FTY720 Increases Doxorubicin Sensitivity in Resistant Acute Myeloid Leukemia Cells

Summary

This protocol evaluates how FTY720 increases doxorubicin sensitivity in resistant acute myeloid leukemia cells by assessing cell viability, apoptosis, intracellular doxorubicin accumulation, drug-efflux transporter expression, and protein kinase B/mechanistic target of rapamycin signaling.

Abstract

Chemoresistance remains a major barrier to effective treatment of acute myeloid leukemia, partly because resistant leukemic cells reduce intracellular drug exposure and evade apoptosis. FTY720, a sphingosine-1-phosphate receptor modulator, has reported antiproliferative and chemosensitizing activity in several tumor models. However, whether FTY720 increases doxorubicin sensitivity in doxorubicin-resistant acute myeloid leukemia cells, and whether this effect is associated with changes in drug-efflux transporters and protein kinase B/mechanistic target of rapamycin signaling, remains insufficiently defined. This study evaluated the effects of FTY720 alone and in combination with doxorubicin in parental acute myeloid leukemia cell lines (HL60 and K562) and their doxorubicin-resistant derivatives (HL60/A and K562/A). Cell viability was assessed using Cell Counting Kit-8 assays, apoptosis was measured by Annexin V/propidium iodide flow cytometry, intracellular doxorubicin accumulation was quantified by flow cytometry, and P-glycoprotein, multidrug resistance-associated protein 1, protein kinase B/mechanistic target of rapamycin pathway proteins, and apoptosis-related proteins were examined by flow cytometry and Western blotting. FTY720 showed dose-dependent cytotoxicity and increased doxorubicin sensitivity in resistant leukemia cells. Combined treatment reduced cell viability, increased apoptotic cell fractions, enhanced intracellular doxorubicin accumulation, and decreased P-glycoprotein and multidrug resistance-associated protein 1 expression. FTY720 treatment was also associated with reduced phosphorylation of protein kinase B and mechanistic target of rapamycin, whereas the protein kinase B activator SC79 partially reversed the observed changes in signaling, transporter expression, and apoptosis. These findings indicate that FTY720 increases doxorubicin sensitivity in HL60/A and K562/A cells and that this effect is associated with reduced protein kinase B/mechanistic target of rapamycin signaling, decreased drug-efflux transporter expression, enhanced intracellular doxorubicin accumulation, and increased apoptosis. Because this study was limited to in vitro cell-line models, further validation in primary leukemia samples and in vivo systems is required before therapeutic conclusions can be drawn.

Introduction

Acute myeloid leukemia is a heterogeneous hematological malignancy arising from the malignant transformation of hematopoietic stem and progenitor cells. It is the most common form of acute leukemia in adults and remains associated with poor long-term survival. Although treatment strategies have improved, induction chemotherapy based on cytarabine combined with an anthracycline remains a cornerstone of treatment. However, many patients develop primary or acquired resistance, leading to relapse, refractory disease, and poor clinical outcomes^1,2,3. Understanding the mechanisms that reduce doxorubicin sensitivity, therefore, remains an important objective in acute myeloid leukemia research.

Doxorubicin resistance is a multifactorial process. Resistant leukemia cells can reduce intracellular drug exposure through increased expression or activity of adenosine triphosphate-binding cassette drug-efflux transporters, including P-glycoprotein, encoded by adenosine triphosphate-binding cassette subfamily B member 1, and multidrug resistance-associated protein 1, encoded by adenosine triphosphate-binding cassette subfamily C member 1⁴. Activation of protein kinase B and mechanistic target of rapamycin signaling can also promote leukemia-cell survival, suppress apoptosis, and contribute to treatment resistance⁵. Together, these mechanisms reduce intracellular doxorubicin accumulation and diminish cytotoxic efficacy. Direct pharmacological targeting of multidrug-resistance transporters has been challenging because of toxicity, limited efficacy, and compensatory resistance mechanisms⁶. Similarly, inhibitors of protein kinase B and mechanistic target of rapamycin signaling face translational challenges related to dose-dependent toxicity, restricted efficacy, and pathway adaptation^7,8.

FTY720, also known as fingolimod, is a sphingosine-1-phosphate receptor modulator approved by the United States Food and Drug Administration for the treatment of multiple sclerosis and has attracted interest as a potential anticancer agent⁹. Beyond its immunomodulatory activity, FTY720 has demonstrated antiproliferative, proapoptotic, and treatment-sensitizing effects in several preclinical tumor models, including prostate cancer, hepatocellular carcinoma, lung cancer, colon cancer, multiple myeloma, breast cancer, ovarian cancer, leukemia, lymphoma, and glioblastoma^{10,11,12,13,14,15,16,17,18,19}. Previous studies have shown that FTY720 can enhance doxorubicin activity in breast cancer, increase cisplatin sensitivity in cisplatin-resistant non-small cell lung cancer cells, and resensitize cisplatin-resistant melanoma cells through modulation of phosphoinositide 3-kinase signaling^20,21,22. These findings suggest that FTY720 may influence both survival signaling and drug-retention mechanisms in resistant cancer cells. However, whether FTY720 increases doxorubicin sensitivity in doxorubicin-resistant acute myeloid leukemia cells, and whether this effect is associated with coordinated changes in intracellular doxorubicin accumulation, drug-efflux transporter expression, apoptosis, and protein kinase B/mechanistic target of rapamycin signaling, remains insufficiently defined.

The present study was designed to address this gap by using the doxorubicin-resistant acute myeloid leukemia cell models HL60/A and K562/A, along with their parental counterparts. It was hypothesized that FTY720 may increase doxorubicin sensitivity by reducing drug-efflux transporter expression, increasing intracellular doxorubicin accumulation, and enhancing apoptosis, with protein kinase B/mechanistic target of rapamycin signaling involved as an associated regulatory pathway. To evaluate this hypothesis, cell viability, half-maximal inhibitory concentration values, apoptosis, intracellular doxorubicin accumulation, transporter expression, and signaling-protein changes following treatment with FTY720 alone or in combination with doxorubicin were assessed. Because all experiments were performed in cell-line models, the findings are interpreted as in vitro mechanistic evidence rather than direct evidence of clinical efficacy.

Protocol

This study was performed using established human acute myeloid leukemia cell lines and their doxorubicin-resistant derivatives. No human participants, animals, or primary patient-derived samples were involved. Therefore, institutional review board approval for human-subject research and animal ethics approval were not required. The cell lines were obtained from the Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China, and maintained at the Fujian Institute of Hematology, Fujian Medical University Union Hospital. Cell identity was confirmed by karyotype and morphological assessment prior to experimentation, and all cell cultures were tested for mycoplasma contamination. The chemicals, equipment, and software used in the protocol are listed in the Table of Materials.

1. Cell culture and confirmation of the resistant phenotype

Human HL60 and K562 acute myeloid leukemia cells and their doxorubicin-resistant derivatives, HL60/A and K562/A, were obtained from the Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China. Cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a humidified incubator containing 5% carbon dioxide.

HL60/A cells were maintained in complete medium containing 1 µg/mL doxorubicin, and K562/A cells were maintained in complete medium containing 5 µg/mL doxorubicin to preserve the resistant phenotype. Doxorubicin was removed from the culture medium 1 month before experimental testing to avoid acute drug-selection effects during treatment assays.

Before the experiments, the resistant phenotype was reconfirmed by comparing doxorubicin half-maximal inhibitory concentration values between parental and resistant cells. Resistance stability after 1 month of drug-free culture was assessed by repeating the doxorubicin-sensitivity assay and examining basal P-glycoprotein and multidrug resistance-associated protein 1 expression. Cultures were used for experiments only when the resistant derivatives retained higher doxorubicin half-maximal inhibitory concentration values and higher transporter expression than their parental counterparts.

2. Drug preparation and treatment design

FTY720, doxorubicin, and SC79 were prepared as stock solutions according to the manufacturers’ instructions. FTY720 and SC79 were dissolved in dimethyl sulfoxide, and doxorubicin was prepared in the recommended solvent and protected from light during handling. Working solutions were freshly diluted in complete culture medium before treatment. The final dimethyl sulfoxide concentration was kept identical across treatment groups and did not exceed 0.1%.

Vehicle-control groups received the same final dimethyl sulfoxide concentration as the corresponding treatment groups. For combination-treatment experiments, 5 µM FTY720 was used as the sensitizing concentration. Doxorubicin was used at 4 µM for HL60/A cells and 8 µM for K562/A cells in apoptosis and intracellular doxorubicin accumulation assays, unless otherwise stated.

3. Cell viability assay and half-maximal inhibitory concentration calculation

Cells were seeded into 96-well plates at 1 × 10⁴ cells per well in 100 µL complete medium. For FTY720 single-drug testing, HL60, K562, HL60/A, and K562/A cells were treated with 0, 3.125, 6.25, 12.5, 25, 50, and 100 µM FTY720 for 48 h. For doxorubicin-sensitivity testing, HL60/A and K562/A cells were treated with serial concentrations of doxorubicin in the presence or absence of 5 µM FTY720 for 48 h.

Cell viability was measured using the Cell Counting Kit-8 assay according to the manufacturer’s instructions. After treatment, Cell Counting Kit-8 reagent was added to each well, and the plate was incubated at 37 °C for 2 h. Absorbance was measured at 450 nm using a microplate reader.

Relative cell viability was calculated as follows: relative cell viability = [(absorbance of treated wells - absorbance of blank wells) / (absorbance of vehicle-control wells - absorbance of blank wells)] × 100%

Half-maximal inhibitory concentration values were calculated using nonlinear regression with a four-parameter logistic dose-response model in GraphPad Prism, version 9.5. Each experiment included three independent biological replicates, with three technical wells per treatment condition.

4. Drug-combination analysis

The effects of FTY720 on doxorubicin sensitivity were evaluated by comparing doxorubicin dose-response relationships in the presence or absence of FTY720. HL60/A and K562/A cells were treated with serial concentrations of doxorubicin alone or doxorubicin combined with FTY720 for 48 h.

Cell viability was assessed using the Cell Counting Kit-8 assay, and half-maximal inhibitory concentration values were calculated using nonlinear regression with a four-parameter logistic dose-response model. Changes in doxorubicin sensitivity were interpreted based on shifts in dose-response curves and differences in half-maximal inhibitory concentration values between treatment groups.

Because the experiments were not designed as a complete fixed-ratio dose-matrix study, formal combination-index analysis was not performed. Therefore, the results were interpreted as evidence of altered doxorubicin sensitivity rather than definitive pharmacological synergy.

5. Apoptosis analysis by Annexin V and propidium iodide flow cytometry

HL60/A and K562/A cells were seeded into 6-well plates at 4 × 10⁵ cells/mL and treated for 48 h with vehicle, FTY720 alone, doxorubicin alone, or doxorubicin combined with FTY720. For combination treatment, 5 µM FTY720 was combined with 4 µM doxorubicin in HL60/A cells or 8 µM doxorubicin in K562/A cells.

After treatment, cells were collected, including cells suspended in the culture medium, and washed twice with cold phosphate-buffered saline. Cells were resuspended in Annexin V binding buffer at the concentration recommended by the apoptosis detection kit manufacturer. Each sample was stained with 5 µL Annexin V-fluorescein isothiocyanate or Annexin V-allophycocyanin and 5 µL propidium iodide, followed by incubation for 15 min at room temperature in the dark.

Samples were analyzed immediately by flow cytometry. Unstained controls, single-stained controls, and fluorescence-minus-one controls were used to set compensation and quadrant gates. Debris was excluded using forward-scatter and side-scatter gating. Early apoptotic cells were defined as Annexin V-positive and propidium iodide-negative cells, and late apoptotic or dead cells were defined as Annexin V-positive and propidium iodide-positive cells. Total apoptosis was calculated as the sum of early and late apoptotic cell fractions. At least 10,000 viable single-cell events were acquired for each sample.

6. Intracellular doxorubicin accumulation assay

HL60/A and K562/A cells were seeded into 6-well plates at 4 × 10⁵ cells/mL and cultured overnight. Cells were then treated with vehicle control, doxorubicin alone, or doxorubicin combined with 5 µM FTY720. HL60/A cells were treated with 4 µM doxorubicin, and K562/A cells were treated with 8 µM doxorubicin. Cells were incubated for 4 h at 37 °C and protected from light.

After treatment, cells were harvested and washed twice with cold phosphate-buffered saline to remove extracellular doxorubicin. Cells were resuspended in phosphate-buffered saline and analyzed by flow cytometry using the intrinsic fluorescence of doxorubicin. Untreated cells were used to define background autofluorescence, and doxorubicin-treated cells were used as the fluorescence-positive reference. Intracellular doxorubicin accumulation was quantified as median fluorescence intensity after debris and aggregate exclusion. Median fluorescence intensity values were normalized to the doxorubicin-alone group within each independent experiment.

7. Flow cytometric analysis of P-glycoprotein and multidrug resistance-associated protein 1 expression

HL60/A and K562/A cells were seeded into 6-well plates at 2 × 10⁶ cells/mL and treated with 5 µM FTY720 or vehicle control for 24 h. After treatment, cells were washed twice with ice-cold phosphate-buffered saline and resuspended in 100 µL cell staining buffer.

Cells were stained with phycoerythrin-conjugated anti-P-glycoprotein antibody and Alexa Fluor 647-conjugated anti-multidrug resistance-associated protein 1 antibody at the manufacturer-recommended dilution. Matched isotype controls, unstained controls, single-stained controls, and fluorescence-minus-one controls were included for gate setting and compensation. Samples were incubated for 30 min at 4 °C in the dark, washed twice with cell staining buffer, and resuspended in 500 µL cell staining buffer before acquisition.

Flow cytometry was performed using a BD FACScan flow cytometer. At least 10,000 viable single-cell events were acquired for each sample. P-glycoprotein and multidrug resistance-associated protein 1 expression levels were quantified as median fluorescence intensity after subtraction of the corresponding isotype-control signal. The normalized values were expressed relative to the vehicle-control group within each independent experiment.

8. Western blotting and densitometric quantification

After the indicated treatments, cells were collected and washed twice with cold phosphate-buffered saline. Total protein was extracted using radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors. Protein concentration was measured using a bicinchoninic acid protein assay kit.

A total of 30 µg protein per lane was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk or 5% bovine serum albumin in Tris-buffered saline containing Tween 20 for 1 h at room temperature.

Membranes were incubated overnight at 4 °C with primary antibodies against total protein kinase B, phosphorylated protein kinase B, total mechanistic target of rapamycin, phosphorylated mechanistic target of rapamycin, cleaved caspase-3, cleaved poly(adenosine diphosphate-ribose) polymerase, P-glycoprotein, multidrug resistance-associated protein 1, and glyceraldehyde-3-phosphate dehydrogenase. After washing, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence detection.

Band intensities were quantified using ImageJ software, version 1.53t. Phosphorylated protein kinase B was normalized to total protein kinase B, and phosphorylated mechanistic target of rapamycin was normalized to total mechanistic target of rapamycin. P-glycoprotein, multidrug resistance-associated protein 1, cleaved caspase-3, and cleaved poly(adenosine diphosphate-ribose) polymerase were normalized to glyceraldehyde-3-phosphate dehydrogenase. Normalized values were expressed as fold changes relative to the vehicle-control or doxorubicin-alone group, depending on the comparison. Western blot analyses were performed using three independent biological replicates. Uncropped Western blot images were deposited in the public data repository or uploaded as supplementary files.

9. SC79 rescue experiment

To examine whether protein kinase B signaling was associated with the observed effects of FTY720, HL60/A and K562/A cells were treated with FTY720 in the presence or absence of SC79, a protein kinase B activator. Cells were pretreated with 5 µM FTY720 for 12 h and then treated with 30 µM SC79 or vehicle control for an additional 12 h.

After treatment, phosphorylated protein kinase B, phosphorylated mechanistic target of rapamycin, P-glycoprotein, and multidrug resistance-associated protein 1 levels were assessed by Western blotting. In parallel, apoptosis was assessed by Annexin V and propidium iodide staining after doxorubicin, FTY720, and SC79 treatment. Rescue effects were interpreted as pathway-associated evidence. Because no genetic overexpression or knockdown experiment was performed, causal conclusions were stated cautiously.

10. Statistical analysis and data reporting

Statistical analyses were performed using GraphPad Prism, version 9.5, and IBM SPSS Statistics, version 23.0. Biological replicates were defined as independent experiments performed on different days using independently cultured cells. Technical replicates were defined as repeated wells or repeated measurements within the same biological experiment.

Data were presented as mean ± standard error of the mean unless otherwise specified. Two-group comparisons were analyzed using an unpaired two-tailed Student’s t-test when the data were approximately normally distributed. Comparisons among three or more groups were analyzed using one-way analysis of variance followed by Tukey’s post hoc test. Experiments involving two independent factors, such as drug concentration and treatment condition, were analyzed using two-way analysis of variance followed by an appropriate multiple-comparison correction.

When normality assumptions were not supported, nonparametric tests were used. Exact p-values were reported whenever possible. Statistical significance was defined as p < 0.05 after correction for multiple comparisons. All figure legends specified the number of biological replicates, the statistical test used, the meaning of error bars, and the significance threshold. Individual biological replicate values were shown in scatter plots or overlaid on bar graphs whenever possible.

Results

FTY720 reduced the viability of parental and doxorubicin-resistant acute myeloid leukemia cells

HL60, K562, HL60/A, and K562/A cells were exposed to increasing concentrations of FTY720 (0, 3.125, 6.25, 12.5, 25, 50, and 100 µM) for 48 h, and cell viability was measured using the Cell Counting Kit-8 assay. FTY720 reduced cell viability in all four cell lines in a concentration-dependent manner (Figure 1A). The calculated half-maximal inhibitory con...

Discussion

Chemoresistance remains one of the major barriers to effective acute myeloid leukemia treatment. In patients who fail to respond to induction therapy or develop relapse after initial response, resistant leukemia cells can persist despite exposure to cytarabine- and anthracycline-based regimens, making the identification of approaches that increase drug sensitivity an important experimental and clinical question²³. Doxorubicin is an anthracycline with broad antitumor activity, but its clinical use ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-well tissue culture plates	Corning / Costar	3516 or laboratory-verified equivalent	Used for treatment, apoptosis, doxorubicin accumulation, and protein-expression experiments.
96-well tissue culture plates	Corning / Costar	3599 or laboratory-verified equivalent	Used for Cell Counting Kit-8 assays.
Alexa Fluor 647 mouse immunoglobulin G1 isotype control	BioLegend	400130 or laboratory-verified equivalent	Isotype control for Alexa Fluor 647-conjugated antibody staining. Use the exact catalog number from the reagent vial.
Alexa Fluor 647-conjugated anti-human multidrug resistance-associated protein 1 / ABCC1 antibody	BioLegend	370103	Used for flow cytometric detection of multidrug resistance-associated protein 1. Product availability should be confirmed because some BioLegend MRP1 formats have been discontinued or replaced.
Annexin V apoptosis detection kit with propidium iodide	BioLegend	640914 or laboratory-verified equivalent	Used for Annexin V and propidium iodide apoptosis analysis by flow cytometry. If Annexin V-allophycocyanin was used instead of Annexin V-fluorescein isothiocyanate, replace this with the exact kit catalog number.
Bicinchoninic acid protein assay kit	Thermo Fisher Scientific / Beyotime / laboratory-verified supplier	Use the actual catalog number	Used for protein concentration measurement.
Carbon dioxide cell culture incubator	Thermo Fisher Scientific / Eppendorf / laboratory-verified instrument	Model to be inserted from laboratory record	Maintained cells at 37 °C and 5% carbon dioxide.
Caspase-3 antibody	Cell Signaling Technology	9662; RRID:AB_331439	Detects full-length and cleaved caspase-3 by Western blot. If a cleaved caspase-3-specific antibody was used, replace with the exact catalog number.
Cell Counting Kit-8	Dojindo Molecular Technologies	CK04	Used for cell viability and half-maximal inhibitory concentration assays.
Cell staining buffer	BioLegend	420201	Used for antibody dilution and washing during flow cytometric staining.
Cleaved poly(adenosine diphosphate-ribose) polymerase antibody	Cell Signaling Technology	94885; RRID:AB_2800237	Western blot antibody for cleaved poly(adenosine diphosphate-ribose) polymerase.
CompuSyn	ComboSyn, Inc.	Version 1.0 or laboratory-verified version	Used for Chou-Talalay combination-index analysis if complete combination-dose data are available.
Dimethyl sulfoxide	Sigma-Aldrich / Merck	D2650 or laboratory-verified equivalent	Vehicle for FTY720 and SC79 stock solutions; final concentration did not exceed 0.1%.
Doxorubicin hydrochloride	Use the actual supplier from the laboratory purchase record	Use the actual catalog number and lot number	Anthracycline used for resistance maintenance and treatment assays. This entry must match the reagent actually used in the experiments.
Enhanced chemiluminescence substrate	Thermo Fisher Scientific / MilliporeSigma / laboratory-verified supplier	Use the actual catalog number	Used for Western blot signal detection.
Fetal bovine serum	Gibco, Thermo Fisher Scientific	10099-141 or laboratory-verified equivalent	Added to culture medium at 10%. Use heat-inactivated or non-heat-inactivated serum consistently across all experiments.
Flow cytometer	BD Biosciences	BD FACScan or actual instrument model	Used for apoptosis, intracellular doxorubicin accumulation, and transporter-expression assays. Record laser/filter configuration in the supplementary methods if available.
Flow cytometry analysis software	BD Biosciences / FlowJo / laboratory-verified software	Version to be inserted from laboratory record	Used for compensation, gating, and median fluorescence intensity analysis.
FTY720 / fingolimod hydrochloride	Use the actual supplier from the laboratory purchase record	Use the actual catalog number and lot number	Drug-treatment reagent. This entry must match the reagent actually used in the experiments. Do not replace with a different supplier unless the experiment was performed with that product.
Glyceraldehyde-3-phosphate dehydrogenase antibody	Abcam	ab181602	Loading control for Western blot. Confirm RRID from the product insert before submission.
GraphPad Prism	GraphPad Software	Version 9.5	Used for dose-response curve fitting, half-maximal inhibitory concentration calculation, and statistical analysis.
HL60 human acute myeloid leukemia cells	Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China	Not commercially catalogued in this study	Human acute myeloid leukemia parental cell line; authenticated by karyotype and morphology before use. If a public reference is required, HL60 corresponds to RRID:CVCL_0002.
HL60/A doxorubicin-resistant cells	Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China	Not commercially catalogued	Doxorubicin-resistant derivative of HL60 cells; maintained at the Fujian Institute of Hematology. Resistance was maintained with 1 µg/mL doxorubicin before drug-free culture.
Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibody	Cell Signaling Technology / Abcam / laboratory-verified supplier	Use the actual catalog number	Secondary antibody for Western blot. Insert exact supplier and catalog number from laboratory records.
IBM SPSS Statistics	IBM	Version 23.0	Used for statistical analysis.
ImageJ	National Institutes of Health	Version 1.53t	Used for Western blot densitometric quantification.
K562 human leukemia cells	Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China	Not commercially catalogued in this study	Human leukemia parental cell line; authenticated by karyotype and morphology before use. If a public reference is required, K562 corresponds to RRID:CVCL_0004.
K562/A doxorubicin-resistant cells	Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China	Not commercially catalogued	Doxorubicin-resistant derivative of K562 cells; maintained at the Fujian Institute of Hematology. Resistance was maintained with 5 µg/mL doxorubicin before drug-free culture.
Microplate reader	BioTek / Thermo Fisher Scientific / Molecular Devices / laboratory-verified instrument	Model to be inserted from laboratory record	Used to measure absorbance at 450 nm for Cell Counting Kit-8 assays.
Multidrug resistance-associated protein 1 antibody for Western blot	Cell Signaling Technology	72202	Used for Western blot detection of multidrug resistance-associated protein 1. Confirm RRID from the product insert or Antibody Registry before submission.
Penicillin-streptomycin solution	Gibco, Thermo Fisher Scientific	15140122 or laboratory-verified equivalent	Added to complete medium at 1%.
P-glycoprotein / MDR1 / ABCB1 antibody for Western blot	Cell Signaling Technology	13978; RRID:AB_2798357	Used for Western blot detection of P-glycoprotein.
Phosphate-buffered saline	Gibco, Thermo Fisher Scientific	10010023 or laboratory-verified equivalent	Used for cell washing and sample preparation.
Phosphorylated mechanistic target of rapamycin antibody	Cell Signaling Technology	2971; RRID:AB_330970	Western blot antibody for phosphorylated mechanistic target of rapamycin at Ser2448.
Phosphorylated protein kinase B antibody	Cell Signaling Technology	4060; RRID:AB_2315049	Western blot antibody for phosphorylated protein kinase B at Ser473.
Phycoerythrin mouse immunoglobulin G2a isotype control	BioLegend	400213	Isotype control for phycoerythrin-conjugated antibody staining. Confirm the isotype matches the anti-P-glycoprotein antibody used.
Phycoerythrin-conjugated anti-human P-glycoprotein / CD243 / ABCB1 antibody	BioLegend	919405	Used for flow cytometric detection of P-glycoprotein. Include clone and RRID if confirmed from the product insert.
Polyvinylidene difluoride membrane	MilliporeSigma	IPVH00010 or laboratory-verified equivalent	Used for Western blot transfer.
Protease and phosphatase inhibitor cocktail	Thermo Fisher Scientific / Roche / laboratory-verified supplier	Use the actual catalog number	Added to lysis buffer before protein extraction.
Radioimmunoprecipitation assay lysis buffer	Beyotime / Thermo Fisher Scientific / laboratory-verified supplier	Use the actual catalog number	Used for total protein extraction.
Roswell Park Memorial Institute 1640 medium	HyClone / Cytiva	SH30809.01 or laboratory-verified equivalent	Basal culture medium for all leukemia cell lines. Use the exact catalog number from the laboratory purchase record if different.
SC79, protein kinase B activator	Use the actual supplier from the laboratory purchase record	Use the actual catalog number and lot number	Used in rescue experiments. The manuscript should consistently report the same final SC79 concentration across Protocol, Results, and Figure 6 legend.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reagents	Bio-Rad / Beyotime / laboratory-verified supplier	Use the actual catalog number	Used for Western blot protein separation.
Total mechanistic target of rapamycin antibody	Cell Signaling Technology	2972; RRID:AB_330978	Western blot antibody for total mechanistic target of rapamycin.
Total protein kinase B antibody	Cell Signaling Technology	4691; RRID:AB_915783	Western blot antibody for total protein kinase B.
Western blot imaging system	Bio-Rad / Cytiva / laboratory-verified instrument	Model to be inserted from laboratory record	Used for enhanced chemiluminescence image acquisition.