The chromosomal translocation products AML1-ETO and PML-RAR contribute to the pathogenesis of leukemias. Here, we demonstrate that both AML1-ETO and PML-RAR are degraded by the ubiquitin-proteasome system and that their turnover critically depends on the E2-conjugase UbcH8 and the E3-ligase SIAH-1. Contrary to its role in HDAC2 degradation, the E3-ligase RLIM does not target AML1-ETO and PML-RAR for ubiquitin-dependent elimination. RLIM rather is a substrate of SIAH-1, which indicates that these E3-ligases operate in a hierarchical order. Remarkably, proteasomal degradation of leukemia fusion proteins, in addition to the block of histone deacetylase (HDAC) enzymatic activity is a consequence of HDAC-inhibitor treatment. The former requires the induction of UbcH8 expression and each of these processes might be beneficial for leukemia treatment. Our observations shed light on the mechanism determining the interplay between E2-conjugases, E3-ligases, and their substrates and suggest a strategy for utilizing the ubiquitylation machinery in a therapeutic setting.—Krämer, O. H., Müller, S., Buchwald, M., Reichardt, S., Heinzel, T. Mechanism for ubiquitylation of the leukemia fusion proteins AML1-ETO and PML-RAR.