Selective protection of human cardiomyocytes from anthracycline cardiotoxicity by small molecule inhibitors of MAP4K4

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  Figure 1. Inhibitors of MAP4K4 confer protection from DOX in rat H9c2 cardiomyocytes. H9c2 cells were treated with the MAP4K4 inhibitors shown, or DMSO as the vehicle control, beginning 1 h prior to DOX. (A–C) Viability, measured by the CellTiter-Glo assay. (A,B) Dose–response for DOX cardiotoxicity at 24 h (A) and 48 h (B), in the absence or presence of 10 μM F1386-0,303, the second-generation MAP4K4 inhibitor11. Data are triplicates, plotted as the mean ± SEM, and are representative of 3 independent dose–response experiments. (C) Protection from 333 nM DOX at 48 h by F1386-0303 versus DMX-5804, a third-generation inhibition11. Data are from a single experiment, plotted as the mean ± SEM, and are representative of ≥ 20 independent dose–response experiments across multiple batches of each compound. (D–F) Activation of executioner caspases. (D,E) Dose–response for caspase-3/7 activity triggered by DOX, at 6 h (D) and 24 h (E), in the absence or presence of 10 μM F1386-0303. Data are triplicates, shown as the mean ± SEM. (F) Dose–response for protection from 1 μM DOX at 24 h by F1386-0303 versus DMX-5804. Data are the mean ± SEM for quadruplicate cultures in three independent experiments.…

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  Figure 2. Inhibitors of MAP4K4 confer protection from DOX in human PSC-derived cardiomyocytes. (A,B) Protection of hPSC-CM viability (CellTiter-Glo). (A) Human vCorv.4U ventricular myocytes were treated for 72 h with 3 μM DOX, 2 μM Idarubicin, or 30 μM menadione, a proven MAP4K4-dependent death signal11, ± 10 μM F1386-0,303. Data were normalised to untreated and 0.1% Triton X-100-treated cells (100% and 0% viability, respectively). Data are triplicates, plotted as the mean ± SEM, and are representative of 3 independent experiments. **P ≤ 0.01; ***P ≤ 0.001. (B) Human IMR-90 cardiomyocytes were treated for 24 h with 0–75 μM DOX ± 10 μM DMX-5804. **P ≤ 0.01. (C–F) Suppression of hPSC-CM apoptosis. (C,D) Human vCor.4U cells were treated for 24 h with 0–10 μM DOX ± 10 μM DMX-5804 and were analyzed by TUNEL staining. (C) Representative images. Bar, 50 μm. (D) Data are the mean ± SEM of ≥ 3 replicates and are representative of 2 independent experiments. ***P ≤ 0.001. (E,F) IMR-90 cardiomyocytes, treated for 24 h as shown, were assayed by Western blotting for cleaved PARP1. (E) Representative Western blot (See also Supplementary Fig. S1). (F) Data are the mean ± SEM of 2 independent experiments.…

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  Figure 3. Inhibitors of MAP4K4 do not impair cancer cell killing by DOX. (A–D) Viability. Human tumor cells from the five indicated lines were treated with DOX and DMX-5804 at the concentrations shown and were assayed at 24 h (CellTiter Glo). Data are the mean ± SEM of ≥ 5 replicates and are representative of 2 independent dose–response experiments, excepting KMS-12-BM (5 replicates, 1 study). (E) Hypodiploid (sub-G1) DNA fragmentation. U266 cells were treated with 0–20 μM DOX for 24 h ± 10 μM DMX-5804, and DNA content was analysed by flow cytometry. Above, representative DNA histograms. Below, results are the mean ± SEM of single determinations in 2–3 independent experiments. (F) Annexin V. U266 cells were treated as in panel E then were analysed by flow cytometry for annexin V-Blue and PI fluorescence. Above, Representative scattergrams. Below, Data are single determinations, representative of two independent experiments. (G) Activation of caspase-3/7 (cf. Fig. 1E). THP1 cells were treated with 1 μM DOX for 24 h ± F1386-0303 at the concentrations shown. Data are the mean ± SEM of 4 replicates in each of 3 independent dose–response experiments, shown relative to the vehicle-treated…