Poster Presentation 44th Lorne Genome Conference 2023

CRISPR-ChIP delineates a Menin-dependent oncogenic DOT1L complex in MLL leukaemia    (#253)

Omer Gilan 1 , Charles Bell 2 , Laure Talarmain 2 , Daniel Neville 1 , Kathy Knezevic 2 , Daniel Ferguson 1 , Marion Boudes 3 , Chih-yih Chan 2 , Chen Davidovich 3 , Enid Lam 2 , Mark Dawson 2
  1. Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia
  2. Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
  3. Biomedicine discovery institute, Monash University, Clayton, VIC, Australia

Acute leukaemias driven by MLL fusion oncoproteins (MLL-FP) are aggressive and often incurable malignancies that have attracted a substantial investment in therapies against essential oncogenic cofactors such as Menin, DOT1L and BRD4.  The success of these novel drugs rests on a detailed understanding of the molecular mechanisms that regulate the recruitment and transcriptional activity of MLL-FP. Here, we develop a dynamic modular system to both map the genome-wide occupancy of MLL-FP in leukaemia cells and rapidly alter its oncogenic potential with an auxin-inducible degron. We use this system to define a core set of genes that are directly regulated by MLL-FP and show that the functional integrity of both DOT1L and Menin but not the BET bromodomain proteins is required for the chromatin localisation of MLL-FP. We find that perturbation of Menin surprisingly results in a dramatic loss of H3K79me2 at a small subset of loci directly linked to MLL-FP activity. To elucidate the regulation of H3K79methylation, we developed a novel CRISPR-screening method with a chromatin readout (CRISPR-ChIP) that unbiasedly identifies the non-redundant factors required for H3K79me2 and other chromatin modifications. Using CRISPR-ChIP in MLL-FP leukaemia, we uncover a functional partitioning of H3K79 methylation into two distinct regulatory units: an oncogenic DOT1L complex, where the malignant driver directs the catalytic activity of DOT1L at MLL-fusion target genes and a separate endogenous DOT1L complex, where catalytic activity is regulated by MLLT10. This functional demarcation has therapeutic implications that may enhance outcomes against MLL-FP leukaemia.