The DOT1L enzyme is hijacked by MLL-Fusion oncoproteins (MLL-FP) to aberrantly deposit H3K79methylation at key target genes to drive leukemogenesis. MLL-FP activity can be disrupted by blocking its chromatin occupancy with Menin inhibitors, or by blocking its biochemical activity through inhibition of DOT1L. While these inhibitors are promising, our understanding of how they elicit specific anti-leukemia effects remains incomplete. Using functional genomics, we uncovered that DOT1L functionally antagonises the PRC1.1 complex to maintain transcription of critical MLL-Fusion target genes. Loss of the PRC1.1 subunits, PCGF1 or BCOR, confers resistance to DOT1L and Menin inhibition by preventing the potent down-regulation of critical genes, differentiation, and apoptosis of murine and human MLL-leukaemia cells in vitro and in vivo. We find that Menin inhibition elicits a PRC1.1-dependent selective induction of H2AK119Ub and H3K27me3 at key MLL-Fusion target genes whilst DOT1L inhibition leads to the widespread increase in H2AK119Ub. Consistent with these findings, we demonstrate that the induction of H2AK119Ub following Menin inhibition is specifically associated with loss of H3K79me2 rather than eviction of the MLL-Fusion protein or reduction in transcription. In addition, temporal analysis of chromatin and transcriptional changes revealed that the deposition of H2AK119Ub is delayed and correlated with the gradual loss of H3K79me2, suggesting a direct functional antagonism between PRC1.1 and DOT1L. Upon this switch in chromatin modifications, polycomb target genes undergo stable repression leading to the irreversible commitment of leukaemia cells towards differentiation and apoptosis even after early drug withdrawal. Taken together, our model establishes a fundamental and conserved function for DOT1L in protecting genes from rapid PRC1-mediated repression. This epigenetic silencing is essential for the efficacy of differentiation therapies and highlights why DOT1L is hijacked in MLL leukaemia with potential implications for future therapeutic strategies using Menin inhibitors.