Interplay of Ubiquitin-Like Modifiers Following Arsenic Trioxide Treatment

Arsenic trioxide (ATO) is a therapeutic agent used to treat acute promyelocytic leukemia (APL), a disease caused by a chromosomal translocation of the retinoic acid receptor α (RARα) gene that can occur reciprocally with the promyelocytic leukemia (PML) gene. The mechanisms through which ATO exerts its effects on cells are not fully characterized though they involve the SUMOylation, the ubiquitylation, and the degradation of the PML/RARα oncoprotein through the PML moiety. To better understand the mechanisms that underlie the cytotoxicity induced with increasing ATO levels, we profiled the changes in protein SUMOylation, phosphorylation, and ubiquitylation on HEK293 cells following exposure to low (1 μM) or elevated (10 μM) ATO for 4 h. Our analyses revealed that a low dose of ATO resulted in the differential modification of selected substrates including the SUMOylation (K380, K394, K490, and K497) and ubiquitylation (K337, K401) of PML. These experiments also highlighted a number of unexpected SUMOylated substrates involved in DNA damage response (e.g., PCNA, YY1, and poly­[ADP-ribose] polymerase 1 (PARP1)) and messenger RNA (mRNA) splicing (e.g., ACIN1, USP39, and SART1) that were regulated at higher ATO concentrations. Interestingly, additional enzymatic assays revealed that SUMOylation of PARP1 impeded its proteolytic cleavage by caspase-3, suggesting that SUMOylation could have a protective role in delaying cell apoptosis.