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S. Demir et al.
TP53mut and TP53wt ALL cells, leading to similar cell death rates (Online Supplementary Figure S4).
Next, we investigated whether induction of oxidative stress is involved in APR-246-mediated cell death in TP53mut ALL. Importantly, methyl quinuclidinone, the active drug spontaneously formed from APR-246, binds covalently to cysteine residues in the core domain of p53, but also to cysteines in the widely used antioxidant and ROS inhibitor N-acetylcysteine (NAC).28,44 Thus, NAC directly blocks APR-246 activity and cannot be used to investigate the role of ROS in APR-246-mediated cell death. Therefore, the synthetic antioxidant compound and ROS inhibitor superoxide dismutase mimetic Mn (III) tetrakis (5, 10, 15, 20-benzoic acid) porphyrin (MnTBAP) was used. Cell death was analyzed together with ROS lev- els in TP53mut (KOPN-8 and RS4;11) and TP53wt (NALM- 6 and UoCB6) leukemia cells exposed to APR-246 with or without NAC or MnTBAP. Similar ROS levels were observed upon APR-246 treatment in both TP53mut and TP53wt ALL (Online Supplementary Figure S5A, C, E, G), however induction of cell death was only seen in TP53mut cells (Online Supplementary Figure S5F, H) but not in TP53wt cells (Online Supplementary Figure S5B, D). Interestingly, ROS inhibition by MnTBAP partially inhibited APR-246- induced cell death in TP53mut ALL, indicating that ROS
contribute to APR-246- induced cell death. It was also interesting that, even in the presence of MnTBAP, i.e. in the absence of ROS, APR-246 retained a statistically significant cytotoxic effect (Online Supplementary Figure S5F, H). In line with previous reports,44 the activity of APR-246 was com- pletely blocked by NAC.
Taken together, these data show that induction of
oxidative stress might contribute to APR-246-mediated
cell death in ALL, in line with previously reported data of
a dual mode of action of APR-246 in other malignan- cies.28,30-33,44-46
APR-246 activity depends on mutant p53
Activity of APR-246 was observed in TP53mut but not TP53wt ALL. Therefore, we analyzed the effect of APR- 246 in TP53mut and TP53wt cell lines upon lentiviral shRNA-mediated knockdown of p53 (Figure 4A-C). In both TP53mut lines (KOPN-8 and RS4;11) depletion of p53 led to APR-246 insensitivity and cell death resistance, in contrast to dose-dependent cell death induction in con- trol-transduced cells (Figure 4D, E). However, TP53wt cells with p53 depletion were unresponsive to APR-246, like the corresponding control transduced cells (Figure 4F). A similar result was observed upon siRNA-mediated p53 downregulation with clearly lower cell death induction in
AB
CD
EF
Figure 2. APR-246 induces apoptosis in TP53-mutated acute lymphoblastic leukemia. (A-F) Induction of cell death (left diagrams, forward/side scatter criteria, flow cytometry), annexin-V/propidium iodide (PI) positivity (middle diagrams) and caspase-3 activation (right diagrams) by APR-246 in TP53mut cell lines KOPN-8 (A) and RS4;11 (B), in contrast to cell death and apoptosis induction by doxorubicin in TP53wt lines NALM-6 (C), UoCB-6 (D), EU-3 (E), and MUTZ-5 (F). Proportions of cells after 48 h exposure to solvent (CTRL), APR-246 (APR, 5 mM), or doxorubicin (DOX, 15 ng/mL). Mean values ± standard deviation of three independent experiments, each performed in triplicate. Student t-test, ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; n.s., not significant.
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