Experiments were conducted on primary patient samples, which were provided by the Vanderbilt-Ingram Cancer Center Hematopoietic Malignancies Repository in accordance with the tenets of the Declaration of Helsinki, and were approved by the Vanderbilt University Medical Center Institutional Review Board (approval number 151710).
AML cell lines THP-1 and MV-4-11 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). The MOLM-13 cell line was purchased from Deutsche Sammelung von Mikroorganismen und Zellkulturen (DSMV) (Braunschweig, Germany).
ATCC and DSMV cell lines are authenticated by short tandem repeat profiling and cytochrome C oxidase gene analysis. Cultured cells were split every 3 days and maintained in exponential growth phase. Cell lines were tested for mycoplasma as per lab standard of practice using the Universal Mycoplasma Detection Kit (ATCC). Cells were used for the experiments presented here within 10–30 passages from thawing. Cell lines were cultured in RPMI (Gibco, Carlsbad, CA) supplemented with 10–20% fetal bovine serum (Gibco) and 100 U/mL penicillin (Gibco) and 100 µg/mL streptomycin (Gibco). Cells were kept at 37 °C in a 5% CO2 incubator.
Cell Viability Assay
Compounds were diluted in dimethyl sulfoxide (DMSO) (< 0.2%) and dispensed into a 384-well plate using the Echo 555 liquid handler (Labcyte, San Jose, CA). Following the addition of compounds, cells were pipetted into the 384-well plates at a concentration of 3000 cells per well in RPMI media, as noted above, supplemented with 10% FBS and incubated at 37 °C, 5% CO2 in a tissue culture incubator. Plates were incubated for 72 h, and cell viability was measured using the Cell TiterGlo reagent (Promega, Madison, WI). Percentage viability was defined as the relative luminescence units (RLU) of each well divided by the RLU of cells in DMSO control. Dose–response curves and the 50% growth inhibition concentration (GI50) values were determined using non-linear regression of double-log transformed data using GraphPad Prism version 6.0 h.
All animal experiments were conducted in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) at Vanderbilt University Medical Center under protocol M1500038-01. The pharmacokinetics of AZA in NSGS (NOD-scid IL2Rgnull3Tg [hSCF/hGM-CSF/hIL3]) male mice (The Jackson Laboratory, Bar Harbor, ME) (n = 4/group) were assessed in whole blood after dosing with AZA alone via oral (10 mg/kg) or i.p. (2.5 mg/kg) administration, and via oral administration (2.5 mg/kg) in combination with CDZ (1–30 mg/kg). Whole blood samples (10 µL) were collected over up to 24 h in tubes containing 10 µL of 19 mg/mL aqueous ethylenediaminetetraacetic acid (EDTA) and 10 µL of 30 µg/mL aqueous THU for stabilization. The pharmacokinetics of AZA in cynomolgus monkeys (n = 3, male, aged 3–4 years, 3.9–4.9 kg) were assessed in plasma using a crossover design after dosing on each occasion with AZA alone, via s.c. or oral (6.25 mg/kg) administration, and oral administration (6.25 mg/kg) in combination with CDZ (10 mg/kg), with serial blood samples for plasma collected over 24 h into THU-spiked EDTA tubes and centrifuged within 30 min of collection (2000g for 10 min at 5 °C) for plasma. The plasma samples from monkeys and diluted whole blood samples from mice were stored at − 70 °C until bioanalysis with a qualified liquid chromatography tandem mass spectrometry (LC–MS/MS) method.
DNA Isolation and Pyrosequencing Method
Global DNA methylation was assessed with LINE-1 methylation bisulfite sequencing analysis. For this, DNA was harvested from blood collected in Paxgene tubes. Cells were resuspended in 1X proteinase K digestion buffer with proteinase K and incubated at 50 °C for 30 min. Cell debris was pelleted by centrifugation at 14,000×g for 10 min, and 20 μL was used in the bisulfite conversion reaction using the EZ DNA Methylation-Direct kit (Zymo Research, Irvine, CA). For DNA methylation analysis, 500 ng of extracted genomic DNA was bisulfite treated using the EZ DNA Methylation kit (Zymo Research). Bisulfite-treated DNA was purified according to the manufacturer’s protocol. Polymerase chain reaction (PCR) was performed using 1 μL of bisulfite-treated DNA and 0.2 μM of each primer. One primer was biotin labeled and high performance liquid chromatography (HPLC) purified in order to purify the final PCR product using Sepharose beads. PCR product was bound to Streptavidin Sepharose HP (GE Healthcare Life Sciences, Chicago, IL). The immobilized PCR products were purified, washed, denatured with a 0.2 μM NaOH solution, and rewashed using the Pyrosequencing Vacuum Prep Tool (Qiagen, Hilden, Germany), as per the manufacturer’s protocol. Next, 0.5 μM of sequencing primer was annealed to the purified single-stranded PCR products. 10 μL of the PCR products was sequenced by pyrosequencing on the PSQ96 HS System (Qiagen) following the manufacturer’s instructions. The methylation status of each CpG site was determined individually as an artificial C/T single nucleotide polymorphism (SNP) using QCpG software (Qiagen). The methylation level at each CpG site was calculated as the percentage of the methylated alleles divided by the sum of all methylated and unmethylated alleles. The mean methylation level was calculated using methylation levels of all measured CpG sites within the targeted region of each gene. Each experiment included non-CpG cytosines as internal controls to detect incomplete bisulfite conversion of the input DNA. In addition, a series of unmethylated and methylated DNA were included as controls in each PCR.
In Vivo Murine Modeling of AML
Female NSGS mice (The Jackson Laboratory), 6–8 weeks old were irradiated with 100 cGy radiation. Twenty-four hours later, mice were transplanted intravenously with cells of interest. In the cell line model experiments, 3 × 103 MOLM-13 cells were transplanted in each irradiated mouse. At day 7 post-transplant, engrafted mice were randomized to receive 2.5 mg/kg i.p. AZA, 2.5 mg/kg oral AZA, 2.5 mg/kg oral AZA + 3 mg/kg CDZ, or CDZ alone at 30 mg/kg (to test for off-target toxicity of CDZ). Oral AZA and CDZ were administered via oral gavage daily for 7 consecutive days; i.p. therapy was similarly given for 7 consecutive days. For the PDX model, 2 × 106 mononucleated cells from primary patient samples were transplanted via tail vein injections.
Prior to treatment, peripheral micro-chimerism was documented at week 1 in the cell line model. For the AML PDX model, peripheral chimerism was established by 2 weeks. Mice showing no peripheral micro-chimerism by 1 week in the cell line model or 4 weeks in the AML PDX model were removed from the study. Chimeric mice were randomized pre-treatment. Upon establishing micro-chimerism, and after 35 days, mice were treated with either AZA (Selleckchem, Houston, TX) by daily gavage or i.p. injection, CDZ by gavage, or VEN (Chemietek, Indianapolis, IN) by gavage. VEN was dissolved in polyethylene glycol (PEG) and ethanol and diluted with Phosal 50 PG for gavage dosing. CDZ was dissolved in 5% methylcellulose. Engrafted mice received 2.5 mg/kg i.p. AZA, 2.5 mg/kg oral AZA, 2.5 mg/kg oral AZA + 3 mg/kg CDZ, or CDZ alone as vehicle at 30 mg/kg. Oral drugs were administered via oral gavage daily for 7 consecutive days; i.p. therapy was similarly given for 7 consecutive days. Peripheral blood was assessed weekly for human chimerism. Spleen/body ratio was calculated as organ weight (gram) per gram of body weight.
For flow cytometry, red blood cells were lysed with EL Buffer on ice (Qiagen), and the remaining cells were washed and resuspended in 1 × phospahte-buffered saline (PBS) with 1% bovine serum albumin (BSA) and stained for 15 min with the following antibodies: human CD45-APC (clone 2D1), human CD33-PE-Cy7 (clone P67.6), murine CD45-PE (clone 30-F11), and DAPI (all from Biolegend, San Diego, CA).
Tissues were fixed in 4% paraformaldehyde for 48 h and stored in 70% ethanol before being embedded in paraffin and sectioned at 5 µm. The bone tissue was decalcified prior to being embedded in paraffin. Sections were de-waxed in xylene and rehydrated in successive ethanol baths. Standard Mayer’s hematoxylin and eosin (H&E) staining was performed. Antigen retrieval using a standard pH 6 sodium citrate buffer (BioGenex, Fremont, CA) was performed, and sections were stained with Monoclonal Mouse Anti-Human CD45 (Dako, Santa Clara, CA, M0701, dilution 1:200) using M.O.M. Kit (Vector, Burlingame, CA).
Measurement of In Vivo Hypomethylation
Global DNA methylation was assessed in murine blood at baseline and 8, 19, and 29 days after treatment using the LINE-1 methylation bisulfite sequencing assay as previously described .
Unless otherwise noted, data were summarized using the mean (± standard deviation [SD]). Per group sample sizes are presented in figures and results reported from two separate experiments, unless stated otherwise. To avoid normality assumptions, pairwise group comparisons were made using the non-parametric Mann–Whitney U test. The non-parametric Spearman correlation was used to assess pairwise variable associations. Data were analyzed using GraphPad Prism 6.0 for Windows (GraphPad Software, www.graphpad.com) and R (R Core Team 2017; R: a language and environment for statistical computing; R Foundation for Statistical Computing, Vienna, Austria, https://www.R-project.org/). Significance was listed as *p < 0.05, **p < 0.01, and ***p < 0.001.