Lung cancer is one of the most common cancers in the world. Non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancers, whereas small cell lung cancer represents 15–20% cases [1
]. Despite advancements in our understanding of the molecular/genetic basis of lung cancer and improvements in therapy, the five-year survival rate (18%) of patients with lung cancer is lower than those with many other types of cancer, such as melanoma of the skin (92%), female breast cancer (90%), and prostate cancer (99%) in the United States [2
]. Currently, platinum-based regimens are the first-line standard chemotherapy for treating NSCLC, and the second-line therapies include docetaxel, pemetrexed, or erlotinib. However, many patients with NSCLC receive third-line therapies because of no response or resistance to those therapies [3
]. Therefore, development of novel drugs or strategies involving combination therapy with the existing drugs is urgently required.
The human genome encodes 58 receptor type protein tyrosine kinases (RTKs), which have been structurally classified into 20 different subfamilies, including class III RTKs. A prototype RTK has an extracellular ligand-binding domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. Dysregulated RTK signaling has been implicated in the development of many human diseases. RTKs are often mutated, aberrantly overexpressed, or excessively activated in various types of cancer, and their signaling cascades affect tumor initiation, progression, malignancy, and metastasis [4
]. The class III RTK family includes platelet-derived growth factor receptor (PDGFR)-α/β, c-KIT, colony stimulating factor 1 receptor (CSF1R), and FMS-like tyrosine kinase 3 (FLT-3), which play critical roles in the proliferation, differentiation, angiogenesis, and malignancy of various types of human cancers [5
]. Numerous RTK inhibitors have been developed to induce cancer cell death in various tumor types.
Drugs targeting class III and class IV RTK for retarding tumor growth are attractive, as they exert their antitumor activity by regulating multiple molecular signaling mechanisms and offer the benefits of combination therapy with other therapeutic agents such as platinum-based drugs in various tumor types [6
]. However, small molecule compounds inhibiting class III RTK have not been well-studied in NSCLC. Only sorafenib and sunitinib, which are multi-kinase inhibitors targeting vascular endothelial growth factor receptor (VEGFR)-2 (class IV RTK), PDGFR-β, c-KIT, and FLT3, have shown significant antitumor activity and anti-angiogenesis in the preclinical models of various tumor types, including NSCLC [7
]. Multi-kinase inhibitors targeting mainly FLT3 are generally accepted for acute myeloid leukemia (AML) treatment but are not clinically used on solid tumors due to low efficacy. Currently, combination treatments, instead of single therapeutic agents, are being extensively investigated for the treatment of solid tumors [9
Recently, Maifrede et al. demonstrated that the mutated FLT3-ITD augmented reactive oxygen species (ROS) levels, which induced DNA damage, resulting in mutations and chromosomal instability, and inhibition of FLT3-ITD activity by a FTL3 inhibitor AC220 (quizartinib) inhibited two major DNA double-strand break (DSB) repair pathways due to inhibiting activities of the DNA repair proteins BRCA1, BRCA2, PALB2, RAD51, and LIG4 [14
]. The drug-mediated “BRCAness/DNA-PKness” phenotype can provide an opportunity to induce synthetic lethality with a poly (ADP-ribose) polymerase (PARP) inhibitor in combination therapy.
In our attempts to identify a small molecule potentially targeting the class III RTK, we designed, synthesized, and evaluated a novel multi-kinase inhibitor, AIU2001. This study aimed to investigate the antitumor effect of AIU2001 and the potential for combination therapy with AIU2001 and a PARP inhibitor or radiotherapy in NSCLC cells
In this study, we screened novel kinase inhibitors targeting class III RTK activity from synthesized in-house compounds using in vitro kinase profiling and cell viability assays in various cancer cell lines. Among the compounds, AIU2001 was identified as a potent class III RTK inhibitor with anti-cancer effects against solid cancer cell lines, including NSCLC cells, as well as AML cells. We observed that AIU2001 inhibited DNA damage repair and induced ROS production. Subsequent experiments demonstrated that AIU2001 induced the “BRCAness/DNA-PKness” phenotype, which contributed to the cytotoxicity of NSCLC cells and enhanced the sensitivity to a PARP inhibitor.
As quizartinib is a more potent and selective second-generation FLT3 inhibitor than the first-generation FLT3 inhibitor midostaurin, which was approved for AML treatment in 2017, it can inhibit multiple class III RTKs, including the structurally similar FLT3 and c-KIT. AIU2001 also exhibited a pan-class III RTK inhibitory effect.
Gain-of-function mutations of FLT3 and KIT play critical roles in the oncogenesis of AML [26
]. In particular, the most prevalent genetic mutations associated with AML have been detected in FLT3. Unlike other AML treatments, quizartinib has not been studied extensively due to its limited efficacy on solid tumors, which harbor few genetic mutations in FLT3, with the exception of gastrointestinal stromal tumors (GISTs), which harbor gain-of-function mutations in the KIT receptor. Most FLT3 inhibitors possess antitumor activity as they can block the activated STAT5 pathway in AML cells with FLT3-ITD, and combination approaches with quizartinib or midostaurin and other anti-cancer drugs were mainly studied in myeloid leukemia and not solid tumors [29
]. Recent studies have shown that inhibition of FLT3(ITD) activity by quizartinib downregulated the DNA repair proteins and indicated that quizartinib sensitized FLT3(TID)-positive AML cells to synthetic lethality triggered by PARP inhibitors via inhibition of the DSB repair pathways. This is because only FLT3(ITD)-positive leukemia cells, but not wild-type FLT3 AML cells, accumulate ROS-induced DSBs, but can survive due to the enhanced DNA repair activities [14
]. According to Maifrede et al., the quizartinib-mediated inhibition of DNA repair might be because of inhibition of the signaling pathway of class III RTK [14
]. Our data also demonstrated that STAT5 knockdown significantly inhibited the DNA repair genes in H1299 and A549 cells.
Class III RKT inhibitors are not considered for NSCLC therapy as FLT3 and c-KIT are not oncogenic drivers of NSCLC. Furthermore, the cellular function of STAT5 signaling has not been extensively studied in NSCLC cells. However, we found common features between FLT3(ITD)-positive AML cells and NSCLC cells, including strong resistance to DNA-damaging therapeutic agents due to high DNA repair system compared to wild-type FLT3 cells or normal cells. Thus, the question arises as to whether a FLT3 inhibitor could be effective on NSCLC cells when DNA repair genes are essential for the survival of NSCLC cells, even those with wild-type FLT3 [14
]. A large number of genetic alterations (> 200) have been identified in human NSCLC; for example, the v-Ki-ras2 Kirsten rat sarcoma virus oncogene (KRAS) and epidermal growth factor receptor (EGFR) are the most commonly mutated oncogenes that drive the pathogenesis of lung cancer [33
]. Amplification of KRAS or EGFR signaling is associated with DNA damage repair. Indeed, activation of mutated KRAS cancer cells is highly dependent on RAD51 for survival, KRAS mutation-dependent AKT1 stimulates HR and NHEJ activities for DNA DSB repair, and nuclear translocation of EGFR is associated with DNA-PKs for DSB repair [36
]. Several studies have demonstrated that combination treatment with inhibitors targeting HR and/or NHEJ with IR or cytotoxic drugs sensitizes NSCLC cells [39
]. We showed that AIU2001 inhibited cell viability, which is probably due to accumulated ROS-mediated DNA damage and suppression of DNA repair via downregulation of STAT5. Subsequently, AIU2001 treatment induced cell cycle arrest at the G2/M phase with accumulation of a significant sub-G1 population in H1299 and A549 cells. The mechanisms responsible for AIU2001-mediated downregulation of HR and NHEJ and the relationship between STAT5 and DNA damage repair genes have not been completely uncovered in this study, which is a limitation of this study. Further studies are required to investigate the mechanisms associated with AIU2001-induced downregulation of DNA damage repair genes.
As one of the clinical applications of synthetic lethal treatment, PARP inhibition has shown promising effect in the treatment of patients with tumors harboring mutations in BRCA1 or BRCA2. Recently, combination therapies with an FDA-approved PARP inhibitor and DNA damaging agents have been studied in NSCLC to expand the clinical indications of FDA-approved PARP inhibitors [41
]. AIU2001 downregulated the expression of HR and NHEJ genes, resulting in “BRCAness” and “DNA-PKness” phenotype, which contributed to synthetic lethality with PARP inhibition or IR in NSCLC cells. Our results demonstrated that the combination of AIU2001 and olaparib or IR significantly inhibited cancer cell growth. It is noteworthy that FLT3 inhibitors can be considered partner drugs of PARP inhibitors or radiotherapy for inducing synthetic lethality in solid tumors.
In summary, our study demonstrated that AIU2001 exhibited a potent pan-class III RKT inhibitor activity and cytotoxicity toward NSCLC cells. AIU2001 treatment suppressed DNA repair genes and induced DNA damage, resulting in induction of cell cycle arrest at the G2/M phase and apoptotic cell death. We have also shown that the combination of AIU2001 with the PARP inhibitor olaparib or IR considerably delayed cell proliferation. In this context, AIU2001 may act as an effective anti-cancer therapeutic for solid tumors as well as for AML although further investigations are warranted.
4. Materials and Methods
4.1. Chemical Synthesis
All chemical reagents were commercially available and were used without further purification. Melting points were determined using a Kruess M5000 melting point apparatus and were not corrected. Proton NMR spectra were recorded on an Avance-500 (Bruker, Billerica, MA, USA) at 500 MHz. Chemical shifts were reported in ppm units with Me4Si as the reference standard. Mass spectra were recorded on a JEOL, JMS-600W VG Trio-2 GC–MS. Reaction products were purified using flash column chromatography with silica gel 60 (230–400 mesh, Merck, Mumbai, India) and monitored using TLC on precoated silica gel 60 F254 (Merck). Spots were visualized under UV light (254 nm) after staining with phosphomolybdic acid (PMA) or Hanessian’s solution. The details have been described in the supplementary materials
and methods section and Supplementary Scheme S1
4.2. Prediction of Drug-Protein Interactions
For the docking study, the targets were prepared and minimized using the Cresset Flare software [42
], the grid box was defined according to the clustered ligand of downloaded FLT3 and c-KIT, and the docking calculations were performed using the Cresset Flare software in normal mode and default settings.
MTT was purchased from Amresco (Solon, OH, USA). The primary antibodies used in this study included the following; anti-PARP1, anti-cleaved caspase 3, anti-phospho-CHK1, anti-CHK1, anti-phospho-CHK2, anti-CHK2, anti-phospo-p53, anti-p53 (Cell Signaling Technology; Danvers, MA, USA), anti-Cyclin B1, anti-CDC25C, anti-phospho-histone H2AX (Santa Cruz Biotechnology; Dakkas, TX, USA), and anti-β-actin (Sigma-Aldrich; St. Louis, MO, USA) antibodies. Olaparib (AZD2281) and quizartinib (AC220) were purchased from Selleckchem (Houston, TX, USA) and dissolved in DMSO (Sigma Aldrich). N-acetyl-L-cysteine (NAC) was purchased from Sigma Aldrich. CM-H2DCFDA was purchased from Invitrogen (Carlsbad, CA, USA).
4.4. Cell Culture
H1299, A549, and H460 human lung cancer cell lines and BEAS2B and CCD18-Lu human normal lung cells (American Type Culture Collection, Manassas, VA, USA) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (H1299, A549, and H460; Welgene, Gyeonsangbukdo, Korea), Dulbecco’s modified Eagles medium (DMEM; BEAS2B; Welgene) or Eagle’s minimum essential medium (EMEM; CCD18-Lu; Welgene) supplemented with 10% fetal bovine serum (FBS; Welgene) and 100 units/mL penicillin streptomycin solution (Gibco, Grand Island, NY, USA) at 37 °C in a humidified 5% CO2 atmosphere.
4.5. Cell Viability Assay
Cell viability was assessed using the MTT colorimetric assay. Cells (1 × 103 cells/well) were seeded into 96-well plates and treated with the various concentrations of each compound or combination of two compounds. After five days of treatment, 10 μL MTT (0.5 mg/mL) was added, and further incubated for 3 h. After removal of the supernatant, the resultant pellet was dissolved in DMSO. The absorbance of the resultant formazan was measured at 540 nm using a plate reader (Multiskan EX; ThermoLabsystems, Waltham, MA, USA).
4.6. In Vitro Kinase Assay
Initial kinase profiling of AIU2001 against a panel of 53 functional kinases was performed by Eurofins (Eurofins Pharma Discovery, UK) and the IC50 values of the kinases were determined by Reaction Biology Corp. (Malvern, PA, USA).
4.7. Immunoblot Analysis
Cell lysates were prepared by extracting proteins with TNN buffer (40 mM Tris-Cl pH 8.0, 0.2% NP-40, 120 mM NaCl) or radioimmunoprecipitation assay (RIPA) lysis buffer (Millipore, Billerica MA, USA) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of proteins were separated using SDS-PAGE on 8–13% gels, and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline-Tween 20 (TBST) (150 mM NaCl, 10 mM Tris, 0.2% Tween20), followed by overnight incubation with primary antibodies at 4 °C. The blots were developed using peroxidase-conjugated secondary antibody and the immunoreactive proteins were visualized using enhanced chemiluminescence (ECL) reagents, according to the manufacturer’s recommendations (Amersham, GE Healthcare, Buckingamshire, UK). The protein bands were visualized using a digital imaging system (ImageQuant LAS 4000 mini; GE Healthcare, UK). The protein levels were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). Experiments were repeated at least thrice.
4.8. Annexin V/PI-Based Flow Cytometric Analysis
Annexin V assays were performed according to the manufacturer’s protocol (BD Pharmingen, San Diego, CA, USA). Briefly, 10,000 cells were plated into 60-mm plates and treated with varying concentrations of AIU2001 for 48 h. The cells were harvested and incubated with 4 μL allophycocyanin (APC)-conjugated annexin V (20 μg/mL) and 4 μL PI (50 μg/mL) for 15 min. Fluorescence analyses were performed using flow cytometry (CyFlow Cube 6; Sysmexpartec, Goerlitz, Germany). Cells were classified as early apoptotic (annexin V-positive/PI-negative), late apoptotic/necrotic (annexin V-positive/PI-positive), necrotic/dead (annexin V-negative/PI-positive), and live (annexin V-negative/PI-negative). Flow cytometry data was analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).
4.9. Cell Cycle Analysis
Samples were collected at the indicated time points and fixed in 70% cold ethanol overnight. For cell cycle analysis, the fixed cells were treated with RNase for 20 min before addition of 50 μg/mL PI and analyzed using FACS Calibur™ (BD Biosciences, San Jose, CA, USA).
4.10. Tumor Xenograft Mouse Models
A549 human lung cancer cell xenografts were initially established by s.c. implanting 1 × 106 cultured cells into the thigh of the right hind leg of six week old mice. When tumor volumes had reached approximately 150 mm3, AIU2001 (10 mg/kg) was administered i.p. once per two or three days for five times in total. All animal experiments were reviewed and approved by the Institutional Animal Care & Use Committee of Korea Institute of Radiological and Medical Sciences (kirams2018-0063, 6 December 2018).
4.11. Tumor Measurement
Two axes of the tumor (L, longest axis; W, shortest axis) were measured twice per week after irradiation using Vernier calipers. Tumor volume was calculated as (L × W2)/2 (mm3).
4.12. RNA Extraction and Quantitative Polymerase Chain Reaction (qPCR) Analysis
RNA was extracted using TRIzol® RNA isolation reagent (ThermoFisher Scientific). RNA was reverse-transcribed into cDNA using the M-MLV reverse transcriptase (Enzynomics, Daejeon, Korea) and RNase inhibitor (Promega, Madison, WI, USA). The sequence of the primers targeting human BRCA1, BRCA2, RAD51, BARD1, XRCC6, and XRCC5 are as follows: BRCA1 sense 5′-CTGAAGACTGCTCAGGGCTATC-3′, BRCA1 antisense 5′-AGGGTAGCTGTTAGAAGGCTGG-3′, BRCA2 sense 5′-GGCTTCAAAAAGCACTCCAGATG-3′, BRCA2 antisense 5′-GGATTCTGTATCTCTTGACGTTCC-3′, RAD51 sense 5′-CTCAGCCTCCCGAGTAGTTG-3′, RAD51 antisense 5′-CATCACTGCCAGAGAGACCA-3′, BARD1 sense 5′-GCCAAAGCTGTTTGATGGAT-3′, BARD1 antisense 5′-CGAACCCTCTCTGGGTGATA-3′, XRCC6 sense 5′-AAAAGACTGGGCTCCTTGGT-3′, XRCC6 antisense 5′-TGTGGGTCTTCAGCTCCTCT-3′, XRCC5 sense 5′-CGACAGGTGTTTGCTGAGAA-3′,and XRCC5 antisense 5′-TCACATCCATGCTCACGATT-3′. β-actin was used as a housekeeping gene for normalization. The cDNA was quantified using real-time PCR with SYBR Green/fluorescein qPCR master mix (ThermoScientific, Carlsbad, CA, USA) on a Lightcycler 96 system (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol.
4.13. Immunofluorescence and Foci Assay
For γ-H2AX and 4′,6-diamidino-2-phenylindole (DAPI) staining, cells were fixed in 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 20 min. The cells were then incubated with 1:200 dilution of anti-phosho-H2AX (Ser139) antibody overnight at 4 °C. AlexaFluor 594-conjugated anti-mouse IgG antibody (Abcam, Cambridge, UK) was used at 1:400 dilution for 1 h at room temperature. The slides were mounted in mounting medium (DAKO, Santa Clara, CA, USA) with DAPI (ThermoFisher Scientific) before imaging. Images were acquired using an LSM880 laser scanning microscope (ZEISS, Jena, Germany). Fluorescent images were captured using the appropriate filters. Images were analyzed using the Image J and ZEN software.
4.14. Detection of Intracellular Reactive Oxygen Species (ROS)
DMSO- or AIU2001-treated cells (5 × 105) were further treated with 10 μM CM-H2DCFH-DA for 30min and then washed with PBS before trypsinization. After detaching with trypsin, the cells were collected, washed, and resuspended in PBS. ROS inhibition was evaluated by treating cells with 5 mM NAC 2 h prior to AIU2001 treatment. Intracellular ROS levels were detected using a flow cytometer (CyFlow cube 6) at excitation/emission wavelengths of 488/525 nm.
4.15. Combination Index (CI)
CI scores were calculated using the CompuSyn software by Chou (CompuSyn Inc., Paramus, NJ) [25
] based on cell viability after treatment with single- and paired-drug concentrations. The CI equation for two drugs was used:
where (Dx)A is the concentration of drug A alone that inhibits x
%, (Dx)B is the concentration of drug B alone that inhibits x
%, (D)A or (D)B is the portion of drug A or drug B in the combination (D)A + (D)B that inhibits x
%. Thus (D)A + (D)B also inhibits x
4.16. Clonogenic Assay
Cells were seeded on 60-mm culture dishes at various densities and then treated with DMSO or 2 μM AIU2001. After 2 h, the cells were treated with the indicated doses of 137Cs γ-radiation. After 10 days, the colonies were fixed and stained with 1.5% methylene blue (Sigma Aldrich) in methanol solution. Colonies containing > 50 cells were counted. The dose enhancement ratio (DER) was calculated as the dose (Gy) of radiation that yielded a surviving fraction of 0.1 for DMSO-treated cells divided by that dose for AIU2001-treated cells. The experiment was performed in triplicate.
4.17. Statistical Analysis
Results are shown as means ± the standard deviations (SDs). Data were analyzed using two-tailed Student’s t-tests. Analysis of variance (ANOVA) and Tukey’s post hoc test were used for the two or three-group comparisons. Differences between groups with p-values < 0.05 were considered statistically significant.