Biomodulatory Treatment With Azacitidine, All- trans Retinoic Acid and Pioglitazone Induces Differentiation of Primary AML Blasts Into Neutrophil Like Cells Capable of ROS Production and Phagocytosis

Abstract

Effective and tolerable salvage therapies for elderly patients with chemorefractory acute myeloid leukemia (AML) are limited and usually do not change the poor clinical outcome. We recently described in several chemorefractory elderly AML patients that a novel biomodulatory treatment regimen consisting of low-dose azacitidine (AZA) in combination with PPARγ agonist pioglitazone (PGZ) and all-trans retinoic acid (ATRA) induced complete remission of leukemia and also triggered myeloid differentiation with rapid increase of peripheral blood neutrophils. Herein, we further investigated our observations and comprehensively analyzed cell differentiation in primary AML blasts after treatment with ATRA, AZA, and PGZ ex vivo. The drug combination was found to significantly inhibit cell growth as well as to induce cell differentiation in about half of primary AML blasts samples independent of leukemia subtype. Notably and in comparison to ATRA/AZA/PGZ triple-treatment, effects on cell growth and myeloid differentiation with ATRA monotherapy was much less efficient. Morphological signs of myeloid cell differentiation were further confirmed on a functional basis by demonstrating increased production of reactive oxygen species as well as enhanced phagocytic activity in AML blasts treated with ATRA/AZA/PGZ. In conclusion, we show that biomodulatory treatment with ATRA/AZA/PGZ can induce phenotypical and functional differentiation of primary AML blasts into neutrophil like cells, which aside from its antileukemic activity may lower neutropenia associated infection rates in elderly AML patients in vivo. Clinical impact of the ATRA/AZA/PGZ treatment regimen is currently further investigated in a randomized clinical trial in chemorefractory AML patients (NCT02942758).

Keywords: acute myeloid leukemia; all-trans retinoic acid; azacitidine; differentiation; pioglitazone.

Figures

FIGURE 1

Treatment of HL-60 and U937 cells. (A) HL-60 cells (1.2 × 106) were incubated in RPMI supplemented with 10% FCS and as indicated with DMSO (open circles), 1 μM ATRA (closed circle), 5 μM PGZ (open square), 0.1 μM AZA (closed square), ATRA/PGZ (open diamond), ATRA/AZA (closed diamond), PGZ/AZA (open triangle), or ATRA/AZA/PGZ (closed triangle). On days 3, 5, and 7 during treatment, cells were counted by trypan blue staining. Data represent means of cell numbers from three independent experiments. Error bars indicate the standard deviation. (B) HL-60 cells were treated as described in (A). On day 5 of treatment, cells were characterized for surface-expression of the differentiation marker CD11b by flow cytometry. Shown are relative MFI values calculated by division of the MFI of antigen staining by the MFI of DMSO staining. Data represent means and standard deviations of three independent experiments. p-Values are shown for samples with significant differences in comparison to DMSO controls. Statistical analysis for group differences were performed using Friedman and Dunn’s multiple comparisons test. (C,D) U937 cells were incubated in RPMI supplemented with 10% FCS and as indicated with DMSO (open circles), 1 μM ATRA (closed circle), 5 μM PGZ (open square), 0.1 μM AZA (closed square), ATRA/PGZ (open diamond), ATRA/AZA (closed diamond), PGZ/AZA (open triangle), or ATRA/AZA/PGZ (closed triangle). From day 0 to day 4 during treatment, cells were analyzed for cell growth (C) and apoptosis (D). Data represent means from two independent experiments. Error bars indicate the standard deviation.

FIGURE 2

Cell growth and apoptosis of primary AML blasts. (A,B) Primary AML blasts (5 × 105) were incubated in AIM-V medium supplemented with 10% HS, G-CSF and SCF (each 50 ng/mL) and as indicated with medium, DMSO, ATRA, or ATRA/AZA/PGZ. On 14 of treatment, cell numbers were measured by conventional trypan blue staining. (A) Shows cell numbers normalized to medium (n = 14 per group), (B) shows Annexin positive cells normalized to medium (medium = 100%; n = 14 per group). Symbols represent individual patient samples and horizontal bars mark median values. Statistical analysis for group differences were performed using Friedman and Dunn’s multiple comparisons test. (C) Primary AML blasts (5 × 105) of patient sample AML144 (left panel) or AML111 (right panel) were incubated as described in (A). On days 7 and 14 of treatment, cell numbers were measured by conventional trypan blue staining. (D) AML blasts as described in (A) were characterized by Annexin V binding 14 days after treatment. Shown are % Annexin positive cells of AML111 (white bars) and AML144 (black bars).

FIGURE 3

Morphological differentiation of primary AML blasts. (A) Primary AML blasts (5 × 105) were treated as described in Figure 2. On day 14 after start of treatment, cells (5 × 104) were analyzed by light microscopy after centrifugation on microscope slides and May-Grünwald and Giemsa staining. Shown are AML cells of sample AML101 after treatment with ATRA/AZA/PGZ (I), ATRA (II), DMSO (III), or medium (IV), respectively. (B) Summary of 12 primary AML patient’s samples on day 14 of treatment as described in (A). Cells with morphological signs of differentiation were normalized to medium controls. Symbols represent individual patient samples and horizontal bars mark median values. (C) On day 14 of treatment with ATRA, PGZ, AZA, ATRA/AZA, ATRA/PGZ, PGZ/AZA, ATRA/AZA/PGZ, or DMSO sample AML101 was analyzed for morphological changes as described in (A). Cells with morphological signs of differentiation were normalized to medium controls. Data represent means of morphological changes from two independent experiments. Error bars indicate the standard deviation. (D) Primary AML samples (n = 14) at day 14 of treatment. Shown are the relative MFIs of CD11b staining normalized to medium. Symbols represent individual patient samples and horizontal bars mark median values. Statistical analysis for group differences were performed using Friedman and Dunn’s multiple comparisons test.

FIGURE 4

Reactive oxygen species production and phagocytosis of AML blasts. (A) Primary AML blasts were treated as described in Figure 2. On day 14 of treatment, 5 × 105 AML blasts were incubated with NBT (1 mg/mL) and PMA (200 ng/mL) for 90 min. Subsequently, cells were centrifuged on microscope slides for light microscopy. Shown are AML blasts of sample AML101 after treatment with ATRA/AZA/PGZ (I), ATRA (II), DMSO (III), or medium (IV), respectively. (B) Summary of primary AML samples (n = 11) on day 14 days of treatment as described in (A). NBT positive cells were counted in a blinded fashion and were normalized to medium. Symbols represent individual patient samples and horizontal bars mark median values. Statistical analysis for group differences were performed using Friedman and Dunn’s multiple comparisons test. (C,D) Primary AML blasts were treated as described in Figure 2. On day 14 of treatment, 2.5 × 105 AML blasts (n = 11 patients) were incubated with 6.25 × 106 GFP-labeled E. coli bacteria for 10 min. Subsequently, cells were analyzed by flow cytometry for GFP positive cells. (C) Summary of data (n = 11 AML samples) representing MFIs of GFP signal normalized to medium controls. Symbols represent individual patient samples and horizontal bars mark median values. Statistical analysis for group differences were performed using Friedman and Dunn’s multiple comparisons test. (D) Individual overlay plots and MFI of GFP signals measured with AML101 and AML147.

FIGURE 5

Biomodulatory treatment of AML cell lines in combination with midostaurin. (A) MV4-11 and (B) MOLM-13 cells were incubated in RPMI supplemented with 10% FCS and treated with single agent DMSO, ATRA, AZA, PGZ, MIDO or combinations thereof. On day 3 during treatment cells were counted by trypan blue staining. Data represent means of cell numbers from three independent experiments. Error bars indicate the standard deviation. Statistical analysis for group differences compared to DMSO were performed using Friedman and Dunn’s multiple comparisons test. ns p > 0.05, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.