A high concentration of arsenic or combination with ATRA was capable of reversing the resistance driven by acquired point mutations. (A) U937 cells were transduced with lentiviral constructs expressing A216T. Infected cells were treated without or with 1 μM, 2.5 μM, 5 μM, 7.5 μM, or 10 μM of As2O3 for 4 hours. Protein levels were detected using the indicated antibody. (B-C) 10 μM of As2O3 was used to treat HeLa cells transfected with A216V, S214L, or A216T mutants in the long (B) and short (C) isoforms of PML-RARA for 2 hours, respectively. Immunoblot analysis was performed as described before. (D) HeLa cells were treated with 1 μM or 10 μM of As2O3 for 1 hour after transfection with A216V, S214L, or A216T mutants in the presence of HA-SUMO. Protein levels in the soluble fraction (top row) and in the detergent-insoluble pellets (middle and bottom rows) were detected using anti-Flag, anti-Tubulin, and anti-HA primary antibodies. (E) U937 cells transduced with the lentiviral mutant (A216T) were treated with 1 μM or 5 μM of As2O3 alone, or together with 1 μM of ATRA, respectively, for 16 hours. (F) Single drug (1 μM, respectively) or the As2O3–ATRA combination was used to treat HeLa cells transfected with A216V, S214L, or A216T mutants for 16 hours. Immunoblot analysis was performed as described before. (G) A schematic representing various responses to As2O3 treatment of PML-RARA with different genetic mutations. Left: The regular dose of As2O3 binds to PML-RARA with L217F or S220G and triggers the degradation of proteins. In contrast, the regular dose of As2O3 might not bind to PML-RARA with A216V, S214L, or A216T and therefore drives resistance to arsenic therapy. Right: A high dose of As2O3 or the As2O3–ATRA combination could induce degradation of PML-RARA with A216V, S214L, or A216T, suggesting that the mutation-driven resistance is at least partly overcome.
