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].

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].

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.

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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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.

[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.

Tomivosertib is under investigation in clinical trial NCT03318562 (A PD Study of Oral eFT508 in Subjects With Advanced TNBC and HCC).

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Tomivosertib is an orally bioavailable inhibitor of mitogen-activated protein kinase (MAPK)-interacting serine/threonine-protein kinase 1 (MNK1) and 2 (MNK2), with potential antineoplastic activity. Upon oral administration, tomivosertib binds to and inhibits the activity of MNK1 and 2. This prevents MNK1/2-mediated signaling, and inhibits the phosphorylation of certain regulatory proteins, including eukaryotic translation initiation factor 4E (eIF4E), that regulate the translation of messenger RNAs (mRNAs) involved in tumor cell proliferation, angiogenesis, survival and immune signaling. This inhibits tumor cell proliferation in MNK1/2-overexpressing tumor cells. MNK1/2 are overexpressed in a variety of tumor cell types and promote phosphorylation of eIF4E; eIF4E is overexpressed in many tumor cell types and contributes to tumor development, maintenance and resistance.

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Dysregulated translation of messenger RNA (mRNA) plays a role in the pathogenesis of multiple solid tumors and hematological malignancies. MNK1 and MNK2 integrate signals from several oncogenic and immune signaling pathways, including 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 a subset of cellular mRNA. eFT508 is a potent, highly selective, and orally bioavailable MNK1 and MNK2 inhibitor. eFT508 has a half-maximal inhibitory concentration (IC50) of 1-2 nM against both MNK isoforms in enzyme assays and inhibits the kinase through a reversible, ATP-competitive mechanism of action. 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. These findings are consistent with MNK1 phosphorylation of specific RNA-binding proteins, eg, hnRNPA1, that regulate the stability and translation of mRNA containing specific AU-rich elements (ARE) in their 3'-untranslated regions (UTR). Pro-inflammatory cytokines are drivers of key hallmarks of cancer including tumor cell survival, migration and invasion, angiogenesis, and immune evasion, while also driving drug resistance. Therefore, 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. In addition, eFT508 combined effectively with components of R-CHOP and with novel targeted agents, including ibrutinib and venetoclax, in human lymphoma models. These results underscore the potential of eFT508 for the treatment of DLBCL. eFT508 has also been characterized in nonclinical safety pharmacology and toxicology studies. Clinical trials in patients with hematological and other malignancies are planned.[1]

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Cancer cells develop mechanisms to escape immunosurveillance, among which modulating the expression of immune suppressive messenger RNAs is most well-documented. However, how this is molecularly achieved remains largely unresolved. Here, we develop an in vivo mouse model of liver cancer to study oncogene cooperation in immunosurveillance. We show that MYC overexpression (MYCTg) synergizes with KRASG12D to induce an aggressive liver tumor leading to metastasis formation and reduced mouse survival compared with KRASG12D alone. Genome-wide ribosomal footprinting of MYCTg;KRASG12 tumors compared with KRASG12D revealed potential alterations in translation of mRNAs, including programmed-death-ligand 1 (PD-L1). Further analysis revealed that PD-L1 translation is repressed in KRASG12D tumors by functional, non-canonical upstream open reading frames in its 5' untranslated region, which is bypassed in MYCTg;KRASG12D tumors to evade immune attack. We show that this mechanism of PD-L1 translational upregulation was effectively targeted by a potent, clinical compound that inhibits eIF4E phosphorylation, eFT508, which reverses the aggressive and metastatic characteristics of MYCTg;KRASG12D tumors. Together, these studies reveal how immune-checkpoint proteins are manipulated by distinct oncogenes at the level of mRNA translation, which can be exploited for 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

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.)