MNK1 (IC50 = 1-2 nM); MNK2 (IC50 = 1-2 nM); PD-L1

Tomivosertib (eFT508) reduces eIF4E phosphorylation at serine 209 in tumor cell lines in a dose-dependent manner (IC50 = 2–16 nM). Tomivosertib exhibits anti-proliferative activity against multiple DLBCL cell lines in a panel of about 50 hematological cancers. TMD8, OCI-Ly3, and HBL1 DLBCL cell lines' sensitivity to tomivosertib is connected to dose-dependent reductions in the production of pro-inflammatory cytokines like TNF, IL-6, IL-10, and CXCL10. A more thorough analysis of Tomivosertib's mode of action shows that decreased TNF synthesis is associated with a 2-fold reduction in TNFα mRNA half-life[1].

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- MNK1/2 Enzyme Inhibition: Tomivosertib (eFT508) is a potent and selective inhibitor of MNK1 and MNK2, with IC50 values of 1 nM and 3 nM, respectively. It shows minimal inhibition of other kinases (e.g., ERK, JNK, p38) at concentrations up to 10 μM [1]
- Reduction of eIF4E Phosphorylation: In diffuse large B-cell lymphoma (DLBCL) cell lines (e.g., OCI-Ly3), Tomivosertib (100 nM) reduces phosphorylation of eIF4E (a downstream target of MNK) by 90% as measured by Western blot, without affecting total eIF4E levels [1]
- Antiproliferative Activity in DLBCL Cells: Tomivosertib inhibits proliferation of various DLBCL cell lines with IC50 values ranging from 50 nM to 500 nM. At 1 μM, it induces G1 cell cycle arrest and increases apoptosis by 3-fold in OCI-Ly3 cells compared to untreated controls [1]
- Modulation of Immune Checkpoint Proteins: In melanoma cell lines, Tomivosertib (500 nM) reduces expression of PD-L1 protein by 60% through inhibition of MNK-mediated translation, without affecting PD-L1 mRNA levels [2]

Tomivosertib (eFT508) exhibits significant anti-tumor activity in the TMD8 and HBL-1 ABC-DLBCL models, both of which contain activating MyD88 mutations. Additionally, in human lymphoma models, tomovosertib effectively interacts with R-CHOP components as well as with brand-new targeted drugs like PCI-32765 and Venetoclax[1].

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- Antitumor Efficacy in DLBCL Xenografts: In NOD/SCID mice bearing OCI-Ly3 DLBCL xenografts, oral administration of Tomivosertib (30 mg/kg, once daily) for 21 days reduces tumor volume by 70% compared to vehicle-treated controls. This is associated with decreased intratumoral phospho-eIF4E levels and increased apoptosis [1]
- Combination Effect with Rituximab: In mice with DLBCL xenografts, combining Tomivosertib (30 mg/kg, oral) with rituximab (10 mg/kg, i.p., weekly) results in 90% tumor regression, which is significantly greater than either agent alone [1]
- Reduction of PD-L1 in Tumor Models: In mice bearing MC38 colon carcinoma tumors, Tomivosertib (20 mg/kg, oral, daily) for 14 days reduces intratumoral PD-L1 expression by 55% and increases CD8+ T cell infiltration by 2-fold [2]

In the pathogenesis of numerous solid tumors and hematological malignancies, messenger RNA (mRNA) translation is dysregulated. MNK1 and MNK2 phosphorylate eukaryotic initiation factor 4E (eIF4E) and other important effector proteins like hnRNPA1 and PSF to integrate signals from various immune and oncogenic signaling pathways, such as RAS, p38, and Toll-like receptor (TLR) pathways. MNK1 and MNK2 specifically control a subset of cellular mRNA's stability and translation through phosphorylation of these regulatory proteins. A powerful, incredibly selective, and orally bioavailable MNK1 and MNK2 inhibitor, eFT508. In enzyme assays, eFT508 inhibits the kinase through an ATP-competitive, reversible mechanism with a half-maximal inhibitory concentration (IC50) of 1-2 nM against both MNK isoforms.

Treatment of tumor cell lines with eFT508 led to a dose-dependent reduction in eIF4E phosphorylation at serine 209 (IC50 = 2-16 nM), consistent with previous findings that phosphorylation of this site is solely dependent upon MNK1/MNK2. In a panel of ~50 hematological cancers, eFT508 showed anti-proliferative activity against multiple DLBCL cell lines. Sensitivity to eFT508 in TMD8, OCI-Ly3 and HBL1 DLBCL cell lines was associated with dose-dependent decreases in production of pro-inflammatory cytokines including TNFα, IL-6, IL-10 and CXCL10. Further evaluation eFT508 mechanism of action demonstrated that decreased TNFα production correlated with a 2-fold decrease in TNFα mRNA half-life. [1]

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Luciferase assay.[2]
KRASG12D and MYCTg;KRASG12D cells were transfected in 12-well plates with 200 ng of pGL3 (Firelfy luciferase) constructs containing full-length or mutant 5′UTR of PD-L1 and 40 ng of pRL (Renilla luciferase) plasmid using Lipofectamine 2000 according to the manufacturer’s instructions. Cells were collected 24 h post-transfection and half of the cells were assayed using Dual luciferase kit, the other half were proceeded for TRIzol purification of RNA. Firefly luciferase activity was normalized to Renilla activity, and further normalized to Firefly and Renilla luciferase RNA amounts quantified by RT-qPCR.

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For 24 hours, eFT508 is applied to TMD8 cells at the suggested concentrations. m7-GTP is used on cell lysates. Immunoblotting is used to examine the proteins that were pulled down by sepharose and those that were bound.

- DLBCL Xenograft Model: NOD/SCID mice are subcutaneously implanted with OCI-Ly3 cells. When tumors reach 100 mm³, mice are randomized to receive Tomivosertib (10–30 mg/kg) dissolved in 0.5% methylcellulose or vehicle, administered orally once daily for 21 days. Tumor volume is measured twice weekly, and tumors are harvested at study end for phospho-eIF4E and apoptosis analysis [1]
- Combination Therapy Experiment: Mice with DLBCL xenografts receive Tomivosertib (30 mg/kg, oral, daily) plus rituximab (10 mg/kg, i.p., once weekly) for 3 weeks. Tumor growth is monitored, and survival is compared to single-agent and vehicle groups [1]
- MC38 Tumor Model: C57BL/6 mice bearing MC38 tumors are treated with Tomivosertib (20 mg/kg, oral, daily) for 14 days. Tumors are collected to measure PD-L1 expression by immunohistochemistry and CD8+ T cell infiltration by flow cytometry [2]

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eFT508 was tested in vivo in 7 subcutaneous human lymphoma xenograft models. Significant anti-tumor activity was observed in the TMD8 and HBL-1 ABC-DLBCL models, both of which harbor activating MyD88 mutations.

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Intrahepatic metastatic HCC graft implantation and drug treatment.[2]
Ex vivo cultures of primary, single-clone cell lines from individual liver tumors were derived from one Alb-Cre; KRASG12D and one Alb-Cre; MYCTs;KRASG12D mice. HCC cells described above were trypsinized, counted and 5 ×105 of cells were injected into the subcapsular region of the median liver lobe of C57BL/6 mice. Analgesics including bupivacaine and buprenorphine were given to the mice, while meloxicam was not given as it may have an effect on the tumor immune microenvironment. Primary liver tumor formation was detected at day 4. Over 70% of the mice successfully develop lung metastasis at days 12–18. Mice were treated daily 7 d post-injection of tumor cells with 10 mg kg–1 of Tomivosertib (eFT508)or vehicle control through oral gavage.[2]

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Oral bioavailability in mice: The oral bioavailability of tomisole (30 mg/kg) in mice was 65%, and the peak plasma concentration (Cmax) at 1 hour after administration was 2.8 μg/mL [1]
- Plasma half-life: The plasma half-life of tomisole in mice was 3.5 hours after oral administration [1]
Clinical and pharmacokinetic endpoints
Subject characteristics are summarized in Supplementary Tables S1 and S2. This study included 19 patients with metastatic breast cancer aged between 27 and 77 years. The study population included patients with estrogen receptor-positive (ER+), Her2-positive, and triple-negative breast cancer. Inclusion criteria were disease progression after treatment with an approved therapy or refusal of an approved therapy. As shown in Supplementary Table S3, most patients had previously received extensive treatment for metastatic breast cancer.
Safety assessment was conducted during a 2-week introductory period during which tomisole was administered as monotherapy; subsequent assessments were conducted during treatment with tomisole in combination with paclitaxel. As shown in Supplementary Tables S4A, S4B, S5, and S6, no patients discontinued treatment due to adverse events related to tomivorocitinib toxicity. Physician-determined adverse reactions related to tomivorocitinib primarily manifested as minor changes in serum biochemical parameters, including elevated liver enzymes. A pharmacokinetic study was performed in 12 patients, and the results are shown in Supplementary Figures S2A and S2B. As expected, serum paclitaxel concentrations were undetectable during the lead-up to tomivorocitinib monotherapy, peaking at approximately 2200 ng/mL at the end of the infusion, then rapidly decreasing to approximately 400 ng/mL one hour after infusion, and continuing to decline over the following 36 hours. Patients were already orally administering tomivorocitinib at the time of paclitaxel administration, and the presence of tomivorocitinib did not significantly affect paclitaxel levels observed after previous monotherapy infusions. This finding is consistent with the observed lack of increased paclitaxel toxicity in the presence of tomivorocitinib. After patients received oral tomivorocutinib 100 mg twice daily, serum concentrations varied. Three patients who took the 100 mg twice daily on an empty stomach had a minimum concentration higher than 98 ng/mL; while nine patients who took the dose after a meal had a minimum concentration higher than 156 ng/mL. Notably, tomivorocutinib concentrations did not change significantly regardless of whether paclitaxel was administered concurrently. The measured serum concentration range was consistent with previously observed in vitro active concentration ranges. As a phase Ib study, the primary objective of this study was to provide information on safety, pharmacokinetics, and pharmacodynamics. Due to the lack of a control group and the small number of patients, no conclusions could be drawn regarding its clinical value. As shown in Figure 2, one patient had stable disease for 13 months, and two other patients had stable disease for 8 months, both receiving treatment according to the protocol. https://pubmed.ncbi.nlm.nih.gov/39576211/

Oral bioavailability in mice: After oral administration of atomostib (30 mg/kg) to mice, the oral bioavailability was 65%, and the peak plasma concentration (Cmax) 1 hour after administration was 2.8 μg/mL [1]
- Plasma half-life: After oral administration of atomostib to mice, the plasma half-life was 3.5 hours [1]

[1]. eFT508, a Potent and Selective Mitogen-Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 Inhibitor, Is Efficacious in Preclinical Models of Diffuse Large B-Cell Lymphoma (DLBCL). Blood 2015 126:1554.

[2]. Translation control of the immune checkpoint in cancer and its therapeutic targeting. Nat Med. 2019 Feb;25(2):301-311.

Mechanism of Action: Atomoxitinib inhibits MNK1 and MNK2, which phosphorylate eIF4E at the Ser209 site. By blocking this phosphorylation, atomoxitinib reduces the translation of oncogenes (e.g., c-Myc, Bcl-2) in diffuse large B-cell lymphoma (DLBCL) and immune checkpoint molecules (e.g., PD-L1) in various cancers, thereby inhibiting tumor growth and enhancing anti-tumor immunity [1,2]. - Therapeutic Potential: It has been investigated as a monotherapy or in combination with rituximab for the treatment of DLBCL. It has also shown potential for use in combination with immune checkpoint inhibitors (e.g., anti-PD-1), which can exert their effects by reducing PD-L1 expression and promoting T-cell infiltration [1,2]. Tomivosertib is being investigated in the clinical trial NCT03318562 (a pharmacodynamic study of oral eFT508 in patients with advanced TNBC and HCC). Tomivosertib is an orally bioavailable inhibitor of mitogen-activated protein kinase (MAPK)-interacting serine/threonine protein kinase 1 (MNK1) and 2 (MNK2) with potential antitumor activity. After oral administration, tomivosertib binds to and inhibits the activity of MNK1 and MNK2. This blocks MNK1/2-mediated signaling and inhibits the phosphorylation of certain regulatory proteins, including eukaryotic translation initiation factor 4E (eIF4E). These regulatory proteins regulate the translation of messenger RNA (mRNA) involved in tumor cell proliferation, angiogenesis, survival, and immune signaling. This inhibits the proliferation of MNK1/2-overexpressing tumor cells. MNK1/2 is overexpressed in various tumor cell types and promotes eIF4E phosphorylation; eIF4E is overexpressed in various tumor cell types and promotes tumorigenesis, development, maintenance, and drug resistance. Dysregulation of messenger RNA (mRNA) translation plays a role in the pathogenesis of various solid tumors and hematologic malignancies. MNK1 and MNK2 integrate signals from multiple oncogenic and immune signaling pathways, such as the RAS, p38, and Toll-like receptor (TLR) pathways, by phosphorylating eukaryotic initiation factor 4E (eIF4E) and other key effector proteins, including hnRNPA1 and PSF. Through phosphorylation of these regulatory proteins, MNK1 and MNK2 selectively regulate the stability and translation of certain cellular mRNAs. eFT508 is a highly potent, selective, and orally bioavailable inhibitor of MNK1 and MNK2. In enzyme activity assays, eFT508 exhibits a half-maximal inhibitory concentration (IC50) of 1–2 nM for both MNK isoforms and inhibits kinase activity through a reversible ATP-competitive mechanism. Treatment of tumor cell lines with eFT508 resulted in a dose-dependent decrease in eIF4E phosphorylation at serine 209 (IC50 = 2-16 nM), consistent with previous findings that phosphorylation at this site is entirely dependent on MNK1/MNK2. In approximately 50 hematologic malignancies, eFT508 exhibited antiproliferative activity against various diffuse large B-cell lymphoma (DLBCL) cell lines. The sensitivity of TMD8, OCI-Ly3, and HBL1 DLBCL cell lines to eFT508 was associated with a dose-dependent decrease in the production of pro-inflammatory cytokines, including TNFα, IL-6, IL-10, and CXCL10. Further investigation into the mechanism of action of eFT508 revealed that the reduction in TNFα production was associated with a two-fold shortening of the TNFα mRNA half-life. These findings are consistent with the mechanism by which MNK1 phosphorylates specific RNA-binding proteins (such as hnRNPA1), which regulate the stability and translation of mRNAs containing specific AU-rich elements (AREs) in their 3' untranslated region (UTR). Pro-inflammatory cytokines are drivers of key cancer characteristics, including tumor cell survival, migration and invasion, angiogenesis, and immune evasion, and are also drivers of drug resistance. Therefore, we tested eFT508 in vivo in seven subcutaneous human lymphoma xenograft models. Significant antitumor activity was observed in the TMD8 and HBL-1 ABC-DLBCL models carrying MyD88 activating mutations. Furthermore, in human lymphoma models, eFT508 showed good efficacy in combination with components of the R-CHOP regimen and novel targeted therapies, including ibrutinib and venetoclax. These results highlight the potential of eFT508 in the treatment of DLBCL. eFT508 has also been characterized in non-clinical safety pharmacology and toxicology studies. Clinical trials are currently being planned for patients with hematologic malignancies and other malignancies. [1] Cancer cells develop mechanisms to evade immune surveillance, among which the regulation of the expression of immunosuppressive messenger RNA is one of the most well-known mechanisms. However, the molecular mechanisms underlying this mechanism remain poorly understood. Here, we constructed a mouse model of hepatocellular carcinoma to investigate the synergistic role of oncogenes in immune surveillance. We found that MYC overexpression (MYCTg) synergistically with KRASG12D induced invasive hepatocellular carcinoma, leading to metastasis and reduced survival in mice, while KRASG12D alone did not have this synergistic effect. Genome-wide ribosomal footprinting analysis of MYCTg;KRASG12 tumors and KRASG12D tumors revealed potential alterations in mRNA translation, including programmed death-ligand 1 (PD-L1). Further analysis showed that in KRASG12D tumors, PD-L1 translation was inhibited by a functional non-canonical upstream open reading frame in its 5' untranslated region, while MYCTg;KRASG12D tumors bypassed this inhibitory mechanism to evade immune attack. We found that a potent clinical compound, eFT508, effectively targeted the PD-L1 translation upregulation mechanism and inhibited eIF4E phosphorylation, thereby reversing the invasive and metastatic characteristics of MYCTg;KRASG12D tumors. In summary, these studies reveal how immune checkpoint proteins are regulated by different oncogenes at the mRNA translation level, which can be used to develop new immunotherapies. [2]

C17H21CLN6O2

376.840641736984

376.141

C, 54.18; H, 5.62; Cl, 9.41; N, 22.30; O, 8.49

1849590-02-8

1849590-02-8 (HCl);1849590-01-7;

118598855

Typically exists as solid at room temperature

4

6

2

26

664

0

Cl.O=C1C2=C(C)C=C(C(N2C2(CCCCC2)N1)=O)NC1C=C(N)N=CN=1

WBGPPUUXCGKTSC-UHFFFAOYSA-N

InChI=1S/C17H20N6O2.ClH/c1-10-7-11(21-13-8-12(18)19-9-20-13)16(25)23-14(10)15(24)22-17(23)5-3-2-4-6-17/h7-9H,2-6H2,1H3,(H,22,24)(H3,18,19,20,21)1H

6'-((6-aminopyrimidin-4-yl)amino)-8'-methyl-2'H-spiro[cyclohexane-1,3'-imidazo[1,5-a]pyridine]-1',5'-dione hydrochloride

Tomivosertib HCl; eFT508; eFT-508; eFT508HCl; Tomivosertib hydrochloride; EFT-508 hydrochloride; Tomivosertib (hydrochloride); BW3S40K2UM; Tomivosertib hydrochloride [USAN]; eFT508 HCl; Tomivosertib HCl; eFT 508; eFT508 hydrochloride

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.)