Canertinib as an FLT3 Inhibitor with Potent Activity Against FLT3-Mutated and AC220-Resistant Acute Myeloid Leukemia

AML is one of the most common types of leukemia in adults¹. It is a biologically heterogeneous malignancy, and its mutational landscape can evolve dynamically throughout disease progression². Among the recurrent genetic alterations, mutations in the FLT3 gene are among the most frequent, occurring in approximately 30% of newly diagnosed cases³. FLT3 is a member of the class III receptor tyrosine kinase (RTK) family, structurally characterized by five extracellular immunoglobulin-like domains, a juxtamembrane segment, and two intracellular kinase domains separated by a kinase insert region⁴. FLT3 tyrosine kinase domain (TKD) mutations are observed in roughly 7% of patients and have shown inconsistent prognostic significance⁵. In contrast, internal tandem duplication (ITD) mutations occur in approximately 25% of cases and are widely recognized as critical drivers of leukemogenesis⁵. FLT3-ITD mutations cause constitutive activation of the receptor, leading to hyperactivation of the Mitogen-activated protein kinase (MAPK)/Extracellular signal-regulated kinase (ERK), Phosphatidylinositol 3 kinases (PI3K)/AKT, and STAT5 pathways, which promote uncontrolled leukemic cell proliferation, block hematopoietic differentiation, and confer anti-apoptotic properties^6,7. Clinically, FLT3-ITD-positive patients typically present with more aggressive disease, shorter overall survival, and higher relapse rates compared with FLT3-ITD-negative counterparts^8,9.

Therapeutic strategies targeting FLT3 have advanced into the second generation, including gilteritinib, quizartinib (AC220), and crenolanib. While these agents have improved patient outcomes, their efficacy and selectivity remain suboptimal^10,11,12. Based on binding mode, FLT3 inhibitors are classified into type I agents, such as gilteritinib, which bind the active conformation and inhibit both ITD and TKD mutations, and type II agents, such as AC220, which bind the inactive conformation and are highly active against ITD but less effective against TKD mutations¹³. In 2023, AC220 in combination with intensive chemotherapy was approved for newly diagnosed FLT3-ITD-positive AML¹⁴. Mechanistically, AC220 selectively stabilizes the inactive conformation of ITD-mutated FLT3, thereby blocking autophosphorylation and downstream signaling, ultimately suppressing proliferation and inducing apoptosis^15,16. Despite substantial clinical benefit, resistance to FLT3 inhibitors -- particularly type II agents -- remains a major issue arising from FLT3-TKD mutations (e.g., D835, Y842), F691 mutation or off-target genetic alterations, with TKD mutations representing a predominant mechanism¹⁷. This has driven the search for new FLT3 inhibitors capable of overcoming resistance. Additionally, these inhibitors tend to exhibit significant off-target effects, which can contribute to adverse side effects and reduce therapeutic effectiveness. The aim of this study is to address these limitations by evaluating the potential of Canertinib, a multi-kinase inhibitor, in targeting FLT3 mutations while also modulating other key pathways involved in leukemogenesis.

Canertinib has been characterized as a type V tyrosine kinase inhibitor with specificity for EGFR (ErbB1), ErbB2, and ErbB4 and forming covalent bonds with cysteine residues^18,19. The antitumour activity has been demonstrated in models of lung, ovarian, and breast cancers and hematologic malignancies, with favorable pharmacokinetic and safety profiles^20,21,22,23. In this study, canertinib was identified as a highly selective FLT3 inhibitor with the notable ability to FLT3-mutated AML. In vitro, canertinib exhibited potent activity against 32D-FLT3-ITD, 32D-FLT3-ITD-TKD, MOLM13, MV4-11, and AC220-resistant MV4-11 cells. These findings indicate that canertinib, as a potent FLT3 tyrosine kinase inhibitor, has the potential to overcome secondary resistance and could be developed as either monotherapy or in combination with chemotherapy for the treatment of AML.

Ethical approval for the study was granted by the Ethics Committee of The First Affiliated Hospital of Soochow University, in accordance with the Declaration of Helsinki.

Compounds
Canertinib (CI-1033), 8-Hydroxybergapten, Emodin anthrone, Kinetin, Rutaecarpine, Thiamine nitrate, Thiabendazole, and AC220 were used.

Virtual screening protocol
The protein complex of inhibitor FF-10101 with the FLT3 kinase domain (PDB ID: 5Y8Y) was used to generate the screening model. This protein was prepared using the Protein Preparation Wizard in Maestro (version 11.5, implemented in Schrödinger). The ligand FF-10101 was extracted from the crystal structure. Based on the FF-10101 crystal structure, a pharmacophore model was constructed using the Develop Pharmacophore Hypothesis module in the Phase program. Four main features were defined: aromatic rings (R14 and R15), a hydrogen bond acceptor (A3), and a hydrogen bond donor (D6). Ligands from the L6020 Selectable Natural Compound Library (Topscience) were prepared using the LigPrep program with default parameters. Ligands were energy-minimized using the OPLS3 force field. Virtual screening was performed using the Ligand and Database Screening module in the Phase program.

Cell lines and culture conditions
Cell lines MOLM13, HL60, THP-1, Kasumi-1, K562, MV4-11, 32D, and HEK-293T cells were used. Lentiviral transduction with mutation-specific plasmids was used to generate 32D cells stably expressing distinct FLT3 variants, following previously published protocols²⁴.

32D cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 5 ng/mL recombinant mouse interleukin-3. MOLM13, HL60, Kasumi-1, K562, MV4-11, and THP-1 were cultured in RPMI 1640 with 10% FBS and 0.05 mM β-mercaptoethanol. HEK-293T cells were maintained in DMEM containing 10% FBS. All cell lines were grown at 37 °C in a humidified 5% CO₂ atmosphere and tested negative for mycoplasma contamination.

Primary human peripheral blood mononuclear cells (PBMCs)
Primary leukemic blasts from one AML patient and peripheral blood mononuclear cells from one healthy donor were collected at The First Affiliated Hospital of Soochow University. PBMCs were separated using a density gradient reagent following the manufacturer's protocol. Cells were maintained in IMDM containing 10% fetal bovine serum (FBS), 50 ng/mL recombinant human stem cell factor (rhSCF), 100 ng/mL recombinant human FLT3 ligand (rhFLT3-L), 100 ng/mL recombinant human thrombopoietin (rhTPO), 25 ng/mL recombinant human interleukin-3 (rhIL-3), and 10 ng/mL recombinant human interleukin-6 (rhIL-6).

Cell viability assay
Cells (5-10 × 10³ per well for cell lines; 3-5 × 10⁴ per well for PBMCs) were seeded into 96-well plates and exposed to serial dilutions of the indicated compounds in triplicate. After 72 h at 37 °C, viability was measured using the Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. Dose-response curves and IC₅₀ values were calculated in GraphPad Prism 8.0.

Apoptosis analysis
For apoptosis quantification, 2 × 10⁵ cells per well were plated in 6-well plates, treated with the indicated compounds for 24 h, and stained using the APC-Annexin V apoptosis detection kit. Samples were analyzed on a flow cytometer, and data were processed with a flow cytometer software.

Western blot analysis
The cultured cells were collected, washed with PBS, and then lysed in radioimmunoprecipitation assay (RIPA) buffer. After lysing, the protein samples were clarified by centrifugation at 12,000 × g for 30 min at 4 °C, and the resulting supernatant was collected. The protein concentration was determined using a bicinchoninic acid (BCA) assay kit. Equal amounts (30 µg per lane) of protein were separated by using 12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and subsequently electrotransferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% bovine serum albumin (BSA) and incubated with primary antibodies at 4 ˚C overnight. The membrane was then incubated with secondary antibodies for 1 h at room temperature, and visualized with the use of an enhanced chemiluminescent western blotting detection reagent via a chemiluminescence imaging system. For the protein phosphorylation assay, the cells were collected after 6 h of compound treatment, and a phosphatase inhibitor cocktail was added when the cells were lysed. Anti-phospho-FLT3, anti-FLT3, anti-phospho-STAT5, anti-STAT5, anti-phospho-AKT, anti-AKT, anti-phospho-ERK, anti-ERK, anti-GAPDH and anti-β-actin were used.All antibodies were diluted according to the manufacturer's instructions.

Cellular thermal shift assay (CETSA)
CETSA was conducted as described previously with modifications²⁵. HEK-293T cells stably expressing FLT3-ITD (1 × 10⁷ cells) were treated overnight with 10 µM canertinib or vehicle (DMSO). Cells were lysed in RIPA buffer, aliquoted, and heated for 3 min across a temperature gradient (40-53.8 °C). Proteins were analyzed by immunoblotting. For isothermal dose-response analysis, lysates were prepared after exposure to canertinib (0-100 µM) at 51 °C. Band intensities were quantified with ImageJ, and statistical analyses were performed in GraphPad Prism 8.0.

In vitro enzymatic assays
The inhibitory effect of the test compounds on FLT3 kinase activity was evaluated by analyzing the phosphorylation of a biotinylated tyrosine kinase substrate (TK substrate) using a homogeneous time-resolved fluorescence (HTRF) assay. Optimal conditions for substrate, ATP, enzyme concentration, and reaction duration were established. The test compounds were initially dissolved in dimethyl sulfoxide (DMSO) and then diluted into a reaction buffer containing 50 mM HEPES (pH 7.0), 0.1 mM sodium orthovanadate, 0.01% BSA, 0.02% NaN3, 5 mM MgCl₂, 5 mM MnCl₂, and 1 mM DTT. Four µL of each test compound at varying concentrations were placed into a white 96-well plate, followed by the addition of 2 µL of FLT3 kinase (1 ng/µL) in kinase buffer to each well. After a 10-min pre-incubation period, the reaction was initiated by adding 2 µL of the TK substrate (final concentration 1 µM) and 2 µL of ATP (final concentration 100 µM) in kinase buffer. After a 60-min incubation at 37 °C, the reaction was stopped by adding 10 µL of a mixed detection solution, which included 5 µL of Eu3+-Cryptate-labeled TK antibody and 5 µL of Streptavidin-XL665 (125 nM final concentration). The phosphorylated peptide was then measured by time-resolved fluorescence resonance energy transfer (TRFRET) at an excitation wavelength of 337 nm and dual emission wavelengths of 665 and 620 nm, using a multimode plate reader after 1 h of incubation at 37 °C.

Direct binding of Canertinib to the FLT3 protein and inhibiting its activity
As shown in Figure 1A, a molecular docking-based virtual screening was initially performed to evaluate an L6020 Selectable Natural Compound Library (Topscience), which contains 19,000 natural compounds, yielding 6,837 candidate compounds. The results of the virtual screening were analysed by cluster analysis and visual inspection, and seven representative compounds were selected for cell viability assays in 32D cells and 32D cells with FLT3-ITD overexpression (32D-ITD) (Figure 1B). The result of the cell viability assay showed that one of the 10 compounds (canertinib) exhibited strong binding affinity for the FLT3 protein, with a 50% inhibiting rate at 9.472 µM. Canertinib contains multiple nitrogen-containing heterocycles (such as pyrimidine and morpholine rings) and halogen atoms (F and Cl), which typically possess strong polarity, enabling them to form hydrogen bonds or other polar interactions with polar amino acid residues in proteins (Figure 1C). A kinase activity inhibition assay using HTRF further revealed that canertinib has potent biochemical activity against FLT3 (Figure 1D). Compared to the DMSO-treated group, the dissolution curve of the Canertinib-treated group displayed a significant thermal shift: FLT3 protein was significantly reduced at 45.9 °C in the DMSO-treated group, while the canertinib-treated group showed no noticeable reduction until 51.9 °C (Figure 1E), confirming the direct interaction between canertinib and FLT3.

Canertinib against cell lines and primary AML blasts with FLT3 mutation
To validate the specificity of canertinib for FLT3-mutated cells, we assessed the survival of 32D-ITD cells. The results showed that canertinib effectively suppressed the growth of 32D-ITD cells. Notably, canertinib had minimal effects on parental 32D cells, indicating that its selective and potent activity stems from its specific inhibition of FLT3-ITD (Figure 2A). To investigate the therapeutic potential and specificity of canertinib for AML, we conducted proliferation assays using multiple human leukemia cell lines. The results demonstrated that canertinib significantly inhibited the proliferation of FLT3-ITD-expressing MV4-11 and MOLM13 cell lines, with half maximal inhibitory concentration (IC50) values of 0.849 µM and 1.509 µM. Meanwhile, canertinib showed some activity against FLT3 wild-type (FLT3-WT) AML cell lines, including K562, HL60, Kasumi-1, and THP-1, compared with PBMCs from healthy donors (Figure 2B). Furthermore, to confirm its inhibitory effect on FLT3-mutated AML, we conducted proliferation assays with human leukemia cells. The results showed that canertinib effectively inhibited the viability of primary AML blasts with FLT3-ITD (Supplementary Table 1) but largely spared healthy donor cells, highlighting the therapeutic potential of canertinib in treating AML patients (Figure 1B). After 24 h of canertinib treatment, the proportion of Annexin V-positive cells in 32D-ITD cells increased in a dose-dependent manner (Figure 2C). One of the common resistance mutations in FLT3-ITD is the D835Y mutation. After 24 h of canertinib treatment, we observed a dose-dependent increase in apoptosis in 32D cells overexpressing FLT3-ITD with D835Y mutation (32D-ITD+TKD) (Figure 2C).

Inhibition of FLT3 phosphorylation and downstream pathways in FLT3-ITD-positive cells by Canertinib
Previous studies have shown that activated FLT3 mutation promotes leukemia cell proliferation and survival by continuously autophosphorylating and activating downstream signaling proteins26. We treated 32D-ITD and 32D-ITD+TKD cells with various concentrations of canertinib for 6 h and analyzed the phosphorylation levels of FLT3 and its downstream target proteins, STAT5, AKT, and ERK, using Western blotting. The results indicated that canertinib dose-dependently inhibited the phosphorylation of FLT3 and its downstream target proteins, which is likely the mechanism underlying its significant antileukemic effect on FLT3-ITD-positive cells (Figure 3A,B). We repeated the same experiment in MV4-11 cells (Figure 3C). In primary cells from patients harboring FLT3-ITD mutations, Canertinib also inhibited the phosphorylation of FLT3 and its downstream pathway proteins (Figure 3D), suggesting that the antileukemic effect of Canertinib in patient-derived primary cells is related to the inhibition of the FLT3 signaling pathway.

Effective activity of Canertinib against AC220-resistant cells
FLT3 inhibitor resistance is one of the major causes of relapse in AML patients after treatment. To evaluate the antileukemic activity of canertinib in FLT3 inhibitor-resistant AML, we established an AC220-resistant cell line (MV4-11-ACR) by gradually increasing the FLT3 inhibitor concentration over a period of 12-16 weeks. Cell viability assays showed that MV4-11-ACR cells exhibited a significant reduction in sensitivity to AC220 compared to the parental MV4-11 cells (Figure 4A). We then assessed the inhibitory effect of Canertinib on MV4-11-ACR cells. The results demonstrated that even after acquiring resistance to AC220, Canertinib effectively inhibited the growth of MV4-11-ACR cells (Figure 4A). We observed a dose-dependent induction of apoptosis in MV4-11-ACR cells, consistent with the response seen in parental MV4-11 cells (Figure 4B). Western blot analysis showed that the antileukemic effect of Canertinib in resistant cells was closely associated with the dephosphorylation of molecules FLT3, STAT5, AKT, and ERK, a mechanism similar to that observed in parental MV4-11 cells (Figure 4C).

DATA AVAILABILITY:
All raw data are publicly available to ensure transparency and reproducibility at Zenodo (10.5281/zenodo.17657624).

Figure 1. The discovery of canertinib as a new FLT3 inhibitor. (A) Virtual Screening Strategy for Potential Compounds. (B) The IC50 concentrations of the candidate compounds in 32D and 32D-ITD cells after 72 h. (C) Structure of Canertinib. (D) Canertinib inhibits FLT3 kinase activity, detected by Homogeneous Time-Resolved Fluorescence. (E) 293T-FLT3-ITD cells were treated with canertinib (10 µM) or DMSO for 16 h. The temperature range tested was 40-58.4 °C (left). FLT3 protein levels were quantified using ImageJ, and the melting curve of FLT3 was plotted (right). Please click here to view a larger version of this figure.

Figure 2. Inhibitory activity of Canertinib against cell lines and primary AML blasts with FLT3 mutations. (A) The inhibitory effect of canertinib on the viability of 32D and 32D-ITD cells. At a concentration of 0, the mean viability from three replicates was normalised to 100% as a control. (B) IC50 of 32D, 32D-ITD and 32D-ITD+TKD; FLT3-WT (K562, HL60, THP-1 and Kasumi-1) and FLT3-ITD (MV4-11 and MOLM13) AML cell lines; PBMCs and blasts following canertinib treatment for 72 h. Cell proliferation was measured via WST-based CCK-8 proliferation assays. (C) Proportion of apoptotic cells in 32D, 32D-ITD, and 32D-ITD+TKD cells after treatment with different concentrations of canertinib for 24 h, ***P < 0.001. Please click here to view a larger version of this figure.

Figure 3. Canertinib inhibits FLT3 phosphorylation and downstream pathway activity. Canertinib inhibits FLT3 phosphorylation. Levels of p-FLT3, FLT3, p-STAT5, STAT5, p-AKT, AKT, p-ERK, and ERK in 32D-ITD, 32D-ITD+TKD, MV4-11, and FLT3-ITD AML blasts from patient following treatment with canertinib at the specified doses for 6 h. Please click here to view a larger version of this figure.

Figure 4. Effective activity of Canertinib against AC220-resistant cells. (A) The cell proliferation curve of Canertinib and AC220 at designated concentrations for 72 h on MV4-11 and MV4-11-ACR cells, cell proliferation was measured via WST-based CCK-8 proliferation assays. (B) Proportion of apoptotic cells in MV4-11 and MV4-11-ACR cells after treatment with different concentrations of canertinib for 24 h, ***P < 0.001, ****P < 0.0001. (C) Levels of p-FLT3, FLT3, p-STAT5, STAT5, p-AKT, AKT, p-ERK, and ERK in MV4-11-ACR. Please click here to view a larger version of this figure.

Supplementary Table 1: Representative clinical result. Please click here to download this File.

We identified canertinib as a selective FLT3 inhibitor with potent antileukemic activity across multiple FLT3-ITD-driven models, including AC220-resistant MV4-11-ACR cells. Thermal-shift assays and kinase activity assays confirmed direct FLT3 engagement, supporting its potential to overcome clinically relevant resistance. These findings underscore canertinib as a promising candidate to address the persistent challenge of FLT3 inhibitor resistance in AML^12,14,27,28.

Current FLT3 tyrosine kinase inhibitors (TKIs) are classified by binding mode into type I agents that recognize the active (DFG-in) conformation (e.g., gilteritinib, crenolanib) and type II agents that stabilize the inactive (DFG-out) conformation (e.g., AC220)²⁹. Type I TKIs generally retain activity against FLT3-ITD and most TKD mutations, whereas type II TKIs are highly potent against ITD but are compromised by activation-loop TKD substitutions-notably D835^11,30. Canertinib is a covalent inhibitor targeting FLT3 with a structure similar to FF-10101. The reason canertinib is able to overcome FLT3-TKD resistance lies in its unique mechanism of action. Unlike traditional Type II inhibitors, which rely on binding to the inactive conformation of FLT3, canertinib is a Type I inhibitor that binds to the active conformation of FLT3. Furthermore, canertinib forms an irreversible covalent bond with the cysteine residue (C695) in the FLT3 kinase domain, allowing it to bypass structural changes caused by TKD mutations (such as D835). This covalent binding and active conformation characteristic enable canertinib to effectively inhibit FLT3-TKD mutations, maintaining potent inhibitory activity even in the presence of structural resistance induced by TKD mutations. In 2023, AC220 combined with intensive chemotherapy received FDA approval for newly diagnosed FLT3-ITD-positive AML, underscoring the clinical value of type II inhibition and simultaneously highlighting the need to address secondary resistance³¹. We observed that canertinib remains active in AC220-resistant cells, suggesting a means to help offset vulnerabilities associated with type II resistance.

Jönsson et al. have previously reported that canertinib targets FLT3-mutated cells, demonstrating its specificity using a variety of primary samples and cell line models³². They performed detailed analyses in FLT3-ITD and FLT3-TKD models, showing effects on cell viability, proliferation, apoptosis, FLT3 phosphorylation, and downstream signaling. However, resistance to FLT3 inhibitors remains a major barrier to long-term survival in FLT3-mutated patients, highlighting the importance of evaluating the sensitivity of new FLT3 inhibitors in resistant settings. Among these, the FLT3-ITD+TKD mutation is one of the most common resistance mechanisms. We established 32D-FLT3-ITD+TKD cells and confirmed that canertinib effectively overcomes this dual mutation. Furthermore, by modeling the clinical acquisition of resistance, we generated an AC220-resistant cell line and demonstrated that canertinib retained efficacy in this context. Finally, we provided direct evidence that FLT3 is the molecular target of canertinib, thereby establishing a clear direction for future investigation. These findings position Canertinib as a promising FLT3-targeting agent with the potential to address two clinically salient challenges -- double-mutant disease and on-therapy resistance to AC220 -- thereby providing a pharmacologic basis for future translational evaluation and rational combination strategies.

The strengths of this study include the use of diverse FLT3-ITD and FLT3-ITD+TKD models, incorporation of primary patient specimens, establishment and validation of an AC220-resistant system, and confirmation of direct target engagement by thermal-shift and biochemical activity assays. Limitations are as follows: (i) the evidence is predominantly in vitro, with no PK/PD or in vivo efficacy data; (ii) the number of primary samples is limited; and (iii) the mutational coverage is incomplete-for example, the gatekeeper F691L Y842C and N676K variants that are clinically relevant resistance mechanisms^33,34. In addition, because canertinib is an irreversible pan-ErbB inhibitor and prior solid-tumor trials have documented class-typical EGFR/HER toxicities such as rash and diarrhea^35,36, comprehensive kinome-wide profiling and hematopoietic safety assessments are warranted to inform dose selection and the design of combination regimens.

The biology of resistance and relapse suggests that combination strategies often outperform single-agent therapy. Prior studies have shown that pairing FLT3 inhibitors with venetoclax is synergistic in preclinical FLT3-mutated AML models and, in early clinical testing, can deepen molecular responses^37,38,39. Canertinib is highly selective for FLT3 mutation cells, and future studies may consider combining it with venecla.

The findings here indicate that canertinib is a potent inhibitor of FLT3 mutations that can overcome secondary resistance in AML, particularly clinically important ITD+TKD composite mutations. Canertinib warrants clinical evaluation as monotherapy or in combination with conventional chemotherapy in patients with FLT3-mutated AML.