All investigated multiple myeloma (MM) cell lines are inhibited in their proliferation by umansertib phosphate; typical GI50 values for AML, MM, DLBCL, MCL, and T-ALL cell lines range from 13.2 nM to 230.0 nM [1]. The phosphorylation of downstream PIM kinase substrates (p70S6K/S6 and 4E-BP1) is inhibited by umansertib phosphate (0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 nM) in a dose-dependent manner in MOLM-16 alteration. AML), KMS-12-PE/BM (MM), and Pfeiffer (DLBCL) cell lines [1]. In MOLM-16 and KMS-12-BM cells, PIM kinase-mediated BAD phosphorylation is especially susceptible to Uzansertib phosphate inhibition (mean IC50 of 4 nM and 27 nM, respectively) [1].

All investigated multiple myeloma (MM) cell lines are inhibited in their proliferation by umansertib phosphate; typical GI50 values for AML, MM, DLBCL, MCL, and T-ALL cell lines range from 13.2 nM to 230.0 nM [1]. The phosphorylation of downstream PIM kinase substrates (p70S6K/S6 and 4E-BP1) is inhibited by umansertib phosphate (0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 nM) in a dose-dependent manner in MOLM-16 alteration. AML), KMS-12-PE/BM (MM), and Pfeiffer (DLBCL) cell lines [1]. In MOLM-16 and KMS-12-BM cells, PIM kinase-mediated BAD phosphorylation is especially susceptible to Uzansertib phosphate inhibition (mean IC50 of 4 nM and 27 nM, respectively) [1].

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INCB053914 inhibited proliferation in a panel of hematologic malignancy cell lines. Mean GI₅₀ values were <100 nM in 50% of tested cell lines.
In multiple myeloma (MM) cell lines, mean GI₅₀ ranged from 13.2 nM to 230.0 nM.
In acute myeloid leukemia (AML) cell lines, mean GI₅₀ was <100 nM in 8 of 15 lines. MOLM-16 and Kasumi-3 cells were particularly sensitive (GI₅₀ = 3.3 nM and 4.9 nM, respectively).
In diffuse large B-cell lymphoma (DLBCL), Pfeiffer cells were most sensitive (GI₅₀ = 19.5 nM).
INCB053914 inhibited phosphorylation of PIM kinase downstream substrates (p70S6K, S6, 4E-BP1) in a dose-dependent manner in MOLM-16 (AML), Pfeiffer (DLBCL), and KMS-12-PE/BM (MM) cell lines.
INCB053914 potently inhibited BAD phosphorylation in MOLM-16 (IC₅₀ = 4 nM) and KMS-12-BM (IC₅₀ = 27 nM) cells.
Treatment with INCB053914 increased PIM2 expression in KG-1a (AML), Pfeiffer (DLBCL), and KMS-12-PE (MM) cells.
In primary bone marrow blasts from AML patients treated ex vivo, INCB053914 decreased phosphorylation of p70S6K and 4E-BP1 and increased PIM2 expression.
In peripheral blood mononuclear cells (PBMCs) treated ex vivo, INCB053914 caused a concentration-dependent increase in PIM2 expression (EC₅₀ = 1.8 nM).
In blood samples from AML patients receiving INCB053914 (100 mg BID) in a phase 1/2 trial, increased PIM2 expression and decreased 4E-BP1 phosphorylation were observed.
INCB053914 inhibited erythroid colony formation ex vivo in primary cell cultures from patients with JAK2 V617F-positive myeloproliferative neoplasms (Polycythemia Vera, Essential Thrombocythemia, Primary Myelofibrosis) at concentrations as low as 0.5 nM.
In combination studies, INCB053914 synergistically enhanced the antiproliferative effects of the PI3Kδ inhibitor INCB050465 in Pfeiffer cells.
INCB053914 showed additive/synergistic effects in combination with 19 out of 65 tested anticancer agents, including PI3K/AKT pathway inhibitors, azacitidine, and ibrutinib, in MM cell viability assays.

In mice with MOLM-16 (AML) or KMS-12-BM (MM) tumors, usansertib phosphate (25–100 mg/kg; oral; twice daily; for 15 days) suppresses tumor growth in a dose-dependent manner[1]. Four hours after injection, uzansertib phosphate showed dose-dependent reduction of BAD phosphorylation in comparison to vehicle (IC50=70 nM for MOLM-16 tumors and IC50=145 nM for KMS-12-BM tumors) [1].

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In SCID mice bearing MOLM-16 (AML) xenografts, a single oral dose of INCB053914 dose-dependently inhibited intratumoral BAD phosphorylation (IC₅₀ = 70 nM at 4 hours post-dose).
In SCID mice bearing KMS-12-BM (MM) xenografts, a single oral dose inhibited intratumoral BAD phosphorylation (IC₅₀ = 145 nM at 4 hours post-dose).
Oral administration of INCB053914 (BID for 15 days) dose-dependently inhibited tumor growth in MOLM-16 xenografts. Maximal growth inhibition (96%) was observed at 30 mg/kg BID, with 6 of 8 mice showing partial regression.
Oral administration of INCB053914 (BID for 15 days) dose-dependently inhibited tumor growth in KMS-12-BM xenografts, with 88% inhibition at 100 mg/kg BID.
In combination with the PI3Kδ inhibitor INCB050465, INCB053914 synergistically inhibited tumor growth in Pfeiffer (DLBCL) xenografts, resulting in complete regression in 1 mouse and partial regression in 7 out of 8 mice.
In combination with cytarabine, INCB053914 additively inhibited tumor growth in KG-1 (AML) xenografts.
In combination with the JAK1-selective inhibitor itacitinib, INCB053914 synergistically inhibited tumor growth in INA-6 (MM) xenografts, with partial responses in 4 of 8 mice.

The inhibitory activity of INCB053914 against PIM1 and PIM3 kinases was measured using a luminescence-based AlphaScreen assay.
The inhibitory activity against PIM2 kinase was measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay.
IC₅₀ values were determined from dose-response curves.
Selectivity profiling was performed against a panel of more than 50 kinases at an ATP concentration of 1 mM.

For cell proliferation assays, hematologic tumor cell lines were cultured according to supplier recommendations. Cells were treated with INCB053914 across a range of concentrations, and cell viability/proliferation was assessed using unspecified assays (details in Supporting Information). GI₅₀ values were calculated.
To assess inhibition of substrate phosphorylation and PIM2 upregulation, cells were incubated with INCB053914 for 2 hours. Cells were then lysed, and protein levels were analyzed by Western blotting using antibodies against phospho-BAD (S112), phospho-4E-BP1 (S65), phospho-p70S6K (T389), phospho-S6 (S235/236), and PIM2.
For quantitative measurement of pBAD inhibition, cells were treated with INCB053914 for 2.5 hours, lysed, and pBAD protein concentration was quantified using a specific ELISA kit.
For ex vivo studies on primary cells, bone marrow blasts or PBMCs were treated with INCB053914 for 2 hours (blasts) or overnight (PBMCs), followed by lysis and Western blot analysis.
For erythroid colony formation assays, primary cells from MPN patients were cultured in methylcellulose-based media with INCB053914. Colonies were counted after a specified period.
For combination synergy assays, cells were treated with INCB053914 and other agents. Effects on cell viability were measured, and synergy was assessed using the Bliss independence model.

Animal/Disease Models: Female immunocompromised (severe combined immunodeficiency [SCID]) mice (5-9 weeks old) [1] carrying MOLM-16 (AML) or KMS-12-BM (MM)
Doses: 25, 50 , 75, 100 mg/kg.
Doses: Oral; twice a day; for 15 days.
Experimental Results: Inhibited tumor growth in mice in a dose-dependent manner.

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For xenograft models, female SCID mice were inoculated subcutaneously with tumor cell suspensions (e.g., MOLM-16, KG-1, KMS-12-BM) mixed with Matrigel, or with tumor fragments (e.g., INA-6, Pfeiffer).
Treatment began when tumors reached a specified volume range (e.g., 158-249 mm³ for efficacy studies).
INCB053914 was administered orally (by gavage) twice daily (BID) as a suspension in 5% dimethylacetamide with 0.5% w/v methylcellulose. Doses ranged from 1 to 100 mg/kg. Treatment duration was 7 to 19 days depending on the study.
For some pharmacodynamic studies, INCB053914 was administered via subcutaneous continuous infusion using osmotic pumps.
For combination studies: The PI3Kδ inhibitor INCB050465 was given orally at 10 mg/kg once daily. Cytarabine was administered intraperitoneally at 20 mg/kg twice weekly. The JAK1 inhibitor itacitinib was given orally at 60 mg/kg BID.
Tumor volume was measured periodically and calculated using the formula: volume = (length × width²) / 2.
Animals were monitored for signs of distress and euthanized according to predefined humane endpoints.

In mice carrying MOLM-16 tumors, plasma concentrations were measured up to 16 hours after oral administration of INCB053914 at doses of 1, 3, 10, and 30 mg/kg (twice daily). Results showed a dose-dependent relationship between plasma concentration and concentration. In xenograft models, maximum antitumor activity was correlated with the IC₅₀ value of plasma trough concentration exceeding pBAD inhibition, determined in whole blood assays supplemented with a pBAD inhibitor.

[1]. Preclinical characterization of INCB053914, a novel pan-PIM kinase inhibitor, alone and in combination with anticancer agents, in models of hematologic malignancies. PLoS One. 2018 Jun 21;13(6):e0199108.

INCB053914 is a novel ATP-competitive small-molecule pan-PIM kinase (PIM1, PIM2, PIM3) inhibitor. It was discovered through a rational medicinal chemistry approach designed to inhibit all three PIM isoenzymes. This compound exhibits unique binding properties, with significantly lower inhibitory potency against PIM2 than against PIM1 and PIM3. The rationale for this study includes the overexpression of PIM kinases in hematologic malignancies, their roles in overlapping signaling pathways (JAK/STAT, PI3K/AKT), and compensatory upregulation among isoenzymes, all of which support the necessity of pan-PIM inhibition. Preclinical data support the initiation of a phase 1/2 dose-escalation trial to evaluate the efficacy of INCB053914 alone or in combination with INCB050465 and azacitidine. Cytarabine or ruxotinib are used to treat advanced malignancies.

C26H29F3N5O7P

611.5067

611.175

2088852-47-3

Uzansertib;1620012-39-6

126673672

White to off-white solid powder

7

14

4

42

853

4

P(=O)(O[H])(O[H])O[H].FC1C([H])=C([H])C(C(N([H])C2=C([H])N=C3[C@@]([H])(C([H])([H])C([H])([H])C3=C2N2C([H])([H])[C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])[H])C2([H])[H])O[H])N([H])[H])O[H])=O)=NC=1C1C(=C([H])C([H])=C([H])C=1F)F

CYYVLFVRZDZABX-SEDYQSMDSA-N

InChI=1S/C26H26F3N5O3.H3O4P/c1-12-10-34(11-17(30)25(12)36)24-13-5-8-20(35)22(13)31-9-19(24)33-26(37)18-7-6-16(29)23(32-18)21-14(27)3-2-4-15(21)28;1-5(2,3)4/h2-4,6-7,9,12,17,20,25,35-36H,5,8,10-11,30H2,1H3,(H,33,37);(H3,1,2,3,4)/t12-,17+,20+,25+;/m0./s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 1.8 mg/mL (2.94 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 18.0 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: ≥ 1.8 mg/mL (2.94 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 18.0 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: ≥ 1.8 mg/mL (2.94 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 18.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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