Zotatifin, an eIF4A-Selective Inhibitor, Blocks Tumor Growth in Receptor Tyrosine Kinase Driven Tumors.

Oncoprotein expression is controlled at the level of mRNA translation and is regulated by the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4A, a component of eIF4F, catalyzes the unwinding of secondary structure in the 5'-untranslated region (5'-UTR) of mRNA to facilitate ribosome scanning and translation initiation. Zotatifin (eFT226) is a selective eIF4A inhibitor that increases the affinity between eIF4A and specific polypurine sequence motifs and has been reported to inhibit translation of driver oncogenes in models of lymphoma. Here we report the identification of zotatifin binding motifs in the 5'-UTRs of HER2 and FGFR1/2 Receptor Tyrosine Kinases (RTKs). Dysregulation of HER2 or FGFR1/2 in human cancers leads to activation of the PI3K/AKT and RAS/ERK signaling pathways, thus enhancing eIF4A activity and promoting the translation of select oncogenes that are required for tumor cell growth and survival. In solid tumor models driven by alterations in HER2 or FGFR1/2, downregulation of oncoprotein expression by zotatifin induces sustained pathway-dependent anti-tumor activity resulting in potent inhibition of cell proliferation, induction of apoptosis, and significant in vivo tumor growth inhibition or regression. Sensitivity of RTK-driven tumor models to zotatifin correlated with high basal levels of mTOR activity and elevated translational capacity highlighting the unique circuitry generated by the RTK-driven signaling pathway. This dependency identifies the potential for rational combination strategies aimed at vertical inhibition of the PI3K/AKT/eIF4F pathway. Combination of zotatifin with PI3K or AKT inhibitors was beneficial across RTK-driven cancer models by blocking RTK-driven resistance mechanisms demonstrating the clinical potential of these combination strategies.

mRNA translation is a therapeutic vulnerability necessary for bladder epithelial transformation.

Using genetically engineered mouse models, this work demonstrates that protein synthesis is essential for efficient urothelial cancer formation and growth but dispensable for bladder homeostasis. Through a candidate gene analysis for translation regulators implicated in this dependency, we discovered that phosphorylation of the translation initiation factor eIF4E at serine 209 is increased in both murine and human bladder cancer, and this phosphorylation corresponds with an increase in de novo protein synthesis. Employing an eIF4E serine 209 to alanine knock-in mutant mouse model, we show that this single posttranslational modification is critical for bladder cancer initiation and progression, despite having no impact on normal bladder tissue maintenance. Using murine and human models of advanced bladder cancer, we demonstrate that only tumors with high levels of eIF4E phosphorylation are therapeutically vulnerable to eFT508, the first clinical-grade inhibitor of MNK1 and MNK2, the upstream kinases of eIF4E. Our results show that phospho-eIF4E plays an important role in bladder cancer pathogenesis, and targeting its upstream kinases could be an effective therapeutic option for bladder cancer patients with high levels of eIF4E phosphorylation.

Targeting mutant KRAS.

The protein KRAS has for decades been considered a holy grail of cancer drug discovery. For most of that time, it has also been considered undruggable. Since 2018, five compounds have entered the clinic targeting a single mutant form of KRAS, G12C. Here, we review each of these compounds along with additional approaches to targeting this and other mutants. Remaining challenges include expanding the identification of inhibitors to a broader range of known mutants and to conformations of the protein more likely to avoid development of resistance.

A Highly Selective MNK Inhibitor Rescues Deficits Associated with Fragile X Syndrome in Mice.

Fragile X syndrome (FXS) is the most common inherited source of intellectual disability in humans. FXS is caused by mutations that trigger epigenetic silencing of the Fmr1 gene. Loss of Fmr1 results in increased activity of the mitogen-activated protein kinase (MAPK) pathway. An important downstream consequence is activation of the mitogen-activated protein kinase interacting protein kinase (MNK). MNK phosphorylates the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E). Excessive phosphorylation of eIF4E has been directly implicated in the cognitive and behavioral deficits associated with FXS. Pharmacological reduction of eIF4E phosphorylation is one potential strategy for FXS treatment. We demonstrate that systemic dosing of a highly specific, orally available MNK inhibitor, eFT508, attenuates numerous deficits associated with loss of Fmr1 in mice. eFT508 resolves a range of phenotypic abnormalities associated with FXS including macroorchidism, aberrant spinogenesis, and alterations in synaptic plasticity. Key behavioral deficits related to anxiety, social interaction, obsessive and repetitive activities, and object recognition are ameliorated by eFT508. Collectively, this work establishes eFT508 as a potential means to reverse deficits associated with FXS.

Targeting Oncogene mRNA Translation in B-Cell Malignancies with eFT226, a Potent and Selective Inhibitor of eIF4A.

The PI3K/AKT/mTOR pathway is often activated in lymphoma through alterations in PI3K, PTEN, and B-cell receptor signaling, leading to dysregulation of eIF4A (through its regulators, eIF4B, eIF4G, and PDCD4) and the eIF4F complex. Activation of eIF4F has a direct role in tumorigenesis due to increased synthesis of oncogenes that are dependent on enhanced eIF4A RNA helicase activity for translation. eFT226, which inhibits translation of specific mRNAs by promoting eIF4A1 binding to 5'-untranslated regions (UTR) containing polypurine and/or G-quadruplex recognition motifs, shows potent antiproliferative activity and significant in vivo efficacy against a panel of diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma models with ≤1 mg/kg/week intravenous administration. Evaluation of predictive markers of sensitivity or resistance has shown that activation of eIF4A, mediated by mTOR signaling, correlated with eFT226 sensitivity in in vivo xenograft models. Mutation of PTEN is associated with reduced apoptosis in vitro and diminished efficacy in vivo in response to eFT226. In models evaluated with PTEN loss, AKT was stimulated without a corresponding increase in mTOR activation. AKT activation leads to the degradation of PDCD4, which can alter eIF4F complex formation. The association of eFT226 activity with PTEN/PI3K/mTOR pathway regulation of mRNA translation provides a means to identify patient subsets during clinical development.

Design of Development Candidate eFT226, a First in Class Inhibitor of Eukaryotic Initiation Factor 4A RNA Helicase.

Dysregulation of protein translation is a key driver for the pathogenesis of many cancers. Eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase, is a critical component of the eIF4F complex, which regulates cap-dependent protein synthesis. The flavagline class of natural products (i.e., rocaglamide A) has been shown to inhibit protein synthesis by stabilizing a translation-incompetent complex for select messenger RNAs (mRNAs) with eIF4A. Despite showing promising anticancer phenotypes, the development of flavagline derivatives as therapeutic agents has been hampered because of poor drug-like properties as well as synthetic complexity. A focused effort was undertaken utilizing a ligand-based design strategy to identify a chemotype with optimized physicochemical properties. Also, detailed mechanistic studies were undertaken to further elucidate mRNA sequence selectivity, key regulated target genes, and the associated antitumor phenotype. This work led to the design of eFT226 (Zotatifin), a compound with excellent physicochemical properties and significant antitumor activity that supports clinical development.

Translation control of the immune checkpoint in cancer and its therapeutic targeting.

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.

Nociceptor Translational Profiling Reveals the Ragulator-Rag GTPase Complex as a Critical Generator of Neuropathic Pain.

Nociceptors, sensory neurons in the DRG that detect damaging or potentially damaging stimuli, are key drivers of neuropathic pain. Injury to these neurons causes activation of translation regulation signaling, including the mechanistic target of rapamycin complex 1 (mTORC1) and mitogen-activated protein kinase interacting kinase (MNK) eukaryotic initiation factor (eIF) 4E pathways. This is a mechanism driving changes in excitability of nociceptors that is critical for the generation of chronic pain states; however, the mRNAs that are translated to lead to this plasticity have not been elucidated. To address this gap in knowledge, we used translating ribosome affinity purification in male and female mice to comprehensively characterize mRNA translation in Scn10a-positive nociceptors in chemotherapy-induced neuropathic pain (CIPN) caused by paclitaxel treatment. This unbiased method creates a new resource for the field, confirms many findings in the CIPN literature and also find extensive evidence for new target mechanisms that may cause CIPN. We provide evidence that an underlying mechanism of CIPN is sustained mTORC1 activation driven by MNK1-eIF4E signaling. RagA, a GTPase controlling mTORC1 activity, is identified as a novel target of MNK1-eIF4E signaling. This demonstrates a novel translation regulation signaling circuit wherein MNK1-eIF4E activity drives mTORC1 via control of RagA translation. CIPN and RagA translation are strongly attenuated by genetic ablation of eIF4E phosphorylation, MNK1 elimination or treatment with the MNK inhibitor eFT508. We identify a novel translational circuit for the genesis of neuropathic pain caused by chemotherapy with important implications for therapeutics.SIGNIFICANCE STATEMENT Neuropathic pain affects up to 10% of the population, but its underlying mechanisms are incompletely understood, leading to poor treatment outcomes. We used translating ribosome affinity purification technology to create a comprehensive translational profile of DRG nociceptors in naive mice and at the peak of neuropathic pain induced by paclitaxel treatment. We reveal new insight into how mechanistic target of rapamycin complex 1 is activated in neuropathic pain pointing to a key role of MNK1-eIF4E-mediated translation of a complex of mRNAs that control mechanistic target of rapamycin complex 1 signaling at the surface of the lysosome. We validate this finding using genetic and pharmacological techniques. Our work strongly suggests that MNK1-eIF4E signaling drives CIPN and that a drug in human clinical trials, eFT508, may be a new therapeutic for neuropathic pain.

Structure-based Design of Pyridone-Aminal eFT508 Targeting Dysregulated Translation by Selective Mitogen-activated Protein Kinase Interacting Kinases 1 and 2 (MNK1/2) Inhibition.

Dysregulated translation of mRNA plays a major role in tumorigenesis. Mitogen-activated protein kinase interacting kinases (MNK)1/2 are key regulators of mRNA translation integrating signals from oncogenic and immune signaling pathways through phosphorylation of eIF4E and other mRNA binding proteins. Modulation of these key effector proteins regulates mRNA, which controls tumor/stromal cell signaling. Compound 23 (eFT508), an exquisitely selective, potent dual MNK1/2 inhibitor, was designed to assess the potential for control of oncogene signaling at the level of mRNA translation. The crystal structure-guided design leverages stereoelectronic interactions unique to MNK culminating in a novel pyridone-aminal structure described for the first time in the kinase literature. Compound 23 has potent in vivo antitumor activity in models of diffuse large cell B-cell lymphoma and solid tumors, suggesting that controlling dysregulated translation has real therapeutic potential. Compound 23 is currently being evaluated in Phase 2 clinical trials in solid tumors and lymphoma. Compound 23 is the first highly selective dual MNK inhibitor targeting dysregulated translation being assessed clinically.

Synthesis and biological activity of N-aryl-2-aminothiazoles: potent pan inhibitors of cyclin-dependent kinases.

N-Aryl aminothiazoles 6-9 were prepared from 2-bromothiazole 5 and found to be CDK inhibitors. In cells they act as potent cytotoxic agents. Selectivity for CDK1, CDK2, and CDK4 was dependent of the nature of the N-aryl group and distinct from the CDK2 selective N-acyl analogues. The N-2-pyridyl analogues 7 and 19 showed pan CDK inhibitory activity. Elaborated analogues 19 and 23 exhibited anticancer activity in mice against P388 murine leukemia. The solid-state structure of 7 bound to CDK2 shows a similar binding mode to the N-acyl analogues.

N-(cycloalkylamino)acyl-2-aminothiazole inhibitors of cyclin-dependent kinase 2. N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4- piperidinecarboxamide (BMS-387032), a highly efficacious and selective antitumor agent.

N-Acyl-2-aminothiazoles with nonaromatic acyl side chains containing a basic amine were found to be potent, selective inhibitors of CDK2/cycE which exhibit antitumor activity in mice. In particular, compound 21 [N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide, BMS-387032], has been identified as an ATP-competitive and CDK2-selective inhibitor which has been selected to enter Phase 1 human clinical trials as an antitumor agent. In a cell-free enzyme assay, 21 showed a CDK2/cycE IC(50) = 48 nM and was 10- and 20-fold selective over CDK1/cycB and CDK4/cycD, respectively. It was also highly selective over a panel of 12 unrelated kinases. Antiproliferative activity was established in an A2780 cellular cytotoxicity assay in which 21 showed an IC(50) = 95 nM. Metabolism and pharmacokinetic studies showed that 21 exhibited a plasma half-life of 5-7 h in three species and moderately low protein binding in both mouse (69%) and human (63%) serum. Dosed orally to mouse, rat, and dog, 21 showed 100%, 31%, and 28% bioavailability, respectively. As an antitumor agent in mice, 21 administered at its maximum-tolerated dose exhibited a clearly superior efficacy profile when compared to flavopiridol in both an ip/ip P388 murine tumor model and in a s.c./i.p. A2780 human ovarian carcinoma xenograft model.

1H-Pyrazolo[3,4-b]pyridine inhibitors of cyclin-dependent kinases: highly potent 2,6-Difluorophenacyl analogues.

Structure-activity studies of 1H-pyrazolo[3,4-b]pyridine 1 have resulted in the discovery of potent CDK1/CDK2 selective inhibitor 21h, BMS-265246 (CDK1/cycB IC(50)=6 nM, CDK2/cycE IC(50)=9 nM). The 2,6-difluorophenyl substitution was critical for potent inhibitory activity. A solid state structure of 21j, a close di-fluoro analogue, bound to CDK2 shows the inhibitor resides coincident with the ATP purine binding site and forms important H-bonds with Leu83 on the protein backbone.

1H-Pyrazolo[3,4-b]pyridine inhibitors of cyclin-dependent kinases.

1H-Pyrazolo[3,4-b]pyridine 3 (SQ-67563) has been shown to be a potent, selective inhibitor of CDK1/CDK2 in vitro. In cells 3 acts as a cytotoxic agent with the ability to block cell cycle progression and/or induce apoptosis. The solid state structure of 3 bound to CDK2 shows 3 resides coincident with the ATP purine binding site and forms important H-bonding interactions with Leu83 on the protein backbone.

Discovery of aminothiazole inhibitors of cyclin-dependent kinase 2: synthesis, X-ray crystallographic analysis, and biological activities.

High throughput screening identified 2-acetamido-thiazolylthio acetic ester 1 as an inhibitor of cyclin-dependent kinase 2 (CDK2). Because this compound is inactive in cells and unstable in plasma, we have stabilized it to metabolic hydrolysis by replacing the ester moiety with a 5-ethyl-substituted oxazole as in compound 14. Combinatorial and parallel synthesis provided a rapid analysis of the structure-activity relationship (SAR) for these inhibitors of CDK2, and over 100 analogues with IC(50) values in the 1-10 nM range were rapidly prepared. The X-ray crystallographic data of the inhibitors bound to the active site of CDK2 protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues displayed potent and broad spectrum antiproliferative activity across a panel of tumor cell lines in vitro. In addition, A2780 ovarian carcinoma cells undergo rapid apoptosis following exposure to CDK2 inhibitors of this class. Mechanism of action studies have confirmed that the phosphorylation of CDK2 substrates such as RB, histone H1, and DNA polymerase alpha (p70 subunit) is reduced in the presence of compound 14. Further optimization led to compounds such as water soluble 45, which possesses a favorable pharmacokinetic profile in mice and demonstrates significant antitumor activity in vivo in several murine and human models, including an engineered murine mammary tumor that overexpresses cyclin E, the coactivator of CDK2.