Targeting the translational machinery in aggressive cancers

Eukaryotic initiation factor 4E (eIF4E) is a key focus in cancer research due to its central role in controlling the translation of tumour-associated proteins that drive an aggressive migratory phenotype. eIF4E activity, modulated via its availability and phosphorylation are regulated by the PI3K/AKT/mTOR and mitogen-activated protein kinase interacting protein kinases (MNK1/2). The latter phosphorylates eIF4E on Ser209 whereas mTORC1 phosphorylates and de-activates the eIF4E inhibitor, 4E-BP1, to release translational repression. The work presented here describes the synthesis and characterisation of 4-((4- fluoro-2-isopropoxyphenyl)amino)-5-methyl thieno[2,3-d] pyrimidine-6- carboylic acid, known as compound 1, a MNK1/2 inhibitor. Further analysis of compound 1 in combination with mTORC1/2 inhibitors show that inhibiting these pathways simultaneously effectively slows the rate of cell migration in MDA-MB-231 triple negative breast cancer (TNBC) cells. As an alternative approach, novel, cleavable dual MNK1/2 and PI3K/mTOR inhibiting hybrids were synthesised and characterised in MDA-MB-231 cells. These were found to be less effective at slowing cell migration than the combination of individual inhibitors. Molecular modelling of compound 1 revealed a large hydrophobic pocket which was exploited with a bulkier ferrocene group. Two novel ferrocene-containing compounds based upon compound 1 were synthesised and screened for MNK1/2 inhibition. To target migration more specifically, work was also carried out with an alternative translational protein, DDX3X. Both genetic knockdown and pharmacological inhibition alone and in combination with compound 1 reveal its potential as an anti-cancer target

Synergistic effects of inhibiting the MNK-eIF4E and PI3K/AKT/mTOR pathways on cell migration in MDA-MB-231 cells

The study of eukaryotic initiation factor 4E (eIF4E) is a key focus in cancer research due to its role in controlling the translation of tumour-associated proteins, that drive an aggressive migratory phenotype. eIF4E is a limiting component of the eIF4F complex which is a critical determinant for the translation of mRNAs. Mitogenactivated protein kinase interacting protein kinases (MNK1/2) phosphorylate eIF4E on Ser209, promoting the expression of oncogenic proteins, whereas mTORC1 phosphorylates and de-activates the eIF4E inhibitor, 4E-BP1, to release translational repression. Here we show that inhibiting these pathways simultaneously effectively slows the rate of cell migration in breast cancer cells. However, a molecular hybridisation approach using novel, cleavable dual MNK1/2 and PI3K/mTOR inhibiting hybrid agents was less effective at slowing cell migration

Dual abrogation of Mnk and mTOR; a novel therapeutic approach for the treatment of aggressive cancers

Targeting the translational machinery has emerged as a promising therapeutic option for cancer treatment. Cancer cells require elevated protein synthesis for cell growth and exhibit augmented activity to meet the increased metabolic demand. Eukaryotic translation initiation factor 4E (eIF4E) is necessary for mRNA translation, its availability and phosphorylation are regulated by the PI3K/AKT/mTOR and Mnk1/2 pathways, respectively. The phosphorylated form of eIF4E drives the expression of oncogenic proteins including those involved in metastasis. This article will review the role of eIF4E in cancer, its regulation, and discuss the benefit of dual-inhibition of upstream pathways. The discernible interplay between the Mnk1/2 and mTOR signaling pathways provides a novel therapeutic opportunity to target aggressive migratory cancers through the development of hybrid molecules

Probing the anticancer action of novel ferrocene analogues of MNK inhibitors

Two novel ferrocene-containing compounds based upon a known MNK1/2 kinase (MAPK-interacting kinase) inhibitor have been synthesized. The compounds were designed to use the unique shape of ferrocene to exploit a large hydrophobic pocket in MNK1/2 that is only partially occupied by the original compound. Screening of the ferrocene analogues showed that both exhibited potent anticancer effects in several breast cancer and AML (acute myeloid leukemia) cell lines, despite a loss of MNK potency. The most potent ferrocene-based compound 5 was further analysed in vitro in MDA-MB-231 (triple negative breast cancer cells). Dose–response curves of compound 5 for 2D assay and 3D assay generated IC50 values (half maximal inhibitory concentration) of 0.55 µM and 1.25 µM, respectively

Synthesis and biological investigation of (+)-JD1, an organometallic BET bromodomain inhibitor

(+)-JD1, a rationally designed ferrocene analogue of the BET bromodomain (BRD) probe molecule (+)-JQ1, has been synthesized and evaluated in biophysical, cell-based assays as well as in pharmacokinetic studies. It displays nanomolar activity against BRD isoforms, and its cocrystal structure was determined in complex with the first bromodomain of BRD4 and compared with that of (+)-JQ1, a known BRD4 small-molecule probe. At 1 μM concentration, (+)-JD1 was able to inhibit c-Myc, a key driver in cancer and an indirect target of BRD4

The helicase, DDX3X, interacts with poly(A) binding protein 1 (PABP1) and caprin-1 at the leading edge of migrating fibroblasts and is required for efficient cell spreading

DDX3X, a helicase, can interact directly with mRNA and translation initiation factors, regulating the selective translation of mRNAs that contain a structured 5′ untranslated region (5’UTR). This activity modulates the expression of mRNAs controlling cell cycle progression and mRNAs regulating actin dynamics, contributing to cell adhesion and motility. Previously, we have shown that ribosomes and translation initiation factors localise to the leading edge of migrating fibroblasts in loci enriched with actively translating ribosomes, thereby promoting steady-state levels of ArpC2 and Rac1 proteins at the leading edge of cells during spreading. As DDX3X can regulate Rac1 levels, cell motility and metastasis, we have examined DDX3X protein interactions and localisation using a number of complementary approaches. We now show that DDX3X can physically interact and co-localise with PABP1 and caprin-1 at the leading edge of spreading cells. Furthermore, as depletion of DDX3X leads to decreased cell motility, this provides a functional link between DDX3X, caprin-1 and initiation factors at the leading edge of migrating cells to promote cell migration and spreading