Abstract
Background/Aim: FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase involved in signal transduction underlying survival, proliferation, and differentiation of hematopoietic cells. An internal tandem duplication (ITD) in FLT3 in the juxtamembrane domain is a common mutation causing human acute myeloid leukemia (AML) and activates constitutive signaling. Materials and Methods: We evaluated the novel FLT3 inhibitor 5-(4-fluorophenyl)-N-(naphthalen-1-yl)oxazol-2-amine (AIU2008) for the treatment of AML. Results: AIU2008 was designed by modifying FLT3 inhibitor 7c, and showed improved anti-leukemic efficacy in FLT3-ITD–positive AML cells. Specifically, AIU2008 inhibited cell growth and apoptotic death. In addition, AIU2008 down-regulated DNA repair genes involved in homologous recombination and non-homologous end joining. It contributed to the synergistic inhibition of AML cell growth in combination treatment with PARP inhibitors. Conclusion: AIU2008 is a promising FLT3 targeting agent, and may be used in combination with PARP inhibitors for the treatment of AML.
- FLT3 inhibitor
- AML
- DNA damage repair
- combination therapy
- PARP1 inhibitor
Mutation in FMS-like tyrosine kinase 3 (FLT3), a type III receptor tyrosine kinase (RTK), is the most common genetic alteration in acute myeloid leukemia (AML) (1, 2). Internal tandem duplicates (ITD) in the juxtamembrane domain and a point mutation at D835 in the tyrosine kinase domain (TKD) are common FLT3 mutations (3). FLT3 mutations cause spontaneous induction of kinase activity with or without ligands.
Signal transducer and activator of transcription 5 (STAT5) is a well-known downstream mediator of RTKs, particularly in hematopoietic cancers. FLT3 harboring an ITD or TKD mutation is constitutively activated even in the absence of the ligand, resulting in aberrant STAT5 activation (4, 5). Aberrant signaling associated with FLT3 and STAT5 causes abnormal hematopoietic cell survival and proliferation, leading to leukemia.
Aberrant STAT5 signaling and Rac family small GTPase 1 (RAC1) activation caused by FLT3 mutation contribute to the generation of reactive oxygen species (ROS) (6). High levels of endogenous ROS increase DNA damage, such as double strand breaks (DSBs) and DNA oxidation (7, 8). Since AML is characterized by high levels of ROS, DNA damage is constantly being induced (6). Due to increased genomic instability, FLT3-ITD–positive AML cells show increased DNA repair activity, including homologous recombination (HR), non-homologous end joining (NHEJ), and nucleoside excision repair (NER) (9). Although this allows for cell survival, error-prone DNA repair mechanisms are also increased (10, 11). Abnormally low DNA repair activity can result in disease pathogenesis, whereas elevated DNA repair activity causes mismatch repair and inhibits apoptosis, causing abnormal cell survival (12). According to previous studies, FLT3 inhibitors down-regulate DNA repair genes associated with HR and NHEJ and show synergistic anti-leukemia activity with PARP inhibitors in combination therapy (13). Thus, decreasing DNA repair activity contributes to the mitigation of malignancy of AMLs. According to reports by Sallmyr et al. and our previous studies, FLT3 inhibition results in suppression of HR and NHEJ (6, 14, 15).
In our previous study, we designed and synthesized several FLT3 inhibitors and screened them by conducting in vitro FLT3 kinase inhibition tests and AML growth inhibition. We then suggested a novel FLT3 inhibitor, 7c (5-(4-fluorophenyl)-N-phenyloxazol-2-amine) that inhibits the FLT3-STAT5 axis and has a synergistic effect in combination treatment with a PARP inhibitor (15). In this study, we developed 5-(4-fluorophenyl)-N-(naphthalen-1-yl)oxazol-2-amine (AIU2008) by modifying compound 7c, which showed improved AML growth inhibition. Therefore, we aimed to identify the anti-leukemic activities of AIU2008 and the combination effect of AIU2008 and PARP-1 inhibitors in FLT3-ITD–positive AML.
Materials and Methods
Synthesis of chemicals. All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Sodium azide and 4-fluorophenacyl bromide in DMSO were stirred at room temperature for 2 h to obtain 2-azido-1-(4-fluorophenyl)ethan-1-one. The synthesized 2-azido-1-(4-fluorophenyl) ethan-1-one was dissolved in 1,4-dioxane, triphenyl phosphine and 1-naphthyl isothiocyanate were added at room temperature, and the mixture was refluxed at 100°C for 4 h. The final compound 5-(4-fluorophenyl)-N-(naphthalene-2yl)oxazol-2-amine was obtained. Proton nuclear magnetic resonance (NMR) spectra were recorded as described previously (15).
5-(4-fluorophenyl)-N-(naphthalen-1-yl)oxazol-2-amine (AIU2008). Yellow solid, mp=165-167°C, 1H NMR (300 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.29-8.37 (m, 1H), 8.12 (d, J=7.32 Hz, 1H), 7.81-7.99 (m, 1H), 7.43-7.671 (m, 7H), 7.33-7.27 (t, J=8.99 Hz, 2H); MS (FAB) m/z 305 (MH+).
Cell culture. Human AML MV4-11 and HL-60 cells were obtained from ATCC (Manassas, VA, USA). Molm-13 cells were kindly provided by Dr. In-Ki Kim (Asan Medical Center, Seoul, Korea). The cells were cultured in RPMI 1640 (Molm-13; Welgene, Korea) and IMDM (MV4-11, HL-60; Gibco, USA) media with 10% FBS (Welgene) and 1% penicillin (Gibco). Cells were incubated at 37°C, 5% CO2, and 95% humidity.
Cell viability assay. Cells were seeded in a 96-well plate. After 24 h, they were treated with AIU2008 at a concentration of 3.9-1000 nM, using vehicle (DMSO) as a control. Vehicle-and compound-treated cells were incubated at 37°C, 5% CO2, and 95% humidity for 5 days. Cell Counting Kit-8 (CCK-8; Dojindo, Japan) solution was added to each well. The 450 nm absorbance was measured by using a 96-well plate reader (Multiskan EX; ThermoLabsystems, Waltham, MA, USA)
Western blotting. Protein preparation, separation, and detection were conducted as described previously (15). Primary antibodies used in this study included the following: anti-cleaved caspase 3 (Cell Signaling Technology (CST #9661; Danvers, MA, USA), anti-phospho-FLT3 (CST #3461), anti-FLT3 (CST #3462), anti-phospho-STAT5a/b (Santa Cruz Biotechnology (sc) #81524; Dallas, TX, USA), anti-STAT5 (sc #271542), anti-BRCA1 (sc #6954), anti-BRCA2 (sc #293185), anti-BARD1 (sc #74559), anti-RAD51 (Abcam (ab) #63801; Cambridge, UK), anti-XRCC4 (sc #8285), anti-Ku80 (CST #2180), anti-Ku70 (sc #5309), anti-phospho-histone H2AX, and anti-β-actin (Sigma-Aldrich #A5316).
Quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was prepared using TRIzol® (Thermo Fisher Scientific, Waltham, MA, USA) and cDNA was synthesized using CycleScript reverse transcriptase (Bioneer, Daejeon, Republic of Korea). cDNA was mixed with target sequence primers and Maxima SYBR Green/Fluorescein qPCR Master Mix (Thermo Scientific). The designed primer sequences of housekeeping genes and target genes have been described previously (15). Thermal cycling conditions were as follows: initial duration at 95°C for 10 min, followed by 50 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Data were obtained and analyzed using the LightCycler 96 system and software (Roche, Germany).
In vitro kinase assay. In vitro kinase profiling of AIU2008 was performed by Reaction Biology Corp. (Malvern, PA, USA).
Molecular docking study. The FLT3 protein structure in PDB format was provided by Zorn et al. (16) and downloaded from the RCSB website. Docking analysis of AIU2008 with the FLT3 protein (PDB: 4XUF) was performed as described previously (15).
Statistical analysis. Results are represented as mean±standard deviations. Student’s t-test was performed using Microsoft Excel (Microsoft Co., Redmon, WA, USA) software program to indicate significant differences between vehicle- and compound-treated groups.
Results
AIU2008 inhibits FLT3 and FLT3 mutant kinases in vitro. To improve the efficacy of compound 7c reported previously (15), we designed and synthesized a novel compound, AIU2008, in which a phenyl group is substituted with a naphthyl group of 7c. Kinase inhibitory activities were determined by an in vitro kinase assay. As shown in Table I, kinase inhibition of wild-type FLT3 and D835Y and ITD mutants was calculated as half maximal inhibitory concentration (IC50), and was 50%, 72%, and 80% lower than compound 7c, respectively (15). In addition, AIU2008 mainly inhibited FLT members and class III RTK family members including cKIT and PDGFR (Table II). To investigate the physical mechanism of FLT3 inhibition by AIU2008, we explored the binding mode of AIU2008 and the crystal structure of FLT3 (PDB code: 4XUF) by in silico docking. AIU2008 was bound to the FLT3 structure by a pi-alkyl bond (Figure 1). The aromatic ring of AIU2008 and pi-alkyl interactions were observed between Val624, Val675, Leu818, Ala642, and Leu616, and pi-pi stacked between Phe830 and Tyr693. It showed hydrogen bonding between Cys694 and the nitrogen of the amine group of the oxazole ring. However, the fluorine interactions were not visible.
AIU2008 induced cell death of FLT3 mutated AML cells. After verifying the in vitro FLT3 inhibitory activity, we investigated the cancer cell-based toxicity effect by CCK-8 proliferation assay and immunoblotting of apoptotic targets. Human AML cell lines, Molm-13, MV4-11, and HL-60 were treated with vehicle or AIU2008 for 5 days, and then CCK-8 solution was added. As shown in Figure 2A, the survival rate of Molm-13 and MV4-11 decreased in proportion to the concentration of AIU2008. The IC50 of cell viability was 45.6 nM and 41.6 nM in Molm-13 and MV4-11, respectively. However, AIU2008-induced growth inhibition did not appear in the HL-60, FLT3-null cells. Only FLT3-ITD–positive cell lines, Molm-13 and MV4-11, showed inhibited cell growth. In addition, AIU2008-treated FLT3-ITD–positive cells had increased levels of cleaved-caspase 3, an apoptotic cell death marker (Figure 2B). Thus, AIU2008 increased apoptotic cell death of FLT3-ITD–positive AML cells.
AIU2008 reduced STAT5 signaling. In FLT3-ITD–positive AMLs, the FLT3 downstream mediator STAT5 is persistent and ligand-independently hyperactivated. High levels of p-STAT5 are a negative prognostic marker in myeloid leukemia and implicated in RTK inhibitor resistance (17). As a secondary confirmation of the inhibitory activity of AIU2008, we determined p-FLT3 and p-STAT5 levels in treated Molm-13 and MV4-11 cells by western blotting. Both p-FLT3 and p-STAT5 were dose-dependently reduced by AIU2008 (Figure 3). Reduction in aberrant STAT5 activation may contribute to alleviating malignant tumors in AML.
AIU2008 suppressed DNA damage repair. To investigate whether AML cell death induced by AIU2008 inhibited DNA repair, we examined the gene expression and protein levels of DNA damage repair genes and proteins in Molm-13 and MV4-11. As a result, HR-related genes including BRCA1, BRCA2, BARD1, and RAD51, and NHEJ-related genes XRCC4, XRCC5, and XRCC6, were significantly down-regulated in both FLT3-ITD–positive cell lines (Figure 4A and B). Consequently, DNA damage accumulated in AIU2008-treated Molm-13 and MV4-11 cells. We also found increased levels of the DSB recognition marker histone variant H2AX (γ-H2AX) (Figure 4C).
Anti-leukemic activities of AIU2008 in combination with a PARP inhibitor. We attempted a combination treatment with a PARP inhibitor, olaparib, to improve lethality. Molm-13 and MV4-11 cells were treated with AIU2008 and olaparib for 5 days, and viability was determined by CCK-8 assay. The combination treatment of AIU2008 and olaparib exerted a synergistic effect, contributing to AML growth inhibition (Figure 5). Therefore, combination treatment with AIU2008 and a PARP inhibitor might be a more effective anti-leukemic therapy.
Discussion
In this study, we devised a novel FTL3 inhibitor AIU2008, which showed improved cell growth inhibition compared to the oxazol-2-amine derivative 7c observed in our previous studies (13). AIU2008 is also an oxazol-2-amine-based small-molecule compound that contains fluorophenyl and naphthyl groups (Figure 1A). To investigate the efficacy of AIU2008, we examined the in vitro kinase profiling and cell viability in Molm-13 and MV4-11. The results showed that AIU2008 was five times more effective than 7c in inhibiting FLT3-ITD kinase in vitro and almost twice as effective as 7c in inhibiting AML cell growth, especially in MV4-11. The improved efficacy of AIU2008 is expected to be caused by chemical properties such as drug dissolution, absorption, and cellular stability (18). Therefore, with further study on the physicochemical properties and pharmacokinetics, AIU2008 could become a promising treatment agent for FLT3-ITD–positive AML.
Due to the high ROS levels caused by STAT5 and RAC1, genomic instability is increased in FLT3-ITD–positive AML. To survive ROS-induced genomic damage, AML cells acquire an enhanced DNA damage repair system. Indeed, human AML cells, including Molm-13 and MV4-11, highly express PARP1, an early DNA damage sensor, and RAD51, a key player in HR (12, 19). An overly-active DNA repair system might induce error-prone DNA repair, leading to mutagenesis and malignancy in leukemia. Kinase inhibitors targeting PARP1 effectively treat cancers with impaired HR and NER due to BRCA and DNA-PK mutations (20, 21). Since AIU2008 can inhibit the DNA repair of HR and NHEJ, we evaluated the effect of the combination treatment with the PARP1 inhibitor olaparib. As shown in our results, decreased DNA repair activity by AIU2008 sensitized Molm-13 and MV4-11 to a PARP inhibitor. Therefore, AIU2008 could be a novel drug targeting FLT3-AML–positive cells and a therapeutic agent for combination treatment with PARP inhibitors. Further investigation into the pharmacokinetics and metabolism of AIU2008 should be conducted.
Acknowledgements
This study was supported by the National Research Foundation of Korea (NRF; 2019R1A2C1009238) and the Korea Institute of Radiological and Medical Sciences (50531-2020) funded by the Korean government Ministry of Science and ICT.
Footnotes
Authors’ Contributions
AJ designed the study. KHJ and RH performed the experiments. AJ, KHJ, and RH performed the data analysis. CHK designed and synthesized the chemical compound and performed the docking analysis. AJ and KHJ wrote the manuscript. SJY and HSG revised the draft of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received December 10, 2020.
- Revision received December 27, 2020.
- Accepted December 28, 2020.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.