Nigel Ribeiro

eIF4F is a nexus of resistance to anti-BRAF and anti-MEK cancer therapies

In BRAF(V600)-mutant tumours, most mechanisms of resistance to drugs that target the BRAF and/or MEK kinases rely on reactivation of the RAS–RAF–MEK–ERK mitogen-activated protein kinase (MAPK) signal transduction pathway, on activation of the alternative, PI(3)K–AKT–mTOR, pathway (which is ERK independent) or on modulation of the caspase-dependent apoptotic cascade. All three pathways converge to regulate the formation of the eIF4F eukaryotic translation initiation complex, which binds to the 7-methylguanylate cap (m7G) at the 5′ end of messenger RNA, thereby modulating the translation of specific mRNAs. Here we show that the persistent formation of the eIF4F complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, is associated with resistance to anti-BRAF, anti-MEK and anti-BRAF plus anti-MEK drug combinations in BRAF(V600)-mutant melanoma, colon and thyroid cancer cell lines. Resistance to treatment and maintenance of eIF4F complex formation is associated with one of three mechanisms: reactivation of MAPK signalling, persistent ERK-independent phosphorylation of the inhibitory eIF4E-binding protein 4EBP1 or increased pro-apoptotic BCL-2-modifying factor (BMF)-dependent degradation of eIF4G. The development of an in situ method to detect the eIF4E–eIF4G interactions shows that eIF4F complex formation is decreased in tumours that respond to anti-BRAF therapy and increased in resistant metastases compared to tumours before treatment. Strikingly, inhibiting the eIF4F complex, either by blocking the eIF4E–eIF4G interaction or by targeting eIF4A, synergizes with inhibiting BRAF(V600) to kill the cancer cells. eIF4F not only appears to be an indicator of both innate and acquired resistance but also is a promising therapeutic target. Combinations of drugs targeting BRAF (and/or MEK) and eIF4F may overcome most of the resistance mechanisms arising in BRAF(V600)-mutant cancers.

The natural anticancer compounds rocaglamides inhibit the Raf-MEK-ERK pathway by targeting prohibitin 1 and 2.

Rocaglamides are potent natural anticancer products that inhibit proliferation of various cancer cells at nanomolar concentrations. We have recently shown that these compounds prevent tumor growth and sensitize resistant cancer cells to apoptosis by blocking the MEK-ERK-eIF4 pathway. However, their direct molecular target(s) remain(s) unknown. In this study, using an affinity chromatography approach we discovered that prohibitin (PHB) 1 and 2 are the direct targets of rocaglamides. Binding of rocaglamides to PHB prevents interaction between PHB and CRaf and, thereby, inhibits CRaf activation and subsequently CRaf-MEK-ERK signaling. Moreover, knockdown of PHB mimicked the effects of rocaglamides on the CRaf-MEK-ERK pathway and cell cycle progression. Thus, our finding suggests that rocaglamides are a new type of anticancer agent and that they may serve as a small-molecular tool for studying PHB-mediated cellular processes.

Prohibitin Ligands in Cell Death and Survival: Mode of Action and Therapeutic Potential

Prohibitins (PHBs) are scaffold proteins that modulate many signaling pathways controlling cell survival, metabolism, and inflammation. Several drugs that target PHBs have been identified and evaluated for various clinical applications. Preclinical and clinical studies indicate that these PHB ligands may be useful in oncology, cardiology, and neurology, as well as against obesity. This review covers the physiological role of PHBs in health and diseases and current developments concerning PHB ligands.

Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser2. This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.

Flavaglines as Potent Anticancer and Cytoprotective Agents

Flavaglines represent a family of plant natural products that display potent anticancer, cardioprotective, and neuroprotective activities. Novel flavagline derivatives were synthesized and examined for their cytotoxicity on a panel of human cancer cell lines, their cardioprotection against doxorubicin-induced apoptosis in cardiomyocytes, and their neuroprotection in culture models of Parkinsons disease and cisplatin-induced neurotoxicity. The structural requirements of flavaglines for cardio- and neuroprotection were for the first time unraveled and appeared to be slightly different from those for cytotoxicity on cancer cells. We provide also the first evidence that flavaglines may alleviate cisplatin-induced neurotoxicity, suggesting a prophylactic potential of these compounds to prevent this frequently encountered adverse effect of cancer chemotherapies.

Flavaglines: potent anticancer drugs that target prohibitins and the helicase eIF4A

Flavaglines are complex natural products that are found in several medicinal plants of Southeast Asia in the genus Aglaia; these compounds have shown exceptional anticancer and cytoprotective activities. This review describes the significance of flavaglines as a new class of pharmacological agents and presents recent developments in their synthesis, structure-activity relationships, identification of their molecular targets and modes of action. Flavaglines display a unique profile of anticancer activities that are mediated by two classes of unrelated proteins: prohibitins and the translation initiation factor eIF4A. The identification of these molecular targets is expected to accelerate advancement toward clinical studies. The selectivity of cytotoxicity towards cancer cells has been shown to be due to an inhibition of the transcription factor HSF1 and an upregulation of the tumor suppressor TXNIP. In addition, flavaglines display potent anti-inflammatory, cardioprotective and neuroprotective activities; however, the mechanisms underlying these activities are yet to be elucidated.

Recent advances in the biology and chemistry of the flavaglines.

The flavaglines are a family of plant natural products that induce potent anticancer and neuroprotective activities. This review summarizes recent synthetic approaches to flavaglines and the current status of their pharmacological properties.

Flavaglines Alleviate Doxorubicin Cardiotoxicity: Implication of Hsp27

Background Despite its effectiveness in the treatment of various cancers, the use of doxorubicin is limited by a potentially fatal cardiomyopathy. Prevention of this cardiotoxicity remains a critical issue in clinical oncology. We hypothesized that flavaglines, a family of natural compounds that display potent neuroprotective effects, may also alleviate doxorubicin-induced cardiotoxicity. Methodology/Principal Findings Our in vitro data established that a pretreatment with flavaglines significantly increased viability of doxorubicin-injured H9c2 cardiomyocytes as demonstrated by annexin V, TUNEL and active caspase-3 assays. We demonstrated also that phosphorylation of the small heat shock protein Hsp27 is involved in the mechanism by which flavaglines display their cardioprotective effect. Furthermore, knocking-down Hsp27 in H9c2 cardiomyocytes completely reversed this cardioprotection. Administration of our lead compound (FL3) to mice attenuated cardiomyocyte apoptosis and cardiac fibrosis, as reflected by a 50% decrease of mortality. Conclusions/Significance These results suggest a prophylactic potential of flavaglines to prevent doxorubicin-induced cardiac toxicity.

Novel flavaglines displaying improved cytotoxicity.

Novel flavagline analogues were synthesized and examined with respect to their cytotoxicity. Structural features critical to the potential of this class of anticancer natural products were unraveled. We demonstrated, in particular, that the introduction of substituants at C-2 has a deleterious effect on multidrug resistance. Replacement of the hydroxy at C-1 by an aminoformyl with the opposite configuration enhances the cytotoxicity and led to a compound that reduces tumors growth in an allograft model at nontoxic doses.

Discovery and Cardioprotective Effects of the First Non-Peptide Agonists of the G Protein-Coupled Prokineticin Receptor-1

Prokineticins are angiogenic hormones that activate two G protein-coupled receptors: PKR1 and PKR2. PKR1 has emerged as a critical mediator of cardiovascular homeostasis and cardioprotection. Identification of non-peptide PKR1 agonists that contribute to myocardial repair and collateral vessel growth hold promises for treatment of heart diseases. Through a combination of in silico studies, medicinal chemistry, and pharmacological profiling approaches, we designed, synthesized, and characterized the first PKR1 agonists, demonstrating their cardioprotective activity against myocardial infarction (MI) in mice. Based on high throughput docking protocol, 250,000 compounds were computationally screened for putative PKR1 agonistic activity, using a homology model, and 10 virtual hits were pharmacologically evaluated. One hit internalizes PKR1, increases calcium release and activates ERK and Akt kinases. Among the 30 derivatives of the hit compound, the most potent derivative, IS20, was confirmed for its selectivity and specificity through genetic gain- and loss-of-function of PKR1. Importantly, IS20 prevented cardiac lesion formation and improved cardiac function after MI in mice, promoting proliferation of cardiac progenitor cells and neovasculogenesis. The preclinical investigation of the first PKR1 agonists provides a novel approach to promote cardiac neovasculogenesis after MI.