IFN-γ induces CD38 expression in AML cells through IRF-1 transcriptional regulation. (A) Representative flow cytometry dot plots for 2 AML BM samples comparing percent of CD38High population in IFN-γ–treated group vs vehicle-treated group. IFN-γ was added overnight at a concentration of 10 ng/mL to total BM MNCs and gated for CD45DimCD34CD38 population. (B) Violin plot comparing percent of CD38High population between vehicle- and IFN-γ–treated groups for patients with AML (n = 5; 4 BM and 1 PB). Each shape represents a different patient with AML. Each patient sample was analyzed in duplicate except for 1 patient. Unpaired Student t test was used to calculate statistical significance between vehicle- and IFN-γ–treated groups; ∗∗∗∗P < .0001. (C) AML BM MNCs were treated overnight with different doses of IFN-γ (1.0, 10.0, and 50.0 ng/mL), and CD38 surface expression was determined in CD45Dim population with flow cytometry. Overlaid dot plots show shift in CD38 expression with IFN-γ treatment, and the table to the right lists mean fluorescence intensity (MFI) for each dose and control. (D) Representative images of colony forming cell (CFC) assay for patient with AML after treatment with IFN-γ (10 ng/mL) or vehicle. Images were acquired in tiles to cover complete well and stitched. Violin plot comparing colony forming units (CFUs) between vehicle- and IFN-γ–treated (10 ng/mL) groups for patients with AML (n = 4: 3 PB and 1 BM). Each shape represents a different patient with AML. Samples from each patient was analyzed in duplicate. Unpaired Student t test was used to calculate statistical significance between vehicle- and IFN-γ–treated groups; ∗∗P < .01. (E) 3D uniform manifold approximation and projection (UMAP) depicting effect of IFN-γ treatment (10 ng/mL, 5 hours) on AML total BM MNCs of 1 patient with relapsed AML. (F) CD34pos cells were purified from 3 patients’ BM MNCs (relapsed AML) and equally divided for vehicle and IFN-γ (10 ng/mL) treatment groups. After overnight treatment, cells were collected, and RNA was extracted and subjected to bulk RNAseq. z score–based hierarchical clustering heat map showing top 75 upregulated genes and 5 downregulated genes in 3 patients with AML upon IFN-γ treatment. (G) Venn diagram analysis showing the highest upregulated genes upon IFN-γ treatment that were commonly found between scRNAseq and bulk RNAseq analysis. (H) CD34pos cells were selected from BM MNCs of 3 patients with relapsed AML and treated overnight with vehicle or 10 ng/mL of IFN-γ, followed by CD38 and IRF-1 quantitative reverse transcription polymerase chain reaction (qRT-PCR). CD38 and IRF-1 messenger RNA (mRNA) expression is shown as fold change (F.C.) relative to vehicle control. Each sample was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analyzed in triplicate. Paired Student t test was used to calculate significance. (I) Schematic cartoon showing cloned CD38 promoter in pGL3 plasmid, which encompassed IRF-1 consensus sequences (CS) and transcription start site (TSS) and shows location of IRF-1 CS relative to TSS. Also, mutagenesis strategy used to delete the IRF-1 binding sites in CD38 promoter is shown. (J-K) THP1 and HEK293 cells were cotransfected with CD38 wild-type (WT) promoter vector and empty expression vector, CD38 WT promoter vector and IRF-1 expression vector, and CD38 mutant promoter vector and empty expression vector, and CD38 mutant promoter vector and IRF-1 expression vector for 24 hours for HEK293 and 48 hours for THP1 cells. All the samples were also cotransfected with pRL-TK as internal control. All the samples were subjected to dual luciferase assay and luciferase/renilla ratio was calculated for each sample. Bar graphs show luciferase activity of CD38 promoter. Each group (WT and mutant CD38 promoter) with IRF-1 expression vector was normalized with its respective control expression vectors. Three independent experiments containing 2 technical duplicates were performed. Paired Student t test was used to compare groups. (L) THP1 cells were transfected twice with 50 nM siRNA control (siCtrl) or siRNA IRF-1 (siIRF-1) at time 0 and 24 hours. At 24 hours after transfection, cells were equally divided between wells for siCtrl and siIRF-1 and half the wells for each siRNA were either subjected to vehicle or IFN-γ (10 ng/mL) treatment overnight. Surface staining was performed for CD38 and subjected to flow cytometry. Histograms represented in MFI show IFN-γ–induced CD38 surface expression, which was rescued by IRF-1 knock down. Violin plot is representation of 3 independent experiments. Ordinary 1-way analysis of variance (ANOVA) with multiple comparisons was used as statistical test.

IFN-γ induces CD38 expression in AML cells through IRF-1 transcriptional regulation. (A) Representative flow cytometry dot plots for 2 AML BM samples comparing percent of CD38High population in IFN-γ–treated group vs vehicle-treated group. IFN-γ was added overnight at a concentration of 10 ng/mL to total BM MNCs and gated for CD45DimCD34CD38 population. (B) Violin plot comparing percent of CD38High population between vehicle- and IFN-γ–treated groups for patients with AML (n = 5; 4 BM and 1 PB). Each shape represents a different patient with AML. Each patient sample was analyzed in duplicate except for 1 patient. Unpaired Student t test was used to calculate statistical significance between vehicle- and IFN-γ–treated groups; ∗∗∗∗P < .0001. (C) AML BM MNCs were treated overnight with different doses of IFN-γ (1.0, 10.0, and 50.0 ng/mL), and CD38 surface expression was determined in CD45Dim population with flow cytometry. Overlaid dot plots show shift in CD38 expression with IFN-γ treatment, and the table to the right lists mean fluorescence intensity (MFI) for each dose and control. (D) Representative images of colony forming cell (CFC) assay for patient with AML after treatment with IFN-γ (10 ng/mL) or vehicle. Images were acquired in tiles to cover complete well and stitched. Violin plot comparing colony forming units (CFUs) between vehicle- and IFN-γ–treated (10 ng/mL) groups for patients with AML (n = 4: 3 PB and 1 BM). Each shape represents a different patient with AML. Samples from each patient was analyzed in duplicate. Unpaired Student t test was used to calculate statistical significance between vehicle- and IFN-γ–treated groups; ∗∗P < .01. (E) 3D uniform manifold approximation and projection (UMAP) depicting effect of IFN-γ treatment (10 ng/mL, 5 hours) on AML total BM MNCs of 1 patient with relapsed AML. (F) CD34pos cells were purified from 3 patients’ BM MNCs (relapsed AML) and equally divided for vehicle and IFN-γ (10 ng/mL) treatment groups. After overnight treatment, cells were collected, and RNA was extracted and subjected to bulk RNAseq. z score–based hierarchical clustering heat map showing top 75 upregulated genes and 5 downregulated genes in 3 patients with AML upon IFN-γ treatment. (G) Venn diagram analysis showing the highest upregulated genes upon IFN-γ treatment that were commonly found between scRNAseq and bulk RNAseq analysis. (H) CD34pos cells were selected from BM MNCs of 3 patients with relapsed AML and treated overnight with vehicle or 10 ng/mL of IFN-γ, followed by CD38 and IRF-1 quantitative reverse transcription polymerase chain reaction (qRT-PCR). CD38 and IRF-1 messenger RNA (mRNA) expression is shown as fold change (F.C.) relative to vehicle control. Each sample was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analyzed in triplicate. Paired Student t test was used to calculate significance. (I) Schematic cartoon showing cloned CD38 promoter in pGL3 plasmid, which encompassed IRF-1 consensus sequences (CS) and transcription start site (TSS) and shows location of IRF-1 CS relative to TSS. Also, mutagenesis strategy used to delete the IRF-1 binding sites in CD38 promoter is shown. (J-K) THP1 and HEK293 cells were cotransfected with CD38 wild-type (WT) promoter vector and empty expression vector, CD38 WT promoter vector and IRF-1 expression vector, and CD38 mutant promoter vector and empty expression vector, and CD38 mutant promoter vector and IRF-1 expression vector for 24 hours for HEK293 and 48 hours for THP1 cells. All the samples were also cotransfected with pRL-TK as internal control. All the samples were subjected to dual luciferase assay and luciferase/renilla ratio was calculated for each sample. Bar graphs show luciferase activity of CD38 promoter. Each group (WT and mutant CD38 promoter) with IRF-1 expression vector was normalized with its respective control expression vectors. Three independent experiments containing 2 technical duplicates were performed. Paired Student t test was used to compare groups. (L) THP1 cells were transfected twice with 50 nM siRNA control (siCtrl) or siRNA IRF-1 (siIRF-1) at time 0 and 24 hours. At 24 hours after transfection, cells were equally divided between wells for siCtrl and siIRF-1 and half the wells for each siRNA were either subjected to vehicle or IFN-γ (10 ng/mL) treatment overnight. Surface staining was performed for CD38 and subjected to flow cytometry. Histograms represented in MFI show IFN-γ–induced CD38 surface expression, which was rescued by IRF-1 knock down. Violin plot is representation of 3 independent experiments. Ordinary 1-way analysis of variance (ANOVA) with multiple comparisons was used as statistical test.

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