CEBPB is upregulated in monocytic leukemia and promotes venetoclax and MDM2 inhibitor resistance. (A) The volcano plot depicts significant differentially expressed TFs (RNA-seq) in FAB M4/M5 when compared with M0/M1 cells from the Beat AML cohort. (B) The heat map shows the relative expression (z-score) of the previously identified TFs in bone marrow mononuclear cells (BM-MNCs), CD34+ HSPCs, and M0-M5 FAB subtypes within the Beat AML cohort. (C) The volcano plots show the log fold changes and FDRs of differentially expressed TFs in the 20% most sensitive and 20% most resistant cell lines for idasanutlin and venetoclax in the Beat AML samples, as determined by 2-tailed t tests with multiple comparison correction. (D) The volcano plots depict the correlation between the TFs and the idasanutlin or venetoclax AUC as determined by Pearson correlation coefficient tests. (E) The volcano plot depicts significant DE TFs at the protein level in the FAB M4/M5 cells when compared with the M0/M1 cells based on proteomics data from the Kramer study. (F) MOLM14 and OCIAML3 cells were infected with overexpressing lentiviruses encoding the TFs of interest or an empty control with a dsred reporter. The competitive drug graphs depict the induced population change of the infected cells (Dsred+) when treated with dose gradients of venetoclax or idasanutlin for 24 hours as measured by flow cytometry Dsred% analysis. (G) Representative drug curves depict the mean ± SEM viabilities of MOLM14 and OCIAML3 cells transduced with CEBPB overexpression vectors after treatment with dose gradients of venetoclax or idasanutlin. Viability was assessed using an MTS assay and was normalized to no drug treatment controls. An empty vector was used as a control. (H) Competitive growth graphs depicting the relative percentage changes in cells transduced with 2 CRISPR/Cas9 guides (GFP+) that targeted CEBPB when treated with venetoclax or idasanutlin, normalized to their initial population. A nontargeting vector was used as a control. Idasanutlin and venetoclax were evaluated across a total of 9 concentrations with the highest dose being 2 and 1 μM, respectively. The remaining 8 doses were prepared using 1:1.5 and 1:2 serial dilutions, respective. (I) Competitive growth graphs depicting the normalized drug-induced increase in OCIAML3 cells that overexpressed CEBPB (dsred%) when treated with other MDM2 and BCL2 inhibitors for 24 hours, normalized to their initial dsred percentages. AMG232 and DS3202b and AZD4320 were assessed across 9 concentrations with the highest dose being 2, 2, and 1 μM, respectively. The remaining 8 doses were prepared using 1:1.5, 1:1.5, and 1:2 serial dilutions, respective. (J) The fold change in the drug AUCs of cells overexpressing CEBPB after treatment with various venetoclax combinations at 2 time points in comparison with cells that expressed an empty vector. (K) The scatterplot depicts the correlations between CEBPB expression and the drug AUCs for venetoclax drug combinations in samples from the Beat AML cohort. Correlations were determined using Pearson correlation coefficient tests. (L) Western blot images demonstrating the cytoplasmic and nuclear CEBPB expression in M4/M5 and non-M4/M5 samples from patients with leukemia.

CEBPB is upregulated in monocytic leukemia and promotes venetoclax and MDM2 inhibitor resistance. (A) The volcano plot depicts significant differentially expressed TFs (RNA-seq) in FAB M4/M5 when compared with M0/M1 cells from the Beat AML cohort. (B) The heat map shows the relative expression (z-score) of the previously identified TFs in bone marrow mononuclear cells (BM-MNCs), CD34+ HSPCs, and M0-M5 FAB subtypes within the Beat AML cohort. (C) The volcano plots show the log fold changes and FDRs of differentially expressed TFs in the 20% most sensitive and 20% most resistant cell lines for idasanutlin and venetoclax in the Beat AML samples, as determined by 2-tailed t tests with multiple comparison correction. (D) The volcano plots depict the correlation between the TFs and the idasanutlin or venetoclax AUC as determined by Pearson correlation coefficient tests. (E) The volcano plot depicts significant DE TFs at the protein level in the FAB M4/M5 cells when compared with the M0/M1 cells based on proteomics data from the Kramer study. (F) MOLM14 and OCIAML3 cells were infected with overexpressing lentiviruses encoding the TFs of interest or an empty control with a dsred reporter. The competitive drug graphs depict the induced population change of the infected cells (Dsred+) when treated with dose gradients of venetoclax or idasanutlin for 24 hours as measured by flow cytometry Dsred% analysis. (G) Representative drug curves depict the mean ± SEM viabilities of MOLM14 and OCIAML3 cells transduced with CEBPB overexpression vectors after treatment with dose gradients of venetoclax or idasanutlin. Viability was assessed using an MTS assay and was normalized to no drug treatment controls. An empty vector was used as a control. (H) Competitive growth graphs depicting the relative percentage changes in cells transduced with 2 CRISPR/Cas9 guides (GFP+) that targeted CEBPB when treated with venetoclax or idasanutlin, normalized to their initial population. A nontargeting vector was used as a control. Idasanutlin and venetoclax were evaluated across a total of 9 concentrations with the highest dose being 2 and 1 μM, respectively. The remaining 8 doses were prepared using 1:1.5 and 1:2 serial dilutions, respective. (I) Competitive growth graphs depicting the normalized drug-induced increase in OCIAML3 cells that overexpressed CEBPB (dsred%) when treated with other MDM2 and BCL2 inhibitors for 24 hours, normalized to their initial dsred percentages. AMG232 and DS3202b and AZD4320 were assessed across 9 concentrations with the highest dose being 2, 2, and 1 μM, respectively. The remaining 8 doses were prepared using 1:1.5, 1:1.5, and 1:2 serial dilutions, respective. (J) The fold change in the drug AUCs of cells overexpressing CEBPB after treatment with various venetoclax combinations at 2 time points in comparison with cells that expressed an empty vector. (K) The scatterplot depicts the correlations between CEBPB expression and the drug AUCs for venetoclax drug combinations in samples from the Beat AML cohort. Correlations were determined using Pearson correlation coefficient tests. (L) Western blot images demonstrating the cytoplasmic and nuclear CEBPB expression in M4/M5 and non-M4/M5 samples from patients with leukemia.

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