Non-Canonical MLL1 Activity Regulates Centromeric Phase Separation and Genome Stability

Epigenetic dysregulation is one of the most prominent features in human cancers as exemplified by frequent mutations or aberrant expression of chromatin regulators, including the MLL/KMT2 family of histone methyltransferases. With a growing body of paradoxical evidence debating the importance of canonical activity of MLLs on transcription in cancer, their non-canonical activity, which may lead to new therapeutic opportunities, has never been explored. Here, using an unbiased proteomics approach, we identify Borealin as an unprecedented non-canonical substrate for MLL1/KMT2A. We find that MLL1 methylates Borealin at K143, a residue resides in the intrinsically disordered region essential for liquid-liquid phase separation (LLPS) of inner centromeric chromosome passenger complex (CPC). Co-crystal structure of the MLL1-Borealin complex highlights distinct binding modes of the MLL1 SET domain between H3K4 and Borealin. Importantly, inhibiting MLL1 activity or mutating Borealin K143 to arginine perturbs CPC phase separation and phosphorylation of Aurora kinase B kinetochore substrates, thus impairing resolution of erroneous kinetochore-microtubule attachments as well as proper sister chromatid cohesion. MLL1 inhibition or depletion significantly increases chromosome instability and mitotic defects in CPC-high hepatocellular carcinoma (HCC) in vitro and in vivo. These results demonstrate a surprising function of MLL1 in regulating inner centromere liquid condensates to maintain cancer genome stability and establish MLL1 as a specific targetable vulnerability in HCC.