Epigenetic vulnerabilities of leukemia harboring inactivating EZH2 mutations.

Epigenetic regulators, such as the polycomb repressive complex 2 (PRC2), play a critical role in both normal development and carcinogenesis. Mutations and functional dysregulation of PRC2 complex components, such as EZH2, are implicated in various forms of cancer and associated with poor prognosis. This study investigated the epigenetic vulnerabilities of acute myeloid leukemia (AML) and myelodysplastic/myeloproliferative disorders (MDS/MPN) by performing a chemical probe screen in patient cells. Paradoxically, we observed increased sensitivity to EZH2 and embryonic ectoderm development (EED) inhibitors in AML and MDS/MPN patient cells harboring EZH2 mutations. Expression analysis indicated that EZH2 inhibition elicited upregulation of pathways responsible for cell death and growth arrest, specifically in patient cells with mutant EZH2. The identified EZH2 mutations had drastically reduced catalytic activity, resulting in lower cellular H3K27me3 levels, and were associated with decreased EZH2 and PRC2 component EED protein levels. Overall, this study provides an important understanding of the role of EZH2 dysregulation in blood cancers and may indicate disease etiology for these poor prognosis AML and MDS/MPN cases.

A chemical probe targeting the PWWP domain alters NSD2 nucleolar localization.

Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.

Probing residues in the pore-forming (M2) domain of the Cys-loop receptor homologue GLIC reveals some unusual features.

Cys-loop receptors play important roles in signal transduction. The Gloeobacter ligand-gated ion channel (GLIC) pore binds similar compounds to Cys-loop receptor pores, but has the advantage of known structures in open and closed states. GLIC is activated by protons with a pEC50 of 5.4, and has a histidine residue (His 11') in its pore-forming α-helix (M2) which is involved in gating. Here we explore the role of this His and other M2 residues using two-electrode voltage clamp of mutant receptors expressed in oocytes. We show that 11'His is very sensitive to substitution; replacement with a range of amino acids ablates function. Similarly altering its location in M2 to the 8', 9', 10', 12', 13' or 14' positions ablated function. Most substitutions of Ser6' or Ile9' were also non-functional, although not Ile9'Leu and Ile9'Val. Unexpectedly, an Ile9'His substitution was constitutively active at pH 7, but closed as [H+] increased, with a pIC50 of 5.8. Substitution at 2', 5' and 7' had little effect on pEC50. Overall the data show Ser6' and His11' are critical for the function of the receptor, and thus distinguish the roles of these M2 residues from those of Cys-loop receptors, where substitutions are mostly well tolerated. These data suggest modellers should be aware of these atypical features when using the GLIC pore as a model for Cys-loop receptor pores.

Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16'A-, F16'D-, F16'S-, and F16'T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16' is small. T6'S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16'S, indicating that the inhibitor binds at position 6', as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16' is taken into consideration.

Cys-loop receptor channel blockers also block GLIC.

The Gloeobacter ligand-gated ion channel (GLIC) is a bacterial homolog of vertebrate Cys-loop ligand-gated ion channels. Its pore-lining region in particular has a high sequence homology to these related proteins. Here we use electrophysiology to examine a range of compounds that block the channels of Cys-loop receptors to probe their pharmacological similarity with GLIC. The data reveal that a number of these compounds also block GLIC, although the pharmacological profile is distinct from these other proteins. The most potent compound was lindane, a GABA(A) receptor antagonist, with an IC50 of 0.2 µM. Docking studies indicated two potential binding sites for this ligand in the pore, at the 9' or between the 0' and 2' residues. Similar experiments with picrotoxinin (IC50 = 2.6 µM) and rimantadine (IC50 = 2.6 µM) reveal interactions with 2'Thr residues in the GLIC pore. These locations are strongly supported by mutagenesis data for picrotoxinin and lindane, which are less potent in a T2'S version of GLIC. Overall, our data show that the inhibitory profile of the GLIC pore has considerable overlap with those of Cys-loop receptors, but the GLIC pore has a unique pharmacology.