MLL rearrangements play a crucial role in leukemogenesis and comprise a poor prognosis. Therefore, new treatment strategies are urgently needed. We used the CRISPR/Cas9 system to generate an innovative leukemia model based on 100% pure MLL-AF4 or -AF9 rearranged cells derived from umbilical cord blood with indefinite growth in cell culture systems. Our model shared phenotypical, morphological and molecular features of patient cells faithfully mimicking the nature of the disease. Thus, it serves as a fundamental basis for pharmacological studies: inhibition of histone methyltransferase disruptor of telomeric silencing 1-like (DOT1L) is one specific therapeutic approach currently tested in clinical trials. However, success was limited by restricted response warranting further investigation of drug combinations. Recently, it has been shown that the inhibition of protein arginine methyltransferase 5 (PRMT5) exhibits anti-tumoral activity against human cell lines and in MLL mouse models. Here, we used DOT1L and PRMT5 inhibitors in our human MLL-rearranged model demonstrating dose-dependent reduced proliferation, impairment of cell cycle, increasing differentiation, apoptosis, downregulation of target genes and sensitization to chemotherapy. Strikingly, the combination of both compounds led to synergistic anti-tumoral effects. Our study provides a strong rationale for novel targeted combination therapies to improve the outcome of MLL-rearranged leukemias.