American Society of Hematology

Figure 4.

$Azacytidine sensitizes AML to DNT-mediated cytotoxicity and produces a viral mimicry response. (A-B) AML cell lines (A; OCI-AML2, OCI-AML3, and KG1a) and primary AML samples (B; n = 7) were treated with azacytidine for 5 days followed by coculture with DNTs at a 2:1 DNT:AML ratio for OCI-AML2, OCI-AML3, and primary AML samples or an 8:1 DNT:AML ratio for KG1a. Two hours after coincubation, percent specific killing of AML cells by DNTs was determined by flow cytometry. (C) AML cell lines (OCI-AML2 and KG1a) and primary AML cells (n = 3) were untreated or treated with azacytidine (0.3-9 μM) for 5 days, followed by coculture with untreated or venetoclax-treated (400 nM for 18 hours) DNTs for 2 hours. AML cell viability was measured by Annexin V and flow cytometry. (D) Left panel: OCI-AML2 cells were untreated or treated with azacytidine (0.33 μM) for 5 days. After the treatment period, cells were cytospun, fixed with 4% paraformaldehyde, and immunostained with anti-dsDNA antibody. DNA was stained by 4′,6-diamidino-2-phenylindole. Right panel: DNA was isolated from the cytoplasmic fraction of the OCI-AML2 cells treated with or without azacytidine. The relative level of cytosolic genomic DNA of transposon origin was analyzed by quantitative polymerase chain reaction by using transposon DNA 14 (P14) and 24 (P24) specific primers and nuclear human globulin gene (HGB). Data represent the mean ± SD relative to untreated cells. (E) OCI-AML2 cells were untreated or treated with azacytidine (0.08-0.33 μM) for 5 days. The relative expression of IL-1β and IFN-1β was analyzed by quantitative reverse transcription polymerase chain reaction. Data are relative mean ± SD (n = 3; untreated = 1.0). (F) Primary AML cells were untreated or treated with azacytidine (3 or 9 μM) for 5 days in the presence or absence of the STING inhibitor H-151. The relative expression of 1L-1β (top) and IFN-1β (bottom) were analyzed by quantitative reverse transcription polymerase chain reaction. The experiments were conducted by using DNTs from 4 different donors. (G) AML cell lines (OCI-AML2, OCI-AML3, and KG1a) and primary AML cells (n = 3) were treated with azacytidine (0.3-3 μM) for 5 days with or without the STING inhibitor (H-151). Data represent the mean ± SD increase in DNT-mediated cytotoxicity by azacytidine-treated AML relative to the untreated control. Student t test or 1-way analysis of variance was used for statistics. *P < .05; **P < .01; ***P < .001; ****P < .0001.$

Azacytidine sensitizes AML to DNT-mediated cytotoxicity and produces a viral mimicry response. (A-B) AML cell lines (A; OCI-AML2, OCI-AML3, and KG1a) and primary AML samples (B; n = 7) were treated with azacytidine for 5 days followed by coculture with DNTs at a 2:1 DNT:AML ratio for OCI-AML2, OCI-AML3, and primary AML samples or an 8:1 DNT:AML ratio for KG1a. Two hours after coincubation, percent specific killing of AML cells by DNTs was determined by flow cytometry. (C) AML cell lines (OCI-AML2 and KG1a) and primary AML cells (n = 3) were untreated or treated with azacytidine (0.3-9 μM) for 5 days, followed by coculture with untreated or venetoclax-treated (400 nM for 18 hours) DNTs for 2 hours. AML cell viability was measured by Annexin V and flow cytometry. (D) Left panel: OCI-AML2 cells were untreated or treated with azacytidine (0.33 μM) for 5 days. After the treatment period, cells were cytospun, fixed with 4% paraformaldehyde, and immunostained with anti-dsDNA antibody. DNA was stained by 4′,6-diamidino-2-phenylindole. Right panel: DNA was isolated from the cytoplasmic fraction of the OCI-AML2 cells treated with or without azacytidine. The relative level of cytosolic genomic DNA of transposon origin was analyzed by quantitative polymerase chain reaction by using transposon DNA 14 (P14) and 24 (P24) specific primers and nuclear human globulin gene (HGB). Data represent the mean ± SD relative to untreated cells. (E) OCI-AML2 cells were untreated or treated with azacytidine (0.08-0.33 μM) for 5 days. The relative expression of IL-1β and IFN-1β was analyzed by quantitative reverse transcription polymerase chain reaction. Data are relative mean ± SD (n = 3; untreated = 1.0). (F) Primary AML cells were untreated or treated with azacytidine (3 or 9 μM) for 5 days in the presence or absence of the STING inhibitor H-151. The relative expression of 1L-1β (top) and IFN-1β (bottom) were analyzed by quantitative reverse transcription polymerase chain reaction. The experiments were conducted by using DNTs from 4 different donors. (G) AML cell lines (OCI-AML2, OCI-AML3, and KG1a) and primary AML cells (n = 3) were treated with azacytidine (0.3-3 μM) for 5 days with or without the STING inhibitor (H-151). Data represent the mean ± SD increase in DNT-mediated cytotoxicity by azacytidine-treated AML relative to the untreated control. Student t test or 1-way analysis of variance was used for statistics. *P < .05; **P < .01; ***P < .001; ****P < .0001.

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