human aml cell line u937  (ATCC)

Journal: Science Advances

Article Title: P300/CBP inhibition with inobrodib in combination with gilteritinib and venetoclax targets leukemia stem cells in epigenetic mutant AML

doi: 10.1126/sciadv.aec9305

Figure Lengend Snippet: ( A ) In vivo CCS1477 treatment schematic for survival analyses. CD45.2 + D/F or T/F AML cells were transplanted into sublethally irradiated CD45.1 + WT recipient mice. Treatment was initiated 14 days posttransplant. CCS1477 (20 mg/kg) or vehicle was dosed by mouth (PO) once daily (QD). ( B ) Average ± SEM percent c-Kit + cells in the CD45.2 + peripheral blood (PB) of CCS1477- and vehicle-treated D/F ( n = 7 per treatment) and T/F ( n = 11 per treatment) transplanted AML mice over 2 weeks. Week 0 time point was immediately before the first treatment, followed by weeks 1 and 2 on treatment. Significant differences were evaluated by two-way analysis of variance (ANOVA) Šídák’s multiple comparisons test. Average ± SEM ( C ) spleen weight, and numbers of ( D ) CD45.2 + and ( E ) CD45.2 + LSK (Lin − Sca1 + Kit + ) bone marrow cells of D/F ( n = 8 per treatment) or T/F ( n = 8 per treatment) AML mice after 2 weeks of CCS1477 or vehicle. Significant differences were evaluated by unpaired t test. ( F ) Kaplan-Meier survival analysis of CCS1477-treated and vehicle-treated D/F AML (CCS1477, n = 12; vehicle, n = 14) and T/F AML (CCS1477, n = 9; vehicle, n = 9) transplant recipient mice. Overall survival (OS) of moribund D/F AML mice was 94 and 73 days for CCS1477 and vehicle, respectively. OS of moribund T/F AML mice was 51 and 39 days for CCS1477 and vehicle, respectively. Significance determined by the log-rank (Mantel-Cox) test. ( G ) Average ± SEM spleen weight at mouse moribundity of CCS1477- or vehicle-treated D/F ( n = 3 per treatment) or T/F ( n = 3 per treatment) AML mice. Significant differences were evaluated by unpaired t test. Individual data points represent biological replicates. For all panels, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For secondary transplants, CD45.1 + C57BL6/J mice (Charles River Laboratories), 7 to 8 weeks of age, were irradiated with 700 rads before tail vein injection of 1 million c-Kit + AML cells from moribund D/F or T/F mice.

Techniques: In Vivo, Irradiation

Journal: Science Advances

Article Title: P300/CBP inhibition with inobrodib in combination with gilteritinib and venetoclax targets leukemia stem cells in epigenetic mutant AML

doi: 10.1126/sciadv.aec9305

Figure Lengend Snippet: ( A ) Schematic of in vitro CCS1477 or vehicle treatments for H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). c-Kit + splenocytes from moribund D/F or T/F AML mice were treated for 1 or 6 hours before ChIP-seq or RNA-seq, respectively. ( B ) Quantification of log 2 fold change (adjusted p < 0.05) of H3K27ac at promoter and enhancer regions in CCS1477 versus vehicle in vitro–treated D/F ( n = 2 per treatment) and T/F ( n = 2 per treatment) c-Kit + AML cells after 1 hour. Statistical differences were evaluated by Wilcoxon test. ( C ) Heatmap of differentially expressed genes (cut-off log 2 fold change <−1, adjusted p < 0.05) by RNA-seq of c-Kit + AML D/F ( n = 3 per treatment) and T/F ( n = 3 per treatment) treated in vitro with CCS1477 or vehicle. ( D ) Heatmap depiction of gene set enrichment analyses (GSEA) pathways in common by H3K27Ac ChIP-seq and RNA-seq in CCS1477- versus vehicle-treated D/F (left) and T/F (right) AML cells. Color corresponds to GSEA normalized enrichment score (NES). ( E ) Representative H3K27ac ChIP-seq tracks at Myc blood enhancer cluster (BENC). ( F ) Average ± SEM Myc expression by RT-qPCR in CCS1477 versus vehicle in vitro–treated D/F ( n = 3 per treatment) and T/F ( n = 3 per treatment) c-Kit + AML cells after 6 hours. Significance determined by unpaired t tests. ( G ) Representative Western blot image for c-Myc in D/F and T/F AML cells after in vitro treatment of CCS1477 or vehicle after 6 hours. ( H ) Average ± SEM expression of apoptosis pathway genes by RT-qPCR in CCS1477- versus vehicle in vitro–treated D/F ( n = 4 per treatment) and T/F ( n = 4 per treatment) c-Kit + AML cells after 6 hours. Significance determined by one-way ANOVA with Tukey’s multiple comparisons test. Individual data points represent biological replicates. For all panels, *** P < 0.001 and **** P < 0.0001.

Article Snippet: For secondary transplants, CD45.1 + C57BL6/J mice (Charles River Laboratories), 7 to 8 weeks of age, were irradiated with 700 rads before tail vein injection of 1 million c-Kit + AML cells from moribund D/F or T/F mice.

Techniques: In Vitro, ChIP-sequencing, RNA Sequencing, Expressing, Quantitative RT-PCR, Western Blot

Journal: Science Advances

Article Title: P300/CBP inhibition with inobrodib in combination with gilteritinib and venetoclax targets leukemia stem cells in epigenetic mutant AML

doi: 10.1126/sciadv.aec9305

Figure Lengend Snippet: ( A ) Flow cytometric analyses of average ± SEM annexin V + cells 3 days after in vitro treatment of CCS1477, venetoclax, gilteritinib, combination, or vehicle in c-Kit + D/F AML cells ( n = 3). Significance determined by one-way ANOVA with Tukey’s multiple comparisons test. ( B ) Average colonies [colony-forming units (CFU)] ± SEM formed by c-Kit + D/F AML cells ( n = 3) treated with CCS1477, venetoclax, gilteritinib, combination, or vehicle. Significance determined by one-way ANOVA with Tukey’s multiple comparisons test. ( C ) Representative Western blot image for Mcl-1 in D/F AML cells after in vitro treatment with CCS1477, venetoclax, gilteritinib, combination, or vehicle after 4 hours in the presence of proteasome inhibitor. ( D ) Average ± SEM annexin V + cells by flow cytometric analyses after 3 days in vitro treatment of CCS1477, venetoclax, gilteritinib, combination, or vehicle in deidentified primary human FLT3 - ITD AML patient samples ( n = 10). Significance determined by one-way ANOVA with Tukey’s multiple comparisons test. ( E ) Schematic of in vivo combination therapy. Mice received either CCS1477 ( n = 6), venetoclax ( n = 6), gilteritinib ( n = 6), combination ( n = 6), or vehicle ( n = 6) for 2 weeks. Mice were sacrificed 3 hours after the last treatment. Bar graphs of average ± SEM ( F ) spleen weight and ( G ) bone marrow CD45.2 + LSK (Lin − Sca1 + Kit + ) cells. Significance determined by one-way ANOVA with Tukey’s multiple comparisons test. Individual data points represent biological replicates. For all panels, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For secondary transplants, CD45.1 + C57BL6/J mice (Charles River Laboratories), 7 to 8 weeks of age, were irradiated with 700 rads before tail vein injection of 1 million c-Kit + AML cells from moribund D/F or T/F mice.

Techniques: In Vitro, Western Blot, In Vivo