High Throughput Screening Identifies Potential Inhibitors of WHSC1/MMSET, a Histone Methyltransferase Oncoprotein in Multiple Myeloma and Acute Lymphocytic Leukemia

Aberrant epigenetic gene regulation is now considered a hallmark of many diseases, including cancer.  Mutations and disruptions of epigenetic modifier enzymes are among the commonest mutation in human cancer. MMSET/NSD2/WHSC1 is a histone methyltransferase overexpressed in many cancers, including a subset of multiple myeloma (MM) harboring the t(4;14) translocation. This increased expression of MMSET is linked to a poorer prognosis and response to therapy than other MM subtypes. More recently activating mutations of MMSET were identified in relapsed pediatric ALL. The major substrate for MMSET in vivo is lysine 36 on histone H3 (H3K36).  The ability of MMSET to methylate H3 depends on the enzymatic activity of its SET domain, found in most histone methyltransferases. Overexpression or activating mutations of MMSET lead to a global increase in dimethylation of H3K36 (H3K36me2) and a concomitant global loss of trimethylated H3K27 (H3K27me3), which leads to a more open chromatin confirmation.  We, and others, have shown that MMSET has a role in a number of pathways including cell cycle, apoptosis, cell adhesion, and DNA repair.  The loss of MMSET in myeloma cells decreases cell proliferation and leads to a reduced clonogenic capacity. These data highlight MMSET as a promising therapeutic target for t(4;14)+ MM.  A small molecule inhibitor could also prove advantageous in our efforts to understand the various mechanisms of MMSET’s enzymatic activity.  Here we performed two high throughput screens on two different small molecule libraries.  The screens were conducted by using the recombinant SET methyltransferase domain of MMSET (aa980-1214), generated in vitro, in combination with H4 (aa36-50) as the peptide substrate and S-adenosylmethionine (SAM) as the methyl donor.  These screens were designed to avoid SAM competitors by screening under high SAM concentrations (50μM).  The first screen used SAMDI technology, which combines mass spectrometry with high-density arrays. This method allows us to identify compounds within the libraries that inhibit MMSET in a rapid, label-free format.  The second screen used alphalisa technology, a luminescent proximity assay that provides a homogeneous, sensitive method to screen for molecules that reduce MMSET activity.  A total of 88 compounds from both libraries were identified as potential MMSET inhibitors (>3-standard deviation) and were counter-screened against the histone demethylase LSD1 to confirm their specificity.  Numerous candidate compounds promiscuously inhibited the enzymatic activity of LSD1, yielding eleven putative MMSET inhibitors, with IC50 values ranging from 6-42μM.  These eleven compounds were then screened against full-length MMSET enzyme using HeLa-derived mono/di-nucleosomes as the substrate.  Eight of the compounds demonstrated a 50% or greater inhibition of MMSET at 10 μM and three compounds exhibited a dose-dependent inhibition upon dilution.  Future in vitro, cellular, and animal based assays will determine the potency and selectivity of these compounds against MMSET and gauge their effectiveness as targeted therapies in cancers characterized by MMSET overexpression and activating mutations.