Anqi Ma

An Orally Bioavailable Chemical Probe of the Lysine Methyltransferases EZH2 and EZH1

EZH2 or EZH1 is the catalytic subunit of the polycomb repressive complex 2 that catalyzes methylation of histone H3 lysine 27 (H3K27). The trimethylation of H3K27 (H3K27me3) is a transcriptionally repressive post-translational modification. Overexpression of EZH2 and hypertrimethylation of H3K27 have been implicated in a number of cancers. Several selective inhibitors of EZH2 have been reported recently. Herein we disclose UNC1999, the first orally bioavailable inhibitor that has high in vitro potency for wild-type and mutant EZH2 as well as EZH1, a closely related H3K27 methyltransferase that shares 96% sequence identity with EZH2 in their respective catalytic domains. UNC1999 was highly selective for EZH2 and EZH1 over a broad range of epigenetic and non-epigenetic targets, competitive with the cofactor SAM and non-competitive with the peptide substrate. This inhibitor potently reduced H3K27me3 levels in cells and selectively killed diffused large B cell lymphoma cell lines harboring the EZH2(Y641N) mutant. Importantly, UNC1999 was orally bioavailable in mice, making this inhibitor a valuable tool for investigating the role of EZH2 and EZH1 in chronic animal studies. We also designed and synthesized UNC2400, a close analogue of UNC1999 with potency >1,000-fold lower than that of UNC1999 as a negative control for cell-based studies. Finally, we created a biotin-tagged UNC1999 (UNC2399), which enriched EZH2 in pull-down studies, and a UNC1999-dye conjugate (UNC2239) for co-localization studies with EZH2 in live cells. Taken together, these compounds represent a set of useful tools for the biomedical community to investigate the role of EZH2 and EZH1 in health and disease.

Selective inhibition of EZH2 and EZH1 enzymatic activity by a small molecule suppresses MLL-rearranged leukemia

Enhancer of zeste homolog 2 (EZH2) and related EZH1 control gene expression and promote tumorigenesis via methylating histone H3 at lysine 27 (H3K27). These methyltransferases are ideal therapeutic targets due to their frequent hyperactive mutations and overexpression found in cancer, including hematopoietic malignancies. Here, we characterized a set of small molecules that allow pharmacologic manipulation of EZH2 and EZH1, which include UNC1999, a selective inhibitor of both enzymes, and UNC2400, an inactive analog compound useful for assessment of off-target effect. UNC1999 suppresses global H3K27 trimethylation/dimethylation (H3K27me3/2) and inhibits growth of mixed lineage leukemia (MLL)-rearranged leukemia cells. UNC1999-induced transcriptome alterations overlap those following knockdown of embryonic ectoderm development, a common cofactor of EZH2 and EZH1, demonstrating UNC1999s on-target inhibition. Mechanistically, UNC1999 preferentially affects distal regulatory elements such as enhancers, leading to derepression of polycomb targets including Cdkn2a. Gene derepression correlates with a decrease in H3K27me3 and concurrent gain in H3K27 acetylation. UNC2400 does not induce such effects. Oral administration of UNC1999 prolongs survival of a well-defined murine leukemia model bearing MLL-AF9. Collectively, our study provides the detailed profiling for a set of chemicals to manipulate EZH2 and EZH1 and establishes specific enzymatic inhibition of polycomb repressive complex 2 (PRC2)-EZH2 and PRC2-EZH1 by small-molecule compounds as a novel therapeutics for MLL-rearranged leukemia.

Exploiting an Allosteric Binding Site of PRMT3 Yields Potent and Selective Inhibitors

Protein arginine methyltransferases (PRMTs) play an important role in diverse biological processes. Among the nine known human PRMTs, PRMT3 has been implicated in ribosomal biosynthesis via asymmetric dimethylation of the 40S ribosomal protein S2 and in cancer via interaction with the DAL-1 tumor suppressor protein. However, few selective inhibitors of PRMTs have been discovered. We recently disclosed the first selective PRMT3 inhibitor, which occupies a novel allosteric binding site and is noncompetitive with both the peptide substrate and cofactor. Here we report comprehensive structure-activity relationship studies of this series, which resulted in the discovery of multiple PRMT3 inhibitors with submicromolar potencies. An X-ray crystal structure of compound 14u in complex with PRMT3 confirmed that this inhibitor occupied the same allosteric binding site as our initial lead compound. These studies provide the first experimental evidence that potent and selective inhibitors can be created by exploiting the allosteric binding site of PRMT3.

Crucial role for the VWF A1 domain in binding to type IV collagen

Von Willebrand factor (VWF) contains binding sites for platelets and for vascular collagens to facilitate clot formation at sites of injury. Although previous work has shown that VWF can bind type IV collagen (collagen 4), little characterization of this interaction has been performed. We examined the binding of VWF to collagen 4 in vitro and extended this characterization to a murine model of defective VWF-collagen 4 interactions. The interactions of VWF and collagen 4 were further studied using plasma samples from a large study of both healthy controls and subjects with different types of von Willebrand disease (VWD). Our results show that collagen 4 appears to bind VWF exclusively via the VWF A1 domain, and that specific sequence variations identified through VWF patient samples and through site-directed mutagenesis in the VWF A1 domain can decrease or abrogate this interaction. In addition, VWF-dependent platelet binding to collagen 4 under flow conditions requires an intact VWF A1 domain. We observed that decreased binding to collagen 4 was associated with select VWF A1 domain sequence variations in type 1 and type 2M VWD. This suggests an additional mechanism through which VWF variants may alter hemostasis.

The lipid kinase PIP5K1C regulates pain signaling and sensitization.

Numerous pain-producing (pronociceptive) receptors signal via phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, it is currently unknown which lipid kinases generate PIP2 in nociceptive dorsal root ganglia (DRG) neurons and if these kinases regulate pronociceptive receptor signaling. Here, we found that phosphatidylinositol 4-phosphate 5 kinase type 1C (PIP5K1C) is expressed at higher levels than any other PIP5K and, based on experiments with Pip5k1c(+/-) mice, generates at least half of all PIP2 in DRG neurons. Additionally, Pip5k1c haploinsufficiency reduces pronociceptive receptor signaling and TRPV1 sensitization in DRG neurons as well as thermal and mechanical hypersensitivity in mouse models of chronic pain. We identified a small molecule inhibitor of PIP5K1C (UNC3230) in a high-throughput screen. UNC3230 lowered PIP2 levels in DRG neurons and attenuated hypersensitivity when administered intrathecally or into the hindpaw. Our studies reveal that PIP5K1C regulates PIP2-dependent nociceptive signaling and suggest that PIP5K1C is a therapeutic target for chronic pain.

Discovery of a Selective, Substrate-Competitive Inhibitor of the Lysine Methyltransferase SETD8

The lysine methyltransferase SETD8 is the only known methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20). Monomethylation of H4K20 has been implicated in regulating diverse biological processes including the DNA damage response. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen (PCNA) and promotes carcinogenesis by deregulating PCNA expression. However, selective inhibitors of SETD8 are scarce. The only known selective inhibitor of SETD8 to date is nahuoic acid A, a marine natural product, which is competitive with the cofactor. Here, we report the discovery of the first substrate-competitive inhibitor of SETD8, UNC0379 (1). This small-molecule inhibitor is active in multiple biochemical assays. Its affinity to SETD8 was confirmed by ITC (isothermal titration calorimetry) and SPR (surface plasmon resonance) studies. Importantly, compound 1 is selective for SETD8 over 15 other methyltransferases. We also describe structure–activity relationships (SAR) of this series.

Genome-wide profiling of histone H3 lysine 27 and lysine 4 trimethylation in multiple myeloma reveals the importance of Polycomb gene targeting and highlights EZH2 as a potential therapeutic target.

Multiple myeloma (MM) is a malignancy of the antibody-producing plasma cells. MM is a highly heterogeneous disease, which has hampered the identification of a common underlying mechanism for disease establishment as well as the development of targeted therapy. Here we present the first genome-wide profiling of histone H3 lysine 27 and lysine 4 trimethylation in MM patient samples, defining a common set of active H3K4me3-enriched genes and silent genes marked by H3K27me3 (H3K27me3 alone or bivalent) unique to primary MM cells, when compared to normal bone marrow plasma cells. Using this epigenome profile, we found increased silencing of H3K27me3 targets in MM patients at advanced stages of the disease, and the expression pattern of H3K27me3-marked genes correlated with poor patient survival. We also demonstrated that pharmacological inhibition of EZH2 had anti-myeloma effects in both MM cell lines and CD138+ MM patient cells. In addition, EZH2 inhibition decreased the global H3K27 methylation and induced apoptosis. Taken together, these data suggest an important role for the Polycomb repressive complex 2 (PRC2) in MM, and highlights the PRC2 component EZH2 as a potential therapeutic target in MM.

Structure-activity relationship studies of SETD8 inhibitors

SETD8 (also known as SET8, PR-SET7, or KMT5A (lysine methyltransferase 5A)) is the only known lysine methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20). In addition to H4K20, SETD8 monomethylates non-histone substrates such as the tumor suppressor p53 and proliferating cell nuclear antigen (PCNA). Because of its role in regulating diverse biological processes, SETD8 has been pursued as a potential therapeutic target. We recently reported the first substrate-competitive SETD8 inhibitor, UNC0379 (1), which is selective for SETD8 over 15 other methyltransferases. We characterized this inhibitor in a battery of biochemical and biophysical assays. Here we describe our comprehensive structure-activity relationship (SAR) studies of this chemical series. In addition to 2- and 4-substituents, we extensively explored 6- and 7-substituents of the quinazoline scaffold. These SAR studies led to the discovery of several new compounds, which displayed similar potencies as compound 1, and interesting SAR trends.

EZH2 inhibition enhances the efficacy of an EGFR inhibitor in suppressing colon cancer cells

Metastatic colon cancer has a 5-year survival of less than 10% despite the use of aggressive chemotherapeutic regimens. As signaling from epidermal growth factor receptor (EGFR) is often enhanced and epigenetic regulation is often altered in colon cancer, it is desirable to enhance the efficacy of EGFR-directed therapy by co-targeting an epigenetic pathway. We showed that the histone methyltransferase EZH2, which catalyzes methylation of histone H3 lysine 27 (H3K27), was upregulated in colon cancers in The Cancer Genome Atlas (TCGA) database. Since co-inhibition of both EGFR and EZH2 has not been studied in colon cancer, we examined the effects of co-inhibition of EGFR and EZH2 on 2 colon cancer cell lines, HT-29 and HCT-15. Co-inhibition of EZH2 and EGFR with the small molecules UNC1999 and gefitinib, led to a significant decrease in cell number and increased apoptosis compared to inhibition of either pathway alone, and similar results were noted after EZH2 shRNA knockdown. Moreover, co-inhibition of EZH2 and EGFR also significantly induced autophagy, indicating that autophagy may play a role in the observed synergy. Together, these findings suggest that inhibition of both EZH2 and EGFR serves as an effective method to increase the efficacy of EGFR inhibitors in suppressing colon cancer cells.

Epigenetic siRNA and Chemical Screens Identify SETD8 Inhibition as a Therapeutic Strategy for p53 Activation in High-Risk Neuroblastoma

Given the paucity of druggable mutations in high-risk neuroblastoma (NB), we undertook chromatin-focused small interfering RNA and chemical screens to uncover epigenetic regulators critical for the differentiation block in high-risk NB. High-content Opera imaging identified 53 genes whose loss of expression led to a decrease in NB cell proliferation and 16 also induced differentiation. From these, the secondary chemical screen identified SETD8, the H4K20me1 methyltransferase, as a druggable NB target. Functional studies revealed that SETD8 ablation rescued the pro-apoptotic and cell-cycle arrest functions of p53 by decreasing p53K382me1, leading to activation of the p53 canonical pathway. In pre-clinical xenograft NB models, genetic or pharmacological (UNC0379) SETD8 inhibition conferred a significant survival advantage, providing evidence for SETD8 as a therapeutic target in NB.