Mouse Model of HH-CTCL.
Bortezomib was administered at the dose of 0.5mg/kg i.p.; Pralatrexate was given at the dose of 15mg/kg i.p. (1/4 of the
maximum tolerated dose); both drugs were administered simultaneously in the combination cohort
cohort.
All the cohorts were treated twice weekly for 2 weeks (day 1, 4, 8, 11).
A
B
Time (day)
C vs BP
B vs BP
P vs BP
3
0.003
0.044
0.004
Tumor Volume (mm3) ± SD
Treatment
Day 3
Day 6
Day 9
Day 12
Day 15
Day 18
110.0±
31.7
120.7±
34.3
125.9±
48.4
136.4±
58.8
176.8±
75.5
230.0±
87.6
Bortezomib
79.3±
23.7
103.3±
28.1
112.9±
37.4
107.2±
50.7
184.4±
67.6
166.2±
72.5
Pralatrexate
72.9±
26.9
59.5±
34.1
45.4±
25.1
49.0±
17.8
45.5±
16.5
45.2±
17.5
B+P
27.4±
26.3
18.0±
18.8
7.1±
21.0
7.8±
12.3
0.0±
15.0
0.0±
18.8
Control
6
0.001
0.001
0.012
9
0.001
0.001
0.012
12
0.001
0.001
0.007
15
0.001
0.001
0.002
18
0.001
0.001
0.002
C
TREATMENT
Weight
Toxic Death
Loss ≥ 10%
Activity (CR)
Control
0
0
0
Pralatrexate 15mg/Kg
0
0
0
Bortezomib 0.5 mg/Kg
0
0
0
B 0.5 mg/Kg + P 15 mg/Kg
0
0
6
Supplementary Data C. Table A presents all the p value for all the possible
comparisons at different time point. Table B reveals the median tumor
volume (mm3) and the standard deviation for all the studied cohorts. Table
C demonstrated any significant toxicity in all the cohorts and highlighted the
highest activity of the combination.