The study reports the development and characterization of EPZ004777, a potent and selective inhibitor of DOT1L, a histone methyltransferase involved in leukemogenesis in mixed lineage leukemia (MLL). EPZ004777 selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes in MLL cells. Treatment with EPZ004777 leads to the selective killing of MLL-positive leukemia cells, with minimal effect on non-MLL-positive cells. In vivo, EPZ004777 extends the survival of MLL-rearranged xenograft mice, demonstrating its therapeutic potential. The compound also causes differentiation and apoptosis in MLL-positive cells, reversing the MLL-rearranged gene signature. These findings support the development of DOT1L inhibitors as a targeted therapy for MLL leukemias.The study reports the development and characterization of EPZ004777, a potent and selective inhibitor of DOT1L, a histone methyltransferase involved in leukemogenesis in mixed lineage leukemia (MLL). EPZ004777 selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes in MLL cells. Treatment with EPZ004777 leads to the selective killing of MLL-positive leukemia cells, with minimal effect on non-MLL-positive cells. In vivo, EPZ004777 extends the survival of MLL-rearranged xenograft mice, demonstrating its therapeutic potential. The compound also causes differentiation and apoptosis in MLL-positive cells, reversing the MLL-rearranged gene signature. These findings support the development of DOT1L inhibitors as a targeted therapy for MLL leukemias.