Keywords
Drug discovery, c-MYC, DNA-PK, protein-protein interaction, small
molecule inhibitor, kinase
Introduction
There are over 500 protein kinases in humans (Wilson et al., 2018).
Dysregulation of kinases by mutations are frequently associated with
cancer initiation, proliferation, progression, and recurrence. These
protein kinases have become crucial targets for developing drugs in the
treatment of various cancers, and thus far, the FDA has approved over 50
kinase inhibitors for cancer therapy (Roskoski, 2020). Protein kinases
that have been successfully targeted are ALK, BCR-Abl, B-Raf, BTK,
CDK’s, c-Met, EGFR, JAK, MEK1/2, PDGFR, RET, Src, and VEGFR (Roskoski,
2020; J. Zhang, Yang, & Gray, 2009). This class of drugs led a
transformation from conventional chemotherapy to targeted cancer
treatment and has overcome the normal cell toxicities of traditional
chemotherapy. Although kinase inhibitors have shown activity in various
types of cancers, there are several challenges to overcome, including
drug resistance, unwanted toxicities, and compromised efficacy.
Kinase inhibitors are categorized based on the mechanisms of catalyzing
the transfer of the terminal phosphate of ATP to the substrates. Type I
kinase inhibitors are defined as small molecules that bind to the active
conformation of a kinase in the ATP pocket. They represent
ATP-competitors that mimic the purine ring of the adenine moiety of ATP
(J. Zhang et al., 2009). Despite their clinical activity, these drugs
display relatively low selectivity because the ATP-binding pocket is
highly preserved among kinases. This low selectivity for specific target
kinases is a possible reason for the cardiotoxicity associated with Type
I kinase inhibitors (Bhullar et al., 2018; Force & Kolaja, 2011). Type
II inhibitors reversibly bind to an inactive conformation (usually
Asp-Phe-Gly (DFG)) of a kinase, and this interaction is proposed to
exhibit better selectivity and lower toxicity than Type I kinase
inhibitors (Garuti, Roberti, & Bottegoni, 2010; Kufareva & Abagyan,
2008). Type III kinase inhibitors bind outside the ATP-binding site and
modulate kinase activity in an allosteric manner. Consequently, these
inhibitors have the potential to be highly selective, as bindings sites
targeted by allosteric inhibitors tend to be exclusive to particular
kinases (Fabbro, 2015; Panicker, Chattopadhaya, Coyne, & Srinivasan,
2019). As allosteric inhibitors are anticipated to overcome limitations
of the previous version of kinase inhibitors, several investigational
agents are in the early stages of development (Fasano et al., 2014).
Inhibitors that bind reversibly outside the ATP binding pocket in the
kinase substrate-binding site are classified as Type IV kinase
inhibitors. These are ATP-noncompetitive inhibitors that offer higher
selectivity (Cox, Shomin, & Ghosh, 2011). Finally, type V kinase
inhibitors are small molecules that form an irreversible covalent bond
with the target enzyme and target a catalytic nucleophile cysteine
within the active site of the enzyme (Bhullar et al., 2018; Cox et al.,
2011).
The majority of the FDA-approved kinase inhibitors target ATP binding
sites of kinases (Breen & Soellner, 2015; Huang, Zhou, Lafleur, Nevado,
& Caflisch, 2010). Despite their anti-tumor activity, the ongoing
challenges for clinical activity of kinase inhibitors are to enhance
their selectivity and to overcome drug resistance (Fabbro, 2015). As
described above, several efforts are underway to investigate ways to
improve kinase inhibitors while minimizing their off-target effects
(Mohiuddin & Kang, 2019). Here, we provide an approach to identify
inhibitors with enhanced selectivity by targeting kinase-substrate
interactions of DNA-PKcs. In our previous studies, we have demonstrated
that OCT4 binds to the promoter/enhancer region to activate c-MYC
transcriptionally, and one of the two kinases identified to
phosphorylate OCT4 in this process is DNA-PKcs (Mohiuddin, Wei, & Kang,
2020; Wei et al., 2020). To overcome the limitation of currently
available kinase inhibitors, we developed a cell-based assay to identify
compounds that selectively inhibit the interaction of OCT4 with
DNA-PKcs. Using the assay, we screened a chemical library of compounds
to identify “hits” that inhibit the ability of DNA-PK to bind to OCT4.
Subsequently, we validated the compounds identified from the chemical
library. The purpose of this paper is to describe a novel cell-based
assay to identify novel modulators of the interaction between DNA-PKcs
and OCT4 and the method of hit validation employed to supplement the
assay that may be applicable across many kinase-substrate interactions.
- Methods
- Mammalian cell culture and transduction
HEK293FT (ThermoFisher) cells were cultured in DMEM (ThermoFisher)
supplemented with 10% FBS, 2 mM Glutamine, 100 units/mL Penicillin, 100
µg/mL Streptomycin Sulfate and 1 mM Sodium Pyruvate (ThermoFisher). The
HEK293FT cells were plated at a cell dose of 1 × 107on a 10-cm tissue culture dish and incubated at 37°C 5% CO2 incubator
until the cells reach 80% confluence. The HEK293FT cells were
co-transfected either lentiviral ORFs, the constructs shown in Fig. 2a,
along with Lenti-vpak Packaging Kit (OriGene) using the
transfection reagent MegaTran 1.0 (OriGene). After 48-72 h transfection,
the virus-containing medium was collected, spun down, filtered (0.45
µm), and used for targeting into NCI-H82 by infection. The
virus-infected stable clones were obtained after at least 2-3 weeks of
selection in 10% FBS/RPMI-1640 with 0.5 µg/mL of Puromycin
(Sigma-Aldrich). Protein lysates extracted from the stable clones were
analyzed by SDS-PAGE/IB using specific antibodies to confirm their
protein expression. The methods were performed in accordance with
relevant guidelines and regulations and approved by the Institutional
Biosafety Committee at Texas Tech University Health Sciences Center. The
current study did not involve animals or human subjects.
Chemical Compounds
A total of 79,671 compounds were provided by the Targeted Therapeutic
Drug Discovery & Development Program at The University of Texas at
Austin (Cho et al., 2018). The compounds were compiled from the
following compound libraries: NIH clinical collection (674 compounds;
Evotec, San Francisco, CA),
Natural product or Natural
product-like (3,280 compounds; MicroSource Discovery, Gaylordsville, CT
and LifeChem, Niagara-on-the-Lake ON, Canada), Lopac (1,280 compounds;
Sigma-Aldrich), fragment sets (18,143 compounds) obtained from
Chembridge (San Diego, CA) and ChemDiv (San Diego, CA), kinase set
(11,250 compounds; Chembridge), and diversity sets (43,158 compounds)
obtained from NCI, ChemDiv, LifeChem, and Maybridge (ThermoFisher).
Additionally, two other libraries were interrogated: 1) A kinase-focused
library (600 compounds), custom selected by the Texas Screening Alliance
for Cancer Therapeutics (TxSACT) from various vendors with known
activity against approximately 100 kinases, and 2) An academic
collection (2,000 unique molecules) with diverse pharmacophores
deposited from chemists at The University of Texas at Austin and the
University of Kansas. Compounds were plated in 384-well plates dissolved
in 100% DMSO at 10 mM concentration.
Identification of compounds interfering kinase-substrate
binding (“Hit ID”)
HEK293FT cells stably expressing SmBiT-tagged DNA-PKcs and LgBiT-tagged
OCT4 were suspended at 1 x 106 cells/mL of Opti-MEM®
cell culture medium with reduced serum and seeded 9 µL of the suspension
per well in a sterile black 384-well plate (Greiner, Cat #788086).
Then, the cells were incubated for ~4 hours, and then 1
µL of the compounds (10 mM stock, 1:1000 dilution with Opti-MEM®) were
added to wells to make the final concentration of compounds at 1 µM.
After 6 hours of incubation with the compounds, Nano-Glo® Live Cell
Assay (Promega) reagent was prepared as instructed by the company and
added 1.3 µL to each well. Then, the plates were incubated for 20-30
minutes at room temperature before luminescence was measured by
SpectraMax iD3 microplate reader (Molecular Devices). The cell counts
per well, incubation time and serum content in the culture medium were
optimized before the screening. All pipetting utilized the BenchSmart 96
semi-automated pipetting system (Rainin).
Luminescence was measured from each plate, and the data were collected
in numerical values. For statistical analyses to identify a significant
reduction in signals by compounds, data normality was tested by using a
Shapiro–Wilk test and also visually examined by using a Q-Q normal
plot. A box-cox transformation was performed when necessary. Compounds
with a value of two standard deviations below the mean are considered
outliers (“Hits”), i.e., inhibition of kinase-substrate binding,
inhibition of kinase activity, or direct cell kill effect. The initial
screening will identify the compounds with any of these three effects.
Custom polyclonal phospho-OCT4S93 antibody
production.
The anti-human phospho-OCT4S93(anti-pOCT4S93) rabbit antibody was produced by
GenScript Biotech. The pOCT4S93 polyclonal antibody
was prepared by immunizing two New Zealand rabbits three times with an
NH2-terminal KLH (keyhole limpet hemocyanin)-conjugated
phosphopeptide GLETSQPEGEAGVG as an antigen. The phospho-specific
antibody was affinity-purified through a phosphopeptide-conjugated
Sepharose CL-4B column. Eluted IgG was then passed through the
corresponding non-phosphorylated peptide (GLETSQPEGEAGVG) column to
deplete any IgG that was not specific to pOCT4S93.
Validation of “hits” by immunoblotting and
immunoprecipitation
The stable cell line was prepared for immunoblotting and
co-immunoprecipitation by infecting NCI-H82, a small cell lung cancer
cell line, with a doxycycline-inducible pCW57.1-POU5F1-mycDDKconstruct using a lentiviral system as previously described (Wei et al.,
2020; Y. Zhang et al., 2014). The cell lines were prepared for
immunoblotting and co-immunoprecipitation to validate the effect on
kinase-substrate binding. Unless otherwise specified, cells grown in a
T75 flask were first dissociated by PUCKs or Trypsin-EDTA and washed
once with ice-cold 1× PBS. Cells were lysed on ice with modified RIPA
buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton
X-100, 1 µg/mL Leupeptin, 1 µg/mL Aprotinin, 1 µg/mL Pepstatin A, 1 mM
PMSF, 1 mM Na3VO4 and 1 mM NaF),
followed by centrifugation at 14,000 × g for 15 min at 4°C. Protein
concentration was determined by BCA assay (Pierce). An equal amount of
proteins from different samples were electrophoretically separated on
4-12% SDS-PAGE, transferred to Hybond membrane (GE Healthcare), blocked
with 1% BSA or 5% skim milk, immunoblotted with the indicated primary
antibodies, and incubated with 1:3,000 HRP-conjugated mouse or rabbit
IgG secondary antibodies followed by detection with enhanced
chemiluminescence (GE Healthcare). Primary antibodies used were:
anti-DNA-PKcs (MBL International), anti-Ku80 (Cell Signaling
Technology), anti-Ku70 (Cell Signaling Technology), anti-c-MYC (EMD
Millipore), anti-p53 (BD Biosciences),
anti-phospho-p53S15 (Cell Signaling Technology),
anti-OCT4 (Abcam), anti-pOCT4S93 (Gentech),
anti-pOCT4S111 (Gentech), anti-HSP27 (BD Biosciences),
anti-pHSP27S78, anti-MK2 (Cell Signaling Technology),
anti-GAPDH (Santa Cruz Biotechnology). For EZView Red anti-FLAG
pull-down, 500 µg of protein lysates as prepared above were pulled down
at 4°C overnight with 40 µL EZview Red anti-FLAG M2 affinity gels
(Sigma-Aldrich), washed 4 times with modified RIPA, and then eluted with
an excess of 3× FLAG peptide (100 μg/mL). Immuno-complexes were resolved
by 4-12% SDS-PAGE and immunoblotted with the indicated antibodies
(anti-HA antibody). For pull-down studies using C8- and D8-conjugated
agarose beads, 1 mg of protein lysates as prepared above for pulled down
at 4°C overnight with 50 µL C8- or D8-conjugated agarose beads
(CellMosaic). Beads were washed 4 times with modified RIPA, then eluted
using glycine buffer elution. Beads were incubated with 100 µL of 0.1 M
HCl + Glycine (pH = 3.5) at room temperature for 20 minutes. Ten µL of
0.5 M Tris-HCl (pH = 7.4) was added to neutralize the acidification.
Immuno-complexes were resolved, as described above.
In vitro DNA-PKcs kinase activity and
ADP-GloTM assays
100 Units of purified DNA-PKcs (Promega) or 200 ng His-MK2 was incubated
at 30°C for 30 minutes in 20 μL kinase buffer containing 40mM Tris (pH
7.5), 20 mM MgCl2, 0.1 mg/mL BSA, activation buffer
(100 μg/mL calf thymus DNA in 1X TE buffer), 150 μM ATP, and inhibitors
of interest. Following the 30-minute incubation, 1 μg of
bacterially-derived OCT4 (ProteinOne), p53 (Creative BioMart), HSP27
(Enzo) protein substrates were added to the reaction. Samples were
incubated again for 30 minutes at 30°C, and then the reactions were
quenched with the addition of 4x NuPAGE LDS and 100 mM dithiothreitol
(DTT) prior to proceeding with immunoblotting, as described above.
For the ADP-GloTM assays, a 25 μL kinase reaction was
prepared to consist of 100 Units of purified DNA-PKcs or 200 ng His-MK2,
40mM Tris (pH 7.5), 20 mM MgCl2, 0.1 mg/mL BSA,
activation buffer (100 μg/mL calf thymus DNA in 1X TE buffer), 150μM
ATP, 0.2 μg BSA, peptide substrate (amino acid sequence:
EPPLSQEAFADLWKK; Promega), and inhibitors of interest or DMSO (control).
Following a 30-minute incubation period at 30°C, 25 μL of
ADP-GloTM reagent was added to stop the reaction and
deplete unconsumed ATP. Samples were incubated at room temperature for
40 minutes. Fifty μL of kinase detection reagent was added to the
samples to introduce luciferase and luciferin to detect the presence of
ATP. After 60 minutes of incubation, luminescence was measured by
SpectraMax iD3 microplate reader (Molecular Devices). Three reactions
per treatment were done, and the luminescence measurements were
averaged. DMSO (control) treatments were standardized to 100% kinase
activity.
In vitro cytotoxicity assays
Human small cell lung cancer cell lines (NCI-H417, NCI-H82, NCI-H2171,
NCI-H847, NCI-H1048, NCI-H146, NCI-H510A, NCI-H1963, NCI-H1876) were
kindly provided by Dr. Adi Gazdar at University of Texas Southwestern.
Cells were cultured in RPMI (GE Lifesciences) supplemented with 10%
heat-inactivated fetal bovine serum. Cell lines were tested for and free
of mycoplasma, and cell line identities were verified using short tandem
repeat genotyping as compared with the original primary sample material
within the CCcells database:
www.CCcells.org. Small cell lung cancer
cells (2 x 10
6 cells) were plated in 96-well plates
for 24 hours prior to treatment with inhibitors (0.001, 0.003, 0.01,
0.03, 0.1, 0.3, 1, 3, 10 μmol/L). Six replicates of each drug
concentration were used. After 96 hours of drug incubation, cellular
viability was measured using the DIMSCAN assay, as outlined in previous
studies(Kang et al., 2011; C. Zhang et al., 2012).
Results
Assay Development and Optimization
Having demonstrated that DNA-PKcs binds to and phosphorylates OCT4 at
its Ser93 residue, we developed a luminescence-based drug screening
assay through the use of the NanoBiT® Protein: Protein Interaction
system, which utilizes two subunit promoters (LgBiT and SmBiT) fused to
genes of interest. Once these gene products are translated, fusion
between the two subunits generates a luminescent signal. We stably
expressed both the region of OCT4 necessary for MYC transcription
(the NTD and POUs domains) tagged with the LgBiT subunit and the region
of DNA-PKcs essential for OCT4 binding and phosphorylation tagged with
the SmBiT subunit in the same vector through the use of the porcine
teschovirus-1 2A (P2A) self-cleaving peptide (Figure 1). The P2A system
follows a “stop-carry on” mode of translation, wherein ribosomes pause
translation at the C-terminus of the 2A peptide before resuming
translation at the end of the 2A sequence. This mode of translation
generates two peptide fragments that are expressed in equal amounts
within the transduced cell.
To determine incubation time, we continuously monitored luminescence up
to 45 minutes after adding Nano-Glo® Live Cell Reagent into NCI-H82
cells stably transduced with the vectors shown in Fig. 1. Also, three
different cell seeding densities (2,500, 5,000, and 10,000 cells/well)
and two FBS content in the culture medium (10% FBS in RPMI or
Opti-MEM®) were examined. It is noted that the cells in culture medium
with reduced serum generates 2-3-fold higher luminescence compared with
RPMI supplemented with 10% FBS in both NCI-H82 cells (Figure S1A) and
HEK-293 FT cells (Fig. S1b). In addition, the cell count of 10,000
cells/well (10,000 cells in 10 µL of reaction volume: 1 x
106 cells/mL) displayed the highest luminescence
signals relative to 5,000 or 2,500 cells/well regardless of the FBS
content (Figure S1A&B). The luminescence signals were reduced in
NCI-H82 cells cultured with Opti-MEM® containing medium at 10,000
cells/well over 45 minutes (34% reduction). In contrast, the signal
reduction over incubation time was not seen with lower cell counts or in
cells cultured in RPMI (Figure S1). The luminescence in HEK-293FT
(DNA-PKcs-OCT4 interaction assay) gradually increased over the initial
15 minutes of incubation. Thus, 15-20 minutes of incubation was employed
for the screening assays. The luminescence signals from NCI-H82 cells
transduced with the empty vector were <10 in all three cell
counts regardless of the FBS content in culture medium over 45 minutes
(Figure S1C).
Compounds Identified from Chemical Library through the Assays
After establishing stable transduction of our two desired fragment
constructs, we screened a drug library of ~80,000
compounds to identify potential inhibitors of the interaction between
DNA-PKcs and OCT4 (Figure 2). Cells were seeded in plates containing
Opti-MEM® culture media with reduced serum for 4 hours. Cells were then
incubated with 1 µM of each compound for 6 hours prior to completion of
the addition of the Nano-Glo® Live Cell Assay reagent (Figure 2A).
Following the analysis of the luminescence data, outliers (decreased
signal) were identified as potential inhibitors of the DNA-PKcs-OCT4
interaction (representative heatmap in Figure 2A). Compounds that
inhibited the luminescence reaction by a factor of 1.96 x standard
deviation were deemed to be hits (Figure 2B). Of the library of
compounds tested, we identified 56 compounds for the DNA-PKcs-OCT4
interaction that significantly reduced the luminescence in the
corresponding cell lines.
Effect on pOCT4S93
Using mass spectrometry data, we previously demonstrated that OCT4
interacts with DNA-PKcs, and confirmed these findings through
subcellular fractionation and co-immunoprecipitation. Further, the
PhosphoMotif Finder software predicted that DNA-PKcs phosphorylates OCT4
at Ser93. To validate these anticipated findings, we first developed a
custom phospho-antibody specific to OCT4 Ser93(Wei et al., 2020). Thus,
the inhibition of the phospho-OCT4 levels was used as the first step of
validation. To validate the effect of our compounds on the
phosphorylation of OCT4, we infected a high c-MYC expressing small cell
lung cancer (SCLC) cell line, NCI-H82, with a doxycycline-inducible
lentiviral vector expressing POUF51 tagged with mycDDK. Using a
custom-produced phospho-OCT4 antibody, we determined the effect of the
previously identified “hits” on the phosphorylation of OCT4 at its
Ser93 residue in our OCT4-overexpressing NCI-H82 construct (Fig. S2a).
After DOX-induction for 12 hours, we treated these cells with 1 µM of
each compound. After 8 hours of treatment, immunoblot analysis was
performed to determine the expression levels of
pOCT4S93. We identified six candidate compounds from
the “hits” that significantly impaired phosphorylation of OCT4 at its
Ser93 residue (Figure S2A&B). We also selected one compound as a
negative control. Two compounds, C8 and D8, demonstrated both a
remarkable inhibition of OCT4 Ser93 phosphorylation and decreased OCT4
expression.
Effect on DNA-PKcs-OCT4 Interaction
After validating the effects of the “hits” on OCT4 phosphorylation, we
sought to better categorize these novel inhibitors as either impairing
the catalytic activity of DNA-PKcs or targeting the binding interaction
between DNA-PKcs and OCT4. To this end, we used the stably infected,
DOX-inducible POU5F1 tagged with mycDDK (FLAG) constructs in
NCI-H82 and treated these cells with our validated hits. We then pulled
down the FLAG-tagged OCT4 protein and detected the presence of DNA-PKcs.
Of the seven validated hits, two compounds: C8 and D8, decreased
expression of DNA-PKcs after OCT4 pull-down (Figure 3A). In line with
the findings from the first hit validation step, treatment with C8 and
D8 decreased the amount of OCT4 protein that was pulled down.
Given that these inhibitors impaired the pull-down of both DNA-PKcs and
OCT4, we sought to elucidate their mechanism of action further. To this
end, we produced a custom D8-conjugated agarose gel that would allow us
to pull down proteins from our DOX-inducible OCT4-overexpressing SCLC
cell line that were bound to D8 and detect their presence via
immunoblot. Our pull-down studies demonstrated that D8 binds
specifically to DNA-PKcs, not OCT4, thereby disrupting the DNA-PKcs-OCT4
interaction (Figure 3B). To better characterize the interaction between
DNA-PKcs and its substrates after treatment with D8, we performedin vitro Ni-NTA pull-down studies using His-tagged OCT4 and p53.
After 30 minutes of treatment, we observed that D8 significantly
impaired the interaction between DNA-PKcs and OCT4 in a dose-dependent
manner but did not affect binding between DNA-PKcs and p53 (Figure 3C).
Further characterization of compounds inhibiting DNA-PKcs
catalytic activity
Having established that C8 and D8 significantly inhibited the
interaction between DNA-PKcs and OCT4, but noting that C8 and D8
impaired OCT4 pull-down, we sought to characterize our validated hits
better. We first determined the effect of our novel inhibitors on c-MYC
expression in our DOX-inducible OCT4-overexpressing SCLC cell line
(Figure 4A). In line with our proposed mechanism, inhibition of OCT4
phosphorylation at Ser93 correlated to a decrease in c-MYC expression.
Notably, C8 and D8 demonstrated remarkable reductions in c-MYC and OCT4
expression.
Next, we focused on in vitro kinase assays to assess our
inhibitors’ specific activity against DNA-PKcs-mediated phosphorylation.
The ADP-GloTM assay utilizes a luciferase reaction
where the amount of luminescence measured correlates to the amount of
ATP consumed by the kinase reaction. All seven of the inhibitors
validated in the first step of hit validation demonstrated activity
against DNA-PKcs-mediated phosphorylation of the peptide substrate
(Figure 4B). C8, D8, and G5 showed drastic reductions in DNA-PKcs kinase
activity. E10, the positive control selected, showed a modest decrease
in kinase activity. We then performed in vitro DNA-PKcs kinase
activity assays by utilizing two known DNA-PKcs substrates: OCT4 and
p53. After incubating the validated compounds with DNA-PKcs for 30
minutes, bacterially-derived (lacking post-translational modification)
OCT4 and p53 proteins were added to the kinase reaction. We then
detected the presence of phosphorylated pOCT4S93 and
phospho-p53S78 by IB (Figure 4C). C8 and D8 treatment
inhibited phosphorylation of both OCT4 and p53, reinforcing their
consideration as novel DNA-PKcs inhibitors with widespread activity.
Interestingly, G5 did not significantly impair OCT4S93phosphorylation but did inhibit p53S78
phosphorylation. One possible explanation is that G5 localizes and
binds to a region of DNA-PKcs crucial for binding to p53 and the peptide
substrate, but not OCT4. To assess whether C8 and D8 had activity
against other kinases, we performed the same set of in vitroassays using MK2 as the targeted enzyme and OCT4 and HSP27 as its
substrates. C8 and D8 are not active against MK2, suggesting that they
are specific inhibitors against DNA-PKcs activity (Figure S3A&B).
Taken together, the pull-down and in vitro assays also
demonstrate that the decreases in pOCT4S93 are not due
to degradation of OCT4; instead, these compounds act directly on
DNA-PKcs. To address whether the decrease in total OCT4 protein by C8
and D8 treatment is due to the proteasomal degradation, we treated the
DOX-inducible OCT4-overexpressing SCLC cell line with C8 and D8.
Although OCT4 expression decreased in a dose-dependent manner,
pretreatment of bortezomib prevented the decrease in OCT4 protein level
(Figure S3C). These results suggest that while C8 and D8 act primarily
to bind to DNA-PKcs, thereby preventing its ability to phosphorylate
substrates, they also serve to upregulate the degradation of its
substrates.
In vitro cytotoxicity
We tested the in vitro cytotoxic activity of C8 and D8 in fiveMYC -amplified and five MYC -nonamplified cell lines to
determine their efficacy as single agents. Both compounds demonstrated
notable activity in both the MYC -amplified andMYC -nonamplified cell lines. C8 was more cytotoxic (mean
IC50 = 3.30 nM in MYC- amplified, = 2.70 nM inMYC -nonamplified, = 3.00 nM in both) in SCLC cell lines relative
to D8 cytotoxic (mean IC50 = 25.92 nM inMYC- amplified, = 15.04 nM in MYC -nonamplified, = 20.48 nM
in both) (Figure 5A&B). These data indicate that both compounds have
potential as single-agent therapies in SCLC. Further testing with
current standard-of-care regiments may also be warranted. Recent studies
have shown that SCLC tumors with high c-MYC expression are particularly
susceptible to aurora kinase A inhibition (Mollaoglu et al., 2017). C8
and/or D8 in conjunction with aurora kinase inhibitors, such as
alisertib, may prove to be an effective targeted therapy.
Discussion
Aberrant kinase activity has been implicated in tumor development and
progression in a variety of cancers (J. Zhang et al., 2009). Thus, the
development of kinase inhibitors has emerged as a therapeutic strategy
in the treatment of malignancy. To date, over 50 kinase inhibitors have
received FDA approval for the treatment of a variety of malignancies
(Bhullar et al., 2018). These novel compounds have shown great promise,
but are limited in their overall efficacy because of off-target
toxicities and the development of chemoresistance. Off-target toxicities
are common in type I inhibitors, given that the ATP binding pocket is
well-conserved throughout the kinome (Bhullar et al., 2018).
Chemoresistance to kinase inhibitors often develops rapidly through a
variety of mechanisms: 1) mutations to the targeted kinase that reduce
the binding affinity of the compound, 2) the activation of malignant
downstream targets that circumvent kinase activity, 3) upregulation of
parallel signaling pathways, 4) mutations in drug uptake and transport,
5) epigenetic changes to cellular processes (Camidge, Pao, & Sequist,
2014; Gross, Rahal, Stransky, Lengauer, & Hoeflich, 2015; Holohan, Van
Schaeybroeck, Longley, & Johnston, 2013; Niederst & Engelman, 2013;
Tam & Weinberg, 2013). These limitations highlight the necessity to
develop new kinase inhibitors with higher specificity towards their
targets that can be paired with other therapeutics to overcome drug
resistance.
We have developed a novel cell-based drug screening assay that
explicitly identifies inhibitors of the kinase-substrate interaction
between DNA-PKcs and OCT4 that ultimately target aberrant c-MYC
expression in SCLC. In our experiments, we identified the regions of
DNA-PKcs that are necessary for OCT4 phosphorylation. We then achieved
stable expression of these regions of PRKDC (labeled with the
LgBiT subunit) and the domains of POU5F1 that are critical to
drive c-MYC expression (labeled with the SmBiT subunit) by utilizing the
P2A self-cleavage system. We then screened ~80,000
compounds and identified 56 compounds that inhibited the luminescence
reaction between the LgBiT and SmBiT subunits, thereby potentially
impairing the interaction between DNA-PKcs and OCT4. Given that we have
applied the same principles to develop a similar system targeting the
interaction between MK2 and OCT4, this cell-based drug screening assay
can apply to a multitude of kinase-substrate interactions.
Our first set of validation experiments indicated that 6 of the 56
compounds inhibited DNA-PKcs-mediated phosphorylation of OCT4 at its
Ser-93 residue. One compound (E10) was used as a positive control. We
then confirmed that two of these compounds, C8 and D8, significantly
impair the ability of DNA-PKcs to bind to OCT4 through our pull-down
studies and led to the degradation of OCT4. To further characterize our
inhibitors, we confirmed that these inhibitors act on our novel
DNA-PKcs/OCT4/c-MYC pathway by inhibiting c-MYC expression in an SCLC
cell line. C8, D8, and G5 significantly inhibited DNA-PKcs kinase
activity, DNA-PKcs-mediated phosphorylation of OCT4, and
DNA-PKcs-mediated phosphorylation of p53. Although our initial aim was
to identify specific inhibitors of the DNA/OCT4 interaction, we have
identified C8 and D8 as potent inhibitors of DNA-PKcs kinase activity
across multiple substrates. An increase in efficacy may balance this
perceived loss in specificity.
After establishing C8 and D8 as novel DNA-PKcs inhibitors, we conductedin vitro cytotoxicity assays to determine their effect on
numerous low and high c-MYC expressing SCLC cell lines. We demonstrated
that C8 and D8 are cytotoxic to SCLC cell lines (IC50 =
3.0 nM and 20.5 nM, respectively). c-MYC expression in SCLC is prevalent
in chemo-refractory disease (Kim et al., 2006), and clinical studies
have focused on targeting its activity through upstream mediators (Hook
et al., 2012; Owonikoko et al., 2016; Sos et al., 2012). Given that our
data show that these compounds are effective in targeting aberrant c-MYC
expression in SCLC and that they are cytotoxic to SCLC cell lines, the
further study focused on their in vivo activity as both single
agents and in combination with other therapies is necessary. These
compounds have the potential to serve as lead compounds that may be
further developed to target DNA-PKcs with higher affinity and potency.