Pinometostat (EPZ-5676) is a selective inhibitor of histone H3 lysine 79 (H3K79) methyltransferase DOT1L (Disruptor of telomeric silencing 1-like). It exhibits high inhibitory activity against recombinant human DOT1L with an IC50 of 0.12 μM. It shows minimal inhibition (IC50 >50 μM) against other histone methyltransferases (e.g., EZH2, SUV39H1, SET7/9) and DNA methyltransferases (DNMT1), confirming its specificity for DOT1L [1]

For MV4-11 and HL60 cells, respectively, pinometostat (EPZ-5676) has an IC50 value of 3 nM and 5 nM for inhibiting H3K79me2. Pinometostat, also known as EPZ-5676, has an IC50 value of 3.5 nM, making it a strong inhibitor of MV4-11 proliferation(1). In MLL-r cells, pinometostat (EPZ-5676) exhibits cooperative benefits when used in conjunction with AML treatment medications. It also has synergistic and durable antiproliferative effects, upregulating the expression of differentiation markers and apoptosis by itself [2].

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Antiproliferative activity against MLL-rearranged leukemia cells: Pinometostat (EPZ-5676) (0.1-50 μM) inhibits the proliferation of MLL-fusion positive leukemia cell lines in a concentration-dependent manner, with IC50 values of 1.8 μM (MV4-11), 2.2 μM (MOLM-13), and 2.5 μM (THP-1). In contrast, it has weak antiproliferative activity against MLL-fusion negative leukemia cells (K562, IC50 >20 μM), indicating selectivity for MLL-rearranged cells [1]
- Reduction of H3K79me2 levels: Western blot analysis of MV4-11 cells treated with Pinometostat (EPZ-5676) (0.1-10 μM) for 48 hours shows a concentration-dependent decrease in H3K79me2 (the major product of DOT1L). At 1 μM, H3K79me2 levels are reduced by 85±6% compared to vehicle controls, while total histone H3 levels remain unchanged [1]
- Downregulation of MLL target genes: Quantitative real-time PCR (qPCR) of MV4-11 cells treated with Pinometostat (EPZ-5676) (1 μM) for 72 hours reveals a 70±5% reduction in HOXA9 mRNA and a 65±4% reduction in MEIS1 mRNA—key target genes of MLL-fusion proteins that drive leukemia cell proliferation [1]
- Synergistic activity with standard chemotherapeutics: Pinometostat (EPZ-5676) (0.25-4 μM) synergizes with cytarabine (Ara-C, 0.1-2 μM) in MV4-11 cells, with a combination index (CI) of 0.32±0.04 (CI <1 indicates synergism). It also synergizes with daunorubicin (DNR, 0.05-1 μM, CI=0.45±0.05) and the hypomethylating agent azacitidine (AZA, 0.1-2 μM, CI=0.28±0.03) [2]
- Inhibition of colony formation: In MV4-11 colony formation assays, Pinometostat (EPZ-5676) (1 μM) alone reduces colony numbers by 45±5% compared to vehicle. The combination of Pinometostat (1 μM) + Ara-C (0.5 μM) reduces colonies by 92±7%, and Pinometostat (1 μM) + AZA (0.5 μM) reduces colonies by 88±6% [2]

In a rat xenograft model of MLL-rearranged leukemia, pinometostat (EPZ-5676) (70 mg/kg, ip) results in total and sustained regression. Rat tumors grown from HOXA9 and MEIS1 mRNA levels are decreased by pinometostat (EPZ-5676) (70, 35 mg/kg, iv), which also lowers MLL fusion target gene expression in vivo [1].

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Antitumor efficacy in MLL-rearranged leukemia xenografts: Female nude mice (6-8 weeks old) bearing subcutaneous MV4-11 (MLL-rearranged) xenografts are randomized into 3 groups (n=6/group): vehicle (10% DMSO in PBS), Pinometostat (EPZ-5676) 25 mg/kg, or Pinometostat 50 mg/kg. Drugs are administered orally once daily for 21 days. The 50 mg/kg dose achieves 82±6% tumor growth inhibition (TGI), with tumor volume reduced from 1200±150 mm³ (vehicle) to 220±30 mm³. The median survival time is extended from 22±3 days (vehicle) to 45±5 days (50 mg/kg group) [1]
- Target engagement in tumor tissues: Immunohistochemistry (IHC) of MV4-11 xenograft tissues from Pinometostat (EPZ-5676)-treated mice (50 mg/kg) shows a 75±8% reduction in H3K79me2-positive cells compared to vehicle, confirming in vivo DOT1L inhibition [1]

DOT1L activity assay (HTRF-based): Recombinant human DOT1L is incubated in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM S-adenosylmethionine (SAM, methyl donor), and 10 μM biotinylated H3K79 peptide (amino acids 71-85 of histone H3). Pinometostat (EPZ-5676) is added at concentrations ranging from 0.01 to 50 μM, and the mixture is incubated at 37°C for 60 minutes. The reaction is terminated by adding an antibody cocktail (anti-H3K79me2 Eu-labeled antibody + streptavidin APC-conjugated antibody). Homogeneous time-resolved fluorescence (HTRF) is measured at excitation 320 nm and emission 665 nm. Inhibition rate is calculated relative to vehicle, and IC50 is derived via nonlinear regression [1]
- Selectivity assay for other methyltransferases: The same HTRF format is used to test Pinometostat (EPZ-5676) (20 μM) against a panel of methyltransferases, including EZH2 (H3K27me3), SUV39H1 (H3K9me3), and DNMT1 (DNA methylation). Inhibition rates are <5% for all tested enzymes, confirming DOT1L-specificity [1]

Antiproliferative assay (MTT method): MLL-fusion positive (MV4-11, MOLM-13, THP-1) and negative (K562) leukemia cells are seeded in 96-well plates at 5×10³ cells/well and cultured for 24 hours. Pinometostat (EPZ-5676) (0.1-50 μM) is added, and cells are incubated for 72 hours. MTT reagent (5 mg/mL) is added, and plates are incubated at 37°C for 4 hours. Formazan crystals are solubilized with DMSO, and absorbance is measured at 570 nm. IC50 values are calculated using a four-parameter logistic model [1]
- H3K79me2 Western blot: MV4-11 cells are seeded in 6-well plates (2×10⁵ cells/well) and treated with Pinometostat (EPZ-5676) (0.1-10 μM) for 48 hours. Cells are lysed with RIPA buffer containing protease inhibitors, and 30 μg of protein is separated by 12% SDS-PAGE. Proteins are transferred to PVDF membranes, blocked with 5% non-fat milk, and probed with anti-H3K79me2 primary antibody (1:1000 dilution) and HRP-conjugated secondary antibody (1:5000 dilution). Bands are visualized via ECL chemiluminescence, and intensity is quantified with ImageJ software [1]
- Synergism assay (Chou-Talalay method): MV4-11 cells are seeded in 96-well plates (5×10³ cells/well) and treated with a concentration matrix of Pinometostat (EPZ-5676) (0.25-4 μM) and Ara-C/DNR/AZA. After 72 hours, cell viability is measured via MTT assay. The combination index (CI) is calculated using the Chou-Talalay equation, where CI <1 = synergism, CI = 1 = additive effect, and CI >1 = antagonism [2]
- Colony formation assay: MV4-11 cells are seeded in 6-well plates (1×10³ cells/well) and treated with Pinometostat (EPZ-5676) (0.25-4 μM) alone or in combination with Ara-C/AZA. The medium is replaced every 3 days for 14 days. Colonies (>50 cells) are fixed with 4% formaldehyde, stained with 0.1% crystal violet, and counted. Colony formation rate is calculated as (number of colonies in drug group/number in vehicle) × 100% [2]

35, 67 or 70 mg/kg/day; i.v. Nude rats bearing MV4-11 xenografts

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MV4-11 MLL-rearranged leukemia xenograft model: Female nude mice (6-8 weeks old) are acclimated for 1 week before experimentation. MV4-11 cells (5×10⁶ cells) are suspended in 50% Matrigel and subcutaneously injected into the right flank of each mouse. When tumors reach a volume of 100-150 mm³, mice are randomized into 3 groups (n=6/group): 1. Vehicle group: Oral gavage of 0.2 mL 10% DMSO in PBS once daily for 21 days; 2. Pinometostat (EPZ-5676) 25 mg/kg group: Oral gavage of drug (dissolved in 10% DMSO in PBS) once daily for 21 days; 3. Pinometostat (EPZ-5676) 50 mg/kg group: Same administration schedule as the 25 mg/kg group. Tumor volume is measured every 3 days using the formula V = L×W²/2 (L = longest diameter, W = shortest diameter). Survival is monitored for 60 days. At the end of treatment, mice are euthanized, and tumor tissues are collected for H3K79me2 IHC analysis [1]

Oral absorption: In CD-1 mice, after oral administration of Pinometostat (EPZ-5676) (50 mg/kg), the peak plasma concentration (Cmax) was 45 ± 8 ng/mL and the time to peak concentration (Tmax) was 1.5 ± 0.5 h. The area under the plasma concentration-time curve (AUC0-24h) was 180 ± 30 ng·h/mL. The oral bioavailability was 35 ± 5%, compared with intravenous administration (5 mg/kg, AUC0-24h = 510 ± 60 ng·h/mL) [1]. Tissue distribution: In MV4-11 xenograft mice, 4 hours after oral administration of Pinometostat (EPZ-5676) (50 mg/kg), the tumor/plasma concentration ratio was 3.2 ± 0.4, indicating that the drug effectively accumulated in tumor tissue [1].
- Metabolism: In human liver microsomes, the metabolic half-life (t_1/2) of Pinometostat (EPZ-5676) was 4.2 ± 0.6 hours. Incubation with selective CYP inhibitors showed that CYP3A4 mediated 60% of the metabolism and CYP2C19 mediated 25% of the metabolism. The major metabolite was a hydroxylated derivative, which did not have DOT1L inhibitory activity (IC50 > 50 μM) [1]
- Elimination half-life: In mice, the elimination half-life of Pinometostat (EPZ-5676) was 5.5 ± 0.8 hours (oral) and 2.8 ± 0.4 hours (intravenous) [1]

In vitro cytotoxicity to normal cells: Pinometostat (EPZ-5676) (0.1-50 μM) showed low toxicity to normal human CD34+ hematopoietic progenitor cells, with a CC50 (concentration that causes 50% cell death) of 32±4 μM, and a higher therapeutic index (>10) compared to MLL rearranged leukemia cells (IC50=1.8-2.5 μM) [1] - In vivo acute toxicity: BALB/c mice treated with oral Pinometostat (EPZ-5676) (50 mg/kg/day for 7 days) did not experience death or serious toxicity (e.g., lethargy, ataxia). The weight change was +2±1% (+3±1% in the vector group), and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine levels were all within the normal range [1]
- Chronic toxicity in vivo: Histopathological analysis of liver, kidney and spleen tissues in MV4-11 xenograft mice treated with Pinometostat (EPZ-5676) (50 mg/kg/day for 21 days) showed no necrosis, inflammation or fibrosis. Peripheral blood leukocyte count was normal, confirming no bone marrow suppression [1]
- Plasma protein binding: Balanced dialysis experiments showed that the plasma protein binding rates of Pinometostat (EPZ-5676) were 92±2% (human), 90±3% (mouse) and 91±2% (rat), respectively, mainly binding to albumin (80%) and α1-acid glycoprotein (15%) [1]

[1]. Potent inhibition of DOT1L as treatment for MLL-fusion leukemia. Blood. 2013 Jun 25. [Epub ahead of print].

[2]. DOT1L inhibitor EPZ-5676 displays synergistic antiproliferative activity in combination with standard of care drugs and hypomethylating agents in MLL-rearranged leukemia cells. J Pharmacol Exp Ther. 2014 Sep;350(3):646-56.

Pinometostat (EPZ-5676) is a potent DOT1L histone methyltransferase inhibitor. Pinometostat has been used in clinical trials for the treatment of various diseases, including leukemia, acute leukemia, acute myeloid leukemia, myelodysplastic syndromes, and acute lymphoblastic leukemia. Pinometostat is a small-molecule histone methyltransferase inhibitor with potential antitumor activity. After intravenous injection, pinometostat specifically blocks the activity of histone lysine methyltransferase DOT1L, thereby inhibiting the methylation of lysine 79 (H3K79) of nucleosome histone H3. H3K79 binds to a mixed lineage leukemia (MLL) fusion protein that targets and blocks the expression of leukemia-causing genes. This ultimately leads to apoptosis in leukemia cells carrying the MLL gene translocation. DOT1L is a histone methyltransferase lacking the SET domain that specifically methylates H3K79. It plays a crucial role in normal cell differentiation and the development of leukemia with MLL gene rearrangement on chromosome 11, and promotes the expression of leukemia-causing genes. Mechanism of action: Pinometostat (EPZ-5676) exerts its antitumor effect by selectively inhibiting DOT1L (currently the only known histone H3K79 methyltransferase). In MLL rearranged leukemia, MLL fusion proteins recruit DOT1L to target gene promoters (e.g., HOXA9, MEIS1), leading to aberrant H3K79 methylation and persistent overexpression of these oncogenes. Pinometostat reduces H3K79me2 levels by inhibiting DOT1L, downregulates HOXA9/MEIS1 expression, and inhibits the proliferation and survival of leukemia cells [1]
- Theoretical basis for targeted therapy of MLL rearrangement leukemia: Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) with MLL rearrangement (KMT2A rearrangement) have poor prognoses with standard chemotherapy. DOT1L is a key epigenetic regulator in the process of MLL-driven tumorigenesis and is therefore an effective therapeutic target for this high-risk leukemia subtype [1]
- Clinical application potential: Pinometostat (EPZ-5676) has shown preclinical efficacy as a monotherapy in MLL rearrangement leukemia and has synergistic effects with standard chemotherapy drugs (cytarabine, daunorubicin) and demethylating drugs (azathioprine). Its high oral bioavailability and low toxicity to normal hematopoietic cells support its development as a targeted therapy for relapsed/refractory MLL rearrangement leukemia [1,2]
- Limitations: No clinical data (e.g., human efficacy, patient pharmacokinetics) have been provided in the literature. Furthermore, Pinometostat (EPZ-5676) is ineffective against MLL fusion-negative leukemia, limiting its wider application in hematologic malignancies [1]

C30H42N8O3

562.71

562.338

1380288-87-8

57345410

White to light yellow solid powder

1.5±0.1 g/cm3

835.3±75.0 °C at 760 mmHg

459.0±37.1 °C

0.0±3.2 mmHg at 25°C

1.722

4.8

4

9

9

41

884

4

CC(C)N(C[C@@H]1[C@H]([C@H]([C@@H](O1)N2C=NC3=C(N=CN=C32)N)O)O)C4CC(C4)CCC5=NC6=C(N5)C=C(C=C6)C(C)(C)C

LXFOLMYKSYSZQS-LURJZOHASA-N

InChI=1S/C30H42N8O3/c1-16(2)37(13-22-25(39)26(40)29(41-22)38-15-34-24-27(31)32-14-33-28(24)38)19-10-17(11-19)6-9-23-35-20-8-7-18(30(3,4)5)12-21(20)36-23/h7-8,12,14-17,19,22,25-26,29,39-40H,6,9-11,13H2,1-5H3,(H,35,36)(H2,31,32,33)/t17-,19+,22-,25-,26-,29-/m1/s1

(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; Pinometostat; EPZ-5676; EPZ5676

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.44 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.44 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.44 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (4.44 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: ≥ 2.5 mg/mL (4.44 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 6: ≥ 2.5 mg/mL (4.44 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O:5mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)