Menin (menin-KMT2A interaction). [1][2]
In a homogeneous time-resolved fluorescence assay, the average IC50 of JNJ-75276617 against human menin was 0.1 ± 0.05 nM across 10 independent experiments. Against mouse menin, IC50 was 0.045 ± 0.002 nM. Against dog menin, IC50 was ≤0.066 ± 0.004 nM. [2]
In a fluorescence polarization binding assay, the Ki values for JNJ-75276617 against wild-type menin, M327I mutant menin, and T349M mutant menin were 0.02 nM, 0.4 nM, and 3.8 nM, respectively. [2]
(S)-Bleximenib targets the menin-MLL (mixed lineage leukemia) protein-protein interaction. Menin is a scaffold protein that binds to MLL fusion proteins and mutant NPM1, which are drivers of acute myeloid leukemia (AML). The menin-MLL interaction is essential for the leukemogenic activity of MLL fusion proteins and mutant NPM1. (S)-Bleximenib is the (S)-enantiomer of Bleximenib and serves as the less active isomer or negative control. In the MEIS1 mRNA expression assay (a pharmacodynamic marker of menin-MLL inhibition), (S)-Bleximenib shows an IC50 >1 microM, indicating significantly weaker activity compared to the active (R)-enantiomer. As a menin-MLL inhibitor, this compound functions by binding to menin and preventing its interaction with MLL fusion proteins, thereby disrupting the leukemic transcriptional program.

JNJ-75276617 decreased menin binding to target gene promoters (MEIS1, HOXA9, HOXA10) in KMT2A-r MOLM-14 and NPM1c OCI-AML3 cells at concentrations from 0.1 to 1.0 μM, with fold decreases ranging from 1.4- to 24.7-fold compared to DMSO control. [2]
JNJ-75276617 suppressed mRNA expression of menin-KMT2A target genes (MEIS1, JMJD1C, FLT3, MEF2C, PBX3) and increased differentiation markers (ITGAM/CD11b, MNDA) in KMT2A-r and NPM1c AML cell lines. [2]
JNJ-75276617 showed antiproliferative activity in AML cell lines: MOLM-14 (IC50 <0.1 μM), MOLM-13 (IC50 <0.1 μM), MV4-11 (IC50 <0.1 μM), THP-1 (IC50 >15 μM), EOL-1 (IC50 = 0.116 μM), OCI-AML3 (IC50 = 0.045 μM), and in B-ALL RS4;11 (IC50 = 0.040 μM). Normal peripheral blood mononuclear cells were not impacted up to 10 μM for 7 days. [2]
JNJ-75276617 induced apoptosis in MOLM-14, OCI-AML3, and RS4;11 cells in a dose-dependent manner after 7 days of treatment. [2]
JNJ-75276617 induced expression of CD11b and CD14 differentiation markers in MOLM-14 and OCI-AML3 cells after 3 and 7 days. In KMT2A-AF9-transduced mouse bone marrow cells, 200 nM JNJ-75276617 for 10 days increased neutrophil-like morphology (28.9% ± 2.6% vs DMSO 10.65% ± 1.35%). [2]
JNJ-75276617 demonstrated antiproliferative activity against 6 of 13 NPM1c primary AML patient samples at days 7 and 14, and induced CD11b expression. It also showed antiproliferative effects and dose-dependent apoptosis in a primary KMT2A-r B-ALL patient sample. [2]
JNJ-75276617 showed synergistic antiproliferative effects with gilteritinib in MOLM-13 cells (p<0.001). [2]
JNJ-75276617 combined with venetoclax or with venetoclax plus azacitidine showed synergistic antiproliferative effects in MOLM-13 cells. [2]
JNJ-75276617 retained potent antiproliferative activity in MEN1M327I and MEN1T349M mutant MV4-11 cells (IC50 = 22.6 nM and 53.59 nM, respectively). [2]
JNJ-75276617 treatment (0.3 μM for 4 days) upregulated HLA-A and HLA-DR mRNA and protein expression in primary AML samples, and this was CIITA-dependent. [1]
JNJ-75276617 pretreatment (0.3 μM for 4 days) enhanced T-cell-mediated cytotoxicity in allogeneic and autologous settings in primary AML samples, particularly those with NPM1c and DNMT3A mutations. [1]

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In vitro, (S)-Bleximenib shows an IC50 >1 microM in the MEIS1 mRNA expression assay. MEIS1 is a direct target gene of the menin-MLL complex and serves as a pharmacodynamic marker for menin-MLL inhibitor activity. This IC50 value is significantly higher than that of the active (R)-enantiomer (Bleximenib), which typically shows IC50 values in the low nanomolar range (e.g., 2-10 nM). (S)-Bleximenib can be used as a negative control in cellular assays to demonstrate that the anti-leukemic effects observed with Bleximenib are due to specific menin-MLL inhibition rather than off-target effects. The compound is less active in inhibiting proliferation of MLL-rearranged and NPM1-mutant AML cell lines.

In subcutaneous MOLM-14 AML xenograft mice, oral JNJ-75276617 (as oxalate salt) at 30, 50, and 100 mg/kg daily for 5 weeks induced dose-dependent tumor regressions of 70%, 97%, and 99%, respectively (p<0.0001 each). At 50 and 100 mg/kg, 10/10 and 9/9 tumors failed to regrow after 3 months off treatment. [2]
In a KMT2A-AF6 AML PDX model (CBAM-68552), JNJ-75276617 treatment significantly increased lifespan by >89% across all dose levels (p<0.05), reduced human CD45+ leukemic cells, and increased differentiation markers (CD11b, CD13, CD14). [2]
In NPM1c AML PDX models (LEXFAM-2734 and AM7577), JNJ-75276617 at 30, 50, or 100 mg/kg resulted in median survival >133 days and >147 days, respectively (vehicle: 74 days and 53.5 days), with significant increased lifespan (>66% to >169%, p<0.0001). [2]
In a KMT2A-AF4 B-ALL PDX model (CBAB-62871), JNJ-75276617 at 30 and 100 mg/kg daily increased lifespan by >48% and >160%, respectively (p=0.002), and reduced leukemia burden in bone marrow. [2]
In a disseminated MOLM-13 model, JNJ-75276617 (10 mg/kg) combined with azacitidine (2 mg/kg) and venetoclax (100 mg/kg) induced a 277% increased lifespan (p<0.0001), superior to the azacitidine+venetoclax regimen (53% ILS). [2]
In a disseminated OCI-AML3 NPM1c model, JNJ-75276617 combinations with azacitidine and venetoclax yielded superior survival vs azacitidine+venetoclax, though efficacy was mostly driven by JNJ-75276617. [2]

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No direct in vivo activity studies have been published for (S)-Bleximenib alone. The active (R)-enantiomer Bleximenib has shown potent in vivo efficacy in mouse xenograft models of MLL-rearranged and NPM1-mutant AML, leading to tumor regression and prolonged survival. (S)-Bleximenib, as the less active isomer, serves as a negative control in in vivo studies. It is not expected to have significant anti-tumor activity in mouse models. The compound can be used in pharmacokinetic or pharmacodynamic studies to confirm that the observed in vivo effects are due to specific menin-MLL inhibition rather than non-specific effects of the compound scaffold.

A homogeneous time-resolved fluorescence (HTRF) assay was used to quantify the ability of JNJ-75276617 to displace a fluorescein isothiocyanate (FITC)-labeled menin-binding motif 1 peptide (derived from KMT2A) from binding to terbium-labeled human, dog, and mouse menin. The compound was incubated with menin and FITC-MBM1 peptide, and the TR-FRET signal was monitored using a plate reader. Inhibitor potency was reported at 300 or 420 minutes when equilibrium was reached. Percentage inhibition and potency were calculated using Prism software. [2]
A fluorescence polarization binding assay was performed to assess binding of JNJ-75276617 to wild-type, M327I mutant, and T349M mutant menin. Dose-dependent displacement of a KMT2A peptide was measured. [2]

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No specific enzyme/receptor binding protocols have been established for (S)-Bleximenib. For menin-MLL binding studies, standard biochemical assays include fluorescence polarization (FP) or TR-FRET (time-resolved fluorescence energy transfer) using recombinant menin protein and a fluorescently labeled MLL peptide. Varying concentrations of (S)-Bleximenib (0.1-10000 nM) are incubated with menin and labeled MLL peptide. FP or TR-FRET signal is measured, and IC50 values are calculated from competition curves. For cellular target engagement assays, the MEIS1 mRNA expression assay is used. AML cells (e.g., MLL-rearranged or NPM1-mutant cell lines) are treated with varying concentrations of (S)-Bleximenib for 24-48 hours. RNA is extracted, and MEIS1 mRNA levels are measured by qRT-PCR. IC50 values are calculated from dose-response curves. (S)-Bleximenib shows IC50 >1 microM in this assay.

For the menin-KMT2A inhibitor screen, cryopreserved mononuclear cells from AML patients were thawed, resuspended in medium with DNase I, MgSO4, and heparin, then cultured in liquid medium with G-CSF, TPO, and IL-3 (all 20 ng/mL) or co-cultured on MS5 stromal cells. Cells were treated with DMSO or 0.03, 0.30, and 3.00 μM JNJ-75276617 for 14 days, with fresh medium and inhibitor added at day 7. On days 7 and 14, cells were stained with CD45-PECy7 and CD117, and fluorescence was measured using a flow cytometer. [1]
For apoptosis detection, cells were stained with annexin V and live/dead dye after treatment with JNJ-75276617 for up to 7 days, then analyzed by flow cytometry. [2]
For differentiation assessment, cells were stained with CD11b and CD14 antibodies and analyzed by flow cytometry. May-Grünwald Giemsa staining was used to assess morphological changes in KMT2A-AF9-transduced mouse bone marrow cells after 10 days of treatment with 200 nM JNJ-75276617. [2]
For T-cell cytotoxicity assays, primary AML samples were pretreated with 0.3 μM JNJ-75276617 for 4 days, then washed and co-cultured with anti-CD3/CD28 activated T cells from healthy donors at different effector:target ratios for 3 days. Viable cell counts were determined by annexin-V and Zombie NIR staining. [1]
For autologous T-cell cytotoxicity, CD45+ blast and CD3+ T-cell populations were sorted from NPM1c AML patient samples, and AML blasts were cultured with or without their own T cells at a fixed ratio in the presence or absence of 0.3 μM JNJ-75276617. [1]
For chromatin immunoprecipitation (ChIP)-qPCR, cells were cross-linked, lysed, and sonicated. Menin antibody was used for immunoprecipitation, and binding to target gene promoters (MEIS1, HOXA9, HOXA10) was quantified. [2]
For the Quantigene multiplex assay, cells were treated with JNJ-75276617 or DMSO for 48-72 hours, then lysed. Target-specific probes were mixed with lysates, and signals were measured on a FLEXMAP 3D system. Absolute IC50 values were calculated by dose-response modeling. [2]

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In vitro cellular protocols for (S)-Bleximenib involve culturing AML cell lines with MLL rearrangements (e.g., MV4-11, MOLM-13, OCI-AML3) or NPM1 mutations (e.g., OCI-AML3) in RPMI-1640 with 10-20% FBS at 37degC in 5% CO2. Cells are seeded in 96-well plates (1-5×10⁴ cells/well) and treated with varying concentrations of (S)-Bleximenib (0.01-10000 nM) for 72-120 hours. Cell viability is assessed by MTT, CellTiter-Glo, or Cell Counting Kit-8 (CCK-8) assays. For MEIS1 mRNA expression, cells are seeded in 6-well plates (5×10⁵ cells/well) and treated with (S)-Bleximenib (0.1-10000 nM) for 24 hours. RNA is extracted, and MEIS1 mRNA levels are quantified by qRT-PCR using specific primers. GAPDH or beta-actin serves as housekeeping control. IC50 values are calculated from dose-response curves. The active (R)-enantiomer is used as a positive control. All experiments are performed in triplicate.

Female 6- to 8-week-old immunocompromised mice were used for xenograft studies. JNJ-75276617 (as oxalate salt) was orally dosed daily at various concentrations (10, 30, 50, 100 mg/kg) for up to 4 weeks. Venetoclax (100 mg/kg) was orally dosed daily. Azacitidine (2 mg/kg) was dosed by intraperitoneal injection daily for the first week only. All experiments were performed in accordance with institutional guidelines. [2]
In the subcutaneous MOLM-14 model, tumor volume was measured, and tumor regression was assessed. In disseminated models, survival was monitored, and leukemic burden was assessed by flow cytometry for human CD45+ cells in bone marrow. [2]

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For in vivo studies of (S)-Bleximenib, standard animal protocols involve xenograft models in immunodeficient mice (NSG or nude mice, 6-8 weeks old). Approximately 5×10⁶ MLL-rearranged or NPM1-mutant AML cells are injected subcutaneously or intravenously (tail vein injection for disseminated leukemia models). When tumors reach ~200 mm3 (subcutaneous) or when leukemia engraftment is confirmed (disseminated), mice are randomized into treatment groups (n=5-10 per group). (S)-Bleximenib is administered orally or intraperitoneally at doses of 10-50 mg/kg in formulation vehicles (10% DMSO, 40% PEG300, 5% Tween 80, 45% saline). Dosing frequency may be daily. Tumor volumes (subcutaneous) are measured every 2-3 days. For disseminated models, survival is monitored, and leukemia burden in blood and bone marrow is assessed by flow cytometry (hCD45+ cells) at study termination. (S)-Bleximenib is expected to have little to no anti-leukemic activity, serving as a negative control. The active (R)-enantiomer shows significant tumor regression and survival benefit. All procedures require approval by institutional animal care committees.

No direct pharmacokinetic studies have been published for (S)-Bleximenib. The active (R)-enantiomer Bleximenib has been reported to have favorable oral bioavailability and pharmacokinetic properties in preclinical species. Computed properties for (S)-Bleximenib include molecular formula C32H50FN7O3, molecular weight 599.78 g/mol, predicted logP of approximately 3.0-4.0, suggesting moderate to high lipophilicity. For Bleximenib (racemic or (R)-enantiomer), reported PK parameters may include half-life (2-4 hours in mice), moderate clearance, and oral bioavailability >50%. The (S)-enantiomer likely has similar physicochemical properties but may differ in clearance and volume of distribution. The compound is soluble in DMSO and can be formulated for oral administration.

Safety data for (S)-Bleximenib is limited. The compound is for research use only and not for human therapeutic applications. Standard laboratory safety precautions should be followed: wear protective gloves, safety goggles, and a lab coat. Work in a well-ventilated fume hood. Avoid inhalation, ingestion, and contact with skin and eyes. The compound should be stored at -20degC, protected from light and moisture. As a menin-MLL inhibitor, it may have activity against MLL-rearranged and NPM1-mutant cells, but the (S)-enantiomer is significantly less potent. Nonetheless, handle with appropriate caution. This product is not for human therapeutic use.

[1]. Substituted straight chain spiro derivatives. WO2021121327A1. WIPO (PCT). 2020-12-17.

(S)-Bleximenib is an enantiomer of Bleximenib (JNJ-75276617) and serves as the less active isomer or negative control in menin-MLL inhibitor research. Bleximenib is a potent, selective, and orally bioavailable inhibitor of the menin-MLL protein-protein interaction, a critical dependency in acute myeloid leukemia (AML) with MLL rearrangements or NPM1 mutations. The (S)-enantiomer shows IC50 >1 microM in the MEIS1 mRNA expression assay, while the active (R)-enantiomer has IC50 in the low nanomolar range (2-10 nM). (S)-Bleximenib is used in research studies of AML, particularly as a control to distinguish specific menin-MLL inhibition from off-target effects. No clinical trial data is available for (S)-Bleximenib; the active enantiomer Bleximenib is in preclinical or early clinical development for the treatment of MLL-rearranged and NPM1-mutant acute leukemia. This product is for research use only and not approved for human therapy.

C32H50FN7O3

599.78

599.3959

C, 64.08; H, 8.40; F, 3.17; N, 16.35; O, 8.00

2654081-27-1

Bleximenib; Bleximenib oxalate; (S)-Bleximenib oxalate; 2866179-95-3 (oxalate); 2654081-35-1

Colorless to light yellow Oil‌

CCN(C(C)C)C(=O)C1=C(C=CC(=C1)F)OC2=C(N=CN=N2)N3CCC4(C3)CN(C4)[C@@H](CCCN(C)CCOC)C(C)C.C(=O)(C(=O)O)O

(S)-JNJ-75276617; (S)-Menin-MLL inhibitor 24

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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