Reagents

Doxorubicin and etoposide were purchased from Pharmachemie (The Netherlands), daunorubicin was obtained from Sanofi-Aventis and epirubicin was obtained from Accord Healthcare Limited (UK), idarubicin was obtained from Pfizer and Santa Cruz Biotechnology (sc-204774). Aclarubicin for in vivo mouse experiments was obtained from Shenzhen Main Luck Pharmaceuticals Inc. Aclarubicin (for in vitro experiments, sc-200160) and amrubicin (sc-207289) were obtained from Santa Cruz Biotechnology.

Cell culture

K562 (B. Pang, LUMC Leiden, The Netherlands, RRID: CVCL_K562), MM6 (RRID: CVCL_1426), MOLM13 (RRID: CVCL_1426), MV4:11 (RRID: CVCL_0064), U937 (RRID: CVCL_0007) (all four lines were a gift from L. Smit, VUMC Amsterdam, The Netherlands) and THP-1 (ATCC, Manassas, VA, RRID: CVCL_0006) were cultured in RPMI-1640 medium supplemented with 8% fetal calf serum (FCS, SERANA, S-FBS-CO-015). OCI-AML-2 (DSMZ, ACC-No 99, RRID: CVCL_1619) and OCI-AML-3 (DSMZ, ACC-No 582, RRID: CVCL_1844) (M. Griffioen, LUMC Leiden, The Netherlands) and OCI-AML-4 (M.L.M. Jongsma, LUMC Leiden, The Netherlands, RRID: CVCL_5224) were cultured in IMDM medium supplemented with 8% FCS and glutamine. MelJuSo (RRID: CVCL_1403) cells were maintained in IMDM supplemented with 8% FCS. All cell lines were maintained in a humidified atmosphere of 5% CO2 at 37 °C, regularly tested for the absence of mycoplasma and STR profile.

Cell line construction

For endogenous tagged GFP-H2B K562 cells, mScarlet was swapped for GFP in the homology repair construct using NheI and BglII and cells were generated as described [5]. Co-transfection into K562 cells was done by electroporation using Lonza SF cell line kit. ABCB1 overexpressing K562 cells were generated as described [14]. Endogenous tagged 3×Flag-TopoIIα K562 cell line was generated using HR-3×Flag construct designed at least 40 base pairs up- and downstream of the genomic TopoIIα stop codon. The gRNA target sequence was designed using the Zhang Lab CRISPR tool (http://crispr.mit.edu/) and cloned into the pX330 vector (RRID: Addgene_110403). Primers used for the HR construct: 5’-CACCGATGATCTGTTTTAAAATGTG-3’ and 5’-AAACCACATTTTAAAACAGATCATC-3’. Co-transfection of ssDNA oligo and CRISPR plasmid (pX459) into K562 cells was performed by electroporation using Lonza SF cell line kit. Primers used for genotyping were forward primer: 5’-TAAGCAGAATTCATGCCACTTATTTGGGCAAT-3’ and reverse primer: 5’-TGCTTAAAGCTTTGCCCATGAGATGGTCACTA-3’.

DNA damage assessed by Western blot and constant field gel electrophoresis

After 2-hour drug treatment of indicated drugs at 5 µM, THP-1 cells were washed with PBS, and then lysed directly in sodium dodecyl sulfate (SDS)-sample buffer (2% SDS, 10% glycerol, 5% β-mercaptoethanol, 60 mM Tris-HCl pH 6.8, and 0.01% bromophenol blue). Lysates were resolved by SDS/polyacrylamide gel electrophoresis followed by Western blotting. Primary antibodies used for blotting were γH2AX (1:1,000, 05-036, Millipore) and β-actin (1:10,000, A5441, Sigma, RRID: AB_476744). CFGE were performed as described [15]. Images were quantified with ImageJ (RRID: SCR_003070).

Fractionation assay

Endogenously tagged GFP-H2B K562 cells (1 × 106) were pre-incubated with protease inhibitor cocktail (P1860-1ML, Sigma-Aldrich) for 1 h followed by 3-hour treatment with 10 µM of the indicated drugs. Cells were dissolved in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl2, 0.5% NP40, 2.5% glycerol supplemented with protease inhibitors and 10 mM NMM) for 5 min on ice, and then centrifuged for 10 min at 15,000 g at 4 °C. Both nucleus (pellet) and cytosol (supernatant) were washed once and then submitted for Western Blot analysis. Primary antibodies used for detection: GFP (1:1,000) [16], Lamin B1 (1:1,000, 12,987-I-AP, Proteintech, RRID: AB_2136290), and Calnexin (1:1,000, #2679, Cell Signaling Technology, RRID: AB_2228381).

Time-lapse confocal microscopy

For time-lapse confocal imaging, MelJuSo cells were seeded in 35-mm glass bottom petri dish (Poly-D-Lysine coated, MatTek Corporation), transfected with TopoIIα-GFP construct using Effectene (301425, QIAGEN) and imaged upon treatment with the indicated drugs [2]. Leica SP8 confocal microscope system, 63× lens, equipped with a climate chamber was used. TopoIIα-GFP distribution was quantified using Leica Application Suite X software (RRID: SCR_013673).

Short-term cell viability assay

Twenty-four hours after seeding into 96-well plates, cells were treated with indicated drugs for 2 h at physiologically relevant concentrations [2]. Subsequently, drugs were removed by extensive washing, and cells were cultured for an additional 72 h, according to their pharmacokinetics. Cell viability was measured using the CellTiter-Blue viability assay (G8080, Promega). Survival was normalized to the untreated control samples after correction for the background signal.

ChIP-seq

Endogenous tagged 3×Flag-TopoIIα K562 cells were treated with 10 µM of indicated drugs for 4 h. Cells were fixed and processed as described [3, 17]. ChIP was done with anti-Flag M2 antibody (F3165, Sigma, RRID: AB_259529), followed by sequencing on an Illumina Hiseq2000 platform (Genome Sequencing Service Center of Stanford Center for Genomics and Personalized Medicine Sequencing Center).

ChIP-seq data were processed identically using the ENCODE Data Coordination Center (DCC) ChIP-seq pipeline (https://github.com/ENCODE-DCC/chip-seq-pipeline2) (v1.9.0). Briefly, the ChIP-seq reads were aligned to the human reference genome (GRCh37/hg19) using Bowtie2 (RRID: SCR_016368) [18]. Duplicate reads were removed using Picard MarkDuplicates (RRID: SCR_006525). Peaks of each sample were called against the whole-cell lysate replicates using SPP (RRID: SCR_001790) [19] with the parameters ‘-npeak 300000 -speak 155 -fdr 0.01’. The blacklisted regions described by ENCODE were discarded [20]. Reproducible peaks were intersected from two biological replicates and annotated with epigenomic signatures of K562 cells, downloaded from the Roadmap Epigenomics Project (RRID: SCR_008924) [21]. Normalized TopoIIα binding affinity matrix: consensus peaks by samples, principle component analysis and differential binding affinity analysis were performed using the R package DiffBind (RRID: SCR_012918) [22].

ATAC-seq

Wild-type K562 cells were treated with 10 µM of indicated drugs for 4 h. Cells were fixed and processed as described [23, 24]. DNA was processed using a customized library preparation method for ATAC-seq and was sequenced using an Illumina HiSeq4000 platform.

ATAC-seq data were processed identically using the ENCODE Data Coordination Center (DCC) ATAC-seq pipeline (https://github.com/ENCODE-DCC/atac-seq-pipeline) (v1.10.0). Briefly, the ATAC-seq reads were aligned to the human reference genome (GRCh37/hg19) using Bowtie2 [18]. Duplicate reads were removed using Picard MarkDuplicates (RRID: SCR_006525). Peaks were called using MACS2 (RRID: SCR_013291) [25] with the setting of ‘-p 0.01 --shift − 75 --extsize 150 --nomodel -B --SPMR --keep-dup all’, then followed by blacklisted regions filtering described by ENCODE [20]. Reproducible peaks were identified from two biological replicates and annotated with epigenomic signatures of K562, downloaded from the Roadmap Epigenomics Project [21]. ATAC-seq signal tracks for all the samples were generated by BEDTools (RRID: SCR_006646) with the command ‘bedtools genomecov -scale’ using the read count per million (CPM) normalization and convert to bigwig files using bedGraphToBigWig. Normalized ATAC-seq read density matrix: consensus peaks by samples, principle component analysis and differential chromatin accessibility analysis were performed using the R package DiffBind [22].

Principal component analysis (PCA)

The reproducible peaks were identified by intersecting two biological replicates of each drug treatment, and then merged into consensus peak set. Subsequently, the read density was quantified for the consensus peaks to create an input matrix for PCA analysis. The dba.plotPCA function of DiffBind package was applied to calculate the principal components through eigenvector decomposition of the covariance matrix from the input matrix. The maximum variance of projected data by principal component 1 and 2 were plotted in the x- and y-axis accordingly.

DNA dye exclusion assay

Circular DNA (1 µg/ml) was incubated with Quant-iT PicoGreen dsDNA reagent (P7581, Thermo Fisher Scientific) for 5 min at room temperature (RT). Subsequently, drug was added to the DNA/PicoGreen mix at indicated concentrations and incubated for another 5 min at RT. Following the reaction, the PicoGreen fluorescence was measured using CLARIOstar plate reader (BMG labtech) with excitation at 480 nm and emission at 520 nm (48020/52010 filter). The fluorescence was quantified relative to untreated controls. Fluorescent signals of all samples were corrected for the corresponding drug concentrations in the absence of DNA.

Bio-distribution of anthracyclines in mice

FVB/NRj mice (RRID: MGI:6364162) ordered from Janvier Labs (Le Genest-Saint-Isle, France) were housed in individually ventilated cages under specific pathogen-free conditions in the animal facility of the NKI (Amsterdam, The Netherlands). All mouse experiments were approved by the Animal Ethics Committee of the NKI and were performed according to institutional and national guidelines. Male mice (8-week old) were i.v. injected with doxorubicin, aclarubicin, amrubicin, epirubicin or idarubicin at 5 mg/kg (n = 5 per group). Four hours post injection, animals were sacrificed, and plasma, heart, lung, liver, kidney, spleen, brain, thymus, axillary + inguinal lymph nodes, and testis + epididymis were collected. Hearts were cut into two pieces with coronal section. One piece was fixed in EAF fixative (ethanol/acetic acid/formaldehyde/saline, 40:5:10:45 v/v/v/v) and processed for Phospho-H2AX (Ser139) IHC (1:100, #2577, Cell Signaling Technology). The other half of the heart and the rest of organs were weighed and frozen.

Calibration samples for each anthracycline with defined concentration were prepared in blank tissue homogenates. Daunorubicin was used as internal standards. Of each sample, a 100 µl tissue homogenate was mixed with 200 µl of 0.1% formic acid, 1 ml of Chloroform:2-propanol (1:1) and 10 µl of internal standard, then vortexed vigorously for 5 min, followed by centrifugation at 5,000 g for 5 min at 4 °C. The aqueous layer and the intermediate layer were removed by suction. The organic layer was decanted into a clean polypropylene tube and evaporated by Savant Speed-Vac SC210A concentrator (Thermo Fisher Scientific, Waltham, USA) at 35 °C. The residue was reconstituted in 150 µl of DMSO, vortexed for 20 sec and then sonicated for 5 min. After centrifuged at > 12,000 g for 2 min, 10 µl of supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS)/MS, which consisted of an API 3500 mass spectrometer (Sciex, Framingham, MA) coupled to an UltiMate 3000 LC System (Dionex, Sunnyvale, CA). Samples were separated using a ZORBAX Extend-C18 column (Agilent, Santa Clara, CA), kept at 50 °C preceded by a Securityguard C18 pre-column (Phenomenex, Utrecht, The Netherlands). Elution was done using a mixture of mobile phase A (0.1% formic acid in water (v/v)) and mobile phase B (methanol) in a 2 min gradient from 20 to 95% B, followed by 95% B that was maintained for 3 min and then re-equilibrated at 20% B. Multiple reaction monitoring parameters were 544.0/397.0 (doxorubicin and epirubicin), 498.1/291 (idarubicin), 812.4/570.3 (aclarubicin), 484.2/333.1 (amrubicin) and 528.1/321.1 (daunorubicin). System control and data analysis were done using Analyst® 1.6.2 software (AB Sciex; Foster City, CA).

The cardiotoxicity of anthracyclines in mice

FVB/NRj mice (10–11-week old, RRID: MGI:6364162) were i.v. injected with 5 mg/kg of doxorubicin, 5 mg/kg of aclarubicin, or 5 ml/kg of saline every 2 weeks for 4 times. After 4-week interval, the animals were i.v. injected with 5 mg/kg of indicated drug or 5 ml/kg of saline every 2 weeks for another 4 times. The mice were monitored every other day. When body weight loss was more than 20%, or circulation failure occurred, animals were euthanized by CO2. Subsequently, full body anatomy was performed. All organs were collected, fixed in EAF fixative and embedded in paraffin. Sections were cut at 2 μm from the paraffin blocks and stained with hematoxylin and eosin, and 4 μm for immunohistochemistry of Desmin (1:200, M0760, DakoCytomation), Vimentin (1:100, #5741, Cell Signaling Technology, RRID: AB_10695459), or Periostin (1:100, ab215199, Abcam, RRID: AB_2924310). Pathology slides were reviewed twice by an expert mouse pathologist who was blind to the treatment. Incidence rate (IR = [number of mice with the specific side effect over a time period]/[sum of mice × time at risk during the same time period]) and cumulative incidence (CI = [number of mice with specific side effect at end time point]/[total number of mice at start]) were calculated for cardiotoxicity.

AML patient data analyses

Patients with refractory or relapsed AML treated between July 2012 and July 2022 at Ruijin hospital, China were enrolled in this retrospective study. Some patients participated in trial ChiCTR-OPC-14,005,712, ChiCTR-OPC-15,006,896, ChiCTR-IIR-16,008,809, ChiCTR-OIC-16,008,952, ChiCTR-IIR-16,008,962 and ChiCTR-IIR-17,011,677. This study was approved by the ethics committee of Ruijin Hospital, all patients provided written informed consent.

Cytogenetic risk was classified according to the modified Southwest Oncology Group criteria [26]: (1) favorable risk, including t(8;21) and inv(16) or t(16;16)(p13;q22); (2) unfavorable risk, including del(5q) or monosomy 5, monosomy 7 or del(7q), abnormal 3q, 9q, 11q, 21q, or 17p, t(6;9), t(9;22), and complex karyotypes (three or more unrelated chromosomes abnormal); and (3) intermediate risk, including normal karyotypes and all other anomalies. FLT3 internal tandem duplication (FLT3-ITD) and mutations in CEBPA, NPM1 and IDH1/2 were tested. Integrated risk was classified as described [27]. Complete remission was defined as bone marrow blasts < 5%, absolute neutrophil count ≥ 1 × 109/L, and platelet count ≥ 100 × 109/L, and absence of extramedullary disease. Partial remission was defined as having < 15% (and a 50% decrease in bone marrow blasts) but > 5% blasts or with < 5% blasts but not reaching the CR criteria for blood cell count or clinical manifestation. The baseline characteristics and clinical outcomes of the patients are summarized in supplemental Tables S1 and S2, respectively.

AML treatments

CAG patients were treated with 15‒25 mg/m2 of cytarabine (Ara-C) injected s.c. every 12 h on days 1‒14, 20 mg/day of aclarubicin infused i.v. on days 1‒4, and 200 µg/m2 of granulocyte stimulating factor (G-CSF) administered s.c. daily on days 1‒14. G-CSF was reduced, or temporarily stopped when neutrophilia was > 5 × 109/L. IA patients were treated with 6‒10 mg/m2 of idarubicin infused i.v. on days 1‒3 and 100‒200 mg/m2 of Ara-C on days 1‒7. VA patients were injected with 75 mg/m2 of azacitidine s.c. daily on days 1‒7, and administered with venetoclax orally, once daily. The dose of venetoclax was 100 mg on day 1 and 200 mg on day 2; and 400 mg on days 3‒28. In all subsequent 28-day cycles, the dose of venetoclax was initiated at 400 mg daily. The other induction chemotherapies for r/rAML patients included IA, DA, FLAG, CLAAG and CHA regimens. For patients treated with DA regimen, 20 mg/m2 of decitabine was administered i.v. daily on days 1‒5, and 1 g/m2 of Ara-C was injected every 12 h on days 6‒7. For patients treated with FLAG regimen, fludarabine was infused i.v. at 30 mg/m2 on days 2‒6; 4 h after fludarabine infusion, Ara-C was injected i.v. at 1.5–2 g/m2 over 3 h on days 2‒6; G-CSF was administered at 5 µg/kg s.c. on days 1‒5; additional G-CSF may be administered since 7 days after the end of chemotherapy until white blood cell count > 500/μL. For patients > 60-year-old, the dose may be reduced to 20 mg/m2 for fludarabine and 0.5–1 g/m2 for cytarabine. For patients treated with CLAAG regimen, 5 mg/m2 of cladribine was infused i.v. over 2 hours on days 1‒5; 15 mg/m2 of Ara-C was injected s.c. every 12 h on days 1‒10; trans retinoic acid (ATRA) was administered orally at 45 mg/m2 on days 4‒6, then at 15 mg/m2 on days 7‒20; 300 µg G-CSF was injected s.c. on day 0. For patients treated with CHA regimen, 5 mg/m2 of cladribine was infused i.v. over 2 hours on days 1‒5; 2 mg/m2 of homoharringtonine was infused i.v. over 2 hours on days 1‒5; 1 g/m2 of Ara-C was injected 2 hours after cladribine on days 1‒5.

Statistical analyses

Results are shown as mean ± SEM or mean ± SD. Statistical analysis was performed using GraphPad Prism (RRID: SCR_002798) unless otherwise specified. All in vitro experiments were performed with a minimum of three independent trials with the exception of ChIP-seq and ATAC-seq which were biological duplicates. All individual animals in mouse experiments are shown in the dot plots. Statistical tests are indicated in each figure legend. Survival curves were analyzed by the log-rank (Mantel-Cox) test. Statistical analysis of pathology quantification was determined using the Student’s t-test or Mann-Whitney test. Kinetic analysis was performed with Two-way ANOVA. Clinical outcomes were analyzed by Fisher’s exact test or Mantel-Haenszel test. P values of < 0.05 were considered statistically significant.