Probing the Cancer Mutational Landscape of KMT2 Regulatory Subunits.

Members of the Lysine MethylTransferase 2 (KMT2) family are often abnormally expressed and mutated in many cancers. Similarly, several mutations listed in cancer databases map to key functional regions of KMT2 regulatory subunits, such as WD repeat domain 5 (WDR5), Retinoblastoma binding protein 5 (RbBP5), absent-small-homeotic-2-like (ASH2L), and DumPY-30 (DPY-30). In this study, we report the systematic characterization of cancer-associated mutations that map to regions important for the WDR5/RbBP5/ASH2L/DPY-30 (WRAD) complex formation. Both binding and thermal stability assays show that several cancer-related mutations do not affect ASH2L binding to DPY-30 or RbBP5. A subset of gain-of-function mutants highlights the role of long-range networks of interactions underlying RbBP5 binding by ASH2L. Parallel analysis of RbBP5 mutations shows additional variants that weaken its interactions with WDR5. Finally, systematic mapping of RbBP5 residues interacting with WDR5 defines the optimal WDR5-binding motif and shows that introducing hydrophobic residues beyond the central VDV sequence increases binding affinity. Overall, these findings reveal surprising gain-of-function mutations in ASH2L and provide a framework for targeting this epigenetic hub therapeutically.