Deubiquitinase USP6 stabilizes oncogenic RUNX1 fusion proteins to promote the leukemic potential and malignant progression.

RUNX1-rearranged leukemia is one of the most common subtypes of leukemia associated with genetic abnormalities. Although the majority of patients respond to chemotherapy, relapse and long-term adverse effects remain significant challenges. RUNX1 fusions, resulting from chromosomal rearrangements, are pivotal oncogenic drivers, with over 70 distinct variants identified. Therefore, elucidating their regulatory mechanisms may help to develop novel therapeutic strategies. Herein, we identify a universal deubiquitinase, USP6, that stabilizes RUNX1 fusion proteins with different partners. Importantly, USP6 is specifically upregulated in RUNX1-rearranged leukemia and strongly correlates with poor patient outcomes. Mechanistically, USP6 stabilizes RUNX1 fusions to facilitate the formation of phase separation, leading to robust transcriptional activation of the fusions. Depletion of USP6 dramatically inhibits proliferation and induces differentiation of RUNX1-rearranged leukemic cells. The marketed drug auranofin is identified as a potential USP6 inhibitor, which induces degradation of different RUNX1 fusions, further triggering myeloid differentiation and arresting xenograft tumor growth. Notably, auranofin exhibits selective therapeutic efficacy in patient-derived leukemia blasts from RUNX1-rearranged cases. Together, we not only uncover a new biological function of USP6 in regulating the transcriptional activity of RUNX1 fusions but also validate USP6 as a promising drug target and auranofin as a candidate therapy for RUNX1-rearranged leukemia.