Extended exposure to low doses of azacitidine induces differentiation of leukemic stem cells through activation of myeloperoxidase

Abstract

Oral azacitidine (oral-Aza) treatment results in longer median overall survival (OS) (24.7 vs. 14.8 months in placebo) in patients with acute myeloid leukemia (AML) in remission after intensive chemotherapy. The dosing schedule of oral-Aza (14 days/28-day cycle) allows for low exposure of Aza for an extended duration thereby facilitating a sustained therapeutic effect. However, the underlying mechanisms supporting the clinical impact of oral-Aza in maintenance therapy remain to be fully understood. In this preclinical work, we explore the mechanistic basis of oral-Aza/extended exposure to Aza through in vitro and in vivo modeling. In cell lines, extended exposure to Aza results in sustained DNMT1 loss, leading to durable hypomethylation, and gene expression changes. In mouse models, extended exposure to Aza, preferentially targets immature leukemic cells. In leukemic stem cell (LSC) models, the extended dose of Aza induces differentiation and depletes CD34+CD38- LSC. Mechanistically, LSC differentiation is driven in part by increased myeloperoxidase (MPO) expression. Inhibition of MPO activity either by using an MPO-specific inhibitor or blocking oxidative stress, a known mechanism of MPO, partly reverses the differentiation of LSC. Overall, our preclinical work reveals novel mechanistic insights into oral-Aza and its ability to target LSC.

Figure 1.

Extended-dose Aza leads to a more sustained hypomethylation and gene expression. (A) Schematic of experimental design of (B) OCI-AML2, (C) MV-4-11, and (D) SKM-1 cell lines DNA methylation profiles as measured by whole-genome bisulfite sequencing (WGBS) in response to the 2 dosing regimens. Compared to day 3, in day 5 extended dose results in a progressing/ sustained hypomethylation. (E) Barplots indicating the total number of significantly differentially expressed genes in all 3 cell lines at the 2 time points (day 3 and day 5). Three replicates per cell line, per time point, per gene was used to generate the barplots using the R limma package. Note the increase in the number of genes in extended dose but not in conventional dose azacitidine (Aza) at the later time point (“down” indicates downregulated and “up” indicates upregulated). (F) DNMT1 levels were measured by protein simple western blotting at the indicated times in SKM-1 cell line. (G) Area under the curve (AUC) barplots representing the amount of Aza incorporation in peripheral blood mononucear cells (PBMC) using the conventional or extended dose of Aza. AUC data was generated from 3 animals and 5 time points for each dosing regimen (H) Biochemical analysis was performed in MV-4-11, SKM-1, and AML-193 cells treated with low (0.2 µM) or high (1 µM) dose of Aza for 24 hours. Protein lysates were prepared and assessed for treatment-mediated activation of ISR and loss of DNMT1. AML-193 experiment was duplicated (right panel) to assess the effect of ISRIB, an ISR inhibitor, in ablating Aza-induced ISR activation. Tubulin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (I) AML-193 cells were treated with Aza in conventional or extended regimen and protein lysates were prepared from days 1-7 and assessed for DNMT1 expression and ISR activation markers. EIF2α was used as a loading control. RNA-seq: RNA sequencing.

Figure 2.

Extended dosing of Aza preferentially targets bone marrow blasts. (A) Survival of B6 albino mice injected with C1498-Luc3-GFP⁺ cells and treated with vehicle, conventional-azacitidine (Aza) (5 mg/kg once daily for 4 days [QDx4]) or extended-Aza (1 mg/kg QDx20); N=10 per group. Representative bioluminescence imaging (right panel) of mice are shown (N=1 representative mouse/group). Blue to red color represents low to high intensity of bioluminescence. (B) Mean spleen weights measured at the end of the study for the patient-derived xenograft (PDX) acute myeloid leukemia (AML) mice treated with vehicle, convention-al-Aza (5 mg/kg QDx5) or extended-Aza dosing (1 mg/kg QDx25) are shown as group mean ± standard deviation (N=5 to 7/group). Red dotted line indicates normal C57BL/6J mouse spleen weight. **P ≤ 0.01. (C) Median spleen weights from GEM model of AML harboring FLT3-ITD and TET2 loss (N=17-19/group) (normal spleen weight of wild-type mice is 72 milligrams). *P≤0.05; **P≤0.01, ***P ≤ 0.001 for Aza relative to vehicle/isotype control using non-parametric one-way ANOVA. (D) Flow cytometry was performed on cells collected at the end of week 4 and cell percentages are presented as group medians (N=12/group). ***P≤0.001 for Aza relative to vehicle/isotype control. P values were calculated using non-parametric one-way ANOVA. NS: not significant.

Figure 3.

Extended dosing of Aza induces greater differentiation of leukemic stem cells. (A) OCI-AML20 cells were cultured on OP9 feeder layer and the cells were treated with dimethyl sulfoxide (DMSO) or azacitidine (Aza) (extended or conventional dosing). At day 7, all cells were collected and subjected to flow cytometry. After discriminating live/dead cells and singlets, human cells (to differentiate from mouse OP9 cells) were identified by CD33⁺/CD45⁺ staining. Within that population the profile of CD34/ CD38 was identified. (B) CD34⁺-enriched primary acute myeloid leukemia (AML) sample was treated with both dosing regimens. Flow cytometry was performed on day 7 to identify CD34 and CD38 population (C) Samples from the above-described experiment were collected and subjected to single-cell RNA sequencing (scRNA-seq) as described in the methods. Violin plots indicating the treatment-specific distribution and the Wilcoxon P value of the GSVA enrichment on Van Galen signatures at days 3, 5, and 7. GMP: granulocyte-monocyte progenitors.

Figure 4.

Extended-dose Aza is equally effective as conventional dosing in targeting acute myeloid leukemia cells and stem/ progenitor cells in vivo. (A) Schematic of experiment. Primary acute myeloid leukemia (AML) cells were injected into the right femurs of sub-lethally irradiated NOD/SCID mice. Twenty days after injection, mice (10 animals per group) were treated with vehicle or 2 different doses of azacitidine (Aza) mimicking conventional/injectable dosing (3 mg/kg 5 doses for 5 consecutive days) or extended dosing (1 mg/kg 15 doses over 21 days [5/7 days per week]). At the end of treatment (42 days from day of injection), flow cytometry was performed in both right and left femurs to measure engraftment. (B) Plots indicate the percentage of human cells that engrafted in the left femur (distal site). The red bar indicates the median values, and each data point indicates a mouse. In order to measure the ability of Aza to target the persistence of leukemic cells at the site injection, human cells in the right femur (site of injection) were assessed with the indicated surface markers to identify (C) granulocyte-monocyte progenitors (GMP), (D) common myeloid progenitors (CMP) and (E) megakaryocyte-erythrocyte progenitors (MEP) (F) CD123 (IL-3 receptor α chain), a marker for AML stem and progenitor cells, was also reduced at the site of injection demonstrating equivalent efficacy for the conventional dosing and the extended dosing of Aza. (G) Animal body weight was monitored during treatment as a proxy for animal health. The dotted line indicates 90% (10% change). Data represent median ± standard deviation. *P<0.05 and **P<0.01 by unpaired t test.

Figure 5.

Extended-dose Aza upregulates myeloperoxidase expression to induce leukemic stem cell differentiation, which can be partly rescued by myeloperoxidase inhibition or reactive oxygen species scavenging. (A) OCI-AML20 cells were sorted to retrieve the CD34⁺38^- population. (B) Sorted cells were treated with azacitidine (Aza) (extended or conventional dose) and bulk RNA sequencing was performed at day 5 followed by pathway enrichment analysis (gene ontology [GO] process) to assess Aza-induced pathways. (C) OCI-AML20 cells were treated with Aza (conventional or extended regimen) and myeloperoxidase (MPO) expression was assessed by capillary electrophoresis on day 7, glyceraldehyde-3-phos phate dehydrogenase (GAPDH) was used as a loading control. (D) Representative flow data from 1 experiment with OCI-AML20 cells were treated with extended or conventional regimen of Aza with or without 10 µM MPO-IN-28 or (E) N-acetylcysteine (NAC) on days 2 and 4 and flow cytometry assessment was conducted on day 7 using live/dead, CD45, CD34 and CD38 staining. DMSO: dimethyl sulfoxide.