Figure Legends
Figure 1. A schematic overview of the initial screening and
two-step hit validation is presented. Initial screening was done after
the co-transfection of wild-type POU5F1 tagged with LgBiT and
fragment 6 of PRKDC tagged with SmBiT. Once stable clones were
established, cells were seeded in a 96-well plate and were treated with
an inhibitor. Decreased luminescence indicated a positive hit. Screening
and hit identification yielded 56 compounds that were selected for
further study. These compounds were validated via determining their
effect on phosphorylation of OCT4 Ser93, kinase activity, and the
protein-protein interaction between OCT4 and DNA-PKcs. 2 compounds were
confirmed to inhibit pOCT4S93 and c-MYC expression,
impair DNA-PKcs kinase activity, and prevent the protein-protein
interaction between OCT4 and DNA-PKcs.
Figure 2. Compound screening and identification of “hits.” aGraphical representation of plates used to identify hits as potential
inhibitors of DNA-PKcs-mediated phosphorylation of OCT4. A blank plate
(top) was used to standardize readings. Compounds that demonstrated
significant decreases in luminescence are shown in red (middle and
bottom). b Compounds were deemed as hits (red triangles) if
they demonstrated a reduction in luminescence greater than 1.96 x the
standard deviation from the mean as calculated in the blank plate panel
(top).
Figure 3. Hit Validation #2. Inhibition of the DNA-PKcs-OCT4
interaction following inhibitor treatment. a Stable clones of NCI-H82
cells expressing a DOX-inducible OCT4-mycDDK construct were treated with
doxycycline for 18 hours to induce OCT4-mycDDK expression. Cells were
then treated with the inhibitors at the concentrations indicated for 6
hours. Protein lysates were pulled down using EZView Red ANTI-FLAG
Affinity Gel. Immunoblotting was done to determine the effects of
inhibitor treatment on the DNA-PKcs-OCT4 interaction. Two compounds, C8
and D8, decreased the expression of both DNA-PKcs and OCT4. bStable clones of NCI-H82 cells expressing a DOX-inducible OCT4-mycDDK
construct were treated with doxycycline for 18 hours to induce
OCT4-mycDDK expression. Protein lysates were pulled-down using
D8-conjugated agarose. D8 bound to DNA-PKcs, not OCT4. cIn vitro Ni-NTA pull down assays. DNA-PKcs was incubated with D8
for 30 minutes before the addition of bacterially-derived OCT4 and p53
(His-tagged). His-tagged substrates were pulled down using Ni-NTA
purification. D8 treatment impaired the interaction between DNA-PKcs and
OCT4 in a dose-dependent manner (left) but did not affect the
interaction between DNA-PKcs and p53 (right).
Figure 4. A second-step validation was conducted to validate
hits and to evaluate the effect on the kinase activity of the hits. aHit validation demonstrating that two compounds: C8 and D8, inhibit
OCT4, pOCT4S93 and c-MYC expression in a DOX-inducible
OCT4-overexpressing SCLC cell line (NCI-H82 pCW57.1-OCT4-mycDDK).b ADP-GloTM assay demonstrating that C8, D8,
and G5 drastically impair DNA-PKcs kinase activity. NU7441 (brown), a
known DNA-PKcs inhibitor, was used as a positive control. cIn vitro kinase activity assays. DNA-PKcs was incubated with
inhibitors for 30 minutes prior to the addition of bacterially-derived
OCT4 (His-tag) and p53 protein. After a second 30 minute incubation, IB
demonstrates that C8 and D8 decrease OCT4S93phosphorylation. C8, D8, and G5 decrease p53S15phosphorylation. NU (NU7441) was used as a positive control. Compounds
(concentration in μM): C8 (0.625), D8 (1.25), E10 (2.5), G2 (1.25), G5
(1.25), G10 (2.5), NU (2.5).
Figure 5. In vitro cytotoxicity of the compounds was
evaluated, and the dose-response curves are presented. a Calculated
IC50 values for C8 (left) and D8 (right) in 10 high and
low c-MYC-expressing SCLC cell lines. b Dose-response curves of
D8 in 10 SCLC cell lines are presented. The cells were treated with
vehicle (DMSO, 0.1% as final concentration), 1 nM, 3 nM, 10 nM, 30 nM,
100 nM, 300 nM, 1 μM, 3 μM, 10 μM for 96 hours before viability was
assessed. Each condition was tested in 6 replicates. Symbols: mean,
error bars: standard deviation.