Inhibition of EYA family tyrosine phosphatase activity reveals a therapeutic vulnerability and enhances Menin and DOT1L inhibitor efficacy in KMT2A-rearranged leukemia.

MLL (KMT2A)-rearranged leukemia (MLL-r) is a highly aggressive hematologic malignancy driven by transcriptional dysregulation. Here, we identify EYA family phosphatase activity, particularly EYA1 and EYA3, as key vulnerabilities in MLL-r leukemia. The small molecule benzbromarone (BBR) selectively reduced viability in MLL-r and EYA-expressing MLL-nonrearranged (MLL-nr) leukemia cells. Inhibition of EYA PTP activity increased global RNA Pol II CTD Tyr1 phosphorylation, linking aberrant EYA PTP activity in responsive leukemia cells to transcriptional dysregulation. In vivo, BBR treatment significantly prolonged survival and reduced leukemia burden without overt toxicity. Furthermore, BBR synergized with the menin-MLL inhibitor VTP50469 and showed additive effects with the DOT1L inhibitor EPZ5676, the latter of which restored BBR sensitivity in previously BBR-unresponsive cells. These findings establish EYA PTP activity as a therapeutic target in MLL-r leukemia, support the use of EYA expression for identifying patients likely to benefit from BBR treatment, and highlight the potential of BBR-based combinations to improve response in this high-risk leukemia subtype.