Edward J. Olhava

An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer

The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin–proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.

A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells

EZH2 catalyzes trimethylation of histone H3 lysine 27 (H3K27). Point mutations of EZH2 at Tyr641 and Ala677 occur in subpopulations of non-Hodgkins lymphoma, where they drive H3K27 hypertrimethylation. Here we report the discovery of EPZ005687, a potent inhibitor of EZH2 (K(i) of 24 nM). EPZ005687 has greater than 500-fold selectivity against 15 other protein methyltransferases and has 50-fold selectivity against the closely related enzyme EZH1. The compound reduces H3K27 methylation in various lymphoma cells; this translates into apoptotic cell killing in heterozygous Tyr641 or Ala677 mutant cells, with minimal effects on the proliferation of wild-type cells. These data suggest that genetic alteration of EZH2 (for example, mutations at Tyr641 or Ala677) results in a critical dependency on enzymatic activity for proliferation (that is, the equivalent of oncogene addiction), thus portending the clinical use of EZH2 inhibitors for cancers in which EZH2 is genetically altered.

Potent inhibition of DOT1L as treatment of MLL-fusion leukemia

Rearrangements of the MLL gene define a genetically distinct subset of acute leukemias with poor prognosis. Current treatment options are of limited effectiveness; thus, there is a pressing need for new therapies for this disease. Genetic and small molecule inhibitor studies have demonstrated that the histone methyltransferase DOT1L is required for the development and maintenance of MLL-rearranged leukemia in model systems. Here we describe the characterization of EPZ-5676, a potent and selective aminonucleoside inhibitor of DOT1L histone methyltransferase activity. The compound has an inhibition constant value of 80 pM, and demonstrates 37 000-fold selectivity over all other methyltransferases tested. In cellular studies, EPZ-5676 inhibited H3K79 methylation and MLL-fusion target gene expression and demonstrated potent cell killing that was selective for acute leukemia lines bearing MLL translocations. Continuous IV infusion of EPZ-5676 in a rat xenograft model of MLL-rearranged leukemia caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity. EPZ-5676 is therefore a potential treatment of MLL-rearranged leukemia and is under clinical investigation.

Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-subunit

The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome used on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin–proteasome system in cells. We show that these compounds are entirely selective for the β5 (chymotrypsin-like) site over the β1 (caspase-like) and β2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S β5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin–luciferase reporter, activation of NFκB (nuclear factor κB) in response to TNF-α (tumour necrosis factor-α) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the β5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the β5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome based [corrected] on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the beta5 (chymotrypsin-like) site over the beta1 (caspase-like) and beta2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S beta5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin-luciferase reporter, activation of NFkappaB (nuclear factor kappaB) in response to TNF-alpha (tumour necrosis factor-alpha) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the beta5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the beta5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.

Targeting epigenetic enzymes for drug discovery

Epigenetic control of gene transcription is the result of enzyme-mediated covalent modifications of promoter-region DNA sites and of histone proteins around which chromosomal DNA is wound. Many of the enzymes that mediate these epigenetic reactions are dysregulated in human diseases. Small molecule inhibitors against two classes of these enzymes have been approved for use in patients: DNA methyltransferase (DNMT) inhibitors and histone deacetylase inhibitors. Other classes of epigenetic enzymes have been demonstrated to have strong disease association and are currently being targeted for small molecule inhibition. In this article we review these enzymes and chemical biology approaches aimed at discovering small molecule inhibitors against them for therapeutic use.

DOT1L Inhibitor EPZ-5676 Displays Synergistic Antiproliferative Activity in Combination with Standard of Care Drugs and Hypomethylating Agents in MLL-Rearranged Leukemia Cells

EPZ-5676 [(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol], a small-molecule inhibitor of the protein methyltransferase DOT1L, is currently under clinical investigation for acute leukemias bearing MLL-rearrangements (MLL-r). In this study, we evaluated EPZ-5676 in combination with standard of care (SOC) agents for acute leukemias as well as other chromatin-modifying drugs in cellular assays with three human acute leukemia cell lines: MOLM-13 (MLL-AF9), MV4-11 (MLL-AF4), and SKM-1 (non–MLL-r). Studies were performed to evaluate the antiproliferative effects of EPZ-5676 combinations in a cotreatment model in which the second agent was added simultaneously with EPZ-5676 at the beginning of the assay, or in a pretreatment model in which cells were incubated for several days in the presence of EPZ-5676 prior to the addition of the second agent. EPZ-5676 was found to act synergistically with the acute myeloid leukemia (AML) SOC agents cytarabine or daunorubicin in MOLM-13 and MV4-11 MLL-r cell lines. EPZ-5676 is selective for MLL-r cell lines as demonstrated by its lack of effect either alone or in combination in the nonrearranged SKM-1 cell line. In MLL-r cells, the combination benefit was observed even when EPZ-5676 was washed out prior to the addition of the chemotherapeutic agents, suggesting that EPZ-5676 sets up a durable, altered chromatin state that enhances the chemotherapeutic effects. Our evaluation of EPZ-5676 in conjunction with other chromatin-modifying drugs also revealed a consistent combination benefit, including synergy with DNA hypomethylating agents. These results indicate that EPZ-5676 is highly efficacious as a single agent and synergistically acts with other chemotherapeutics, including AML SOC drugs and DNA hypomethylating agents in MLL-r cells.

Nonclinical pharmacokinetics and metabolism of EPZ-5676, a novel DOT1L histone methyltransferase inhibitor.

(2R,3R,4S,5R)‐2‐(6‐Amino‐9H‐purin‐9‐yl)‐5‐((((1r,3S)‐3‐(2‐(5‐(tert‐butyl)‐1H‐benzo[d]imidazol‐2‐yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran‐3,4‐diol (EPZ‐5676) is a novel DOT1L histone methyltransferase inhibitor currently in clinical development for the treatment of MLL‐rearranged leukemias. This report describes the preclinical pharmacokinetics and metabolism of EPZ‐5676, an aminonucleoside analog with exquisite target potency and selectivity that has shown robust and durable tumor growth inhibition in preclinical models. The in vivo pharmacokinetics in mouse, rat and dog were characterized following i.v. and p.o. administration; EPZ‐5676 had moderate to high clearance, low oral bioavailability with a steady‐state volume of distribution 2–3 fold higher than total body water. EPZ‐5676 showed biexponential kinetics following i.v. administration, giving rise to a terminal elimination half‐life (t1/2) of 1.1, 3.7 and 13.6 h in mouse, rat and dog, respectively. The corresponding in vitro ADME parameters were also studied and utilized for in vitro–in vivo extrapolation purposes. There was good agreement between the microsomal clearance and the in vivo clearance implicating hepatic oxidative metabolism as the predominant elimination route in preclinical species. Furthermore, low renal clearance was observed in mouse, approximating to fu‐corrected glomerular filtration rate (GFR) and thus passive glomerular filtration. The metabolic pathways across species were studied in liver microsomes in which EPZ‐5676 was metabolized to three monohydroxylated metabolites (M1, M3 and M5), one N‐dealkylated product (M4) as well as an N‐oxide (M6). Copyright

Mechanisms of Pinometostat (EPZ-5676) Treatment–Emergent Resistance in MLL-Rearranged Leukemia

DOT1L is a protein methyltransferase involved in the development and maintenance of MLL-rearranged (MLL-r) leukemia through its ectopic methylation of histones associated with well-characterized leukemic genes. Pinometostat (EPZ-5676), a selective inhibitor of DOT1L, is in clinical development in relapsed/refractory acute leukemia patients harboring rearrangements of the MLL gene. The observation of responses and subsequent relapses in the adult trial treating MLL-r patients motivated preclinical investigations into potential mechanisms of pinometostat treatment-emergent resistance (TER) in cell lines confirmed to have MLL-r. TER was achieved in five MLL-r cell lines, KOPN-8, MOLM-13, MV4-11, NOMO-1, and SEM. Two of the cell lines, KOPN-8 and NOMO-1, were thoroughly characterized to understand the mechanisms involved in pinometostat resistance. Unlike many other targeted therapies, resistance does not appear to be achieved through drug-induced selection of mutations of the target itself. Instead, we identified both drug efflux transporter dependent and independent mechanisms of resistance to pinometostat. In KOPN-8 TER cells, increased expression of the drug efflux transporter ABCB1 (P-glycoprotein, MDR1) was the primary mechanism of drug resistance. In contrast, resistance in NOMO-1 cells occurs through a mechanism other than upregulation of a specific efflux pump. RNA-seq analysis performed on both parental and resistant KOPN-8 and NOMO-1 cell lines supported two unique candidate pathway mechanisms that may explain the pinometostat resistance observed in these cell lines. These results are the first demonstration of TER models of the DOT1L inhibitor pinometostat and may provide useful tools for investigating clinical resistance. Mol Cancer Ther; 16(8); 1669–79. ©2017 AACR.

Abstract 2701: Characterization of acquired EPZ-5676 resistance in cell line models of MLL rearranged leukemia

DOT1L inhibitor EPZ-5676 is currently under Phase 1 clinical trial investigation in relapsed/refractory patients with acute leukemia, including those with an MLL-rearrangement (MLL-r). Early clinical results, including complete remissions, support ongoing clinical development and preclinical investigation into mechanisms precipitating EPZ-5676 treatment induced resistance. MLL-r cell lines KOPN-8 (MLL-ENL) and NOMO-1 (MLL-AF9) were exposed to an EPZ-5676 concentration above the pre-determined 14 day proliferation assay IC90. Initial treatment of the cell lines led to the expected inhibition of H3K79 dimethylation (H3K79me2) and MLL-r target genes HOXA9 and MEIS1 as outlined in previous work (Daigle et al, Cancer Cell 2011). Resistance to EPZ-5676 in both cell lines emerged following three weeks of continued treatment with EPZ-5676 and was defined by increased growth rates in the presence of inhibitor. Mechanisms of resistance for both cell lines were investigated using RNASeq and ChIPSeq on parental and resistant cell line pools. Our analysis identified common characteristics between the resistant cell lines, but mechanisms by which they became resistant differed. Global H3K79me2 inhibition was maintained in both refractory cell lines, yet ChIP-seq analysis of resistant pools identified specific loci with H3K79me2 recovery in KOPN-8 cells. In resistant KOPN-8 cells recovery of H3K79me2 was concentrated at the HOXA locus and other MLL-r target genes (e.g. MEIS1 and RUNX2), with the remainder of actively transcribed genes maintaining H3K79me2 inhibition at levels observed in parental cells. In contrast, resistant NOMO-1 cells did not recover H3K79me2 at any actively transcribed genes, including those of the MLL-r signature. Only resistant KOPN-8 cells regained expression of the MLL-r target genes HOXA9 and MEIS1. Of note both resistant NOMO-1 and KOPN-8 cell lines had 8 and 40 fold upregulation of the ABCB1 (MDR1, P-gp) tranporter respectively when compared to a matched control cell line. To explore the role of drug efflux transporter ABCB1 on resistance, we treated cells with Valspodar, a known inhibitor of ABCB1. Following treatment with 1 μM Valspodar, KOPN-8 cells showed decreased cell growth similar to the naive control cell line. Supporting the presence of an alternative resistance mechanism in NOMO-1 cells in addition to MDR1 upregulation, NOMO-1 cells remained resistant upon Valspodar treatment. Detailed gene expression and pathway analysis will be presented supporting mechanisms of treatment emergent resistance to EPZ-5676. In summary, we have identified two mechanisms of EPZ-5676 resistance in MLL-r cell lines, one mechanism dependent on and the other independent of ABCB1. Further refinement of these mechanisms will aid in providing hypotheses for testing mechanisms of EPZ-5676 treatment emergent resistance in patients and may support designing future rational clinical combinations. Citation Format: Scott R. Daigle, Carly T. Campbell, Nigel J. Waters, Edward J. Olhava, Robert A. Copeland, Stephen J. Blakemore, Roy M. Pollock, Jesse J. Smith. Characterization of acquired EPZ-5676 resistance in cell line models of MLL rearranged leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2701. doi:10.1158/1538-7445.AM2015-2701

Abstract 5383: DOT1L inhibitor EPZ-5676 synergizes with cytarabine and azacitidine in preclinical models of MLL-rearranged leukemia

EPZ-5676 is a small molecule inhibitor of the histone methyltransferase DOT1L currently in clinical development and represents a first in class novel therapeutic agent for the treatment of MLL-rearranged (MLL-r) leukemia. In preclinical studies, EPZ-5676 selectively inhibited intracellular histone H3K79 methylation, downstream target gene expression and demonstrated complete tumor regression in a MLL-r leukemia xenograft model. We previously reported synergistic and durable anti-proliferative activity when EPZ-5676 was combined with current AML standard of care drugs, cytarabine and daunorubicin in MLL-r leukemia models MOLM-13 (MLL-AF9) and MV4-11 (MLL-AF4). Combination benefit was also observed when MLL-r cells were treated with cytarabine, prior to co-treatment with EPZ-5676. Additionally, both cytarabine and the DNA methyltransferase inhibitor azacitidine, displayed synergistic anti-leukemic activity in MLL-r rearranged cells in a 7 day co-treatment model (7 days of continuous treatment with EPZ-5676 and second agent; see Klaus et al, JPET, 2014). In this report we discuss results of investigating additional treatment schedules using EPZ-5676 in combination with azacitidine in MLL-r cells. Cells were pretreated with azacitidine at nanomolar concentrations known to reverse promoter DNA-hypermethylation and alter the chromatin state (Tsai et al., Cancer Cell, 2012). We found treating MV4-11 and MOLM-13 cells once daily for three consecutive days followed by sequential treatment with EPZ-5676 elicited a synergistic anti-proliferative effect using the Chou-Talalay method (Chou, Pharmacol Rev., 2006). Results of studies to investigate the mechanism of this synergistic cell killing, including evaluation of differentiation markers and Annexin V staining will be reported. To determine if combinations of EPZ-5676 with cytarabine or azacitidine were tolerable and efficacious in vivo, nude rats implanted subcutaneously with MV4-11 tumors were treated using a range of doses and schedules. Azacitidine and cytarabine were delivered by intraperitoneal injection once daily for 14 days at their respective maximum tolerated doses of 2 and 200 mg/kg. Dosing at the established MTD, these agents inhibited the subcutaneous MV4-11 tumor growth by 50% compared to vehicle controls. Efficacy results from the EPZ-5676 combination studies with cytarabine or azacitidine will be presented. In summary, our results indicate that EPZ-5676 in combination with cytarabine or azacitidine revealed a synergistic effect, regardless of the treatment schedule used in preclinical models of MLL-r leukemia. Tolerable in vivo rat combination doses for EPZ-5676 with both cytarabine and azacitidine have been determined in support of potential future assessment of these combinations in MLL-r leukemia patients. Citation Format: Christine R. Klaus, Scott R. Daigle, Vivek Chopra, Jeffrey A. Keats, Carly T. Campbell, Dorothy Iwanowicz, Edward J. Olhava, Margaret P. Scott, Roy M. Pollock, Robert A. Copeland, Jesse J. Smith, Jorge DiMartino, Stephen J. Blakemore, Alejandra Raimondi. DOT1L inhibitor EPZ-5676 synergizes with cytarabine and azacitidine in preclinical models of MLL-rearranged leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5383. doi:10.1158/1538-7445.AM2015-5383