Transcript Supplementary Information (ppt 792K)

NSC No.
595sp P (%)
595sp VR (%)
595sp VR + Btz (%)
Annotation
105827
15
16
16
Sangivamycin derivative
94600
78
44
10
Camptothecin
102742
1
1
6
622608
85
33
15
36758
2
9
12
Toluidine Blue
47147
25
25
38
Prodigiosin
45383
33
4
3
Rufochromycin, Streptonigrin
122819
35
8
7
Teniposide
345647
1
29
16
Chaetochromin A
129220
50
48
35
Cytarabine derivative
93427
42
36
37
93945
42
59
26
Table S1. HTS positive hits.
Figure S1. Isogenic bortezomib (Btz) resistant mouse and human cell models.
The indicated human (MM.1S and U266) and mouse (595sp and 589bm) were exposed
to increasing concentrations of Btz for greater than 6 months in culture, eventually
giving rise to Btz resistant cell populations. The indicated cell lines were exposed to
increasing concentrations of Btz for 24 (human) or 48 (mouse) hours, at which time cell
viability was measured. The parental, sensitive cell lines show EC50 values between 510 nM, whereas EC50s for resistant cell lines ranged from 60-200 nM.
Figure S2. HTS assay Z’ factor calculation. The Z’ factor, a statistical measure
of assay performance, was calculated using Btz as a positive control/test
compound in the sensitive 595 BzS cells. The assay Z’ factor at an Effective
Concentration 60 (EC60) of Btz was calculated at 0.49. Z’ factor values greater
than 0.40 are considered acceptable for HTS assays, as they demonstrate
statistical significance of the given effect size.
Figure S3. VRC2 gene expression signatures predict p53 pathway activation.
Treatment of human MM1.S BzR cell lines over 24 hours shows downstream evidence
of p53 pathway activation through CCND1 inhibition by Ingenuity Pathway Analysis (see
Figure 2A). Molecules in orange are predicted as activated and those in blue are
inhibited. Orange lines lead to activation, blue lines lead to inhibition, yellow lines are
findings inconsistent between the input dataset and the database, and gray lines have
no effect prediction. Solid lines are known direct effects and dashed lines are predicted
effects based on the literature.
VRC2 (hrs):
0 2 4 6 8
Nutlin
A
p53
Ran
B
P21
PUMA
NOXA
Figure S4. VRC2 induces p53 pathway activation. (A) Wild-type p53 expressing NCIH929 cells were treated with 500nM VRC2 for the indicated time points. Western blots
are shown. The MDM2 inhibitor, Nutlin3a (5μM), was included as a positive control. (B)
NCI-H929 cells were treated with VRC2 (500nM) and mRNA levels of the p53 target
genes P21, PUMA, and NOXA were measured by qPCR. Nutlin3a (5μM) and the DNAdamaging topoisomerase inhibitor, CPT-11 (33μM), were included as positive controls.
A
B
Figure S5. Nutlin3a restores sensitivity to proteasome inhibitors in resistant MM cells. (A)
PI resistant MM.1S VR cells were treated with increasing concentrations of Btz (left) or
carfilzomib (right) in the presence or absence of Nutlin3a (5μM) for 24 hours. Cell viability data
are shown. Nutlin3a had no effect at this time point as a single agent, and therefore any
separation of the dose curves is an indication of a superadditive/synergistic drug interaction.
Parental MM.1s, which are highly sensitive to both Btz and Crflz were included for comparison.
(B) PI resistant mouse 595sp VR cells were treated as in (A) for 48 hours. Cell viability data are
shown.