Comet Assay to Quantify DNA Damage in FLT3 Mutant-expressing 32D Cells after Exposure to Type I and Type II FLT3 Inhibitors

Summary

This protocol describes an effective method for quantifying the difference in DNA damage induced by type I and type II inhibitors in FLT3 mutant cells through the application of the comet assay.

Abstract

FMS-like tyrosine kinase 3 (FLT3), a class III receptor tyrosine kinase, exhibits a mutation frequency of approximately 30% in acute myeloid leukemia (AML) patients and constitutes a critical therapeutic target. As representative type I and type II FLT3 inhibitors, respectively, gilteritinib and Quizartinib (AC220) are clinically employed in FLT3-mutant AML management.

Gilteritinib inhibits both activated and inactivated FLT3 proteins, with additional inhibition of AXL targets to help overcome resistance. AC220 inhibits activated FLT3. The comet assay was systematically employed to quantify and compare DNA damage patterns induced by type I versus type II FLT3 inhibitors in mutant cells. The experimental results revealed that the DNA damage caused by AC220 was significantly higher than that caused by Gilteritinib. For strong DNA damage, FLT3 inhibitors can be combined with DNA damage repair inhibitors to target DNA repair defects. The results provide experimental support for the rational combination strategy of DNA damage-targeting drugs.

Introduction

Acute myeloid leukemia (AML) represents a clonal hematopoietic stem cell disorder defined by pathological expansion of undifferentiated myeloid progenitors, resulting in hematopoietic suppression and bone marrow failure. The inherent molecular heterogeneity of AML poses significant therapeutic challenges¹. Of particular clinical significance, FMS-like tyrosine kinase 3 (FLT3) mutations constitute one of the most prevalent genetic alterations in AML, occurring in approximately 30% of cases, and demonstrate a strong correlation with adverse clinical outcomes². The FLT3 receptor tyrosine kinase undergoes activation at the plasma membrane to mediate PI3K/AKT and RAS/MAPK signaling cascades, whereas the FLT3-ITD mutant variant is retained within the endoplasmic reticulum, inducing persistent Signal Transducer and Activator of Transcription 5 (STAT5) phosphorylation. These genetic alterations drive leukemogenesis through multiple oncogenic mechanisms, principally through signal transduction dysregulation, uncontrolled proliferative signaling, and apoptotic resistance³.

AC220 and gilteritinib, as typical FLT3 inhibitors, inhibit cell proliferation and induce apoptosis by suppressing FLT3 expression, but with different mechanisms of action. Gilteritinib (a type I inhibitor) not only covers a broader spectrum of FLT3-activating mutations but also inhibits the AXL tyrosine kinase, which is closely related to FLT3. Through dual inhibition, gilteritinib can block the growth of cancer cells more comprehensively⁴. AC220 (a type II inhibitor) exerts its inhibitory effect through competitive binding to the activated kinase conformation, specifically targeting the ATP-binding pocket with high specificity⁵. This inhibition may lead to cell cycle arrest and increased DNA replication stress.

Gilteritinib is associated with a low risk of resistance and is indicated for monotherapy in relapsed/refractory FLT3-mutant AML. AC220 is effective and selective for FLT3-ITD mutations, with good clinical response rates, but is ineffective for FLT3-TKD mutations, and may be resistant to FLT3-TKD mutations due to secondary FLT3-TKD mutations (e.g., D835 or F691L) to developing resistance. These inhibitors have gained prominence in AML therapeutics based on demonstrated clinical efficacy and improved therapeutic indices⁶. However, the clinical use of these drugs is challenged by acquired resistance and off-target effects. Long-term use of AC220 in the clinic leads to resistance due to FLT3-TKD mutations, whereas long-term use of gilteritinib may have limitations for compound mutations (e.g., FLT3-ITD combined with TKD) even if it does not result in resistance due to FLT3-TKD mutations^7,8.

These two classes of FLT3 inhibitors exert a dual therapeutic effect by indirectly inducing replicative stress through inhibition of FLT3 and blockade of the proliferative signaling pathway-RAS-MAPK, PI3K-AKT-mTOR, STAT5 signaling. Blockade of cell proliferation usually leads to cell cycle arrest, metabolic disturbances (dysregulation of redox homeostasis), and ultimately to apoptosis^9,10. This mechanism ultimately generates DNA lesions that activate compensatory DNA repair adaptations in resistant cell populations. The comet assay enables quantitative comparison of inhibitor-specific DNA damage profiles in FLT3-mutant cells. This experimental approach provides critical insights for developing rational combination therapies that exploit DNA damage vulnerabilities, thereby overcoming acquired therapeutic resistance.

Contemporary methodologies for DNA damage detection encompass immunohistochemical analysis, agarose gel electrophoresis of DNA fragments, polymerase chain reaction (PCR), and next-generation sequencing (NGS), each demonstrating high analytical sensitivity and specificity¹¹. However, these approaches are constrained by substantial financial requirements, prolonged processing durations, and stringent experimental conditions. This underscores the imperative for adopting detection strategies that balance analytical precision with operational simplicity.

The comet assay has been extensively utilized in environmental toxicology and genotoxicity assessment due to its superior sensitivity, technical accessibility, visualized DNA damage quantification, and broad applicability across biological systems^12,13. Three principal methodological variants have been established: alkaline comet assay for single/double-strand break detection, neutral comet assay for double-strand break analysis, and enzyme-modified comet assay for specific DNA lesion identification.¹⁴

The methodological principle involves: (1) Induction of DNA strand breaks through experimental treatment; (2) Lysis buffer-mediated dissolution of cellular and nuclear membranes, allowing cytoplasmic/nuclear protein and RNA diffusion into electrophoresis buffer, while high-molecular-weight DNA remains immobilized in agarose matrix; (3) Alkaline denaturation (pH > 13) inducing DNA unwinding and liberation of damaged DNA fragments; (4) Electrophoretic field-driven anodic migration of fragmented DNA creating characteristic comet morphology, with intact DNA retaining spherical nucleoid structure^15,16. DNA damage quantification is achieved through computerized analysis of tail DNA percentage (TDP), providing precise measurement of genotoxic impact at single-cell resolution¹⁷.

This investigation employed a systematic experimental framework to compare the differential genotoxic effects of gilteritinib and AC220 in validated FLT3-mutant 32D cells. The experimental design comprised two principal components: (i) establishment of an FLT3 mutation-specific DNA damage model, and (ii) quantitative assessment of drug-induced DNA damage through alkaline comet assay methodology. The FLT3-mutant 32D cell line was maintained under standard in vitro conditions prior to exposure to serially diluted FLT3 inhibitors. Cellular viability was assessed using CCK-8 assays, with subsequent calculation of half-maximal inhibitory concentration (IC₅₀) values through nonlinear regression analysis. A five-fold IC₅₀ value was chosen as the DNA damage induction threshold. Drug-treated cells were harvested and embedded in low-melting-point agarose on slides for subsequent analysis. Electrophoretic separation was performed at 24 V (~0.74 V/cm) and 300 mA for 30 min in alkaline buffer, during which fragmented DNA migrated anodically to form characteristic comet morphology, while intact DNA retained spherical nucleoid configuration. DNA damage quantification was achieved through computerized image analysis comet assay software project (CASP) measuring tail moment, with increased Olive tail moment values directly correlating with the extent of DNA fragmentation.

Protocol

1. Construction of a DNA damage model in 32D cells with FLT3-ITD mutations ¹⁸

1. Cell recovery
   1. Using forceps, rapidly transfer cryopreserved FLT3-ITD mutant 32D cells (stored at -80 °C). Retrieve cryovials using forceps and immediately immerse them in a temperature-regulated water bath (37 °C ± 0.5 °C) for precisely 60 s to complete controlled thawing.
      NOTE: During thawing, intermittently shake the cryovials in the water bath to ensure uniform heating.
   2. Centrifuge the cryopreserved samples at 150 × g for 5 min at 25 °C ± 1 °C. Carefully aspirate the supernatant using sterile serological pipettes while retaining the cellular pellet.
   3. Add 1 mL of complete growth medium (RPMI 1640 + 10% fetal bovine serum + 1% penicillin/streptomycin) to the cryopreservation tube. Perform gentle trituration through 10 sequential pipetting cycles (1 mL volume) to ensure complete resuspension. Transfer the cell suspension to a 100 mm cell culture dish and add 7 mL of complete medium to achieve a final volume of 8 mL.
   4. Gently agitate the culture vessel with five horizontal and five rotational movements. Maintain cells in a humidified incubator at 37.0 °C ± 0.2 °C with 5% CO₂ and 95% humidity. Incubate for 24 H prior to experimental processing.
2. Cell viability assessment
   1. Select FLT3-ITD mutant 32D cells in the logarithmic growth phase with good cellular status for the experiment.
      ​​NOTE: Cells in the logarithmic growth phase usually have a short division cycle and a high frequency of cell division.
      1. Observe the morphology and growth state of FLT3-ITD-mutated 32D cells using an electron microscope. Ensure that the criteria for cellular proliferation thresholds are met: Look for che cells that are in the form of a single globule with smooth edges, uniform dispersion, few aggregates, uniform cell size, intact plasma membrane integrity, and cellular debris < 5%.
   2. Use a cell counter to determine the concentration of the cell suspension.
      NOTE: Mix the cell suspensions (10 sequential pipetting cycles using 1 mL sterile tips at 50% maximum pipette speed).
   3. Standardize the cell suspension to 2.0 × 10⁵ cells/mL in complete growth medium using iterative dilution. Using a pipette, dispense 50 µL aliquots into the central wells (B2-G7) of a 96-well flat-bottom plate. Fill the peripheral wells (A1-H12) with 100 µL of sterile PBS to maintain humidity and minimize edge evaporation effects.
   4. Preparation of FLT3 inhibitor-containing medium: Select nine wells in a 12-well cell culture plate and label them according to the FLT3 inhibitor concentration ladder (0, 3.90625, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 nM). Add 400 µL of complete medium to the wells labeled 10.0 µM and 200 µL of complete medium to the remaining wells.
      1. Gilteritinib dilution: Add 4 µL of 100 µM Gilteritinib reagent (Gilteritinib in Dimethyl Sulfoxide) to each of the wells labeled 1000 nM using a pipette gun and mix the solution thoroughly. Pipette 200 µL of 1000 nM Gilteritinib into the wells labeled 500 nM and mix; then pipette 200 µL of 500 nM Gilteritinib into the wells labeled 250 nM and mix. Each time, take 200 µL of the previous dilution into the next well and mix to achieve the desired concentration.
      2. AC220 dilution: Add 4 µL of 100 µM AC220 reagent (AC220 in Dimethyl Sulfoxide) to each of the wells labeled 1000 nM using a pipette gun. Follow step 1.2.4.1 for the other wells.
         NOTE: Ensure that the FLT3 inhibitor stock solution in the pipette is completely transferred to the wells to avoid residual solution. Ensure that the solution is well mixed before proceeding with dilution.
   5. Incubation: Dispense FLT3 inhibitor-containing media into designated wells (50 µL/well) using a pipette. Maintain the 96-well plate under physiological culture conditions (37.0 °C ± 0.2 °C, 5% CO₂, 95% humidity) for precisely 72 H ± 15 min.
   6. Aliquot 10 µL of CCK-8 reagent to all experimental and blank control wells using a pipette. Protect the plate from light and incubate it under normoxic conditions (37 °C, 5% CO₂) for 2.0 H ± 10 min.
      NOTE: Dispense the reagent at low pipette speed with a 2 mm tip immersion depth to minimize bubble formation. Avoid air bubbles during sample addition to prevent interference with OD readings.
   7. Remove the 96-well plate from the incubator and measure the absorbance at 450 nm using a microplate reader. Record the results.
   8. Calculate the % inhibition according to the IC₅₀ calculation formula:
      Inhibition (%) = × 100%
      ​The drug experimental well denotes cell wells treated with the drug. Cell control well denotes cell wells that were not treated with the drug. Blank control wells indicate medium only without cells.
   9. Analyze the normalized dose-response data through nonlinear regression, employing a four-parameter logistic model. Click the Fitting Curve button for curve fitting. Derive the half-maximal inhibitory concentration (IC₅₀) from model convergence (R² > 0.98, residual sum of squares < 0.15) and calculate the 95% confidence intervals through 1000-iteration bootstrap resampling.
3. DNA damage experiment of 32D cells with FLT3-ITD mutation induced by FLT3 inhibitor
   1. Select FLT3-ITD mutant 32D cells exhibiting growth spurt (population doubling time < 24 h) for DNA damage assessment.
   2. Count the cells (see step 1.2.2 for details).
   3. Cell seeding: Adjust the density of the cell suspension to 1 × 10⁶ cells per dish, using a 6 cm diameter dish and 4 mL of cell suspension per dish.
   4. Prepare three replicates for each inhibitor concentration: 55 nM gilteritinib: 4 mL of medium + 2.2 µL of 100 µM stock, 0.15 nM AC220: 4 mL of medium + 6 µL of 100 nM stock. Vortex-mix the solutions and equilibrate them at 37 °C for 15 min; gently shake the tubes to ensure adequate mixing. Aspirate the original medium and replace it with 4 mL/dish of freshly prepared inhibitor-containing medium using sterile serological pipettes.
   5. Incubate the treated cells under physiological conditions (37 °C, 5% CO₂, 95% humidity) for defined intervals (0, 2, 4, 6 H). Process time-zero controls immediately for baseline DNA damage assessment.
   6. Trypan blue exclusion assay
      1. Centrifuge the collected cells at 150 × g for 1 min. Discard the supernatant and resuspend the cells with 1 mL of culture medium.
      2. Pipette 100 µL of resuspended cells into a plastic centrifuge tube, add 100 µL of trypan blue stain (2x), mix gently, and stain for 3 min.
      3. Count the cells (see step 1.2.2 for details).
      4. Calculate cell viability as follows:
         
         

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Results

The comet assay was systematically employed to quantify differential DNA damage profiles induced by gilteritinib and AC220 in FLT3-mutant cell lines. Analyses showed that the difference in DNA damage between cell populations untreated with gilteritinib and AC220 was not statistically significant (P > 0.05). Dose-dependent increases in Tail DNA (%) and Olive Moment Tail (OMT) values achieved statistical significance (P < 0.05) following 2-6 H exposures. OMT represents the product of the % DNA in the tail and the 'di...

Discussion

The comet assay represents a significant advancement in genotoxicity assessment methodologies. This technique offers distinct advantages over conventional approaches through its technical accessibility, single-cell resolution, and direct visualization of damage patterns via electrophoretic migration profiles. In AML therapeutic development, the comet assay enables systematic evaluation of candidate agents' genotoxic profiles during target validation phases, providing critical pharmacodynamic data for preclinical optimiza...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 M EDTA	Beyotime	ST066
1x YeaRed nucleic acid stain	Yeasen	10202ES
32D CELL	Cobioer	CBP60995
AC220	TargetMol	T2066
Cell Counting Kit-8	Dojindo	CK04
Cell Counting Plate	QiuJing	XB-K-25
CO2 incubator	Thermo	51032872
Comet assay software project (CASP)
CometAssay Kit	Trevigen	4250-050-K
Cytation 5	BioTek	16280004
FBS	PAN	ST30-3302
Gilteritinib	TargetMol	T4409
GraphPad Prism 9.0
high-speed centrifuge	Thermo	9AQ2861
L-1000XLS+ Pipettes	Rainin	17014382
L-20XLS+ Pipettes	Rainin	17014392
liquid nitrogen tank	Mvecryoge	YDS-175-216
Multiskan FC microplate photometer	Thermo	1410101
Na2OH	DAMAO	1588
PBS	Solarbio	P1020
Penicillin-Streptomycin Solution, 100x	Beyotime	C0222
RPMI 1640 medium	Gibco	C22400500BT
Trinocular live cell microscope	Motic	1.1001E+12
Ultra-low temperature freezer	Haire	V118574