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Pharmacological and genetic inhibition of CEBPA synergizes with VEN-induced apoptosis in TP53mut/mut-like AMLs (A) MOLM13 (VEN-sensitive) and KG1 (VEN-resistant) cells were treated for 72 h with increasing concentrations of VEN and GFC. Synergy was determined by Bliss coefficient (ZIP score >10 indicates synergism). (B and C) Drug-induced apoptosis (B) and viable cell counts (C) in MOLM13 shCEBPA/shScr cells treated with VEN alone or in combination with GFC (concentrations indicated in the plots, 72 h) detected by flow cytometry ( n = 4). (D and E) Apoptosis was detected by flow cytometry in gated human <t>CD45</t> dim CD34 + (or CD117 + cells for CD34 − AMLs) of ex vivo -treated AML samples categorized as TP53 mut-like ( n = 8) (D) and TP53 mut ( n = 9) (E) in a co-culture system using an FITC-annexin V/DAPI staining method. Cells were treated with vehicle, VEN (100 and 500 nM), and VEN+Aza (VEN 100 nM + 5′ Aza 1.5 μM), in the presence or absence of GFC (30 μM) for 72 h. Bar graphs represent the mean ± SEM of all the independent patients screened; each point represents a patient. (F and G) Mitochondrial membrane potential (F) (measured by TMRE staining, n = 18) and total cytoplasmatic ROS levels (G) (measured using the CellROX Red probe, via flow cytometry, n = 6) for the data included in (D) and (E). TP53 mut AMLs are depicted in red, and TP53 mut-like AMLs are depicted in black. APR-246, eprenetapopt. Data are reported as mean ± SEM for (B)–(G). The p values and cell types are indicated in the graphs; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ANOVA and Bonferroni post-test.
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Pharmacological and genetic inhibition of CEBPA synergizes with VEN-induced apoptosis in TP53mut/mut-like AMLs (A) MOLM13 (VEN-sensitive) and KG1 (VEN-resistant) cells were treated for 72 h with increasing concentrations of VEN and GFC. Synergy was determined by Bliss coefficient (ZIP score >10 indicates synergism). (B and C) Drug-induced apoptosis (B) and viable cell counts (C) in MOLM13 shCEBPA/shScr cells treated with VEN alone or in combination with GFC (concentrations indicated in the plots, 72 h) detected by flow cytometry ( n = 4). (D and E) Apoptosis was detected by flow cytometry in gated human <t>CD45</t> dim CD34 + (or CD117 + cells for CD34 − AMLs) of ex vivo -treated AML samples categorized as TP53 mut-like ( n = 8) (D) and TP53 mut ( n = 9) (E) in a co-culture system using an FITC-annexin V/DAPI staining method. Cells were treated with vehicle, VEN (100 and 500 nM), and VEN+Aza (VEN 100 nM + 5′ Aza 1.5 μM), in the presence or absence of GFC (30 μM) for 72 h. Bar graphs represent the mean ± SEM of all the independent patients screened; each point represents a patient. (F and G) Mitochondrial membrane potential (F) (measured by TMRE staining, n = 18) and total cytoplasmatic ROS levels (G) (measured using the CellROX Red probe, via flow cytometry, n = 6) for the data included in (D) and (E). TP53 mut AMLs are depicted in red, and TP53 mut-like AMLs are depicted in black. APR-246, eprenetapopt. Data are reported as mean ± SEM for (B)–(G). The p values and cell types are indicated in the graphs; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ANOVA and Bonferroni post-test.
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Pharmacological and genetic inhibition of CEBPA synergizes with VEN-induced apoptosis in TP53mut/mut-like AMLs (A) MOLM13 (VEN-sensitive) and KG1 (VEN-resistant) cells were treated for 72 h with increasing concentrations of VEN and GFC. Synergy was determined by Bliss coefficient (ZIP score >10 indicates synergism). (B and C) Drug-induced apoptosis (B) and viable cell counts (C) in MOLM13 shCEBPA/shScr cells treated with VEN alone or in combination with GFC (concentrations indicated in the plots, 72 h) detected by flow cytometry ( n = 4). (D and E) Apoptosis was detected by flow cytometry in gated human <t>CD45</t> dim CD34 + (or CD117 + cells for CD34 − AMLs) of ex vivo -treated AML samples categorized as TP53 mut-like ( n = 8) (D) and TP53 mut ( n = 9) (E) in a co-culture system using an FITC-annexin V/DAPI staining method. Cells were treated with vehicle, VEN (100 and 500 nM), and VEN+Aza (VEN 100 nM + 5′ Aza 1.5 μM), in the presence or absence of GFC (30 μM) for 72 h. Bar graphs represent the mean ± SEM of all the independent patients screened; each point represents a patient. (F and G) Mitochondrial membrane potential (F) (measured by TMRE staining, n = 18) and total cytoplasmatic ROS levels (G) (measured using the CellROX Red probe, via flow cytometry, n = 6) for the data included in (D) and (E). TP53 mut AMLs are depicted in red, and TP53 mut-like AMLs are depicted in black. APR-246, eprenetapopt. Data are reported as mean ± SEM for (B)–(G). The p values and cell types are indicated in the graphs; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ANOVA and Bonferroni post-test.
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Pharmacological and genetic inhibition of CEBPA synergizes with VEN-induced apoptosis in TP53mut/mut-like AMLs (A) MOLM13 (VEN-sensitive) and KG1 (VEN-resistant) cells were treated for 72 h with increasing concentrations of VEN and GFC. Synergy was determined by Bliss coefficient (ZIP score >10 indicates synergism). (B and C) Drug-induced apoptosis (B) and viable cell counts (C) in MOLM13 shCEBPA/shScr cells treated with VEN alone or in combination with GFC (concentrations indicated in the plots, 72 h) detected by flow cytometry ( n = 4). (D and E) Apoptosis was detected by flow cytometry in gated human <t>CD45</t> dim CD34 + (or CD117 + cells for CD34 − AMLs) of ex vivo -treated AML samples categorized as TP53 mut-like ( n = 8) (D) and TP53 mut ( n = 9) (E) in a co-culture system using an FITC-annexin V/DAPI staining method. Cells were treated with vehicle, VEN (100 and 500 nM), and VEN+Aza (VEN 100 nM + 5′ Aza 1.5 μM), in the presence or absence of GFC (30 μM) for 72 h. Bar graphs represent the mean ± SEM of all the independent patients screened; each point represents a patient. (F and G) Mitochondrial membrane potential (F) (measured by TMRE staining, n = 18) and total cytoplasmatic ROS levels (G) (measured using the CellROX Red probe, via flow cytometry, n = 6) for the data included in (D) and (E). TP53 mut AMLs are depicted in red, and TP53 mut-like AMLs are depicted in black. APR-246, eprenetapopt. Data are reported as mean ± SEM for (B)–(G). The p values and cell types are indicated in the graphs; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ANOVA and Bonferroni post-test.
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Pharmacological and genetic inhibition of CEBPA synergizes with VEN-induced apoptosis in TP53mut/mut-like AMLs (A) MOLM13 (VEN-sensitive) and KG1 (VEN-resistant) cells were treated for 72 h with increasing concentrations of VEN and GFC. Synergy was determined by Bliss coefficient (ZIP score >10 indicates synergism). (B and C) Drug-induced apoptosis (B) and viable cell counts (C) in MOLM13 shCEBPA/shScr cells treated with VEN alone or in combination with GFC (concentrations indicated in the plots, 72 h) detected by flow cytometry ( n = 4). (D and E) Apoptosis was detected by flow cytometry in gated human <t>CD45</t> dim CD34 + (or CD117 + cells for CD34 − AMLs) of ex vivo -treated AML samples categorized as TP53 mut-like ( n = 8) (D) and TP53 mut ( n = 9) (E) in a co-culture system using an FITC-annexin V/DAPI staining method. Cells were treated with vehicle, VEN (100 and 500 nM), and VEN+Aza (VEN 100 nM + 5′ Aza 1.5 μM), in the presence or absence of GFC (30 μM) for 72 h. Bar graphs represent the mean ± SEM of all the independent patients screened; each point represents a patient. (F and G) Mitochondrial membrane potential (F) (measured by TMRE staining, n = 18) and total cytoplasmatic ROS levels (G) (measured using the CellROX Red probe, via flow cytometry, n = 6) for the data included in (D) and (E). TP53 mut AMLs are depicted in red, and TP53 mut-like AMLs are depicted in black. APR-246, eprenetapopt. Data are reported as mean ± SEM for (B)–(G). The p values and cell types are indicated in the graphs; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ANOVA and Bonferroni post-test.
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Pharmacological and genetic inhibition of CEBPA synergizes with VEN-induced apoptosis in TP53mut/mut-like AMLs (A) MOLM13 (VEN-sensitive) and KG1 (VEN-resistant) cells were treated for 72 h with increasing concentrations of VEN and GFC. Synergy was determined by Bliss coefficient (ZIP score >10 indicates synergism). (B and C) Drug-induced apoptosis (B) and viable cell counts (C) in MOLM13 shCEBPA/shScr cells treated with VEN alone or in combination with GFC (concentrations indicated in the plots, 72 h) detected by flow cytometry ( n = 4). (D and E) Apoptosis was detected by flow cytometry in gated human <t>CD45</t> dim CD34 + (or CD117 + cells for CD34 − AMLs) of ex vivo -treated AML samples categorized as TP53 mut-like ( n = 8) (D) and TP53 mut ( n = 9) (E) in a co-culture system using an FITC-annexin V/DAPI staining method. Cells were treated with vehicle, VEN (100 and 500 nM), and VEN+Aza (VEN 100 nM + 5′ Aza 1.5 μM), in the presence or absence of GFC (30 μM) for 72 h. Bar graphs represent the mean ± SEM of all the independent patients screened; each point represents a patient. (F and G) Mitochondrial membrane potential (F) (measured by TMRE staining, n = 18) and total cytoplasmatic ROS levels (G) (measured using the CellROX Red probe, via flow cytometry, n = 6) for the data included in (D) and (E). TP53 mut AMLs are depicted in red, and TP53 mut-like AMLs are depicted in black. APR-246, eprenetapopt. Data are reported as mean ± SEM for (B)–(G). The p values and cell types are indicated in the graphs; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ANOVA and Bonferroni post-test.
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Pharmacological and genetic inhibition of CEBPA synergizes with VEN-induced apoptosis in TP53mut/mut-like AMLs (A) MOLM13 (VEN-sensitive) and KG1 (VEN-resistant) cells were treated for 72 h with increasing concentrations of VEN and GFC. Synergy was determined by Bliss coefficient (ZIP score >10 indicates synergism). (B and C) Drug-induced apoptosis (B) and viable cell counts (C) in MOLM13 shCEBPA/shScr cells treated with VEN alone or in combination with GFC (concentrations indicated in the plots, 72 h) detected by flow cytometry ( n = 4). (D and E) Apoptosis was detected by flow cytometry in gated human CD45 dim CD34 + (or CD117 + cells for CD34 − AMLs) of ex vivo -treated AML samples categorized as TP53 mut-like ( n = 8) (D) and TP53 mut ( n = 9) (E) in a co-culture system using an FITC-annexin V/DAPI staining method. Cells were treated with vehicle, VEN (100 and 500 nM), and VEN+Aza (VEN 100 nM + 5′ Aza 1.5 μM), in the presence or absence of GFC (30 μM) for 72 h. Bar graphs represent the mean ± SEM of all the independent patients screened; each point represents a patient. (F and G) Mitochondrial membrane potential (F) (measured by TMRE staining, n = 18) and total cytoplasmatic ROS levels (G) (measured using the CellROX Red probe, via flow cytometry, n = 6) for the data included in (D) and (E). TP53 mut AMLs are depicted in red, and TP53 mut-like AMLs are depicted in black. APR-246, eprenetapopt. Data are reported as mean ± SEM for (B)–(G). The p values and cell types are indicated in the graphs; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ANOVA and Bonferroni post-test.

Journal: Cell Reports Medicine

Article Title: ΔNp73 isoform defines a TP53 -mutant-like poor-risk subgroup of acute myeloid leukemia

doi: 10.1016/j.xcrm.2025.102540

Figure Lengend Snippet: Pharmacological and genetic inhibition of CEBPA synergizes with VEN-induced apoptosis in TP53mut/mut-like AMLs (A) MOLM13 (VEN-sensitive) and KG1 (VEN-resistant) cells were treated for 72 h with increasing concentrations of VEN and GFC. Synergy was determined by Bliss coefficient (ZIP score >10 indicates synergism). (B and C) Drug-induced apoptosis (B) and viable cell counts (C) in MOLM13 shCEBPA/shScr cells treated with VEN alone or in combination with GFC (concentrations indicated in the plots, 72 h) detected by flow cytometry ( n = 4). (D and E) Apoptosis was detected by flow cytometry in gated human CD45 dim CD34 + (or CD117 + cells for CD34 − AMLs) of ex vivo -treated AML samples categorized as TP53 mut-like ( n = 8) (D) and TP53 mut ( n = 9) (E) in a co-culture system using an FITC-annexin V/DAPI staining method. Cells were treated with vehicle, VEN (100 and 500 nM), and VEN+Aza (VEN 100 nM + 5′ Aza 1.5 μM), in the presence or absence of GFC (30 μM) for 72 h. Bar graphs represent the mean ± SEM of all the independent patients screened; each point represents a patient. (F and G) Mitochondrial membrane potential (F) (measured by TMRE staining, n = 18) and total cytoplasmatic ROS levels (G) (measured using the CellROX Red probe, via flow cytometry, n = 6) for the data included in (D) and (E). TP53 mut AMLs are depicted in red, and TP53 mut-like AMLs are depicted in black. APR-246, eprenetapopt. Data are reported as mean ± SEM for (B)–(G). The p values and cell types are indicated in the graphs; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ANOVA and Bonferroni post-test.

Article Snippet: To analyze the cytotoxicity in the different fractions of the bulk treated AML cells, treated MNCs were blocked with human FcR blocking reagent (Miltenyi Biotec) for 5 min and stained with the following antibodies: CD45-APC-Cy7, TMRE, CD14-PerCP, CD34-PE-Cy7 (or CD117-PE-Cy7 for CD34 − samples), and CD11b-APC for 20 min at 4°C.

Techniques: Inhibition, Flow Cytometry, Ex Vivo, Co-Culture Assay, Staining, Membrane