Actinomycin D enhances cytotoxicity and reverses resistance to Ven in NPM1c+ preleukemic and leukemic cells. (A) Counts of preleukemic Npm1cA/+ lin– cells treated with DMSO, ActD, venetoclax (Ven), or a combination (mean ± SD, n = 3 independent experiments). (B) Representative CFU assays of preleukemic Npm1cA/+ lin– cells treated with DMSO, ActD, Ven, or a combination. (C) Total CFU colony numbers of preleukemic Npm1cA/+ lin– cells treated with DMSO, ActD, Ven, or a combination (n = 2 biological replicates, each performed in duplicate). (D) Counts of murine Npm1cA/+; Flt3ITD/+AML cells treated with DMSO, ActD, Ven, or a combination (mean ± SD, n = 3 independent experiments). (E) Representative synergy plot revealing the interaction between ActD and Ven in inhibiting the proliferation of murine Npm1cA/+; Flt3ITD/+AML cells after 4 days of drug incubation (MTS assay) (representative examples from 1 of 3 independent experiments). (F) OCI-AML3 cells were pretreated with 0.5 nM ActD vs DMSO for 24 hours and then tested for sensitivity to venetoclax. (G) IC50 values of DMSO or ActD pretreated OCI-AML3 cells after 4 days of incubation with Ven at the indicated doses (MTS assay). (H) Outline of approach to generate Ven-resistant murine Npm1c-mutant AML cells by treatment with escalating concentrations of Ven. Ven-resistant cells were then pretreated with ActD for 24 hours and their sensitivity to Ven was assessed using the MTS assay. (I) Relative proliferation of DMSO or ActD pretreated murine NPM1c+ AML cells after 4 days incubation with Ven at the indicated doses. Absorbance values were normalized to DMSO-treated cells (mean ± SD, data from 1 of 2 biological replicates, performed in triplicate). (J) IC50 values of DMSO or ActD pretreated murine NPM1c+ AML cells after 4 days of incubation with Ven (mean ± SD, data from 1 of 2 biological replicates, performed in triplicate). (A,C-D,G,J) t test was used to calculate P values between groups. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001. ZIP, zero interaction potency. Panels F and H were created with BioRender.com. Vassiliou, G. (2025) https://BioRender.com/9tt8kww.
Figure 5.

Actinomycin D enhances cytotoxicity and reverses resistance to Ven in NPM1c+ preleukemic and leukemic cells. (A) Counts of preleukemic Npm1cA/+ lin– cells treated with DMSO, ActD, venetoclax (Ven), or a combination (mean ± SD, n = 3 independent experiments). (B) Representative CFU assays of preleukemic Npm1cA/+ lin– cells treated with DMSO, ActD, Ven, or a combination. (C) Total CFU colony numbers of preleukemic Npm1cA/+ lin– cells treated with DMSO, ActD, Ven, or a combination (n = 2 biological replicates, each performed in duplicate). (D) Counts of murine Npm1cA/+; Flt3ITD/+AML cells treated with DMSO, ActD, Ven, or a combination (mean ± SD, n = 3 independent experiments). (E) Representative synergy plot revealing the interaction between ActD and Ven in inhibiting the proliferation of murine Npm1cA/+; Flt3ITD/+AML cells after 4 days of drug incubation (MTS assay) (representative examples from 1 of 3 independent experiments). (F) OCI-AML3 cells were pretreated with 0.5 nM ActD vs DMSO for 24 hours and then tested for sensitivity to venetoclax. (G) IC50 values of DMSO or ActD pretreated OCI-AML3 cells after 4 days of incubation with Ven at the indicated doses (MTS assay). (H) Outline of approach to generate Ven-resistant murine Npm1c-mutant AML cells by treatment with escalating concentrations of Ven. Ven-resistant cells were then pretreated with ActD for 24 hours and their sensitivity to Ven was assessed using the MTS assay. (I) Relative proliferation of DMSO or ActD pretreated murine NPM1c+ AML cells after 4 days incubation with Ven at the indicated doses. Absorbance values were normalized to DMSO-treated cells (mean ± SD, data from 1 of 2 biological replicates, performed in triplicate). (J) IC50 values of DMSO or ActD pretreated murine NPM1c+ AML cells after 4 days of incubation with Ven (mean ± SD, data from 1 of 2 biological replicates, performed in triplicate). (A,C-D,G,J) t test was used to calculate P values between groups. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001. ZIP, zero interaction potency. Panels F and H were created with BioRender.com. Vassiliou, G. (2025) https://BioRender.com/9tt8kww.

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