The Function of the ASH1L Histone Methyltransferase in Cancer: A Chemical Biology Approach

Abstract

ASH1L (absent, small, or homeotic-like 1) is a histone lysine methyltransferase (KMTase) that is overexpressed in cancer and activates oncogenic HOX genes. Small molecule inhibitors of ASH1L would be invaluable tools to investigate the role of ASH1L in cancer; however, no ASH1L inhibitors have been reported to date. Previous studies of ASH1L’s catalytic SET domain identified an autoinhibitory loop that blocks access of histone substrate to the enzyme active site. We used nuclear magnetic resonance (NMR) and X-ray crystallography to identify conformational heterogeneity in the ASH1L autoinhibitory loop and nearby SET-I loop, two structural features that regulate the enzymatic activity of the SET domain. These studies suggested that conformational heterogeneity in the autoinhibitory loop region of the ASH1L SET domain may create transient pockets into which small molecule ligands could bind.

We took a fragment-based drug discovery (FBDD) approach to probe the ASH1L SET domain for potential small molecule binding sites and then construct new ligands specific to ASH1L. FBDD identified a ligand that binds to the ASH1L autoinhibitory loop region. We used information from NMR and crystallographic studies to optimize the fragment-like ligand into a first-in-class potent and specific ASH1L inhibitor. Structural studies indicate that ASH1L inhibitors block enzymatic activity by stabilizing the autoinhibited conformation of the SET domain. Our current most potent compounds inhibit ASH1L activity with IC50 of ~4 μM and bind to ASH1L with ~1 μM affinity, representing greater than 1000-fold improvement over the fragment screening hit. Our work identified the first ASH1L inhibitor and represents the first example of successfully applying FBDD to KMTases.

Using our ASH1L inhibitors, we took a combined genetic and pharmacologic approach to investigate the role of ASH1L in leukemia and breast cancer. We found that ASH1L is required for proliferation of breast cancer and leukemia cells, and that ASH1L activates HOXA genes and MEIS1 in leukemia. Moreover, inhibition of the ASH1L SET domain downregulated HOX gene expression and induced differentiation of leukemia cells transformed by MLL-AF9. Our results demonstrate cellular efficacy of ASH1L inhibitors and uncover a new role for the ASH1L SET domain in acute leukemia.