CARM1 Preferentially Methylates H3R17 over H3R26 through a Random Kinetic Mechanism

CARM1 is a type I arginine methyltransferase involved in the regulation of transcription, pre-mRNA splicing, cell cycle progression, and the DNA damage response. CARM1 overexpression has been implicated in breast, prostate, and liver cancers and therefore is an attractive target for cancer therapy. To date, little about the kinetic properties of CARM1 is known. In this study, substrate specificity and the kinetic mechanism of the human enzyme were determined. Substrate specificity was examined by testing CARM1 activity with several histone H3-based peptides in a radiometric assay. Comparison of <i>k</i>_cat/<i>K</i>_M values reveals that methylation of H3R17 is preferred over that of H3R26. These effects are <i>K</i>_M-driven as <i>k</i>_cat values remain relatively constant for the peptides tested. Shortening the peptide at the C-terminus by five amino acid residues greatly reduced binding affinity, indicating distal residues may contribute to substrate binding. CARM1 appears to bind monomethylated peptides with an affinity similar to that of unmethylated peptides. Monitoring of the CARM1-dependent production of monomethylated and dimethylated peptides over time by self-assembled monolayer and matrix-assisted laser desorption ionization mass spectrometry revealed that methylation by CARM1 is distributive. Additionally, dead-end and product inhibition studies suggest CARM1 conforms to a random sequential kinetic mechanism. By defining the kinetic properties and mechanism of CARM1, these studies may aid in the development of small molecule CARM1 inhibitors

Synergistic Anti-Tumor Activity of EZH2 Inhibitors and Glucocorticoid Receptor Agonists in Models of Germinal Center Non-Hodgkin Lymphomas

<div><p>Patients with non-Hodgkin lymphoma (NHL) are treated today with a cocktail of drugs referred to as CHOP (Cyclophosphamide, Hydroxyldaunorubicin, Oncovin, and Prednisone). Subsets of patients with NHL of germinal center origin bear oncogenic mutations in the EZH2 histone methyltransferase. Clinical testing of the EZH2 inhibitor EPZ-6438 has recently begun in patients. We report here that combining EPZ-6438 with CHOP in preclinical cell culture and mouse models results in dramatic synergy for cell killing in <i>EZH2</i> mutant germinal center NHL cells. Surprisingly, we observe that much of this synergy is due to Prednisolone – a glucocorticoid receptor agonist (GRag) component of CHOP. Dramatic synergy was observed when EPZ-6438 is combined with Prednisolone alone, and a similar effect was observed with Dexamethasone, another GRag. Remarkably, the anti-proliferative effect of the EPZ-6438+GRag combination extends beyond EZH2 mutant-bearing cells to more generally impact germinal center NHL. These preclinical data reveal an unanticipated biological intersection between GR-mediated gene regulation and EZH2-mediated chromatin remodeling. The data also suggest the possibility of a significant and practical benefit of combining EZH2 inhibitors and GRag that warrants further investigation in a clinical setting.</p></div

Identification of a peptide inhibitor for the histone methyltransferase WHSC1

<div><p>WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.</p></div

Combination benefit with CHOP components and EPZ-6438 in <i>EZH2</i> mutant germinal center B-cell lymphoma cell lines.

<p>Combination index (CI) graphs, generated in Calcusyn, of EPZ-6438 with Mafosfamide, Doxorubicin, or Vincristine in the <i>EZH2</i> Y646F mutant cell lines WSU-DLCL2 (A–C) or SUDHL10 (D–F). The 95% confidence interval is displayed in each graph (representative of 2 biological replicates for each cell line). The fractional effect (Fa) plotted is the fraction of cell growth (inhibition) resulting from a compound treatment, calculated from the DMSO control. A) Additivity was induced for the EPZ-6438/Mafosfamide combination at a 1∶20 constant ratio (doses were 16–125 nM for EPZ-6438 and 313–2500 nM for Mafosfamide). B) Synergy was induced for the EPZ-6438/Doxorubicin combination at a 50∶1 constant ratio (doses were 16–500 nM for EPZ-6438 and 0.3–10 nM for Doxorubicin). C) Additivity was induced for the EPZ-6438/Vincristine combination at a 400∶1 constant ratio (doses were 16–1000 nM for EPZ-6438 and 0.39–2.5 nM for Vincristine). D) Additivity was induced for the EPZ-6438/Mafosfamide combination at a 4∶25 constant ratio (doses were 12.5–200 nM for EPZ-6438 and 78–1250 nM for Mafosfamide). E) Additivity was induced for the EPZ-6438/Doxorubicin combination at a 10∶3 constant ratio (doses were 3–50 nM for EPZ-6438 and 0.94–15 nM for Doxorubicin). F) Additivity was shown for the EPZ-6438/Vincristine combination at an 800∶1 constant ratio (doses were 12.5–200 nM for EPZ-6438 and 15.6–250 pM for Vincristine).</p

EPZ-6438/CHOP combinations show enhanced anti-tumor activity compared to single agents in several <i>EZH2</i> mutant lymphoma xenograft models.

<p>A) WSU-DLCL2 (<i>EZH2</i> Y646F) xenograft-bearing mice were treated for 28 days as indicated. Mean tumor volumes ± SEM (n = 12) are plotted. Treatment with EPZ-6438 at 225 mg/kg BID plus CHOP induced the highest tumor growth inhibition (93%). B) SUDHL6 (<i>EZH2</i> Y646N) xenograft-bearing mice were treated for 28 days as indicated. Mean tumor volumes ± SEM (n = 12) are plotted in the top panel. * <i>p</i><0.05, *** <i>p</i><0.001 vs. vehicle, repeated measures ANOVA, Dunnett's post test. Kaplan-Meyer survival curves (bottom panel) out to 60 days demonstrate significant tumor growth delay in animals treated with EPZ-6438+CHOP (** <i>p</i><0.01). C) SUDHL10 (<i>EZH2</i> Y646F) xenograft-bearing mice were treated with EPZ-6438, COP (chemotherapy without the Doxorubicin component), or their combination for 28 days. Mean tumor volumes ± SEM (n = 8) are plotted in top panel. Percent survival out to 60 days is plotted in the bottom panel (note: 500 mg/kg and 250 mg/kg+COP survival curves overlap). D) SUDHL10 (<i>EZH2</i> Y646F) xenograft-bearing mice were treated for 28 days as indicated (Pred-1 = Prednisone at 0.15 mg/kg BID×5 on days 1–5 and 22–26; Pred-2 = Prednisone at 0.15 mg/kg BID×28). The scatter blot shows the tumor volumes on day 28, and the inset shows the mean tumor volumes ±SEM (n = 10) followed over 28 days (also presented in figure S6B). * <i>p</i><0.05, ** <i>p</i><0.01; two-tailed <i>t</i> test. All groups administered EPZ-6438 show statistically significant smaller tumor volumes on day 28 (<i>p</i><0.01 at least, vs. vehicle or Prednisone single agent at both schedules; two-tailed <i>t</i> test). CHOP: Cyclophosphamide, Hydroxyldaunorubicin (Doxorubine), Oncovin (Vincristine), Prednisone; COP: Cyclophosphamide, Oncovin (Vincristine), Prednisone; BID: two times a day every 12 hours; QD: once a day; TID: three times a day every 8 hours.</p

Effects of EZH2 inhibition in synovial sarcoma cell line xenograft models.

<p>(A) Tumor growth inhibition in Fuji xenograft induced by twice daily (BID) administration of tazemetostat for 35 days at the indicated dosage, with or without doxorubicin (10 mg/kg) treatment on days 1 and 22. An alternative EZH2 inhibitor, EPZ011989, was also included at 500 mg/kg BID. Treatment was stopped after 35 days and tumor regrowth was monitored. Data shown as mean values ±SEM; n = 7. Arrowheads indicate the administration of doxorubicin, lines indicate the dosing period for tazemetostat or EPZ011989. (B) EZH2 target inhibition in Fuji xenograft samples from mice treated with tazemetostat for seven days at the indicated doses in relationship to systemic C_trough levels of tazemetostat measured 5 minutes before the last dose on day 7. H3K27Me3 and H3 levels were measured in histones preparations by ELISA and data represents the ratio of H3K27Me3 to total H3. The horizontal line represents the mean. (C) Assessment of tumor growth in HS-SY-II xenograft model. Mice were treated with tazemetostat for 28 days at the indicated dosage, with or without doxorubicin (10 mg/kg) treatment on days 1 and 22. Data are shown as mean values ±SEM; n = 6 and representative of two independent experiments. Arrowheads indicate the administration of doxorubicin, horizontal arrows indicate the dosing period for tazemetostat. (D) EZH2 target inhibition in HS-SY-II xenograft samples from mice treated with tazemetostat for seven days at the indicated doses in relationship to systemic C_trough levels of tazemetostat measured 5 minutes before the last dose on day 7. H3K27Me3 and H3 levels were measured in histones preparations by ELISA and data represents the ratio of H3K27Me3 to total H3. The horizontal line represents the mean. * <i>P</i><0.05 vs. vehicle, # <i>P</i><0.05 vs. both 250 mg/kg tazemetostat and doxorubicin; one-way analysis of variance followed by Tukey’s multiple comparison test after logarithmic transformation.</p

Norleucine-containing peptides can inhibit WHSC1 and WHSC1L1 activity in vitro.

<p>Representative peptide inhibitor biochemical dose-response curves for (A) WHSC1 941–1240 and (B) WHSC1L1 1054–1285. Error bars represent the standard deviation of three independent replicates. Resulting IC₅₀ values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197082#pone.0197082.t002" target="_blank">Table 2</a>.</p

Biochemical and biophysical peptide inhibitor potency values for WHSC1 941–1240 and WHSC1L1 1054–1285^a.

<p>Biochemical and biophysical peptide inhibitor potency values for WHSC1 941–1240 and WHSC1L1 1054–1285<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197082#t002fn001" target="_blank">^a</a>.</p

Representative sensorgram for PTD2 binding to Avi-tagged WHSC1 941–1240 from single-cycle kinetic SPR measurements.

<p>WHSC1 was immobilized on a streptavidin-coated chip and peptide inhibitor was co-injected with SAM utilizing a 3-fold, 5-point dilution series ending at a 20 μM top concentration. Data reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197082#pone.0197082.t002" target="_blank">Table 2</a> is presented as the standard deviation of three independent experiments.</p

The Importance of Being Me: Magic Methyls, Methyltransferase Inhibitors, and the Discovery of Tazemetostat

Posttranslational methylation of histones plays a critical role in gene regulation. Misregulation of histone methylation can lead to oncogenic transformation. Enhancer of Zeste homologue 2 (EZH2) methylates histone 3 at lysine 27 (H3K27) and abnormal methylation of this site is found in many cancers. Tazemetostat, an EHZ2 inhibitor in clinical development, has shown activity in both preclinical models of cancer as well as in patients with lymphoma or INI1-deficient solid tumors. Herein we report the structure–activity relationships from identification of an initial hit in a high-throughput screen through selection of tazemetostat for clinical development. The importance of several methyl groups to the potency of the inhibitors is highlighted as well as the importance of balancing pharmacokinetic properties with potency