Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Nov 17.
Published in final edited form as: Biotechnol Bioeng. 2021 Mar 1;118(5):1987–2000. doi: 10.1002/bit.27712

Development of cell-based high throughput luminescence assay for drug discovery in inhibiting OCT4/DNA-PKcs and OCT4–MK2 interactions

Ismail S Mohiuddin 1,2, Sung-Jen Wei 1,2, In-Hyoung Yang 1,2, Gloria M Martinez 1,2, Shengping Yang 3, Eun J Cho 4, Kevin N Dalby 4, Min H Kang 1,2
PMCID: PMC8597627  NIHMSID: NIHMS1706052  PMID: 33565603

Abstract

Amplification-independent c-MYC overexpression is suggested in multiple cancers. Targeting c-MYC activity has therapeutic potential, but efforts thus far have been mostly unsuccessful. To find a druggable target to modulate c-MYC activity in cancer, we identified two kinases, MAPKAPK2 (MK2) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylate the Ser111 and the Ser93 residues of OCT4, respectively, to transcriptionally activate c-MYC. Using these observations, we present here a novel cell-based luminescence assay to identify compounds that inhibit the interaction between these kinases and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds (“hits”) that inhibited the luminescence reaction between DNA-PKcs and OCT4, and 65 hits inhibiting the MK2–OCT4 interaction. Using custom antibodies specific for pOCT4S93 and pOCT4S111, the “hits” were validated for their effect on OCT4 phosphorylation and activation. Using a two-step method for validation, we identified two candidate compounds from the DNA-PKcs assay and three from the MK2 assay. All five compounds demonstrate a significant ability to kill cancer cells in the nanomolar range. In conclusion, we developed a cell-based luminescence assay to identify novel inhibitors targeting c-MYC transcriptional activation, and have found five compounds that may function as lead compounds for further development.

Keywords: c-MYC, DNA-PKcs, drug discovery, kinase modulation, MK2, protein–protein interaction

1 |. 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 as of June 2020, 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; Zhang et al., 2009). This class of drugs led to a transformation from conventional chemotherapy to targeted cancer treatment and has overcome the normal cell toxicities of traditional chemotherapy. To date, the majority of the FDA-approved drugs that inhibit protein kinases bind at the active site and compete with ATP. Depending on targeting mechanisms, the kinase inhibitors are classified into four types: ATP competitors, binders of the catalytic sites of the inactive conformation of kinases, binders of noncatalytic subunit/ATP-binding sites (also called allosteric inhibitors), reversible inhibitors of substrate binding sites, and covalent kinase inhibitors (Breen & Soellner, 2015). 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 (Bhullar et al., 2018).

c-MYC overexpression plays a vital role in the oncogenic transformation of cells in various cancers (Eischen et al., 2001; Kaur & Cole, 2013; Stine et al., 2015). It is a transcription factor with a short half-life, and its expression is low and tightly expressed in normal cells (Dang, 2013; Farrell & Sears, 2014). On the other hand, either gene amplification or transcriptional/posttranscriptional regulation keeps the expression of c-MYC unusually high in tumor cells (Miller et al., 2012). Over a thousand c-MYC responsive genes have been reported, including genes involved in cell proliferation and metabolism (Miller et al., 2012). Given that c-MYC overexpression is frequent in cancers, and that it contributes to approximately 40% of all cancers in humans, regulation of c-MYC would benefit cancer treatment. However, strategies to directly target c-MYC have been challenging, mainly due to c-MYC being a transcription factor (Dang et al., 2017).

In our previous studies, we have reported that OCT4 binds to the promoter/enhancer region to activate c-MYC transcriptionally in progressive disease neuroblastoma, and demonstrated that MK2 phosphorylates OCT4 at its Ser111 residue (Mohiuddin et al., 2020; Wei et al., 2020). Also, we also identified DNA-PKcs as a potential mediator of this novel pathway and predicted that it phosphorylates OCT4 at its Ser93 residue. Here, we provide an approach to identify inhibitors with enhanced selectivity by targeting kinase-substrate interactions of two kinases, DNA-PKcs and MAPKAPK2 (MK2), with OCT4. To overcome the limitation of currently available kinase inhibitors, we developed a cell-based assay to identify compounds that selectively inhibit the kinase-substrate interaction between DNA-PKcs or MK2 with OCT4. Given the large size of DNA-PKcs, we first identified the OCT4 binding domain on the full-length protein. We then stably co-expressed the full-length OCT4 with either the crucial DNA-PKcs fragments or full-length MK2 tagged with luminescence probes in mammalian cells. During the process, all possible sequences of the genes and probes were tested to select the ones with optimal luminescence for the assay. Using the assay, we screened a library of compounds to identify “hits” that inhibit the ability of either DNA-PKcs or MK2 to bind to OCT4. Subsequently, we validated the compounds identified from the chemical library. This describes a novel cell-based assay to identify modulators of the DNA-PKcs- or MK2–OCT4 interaction and the method of hit validation employed to supplement our findings.

2 |. METHODS

2.1 |. Mammalian cell culture and transduction

HEK-293FT (Thermo Fisher Scientific) cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 2 mM Glutamine, 100 units/ml Penicillin, 100 μg/ml streptomycin sulfate, and 1 mM sodium pyruvate (Thermo Fisher Scientific). The HEK-293FT cells were plated at a cell dose of 1 × 107 on a 10-cm tissue culture dish and incubated at 37°C 5% CO2 incubator until the cells reach 80% confluence. The HEK-293FT cells were cotransfected either lentiviral ORFs, the constructs shown in Figure 1a, 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 expression in stable clones was confirmed by Western blot analysis.

FIGURE 1.

FIGURE 1

Open in a new tab

Compound screening and identification of “hits.” (a) Graphical 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 × the standard deviation from the mean as calculated in the blank plate panel (top)

2.2 |. 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), Natural product or Natural product-like (3280 compounds; MicroSource Discovery and LifeChem), Lopac (1280 compounds; Sigma-Aldrich), fragment sets (18,143 compounds) obtained from ChemBridge and ChemDiv, kinase set (11,250 compounds; ChemBridge), and diversity sets (43,158 compounds) obtained from NCI, ChemDiv, LifeChem, and Maybridge (Thermo Fisher Scientific). 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 (2000 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.

2.3 |. Identification of compounds interfering kinase-substrate binding (“hit ID”)

HEK-293FT cells stably expressing SmBiT-tagged DNA-PKcs and LgBiT-tagged OCT4, and NCI-H82 cells stably expressing SmBiT-tagged MK2 and LgBiT-tagged OCT4 were suspended at 1 × 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 h, 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 h 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 min 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”), that is, 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.

2.4 |. Custom polyclonal phospho-OCT4S93 and phospho-OCT4S111 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 pOCT4S111 polyclonal antibody was prepared by immunizing two New Zealand rabbits three times with an NH2-terminal KLH (keyhole limpet hemacyanin)-conjugated phosphopeptide SNSDGAPEPCTVT 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 for pOCT4S93 and SNSDGASPEPCTVT for pOCT4S111) column to deplete any IgG that was not specific to pOCT4S93 or pOCT4S111.

2.5 |. 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-mycDDK construct using a lentiviral system as previously described (Wei et al., 2020; Zhang et al., 2014). The cell lines were prepared for immunoblotting and co-immunoprecipitation to validate the effect on kinase-substrate binding as described previously (Kang et al., 2007). 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 (Cell Signaling Technology), anti-MK2 (Cell Signaling Technology), and 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 four 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 four 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 min. About 10 μl of 0.5 M Tris–HCl (pH = 7.4) was added to neutralize the acidification. Immuno-complexes were resolved, as described above.

2.6 |. In vitro DNA-PKcs kinase activity and ADP-Glo™ assays

About 100 units of purified DNA-PKcs (Promega) or 200 ng His-MK2 was incubated at 30°C for 30 min in 20 μl; kinase buffer containing 40 mM Tris (pH = 7.5), 20 mM MgCl2, 0.1 mg/ml BSA, activation buffer (100 μg/ml calf thymus DNA in 1× TE buffer), 150 μM ATP, and inhibitors of interest. Following the 30-min incubation, 1 μg of bacterially derived OCT4 (ProteinOne), p53 (Creative BioMart), and HSP27 (Enzo) protein substrates were added to the reaction. Samples were incubated again for 30 min at 30°C, and then the reactions were quenched with the addition of 4× NuPAGE LDS and 100 mM dithiothreitol (DTT) before proceeding with immunoblotting, as described above.

ADP-Glo™ assays were performed using the ADP-Glo™ Kinase Assay kits, as described in the instruction manual of Promega (#TM313; Stokoe et al., 1993). For the DNA-PKcs-related assays, a 25 μl kinase reaction was prepared to consist of purified DNA-PKcs (100 units) or His-MK2 (200 ng), Tris (40 mM, pH = 7.5), MgCl2 (20 mM), BSA (0.1 mg/ml), activation buffer (100 μg/ml calf thymus DNA in 1× TE buffer), ATP (150 μM), BSA (0.2 μg), peptide substrate (amino acid sequence: EPPLSQEAFADLWKK; Promega), and inhibitors of interest or DMSO (control). Reactions were pretreated with the inhibitors before the addition of the peptide substrate. For the MK2-related assays, the kinase reaction was conducted at room temperature for 60 min in a total volume of 25 μl of 1× kinase buffer with components of fully active GST-MK2 enzyme (0.4 ng), HSP27tide synthetic peptide substrate RRLNRQLSVA-amide (0.4 μg), ATP (50 μM), and 1% DMSO vehicle or inhibitor. Reactions were pretreated with inhibitors, including PF3644022 (100 nM or 1 μM), B5 (0.3 or 1 μM), C5 (30 or 100 nM), and E5 (10 or 30 nM), before the addition of HSP27 synthetic peptide in the reaction mixture. Following a 30-min incubation period at 30°C, 25 μl of ADP-Glo™ reagent was added to stop the reaction and deplete unconsumed ATP. Samples were incubated at room temperature for 40 min. About 50 μl of kinase detection reagent was added to the samples to introduce luciferase and luciferin to detect the presence of ATP. After 60 min 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.

2.7 |. 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, and NCI-H1876) were kindly provided by Dr. Adi Gazdar at the University of Texas Southwestern. Cells were cultured in RPMI (GE Lifesciences) supplemented with 10% heat-inactivated FBS. 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 × 106 cells) were plated in 96-well plates for 24 h before treatment with inhibitors (1 nM–10 μM in 3× increment). Six replicates of each drug concentration were used. After 96 h of drug incubation, cellular viability was measured using the DIMSCAN assay, as outlined in previous studies (Kang et al., 2011; Zhang et al., 2012).

3 |. RESULTS

3.1 |. Assay development and optimization

Having demonstrated that both DNA-PKcs and MK2 bind to and phosphorylate OCT4 at its Ser93 and Ser111 residues, respectively, we developed a luminescence-based drug screening assay using the NanoBiT® Protein:Protein Interaction system, which utilizes two subunit promoters (LgBiT and SmBiT) fused to genes of interest. The detailed cloning information is included in Supporting Information. 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 into HEK-293FT cells. We created a similar construct for MK2, utilizing the same regions of OCT4 tagged with SmBiT and the full-length MK2 sequence tagged with LgBiT, and achieved stable expression in NCI-H82 cells (Figure 2). 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, generating two peptide fragments in equal amounts in the transduced cell. To determine incubation time, we continuously monitored luminescence up to 45 min after adding Nano-Glo® Live Cell Reagent into HEK-293FT cells (for DNA-PKcs) and NCI-H82 cells (for MK2) stably transduced with the vectors shown in Figure 2. Also, three different cell seeding densities (2500, 5000, and 10,000 cells/well) and two FBS content parameters in the culture medium (10% FBS in RPMI or Opti-MEM®) were examined. 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 the culture medium over 45 min (Figure S1a). Culture medium with reduced serum generated 2- to 3-fold higher luminescence compared with DMEM or RPMI supplemented with 10% FBS in both HEK-293FT cells (Figure S1b) and NCI-H82 cells (Figure S1c). In addition, the cell count of 10,000 cells/well (10,000 cells in 10 μl of reaction volume: 1 × 106 cells/ml) displayed the highest luminescence signals relative to 5000 or 2500 cells/well regardless of the FBS content with both HEK-293FT cells and NCI-H82 cells. The luminescence signals sustained over the duration of the measurement both in NCI-H82 cells and in HEK-293FT with different peaks of signal. Thus, 15–20 min of incubation was employed for the screening assays.

FIGURE 2.

FIGURE 2

Open in a new tab

A schematic overview of the initial screening and two-step hit validation is presented. Initial screening was done after the cotransfection of wild-type POU5F1 (nt1-648) tagged with LgBiT and fragment 6 of PRKDC tagged with SmBiT in HEK-293FT cells or with wild-type POU5F1 (nt1-648) tagged with LgBiT and MAPKAPK2 (full length) tagged with SmBiT in NCI-H82 cells. Once stable clones were established, cells were seeded in a 384-well plate and were treated with an inhibitor. Decreased luminescence indicated a positive hit. Screening and hit identification yielded 56 compounds inhibiting the DNA-PKcs/OCT4 interaction and 65 compounds inhibiting the MK2–OCT4 interaction that were selected for further study. These compounds were validated via determining their effect on phosphorylation of OCT4 Ser93 or Ser111, kinase activity, and the protein–protein interaction between OCT4 and DNA-PKcs or MK2. Two 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. Three compounds were confirmed to inhibit pOCT4S111 and c-MYC expression and prevent the protein–protein interaction between OCT4 and MK2

3.2 |. 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 as well as MK2 and OCT4 (Figure 1 for DNA-PKcs and Figure S2a for MK2). The cells were seeded in plates containing Opti-MEM® culture media with reduced serum for 4 h. The cells were then incubated with 1 μM of each compound for 6 h before completion of the addition of the Nano-Glo® Live Cell Assay Reagent (Figure 1a). Following the analysis of the luminescence data, outliers (decreased signal) were identified as potential inhibitors of the DNA-PKcs/OCT4 or MK2–OCT4 interaction (representative heatmap in Figures 1a and S2a). Compounds that inhibited the luminescence reaction by a factor of 1.96 × standard deviation were deemed to be hits (Figures 1b and S2b). 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. For the MK2 assay, 65 compounds significantly reduced the luminescence, indicating inhibition of the interaction between MK2 and OCT4.

3.3 |. Effect on pOCT4S93 and pOCT4S111

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. Similarly, we have demonstrated that MK2 phosphorylates OCT4 at its Ser111 residue. To validate these anticipated findings, we first developed two custom phospho-antibodies specific to OCT4 Ser93 and OCT4 Ser111 (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 the custom-produced phospho-OCT4 antibody, we determined the effect of the previously identified “hits” on the phosphorylation of OCT4 at its Ser93 residue, for DNA-PKcs, and Ser111, for MK2, in our OCT4-overexpressing NCI-H82 construct (Figures S3 and S4). After DOX-induction for 12 h, we treated these cells with 1 μM of each compound. After 8 h of treatment, immunoblot analysis was performed to determine the expression levels of pOCT4S93 and pOCT4S111. We identified six candidate compounds from the screened DNA-PKcs “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 the inhibition of OCT4 Ser93 phosphorylation and decreased OCT4 expression while C9 and C10 reduced the phosphorylation of the OCT4 without affecting total OCT4. We selected C8 and D8 for the next step of validation due to the availability of the compounds for individual purchases. Three compounds were identified from the MK2 “hits” that impaired phosphorylation of OCT4 at Ser111 (Figure S3a,b).

3.4 |. 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, indicating that C8 and D8 interfered with the interaction between OCT4 and DNA-PKcs (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. Similarly, all three of the validated hits from the MK2 assay demonstrated a decreased association of MK2 after OCT4 pull-down at low nanomolar concentrations in a dose-dependent manner (Figure 3b).

FIGURE 3.

FIGURE 3

Open in a new tab

Hit Validation #2. Inhibition of the DNA-PKcs/OCT4 or MK2–OCT4 interaction following inhibitor treatment. (a) A stable clone of NCI-H82 cells expressing a DOX-inducible OCT4-mycDDK construct was treated with doxycycline for 18 h to induce OCT4-mycDDK expression. Cells were then treated with the inhibitors at the concentrations indicated for 6 h. 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. Total DNA-PKcs expression input is assessed. (b) Hit validation by pull-down studies in the DOX-inducible OCT4-overexpressing SCLC cell line demonstrates that treatment with B5, C5, and E5 disrupt the protein–protein interaction between OCT4 and MK2 in a dose-dependent manner. (c) Stable clones of NCI-H82 cells expressing a DOX-inducible OCT4-mycDDK construct were treated with doxycycline for 18 h to induce OCT4-mycDDK expression. Protein lysates were pulled-down using C8- or D8-conjugated agarose. C8 and D8 bound to DNA-PKcs, not OCT4. (d) In vitro Ni-NTA pull-down assays. DNA-PKcs was incubated with D8 for 30 min 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)

Given that the DNA-PKcs inhibitors C8 and D8 impaired the pull-down of both DNA-PKcs and OCT4, we sought to elucidate their mechanism of action further. To this end, we produced custom C8- and 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 either C8 or D8 and detect their presence via immunoblot. Our pull-down studies demonstrated that both C8 and D8 binds specifically to DNA-PKcs, not OCT4, thereby disrupting the DNA-PKcs/OCT4 interaction (Figure 3c). To better characterize the interaction between DNA-PKcs and its substrates after treatment with D8, we performed in vitro Ni-NTA pull-down studies using His-tagged OCT4 and p53. After 30 min 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 3d).

3.5 |. Further characterization of hits

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, and that B5, C5, and E5 inhibited the interaction between MK2 and OCT4, 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.

FIGURE 4.

FIGURE 4

Open in a new tab

Hit validation, in vitro assays, and kinase activity assays for DNA-PKcs hits. (a) Hit validation demonstrating that two compounds: C8 and D8, inhibit OCT4, pOCT4S93, and c-MYC expression, but not DNA-PKcs, in a DOX-inducible OCT4-overexpressing SCLC cell line (NCI-H82 pCW57.1-OCT4-mycDDK). (b) ADP-Glo™ 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. (c) In vitro kinase activity assays. DNA-PKcs was incubated with inhibitors for 30 min before the addition of bacterially derived OCT4 (His-tag) and p53 protein. After a second 30-min incubation, IB demonstrates that C8 and D8 decrease OCT4S93 phosphorylation. C8, D8, and G5 decrease p53S15 phosphorylation. 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), and NU (2.5)

Next, we focused on in vitro kinase assays to assess our inhibitors’ specific activity against DNA-PKcs-mediated phosphorylation. The ADP-Glo™ 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 min, bacterially derived (lacking posttranslational modification) OCT4 and p53 proteins were added to the kinase reaction. We then detected the presence of phosphorylated pOCT4S93 and phospho-p53S15 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 OCT4S93 phosphorylation but did inhibit p53S15 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 vitro assays 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 S5a,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 partly 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 S5c). 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.

We conducted similar experiments to determine the effect of B5, C5, and E5 on MK2 catalytic activity. Like the DNA-PKcs inhibitors, B5, C5, and E5 inhibited c-MYC expression in our DOX-inducible OCT4-overexpressing SCLC cell line (Figure 5a). Interestingly, these inhibitors did not impair phosphorylation of HSP27 at its Ser78 residue, indicating that these inhibitors are highly specific against the interaction between MK2 and OCT4, and may not affect MK2 catalytic activity.

FIGURE 5.

FIGURE 5

Open in a new tab

Hit validation, in vitro assays, and kinase activity assays for DNA-PKcs hits. (a) Hit validation demonstrating that three compounds: B5, C5, and E5 inhibit OCT4, pOCT4S111, and c-MYC expression, but not phosphorylation of HSP27, in a DOX-inducible OCT4-overexpressing SCLC cell line (NCI-H82 pCW57.1-POU5F1-mycDDK). Followed by DOX-induction, the cells for 8 h with the compounds at 2 μM, and subjected to immunoblotting. (b) ADP-Glo™ assay demonstrates that B5, C5, and D5 do not affect MK2 kinase activity. (c) In vitro kinase activity assays. MK2 (GST-tag) was incubated with inhibitors for 30 min before the addition of bacterially derived OCT4 (His-tag) or HSP27 protein. After a second 30-min incubation, IB demonstrates that OCT4S111 phosphorylation, but not HSP27S78 phosphorylation, was decreased. PF3644022 (known ATP-competitive MK2 inhibitor) was used as a positive control

To further explore the inhibitory mechanism of the compounds that impaired the ability of MK2 to phosphorylate OCT4, we first utilized the ADP-Glo™ Kinase Assay. When GST-MK2 was incubated with HSP27tide, the MK2 kinase activity was inhibited by the positive control. In contrast, none of the three compounds significantly affects the ability of MK2 to phosphorylate HSP27tide (Figure 5b). These data indicate that the three compounds identified here are not typical inhibitors of MK2 enzymatic activity in preventing the phosphorylation of OCT4. We then conducted in vitro kinase activity assays by using purified recombinant human GST-MK2 kinase and either Escherichia coli-expressed recombinant human full-length His-OCT4 or HSP27 protein. A known MK2 inhibitor, PF3644022, was used as a positive control. Compared with vehicle control, all three compounds reduced phospho-His-OCT4S111 levels. However, the three compounds did not affect the expression of pHSP27S78, suggesting that none of the compounds affect the enzymatic activity of MK2 (Figure 5c).

3.6 |. In vitro cytotoxicity

We tested the in vitro cytotoxic activity of C8 and D8 in nine SCLS, and both compounds demonstrated notable activity in the cell lines. C8 was more cytotoxic (mean IC50 = 3 nM) in SCLC cell lines relative to D8 (mean IC50 = 20 nM; Figure 6a,b). The dose–response curves of D8 are presented in Figure 6b. These data indicate that both compounds have potential as single-agent therapies in SCLC. Further testing with current standard-of-care regimens may also be warranted. Recent studies have shown that SCLC tumors with high c-MYC expression are particularly susceptible to aurora kinase A inhibition (Mollaogluoglu et al., 2017). C8 and/or D8 in conjunction with aurora kinase inhibitors, such as alisertib, may prove to be an effective targeted therapy.

FIGURE 6.

FIGURE 6

Open in a new tab

In vitro cytotoxicity assays of the validated DNA-PKcs compounds. (a) Calculated IC50 values for C8 (left) and D8 (right) in nine high and low c-MYC-expressing SCLC cell lines. (b) dose–response curves of D8 in nine 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 h before viability was assessed. Each condition was tested in six replicates. Symbols: mean, error bars: standard deviation. SCLC, small cell lung cancer

The cytotoxicity of B5, C5, and E5 was assessed in the same ten SCLC cell lines. IC50 concentrations of B5 ranged from 3 to 740 nM in the cell lines tested. The mean IC50 values of C5 and E5 were 25 nM and 3.3 nM, respectively (Figure 7a). The dose–response curves of E5 in all nine cell lines are shown in Figure 7b. IC50 values were lower than the lowest concentration tested (1 nM) in four of the nine cell lines (Figure 7b). In three cell lines (NCI-H510A, NCI-H1963, and NCI-H1876), the viability of cells treated with E5 was less than 10% (dose–response curves not included). All three compounds tested showed significant in vitro cytotoxicity at sub-nanomolar concentrations, depending on cell lines and the compounds.

FIGURE 7.

FIGURE 7

Open in a new tab

In vitro cytotoxicity assays of the validated MK2 compounds. (a) Calculated IC50 values for B5 (left), C5 (middle), and E5 (right) in nine high and low c-MYC-expressing SCLC cell lines. (b) E5 demonstrates cytotoxic activity toward nine SCLC cell lines. Similarly, B5 and C5 demonstrate significant activity (not shown). In three cell lines (NCI-H510A, NCI-H1963, and NCI-H1876), the viability of cells treated with E5 was less than 10%, and thus the dose–response curves are not shown. 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 h before viability was assessed. Each condition was tested in six replicates. Symbols: mean, error bars: standard deviation. SCLC, small cell lung cancer

3.7 |. MK2 inhibitors effect on inflammatory cytokines

As MK2 is a downstream substrate of the p38MAPK pathway and posttranscriptionally regulates cytokines, it is a pro-inflammatory mediator (Lee et al., 1994). Therefore, we evaluated the anti-inflammatory activity of the compounds at the concentrations with cytotoxic activity. While dexamethasone, a positive control, showed a significant reduction in TNF-α and IL-6 levels in a human monocytic leukemia cell line, no reduction in inflammatory cytokines was seen with the three compounds we identified, except under one condition: IL-6 level by 100 nM B5 (Figure S6a,b). These data suggest that the reduction of pOCT4S111 or c-MYC is not the consequence of the anti-inflammatory effect.

4 |. DISCUSSION

Chemoresistance to kinase inhibitors 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 et al., 2014; Gross et al., 2015; Holohan et al., 2013; Niederst & Engelman, 2013; Tam & Weinberg, 2013). These limitations highlight the necessity to develop new kinase inhibitors with higher specificity toward their targets that can be paired with other therapeutics to overcome drug resistance.

Based on our previous observation of novel c-MYC activation pathway via DNA-PK or MK2, we considered inhibiting kinase–protein interactions in the pathway (Wei et al., 2020). Targeting DNA-PKcs in malignancy has shown some promise, as a number of single- and dual-function inhibitors have demonstrated efficacy in preclinical studies. However, the vast majority of these inhibitors been tested in conjunction with chemoradiotherapy (Mohiuddin & Kang, 2019). MK2 is activated by the p38 MAPK (p38) pathway, which has been investigated as a therapeutic target in inflammatory diseases due to its role in the regulation of TNF-α and other mediators with unknown immune responses (Geng et al., 1996). Despite the robust preclinical data, p38 inhibitors did not advance to phase III clinical studies due to the incidences of systemic toxicities, including hepato- and cardiotoxicities, and CNS disorders (Emami et al., 2015; O’Donoghue et al., 2016), possibly due to the involvement of p38 in the regulation of more than 60 substrates with various physiological roles (Trempolec et al., 2013). For this reason, MK2 (the first identified substrate of p38 (Fiore et al., 2016) is being tested as an alternative target to p38 for treating inflammatory diseases (Fiore et al., 2016).

We have developed a novel cell-based drug screening assay that explicitly identifies inhibitors of the kinase-substrate interactions between DNA-PKcs and OCT4 as well as MK2 and OCT4 that ultimately target aberrant c-MYC expression in SCLC. In identifying hits and validating them, several factors facilitated the steep selection process. First, to bypass an additional step of confirming cell penetration, a cell-based assay was implemented by exogenously expressing proteins in cancer cells. Thus, any compounds that are impermeable to cancer cells are eliminated at the first screening stage. Another strategy to expedite the process was to employ a lower initial concentration to limit the number of hits. Generally, the hit rate of a screening assay is approximately 0.4%–1% (Newbatt et al., 2006; Yarrow et al., 2005). The hit rate of our assay was <0.1%, allowing for an efficient validation process.

Another unique feature of the screening method is the utilization of a single CMV promoter to generate the two proteins tagged with luminescence probes at an equal proportion. A commercially available product for a protein–protein interaction assay uses a bidirectional vector with two promoters to generate two proteins. This approach may produce two proteins with a different transcription/translation yield depending on the proficiency of the promoter. Of the two, the protein with a low molar concentration will be rate-limiting. To maximize the interaction and thus, the signals generated from the interaction, we used P2A, a peptide sequence of poliovirus, that cleaved the two proteins post-transcriptionally (Liu et al., 2017). Our experiments demonstrated that this strategy could be utilized to confirm the protein–protein interaction, which can be utilized in screening assays.

After screening a chemical library of nearly 80,000 compounds, we identified 56 hits that demonstrated activity against the interaction between DNA-PKcs and OCT4 and 65 hits that inhibited the MK2–OCT4 interaction. After our two-step validation process, we identified two candidate compounds that significantly impair the novel DNA-PKcs/OCT4/c-MYC pathway we have identified: a cardioglycoside and an isocarbostyril alkaloid. One of them has demonstrated activity in c-MYC overexpressing cancer, but the mechanism was not reported. Similarly, after validation, three compounds demonstrated significant inhibition of the MK2/OCT4/c-MYC pathway. All three compounds showed cholesterol-based structures, and their activity toward c-MYC or OCT4 has not been reported. 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 the five candidate compounds are effective in targeting aberrant c-MYC expression in SCLC and that they demonstrate in vitro anticancer activity at nanomolar concentrations, 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 aberrant c-MYC activity in cancer with higher affinity and potency.

Supplementary Material

BioTech and BioEng_Suppl

ACKNOWLEDGMENTS

The authors thank Dr. Adi Gazdar at the University of Texas Southwestern for providing small cell lung cancer cell lines with gene expression data. This study was funded by the National Institute of Health (R01 CA168699 to Min H. Kang) and the Cancer Prevention and Research Institute of Texas (RP170470 and RP130547 to Min H. Kang; RP160657 to Kevin N. Dalby).

Funding information

Cancer Prevention and Research Institute of Texas, Grant/Award Numbers: RP130547, RP160657, RP170470; National Cancer Institute, Grant/Award Number: R01CA168699

Footnotes

CONFLICT OF INTERESTS

The authors declare that there are no conflict of interests.

SUPPORTING INFORMATION

Additional Supporting Information may be found online in the supporting information tab for this article.

DATA AVAILABILITY STATEMENT

The resources used in the current study will be available to other researchers in the field on request.

REFERENCES

  1. Bhullar KS, Lagaron NO, McGowan EM, Parmar I, Jha A, Hubbard BP, & Rupasinghe HPV (2018). Kinase-targeted cancer therapies: Progress, challenges and future directions. Molecular Cancer, 17(1), 48. 10.1186/s12943-018-0804-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Breen ME, & Soellner MB (2015). Small molecule substrate phosphorylation site inhibitors of protein kinases: Approaches and challenges. ACS Chemical Biology, 10(1), 175–189. 10.1021/cb5008376 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Camidge DR, Pao W, & Sequist LV (2014). Acquired resistance to TKIs in solid tumours: Learning from lung cancer. Nature Reviews Clinical Oncology, 11(8), 473–481. 10.1038/nrclinonc.2014.104 [DOI] [PubMed] [Google Scholar]
  4. Cho EJ, Devkota AK, Stancu G, Edupunganti R, Powis G, & Dalby KN (2018). A Fluorescence-based high-throughput assay for the identification of anticancer reagents targeting fructose-1,6-bisphosphate aldolase. SLAS Discovery, 23(1), 1–10. 10.1177/2472555217726325 [DOI] [PubMed] [Google Scholar]
  5. Dang CV (2013). MYC, metabolism, cell growth, and tumorigenesis. Cold Spring Harbor Perspectives in Medicine, 3(8), a014217. 10.1101/cshperspect.a014217 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dang CV, Reddy EP, Shokat KM, & Soucek L (2017). Drugging the undruggable cancer targets. Nature Reviews Cancer, 17, 502–508. 10.1038/nrc.2017.36 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eischen CM, Roussel MF, Korsmeyer SJ, & Cleveland JL (2001). Bax loss impairs Myc-induced apoptosis and circumvents the selection of p53 mutations during Myc-mediated lymphomagenesis. Molecular and Cellular Biology, 21(22), 7653–7662. 10.1128/mcb.21.22.7653-7662.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Emami H, Vucic E, Subramanian S, Abdelbaky A, Fayad ZA, Du S, & Tawakol A (2015). The effect of BMS-582949, a P38 mitogen-activated protein kinase (P38 MAPK) inhibitor on arterial inflammation: A multicenter FDG-PET trial. Atherosclerosis, 240(2), 490–496. http://www.sciencedirect.com/science/article/pii/S0021915015001999 [DOI] [PubMed] [Google Scholar]
  9. Farrell AS, & Sears RC (2014). MYC degradation. Cold Spring Harbor Perspectives in Medicine, 4(3), a014365. 10.1101/cshperspect.a014365 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fiore M, Forli S, & Manetti F (2016). Targeting mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2, MK2): Medicinal chemistry efforts to lead small molecule inhibitors to clinical trials. Journal of Medicinal Chemistry, 59(8), 3609–3634. 10.1021/acs.jmedchem.5b01457 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Geng Y, Valbracht J, & Lotz M (1996). Selective activation of the mitogen-activated protein kinase subgroups c-Jun NH2 terminal kinase and p38 by IL-1 and TNF in human articular chondrocytes. The Journal of Clinical Investigation, 98(10), 2425–2430. 10.1172/JCI119056 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gross S, Rahal R, Stransky N, Lengauer C, & Hoeflich KP (2015). Targeting cancer with kinase inhibitors. The Journal of Clinical Investigation, 125(5), 1780–1789. 10.1172/JCI76094 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holohan C, Van Schaeybroeck S, Longley DB, & Johnston PG (2013). Cancer drug resistance: An evolving paradigm. Nature Reviews Cancer, 13(10), 714–726. 10.1038/nrc3599 [DOI] [PubMed] [Google Scholar]
  14. Hook KE, Garza SJ, Lira ME, Ching KA, Lee NV, Cao J, Yuan J, Ye J, Ozeck M, Shi ST, Zheng X, Rejto PA, Kan JLC, Christensen JG, & Pavlicek A (2012). An integrated genomic approach to identify predictive biomarkers of response to the aurora kinase inhibitor PF-03814735. Molecular Cancer Therapeutics, 11(3), 710–719. 10.1158/1535-7163.MCT-11-0184 [DOI] [PubMed] [Google Scholar]
  15. Kang MH, Kang YH, Szymanska B, Wilczynska-Kalak U, Sheard MA, Harned TM, Lock RB, & Reynolds CP (2007). Activity of vincristine, L-ASP, and dexamethasone against acute lymphoblastic leukemia is enhanced by the BH3-mimetic ABT-737 in vitro and in vivo. Blood, 110(6), 2057–2066. http://bloodjournal.hematologylibrary.org/cgi/content/abstract/bloodjournal110/6/2057 [DOI] [PubMed] [Google Scholar]
  16. Kang MH, Smith MA, Morton CL, Keshelava N, Houghton PJ, & Reynolds CP (2011). National Cancer Institute pediatric preclinical testing program: Model description for in vitro cytotoxicity testing. Pediatric Blood & Cancer, 56(2), 239–249. 10.1002/pbc.22801 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kaur M, & Cole MD (2013). MYC acts via the PTEN tumor suppressor to elicit autoregulation and genome-wide gene repression by activation of the EZH2 methyltransferase. Cancer Research, 73(2), 695–705. 10.1158/0008-5472.Can-12-2522 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kim YH, Girard L, Giacomini CP, Wang P, Hernandez-Boussard T, Tibshirani R, Minna JD, & Pollack JR (2006). Combined microarray analysis of small cell lung cancer reveals altered apoptotic balance and distinct expression signatures of MYC family gene amplification. Oncogene, 25(1), 130–138. 10.1038/sj.onc.1208997 [DOI] [PubMed] [Google Scholar]
  19. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Keys JR, Land vatter SW, Strickler JE, McLaughlin MM, Siemens IR, Fisher SM, Livi GP, White JR, Adams JL, & Young PR (1994). A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature, 372(6508), 739–746. 10.1038/372739a0 [DOI] [PubMed] [Google Scholar]
  20. Liu Z, Chen O, Wall JBJ, Zheng M, Zhou Y, Wang L, Ruth Vaseghi H, Qian L, & Liu J (2017). Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Scientific Reports, 7(1), 2193. 10.1038/s41598-017-02460-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Miller DM, Thomas SD, Islam A, Muench D, & Sedoris K (2012). c-Myc and cancer metabolism. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 18(20), 5546–5553. 10.1158/1078-0432.CCR-12-0977 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mohiuddin IS, & Kang MH (2019). DNA-PK as an emerging therapeutic target in cancer. Frontiers in Oncology, 9, 635. 10.3389/fonc.2019.00635 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mohiuddin IS, Wei SJ, & Kang MH (2020). Role of OCT4 in cancer stem-like cells and chemotherapy resistance. Biochimica et Biophysica Acta, Molecular Basis of Disease, 1866(4), 165432. 10.1016/j.bbadis.2019.03.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mollaogluoglu G, Guthrie MR, Böhm S, Brägelmann J, Can I, Ballieu PM, Marx A, George J, Heinen C, Chalishazar MD, Cheng H, Ireland AS, Denning KE, Mukhopadhyay A, Vahrenkamp JM, Berrett KC, Mosbruger TL, Wang J, Kohan JL, … Oliver TG (2017). MYC drives progression of small cell lung cancer to a variant neuroendocrine subtype with vulnerability to aurora kinase inhibition. Cancer Cell, 31(2), 270–285. 10.1016/j.ccell.2016.12.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Newbatt Y, Burns S, Hayward R, Whittaker S, Kirk R, Marshall C, Springer C, Mcdonald E, Marais R, Workman P, & Aherne W (2006). Identification of inhibitors of the kinase activity of oncogenic V600E BRAF in an enzyme cascade high-throughput screen. Journal of Biomolecular Screening, 11(2), 145–154. 10.1177/1087057105283584 [DOI] [PubMed] [Google Scholar]
  26. Niederst MJ, & Engelman JA (2013). Bypass mechanisms of resistance to receptor tyrosine kinase inhibition in lung cancer. Science Signaling, 6(294), re6. 10.1126/scisignal.2004652 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. O’Donoghue ML, Glaser R, & Cavender MA (2016). Effect of losmapimod on cardiovascular outcomes in patients hospitalized with acute myocardial infarction: A randomized clinical trial. Journal of the American Medical Association, 315(15), 1591–1599. 10.1001/jama.2016.3609 [DOI] [PubMed] [Google Scholar]
  28. Owonikoko TK, Nackaerts K, Csoszi T, Ostoros G, Baik C, Mark Z, Sheldon-Waniga E, Huebner D, Leonard EJ, & Spigel DR (2016). 1423O – Randomized phase 2 study of investigational aurora A kinase (AAK) inhibitor alisertib (MLN8237) + paclitaxel (P) vs placebo + P as second line therapy for small-cell lung cancer (SCLC). Annals of Oncology, 27, vi493. 10.1093/annonc/mdw389.01 [DOI] [Google Scholar]
  29. Roskoski R Jr. (2020). Properties of FDA-approved small molecule protein kinase inhibitors: A 2020 update. Pharmacological Research, 152, 104609. 10.1016/j.phrs.2019.104609 [DOI] [PubMed] [Google Scholar]
  30. Sos ML, Dietlein F, Peifer M, Schottle J, Balke-Want H, Muller C, Koker M, Richters A, Heynck S, Malchers F, Heuckmann JM, Seidel D, Eyers PA, Ullrich RT, Antonchick AP, Vintonyak VV, Schneider PM, Ninomiya T, Waldmann H, … Thomas RK (2012). A framework for identification of actionable cancer genome dependencies in small cell lung cancer. Proceedings of the National Academy of Sciences of the United States of America, 109(42), 17034–17039. 10.1073/pnas.1207310109 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stine ZE, Walton ZE, Altman BJ, Hsieh AL, & Dang CV (2015). MYC, metabolism, and cancer. Cancer Discovery, 5(10), 1024–1039. 10.1158/2159-8290.Cd-15-0507 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stokoe D, Caudwell B, Cohen PT, & Cohen P (1993). The substrate specificity and structure of mitogen-activated protein (MAP) kinase-activated protein kinase-2. Biochemical Journal, 296(Pt 3), 843–849. 10.1042/bj2960843 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tam WL, & Weinberg RA (2013). The epigenetics of epithelial-mesenchymal plasticity in cancer. Nature Medicine, 19(11), 1438–1449. 10.1038/nm.3336 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Trempolec N, Dave-Coll N, & Nebreda AR (2013). SnapShot: p38 MAPK signaling. Cell, 152(3), 656. http://www.sciencedirect.com/science/article/pii/S0092867413000810 [DOI] [PubMed] [Google Scholar]
  35. Wei SJ, Nguyen TH, Yang IH, Mook DG, Makena MR, Verlekar D, Hindle A, Martinez GM, Yang S, Shimada H, Reynolds CP, & Kang MH (2020). MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13-cisRA-mediated differentiation in neuroblastoma. Cell Death & Disease, 11(5), 368. 10.1038/s41419-020-2563-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wilson LJ, Linley A, Hammond DE, Hood FE, Coulson JM, MacEwan DJ, Ross SJ, Slupsky JR, Smith PD, Eyers PA, & Prior IA (2018). New perspectives, opportunities, and challenges in exploring the human protein kinome. Cancer Research, 78(1), 15–29. 10.1158/0008-5472.CAN-17-2291 [DOI] [PubMed] [Google Scholar]
  37. Yarrow JC, Totsukawa G, Charras GT, & Mitchison TJ (2005). Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor. Chemistry & Biology, 12(3), 385–395. 10.1016/j.chembiol.2005.01.015 [DOI] [PubMed] [Google Scholar]
  38. Zhang C, Ryu YK, Chen TZ, Hall CP, Webster DR, & Kang MH (2012). Synergistic activity of rapamycin and dexamethasone in vitro and in vivo in acute lymphoblastic leukemia via cell-cycle arrest and apoptosis. Leukemia Research, 36(3), 342–349. 10.1016/j.leukres.2011.10.022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhang J, Yang PL, & Gray NS (2009). Targeting cancer with small molecule kinase inhibitors. Nature Reviews Cancer, 9(1), 28–39. 10.1038/nrc2559 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhang Y, Chang FM, Huang J, Junco JJ, Maffi SK, Pridgen HI, Catano G, Dang H, Ding X, Yang F, Kim DJ, Slaga TJ, He R, & Wei SJ (2014). DSSylation, a novel protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells. Protein & Cell, 5(2), 124–140. 10.1007/s13238-013-0018-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

BioTech and BioEng_Suppl
NIHMS1706052-supplement-BioTech_and_BioEng_Suppl.pdf (624.2KB, pdf)

Data Availability Statement

The resources used in the current study will be available to other researchers in the field on request.

RESOURCES