Developing anti-neoplastic biotherapeutics against eIF4F

Biotherapeutics have revolutionized modern medicine by providing medicines that would not have been possible with small molecules. With respect to cancer therapies, this represents the current sector of the pharmaceutical industry having the largest therapeutic impact, as exemplified by the development of recombinant antibodies and cell-based therapies. In cancer, one of the most common regulatory alterations is the perturbation of translational control. Among these, changes in eukaryotic initiation factor 4F (eIF4F) are associated with tumor initiation, progression, and drug resistance in a number of settings. This, coupled with the fact that systemic suppression of eIF4F appears well tolerated, indicates that therapeutic agents targeting eIF4F hold much therapeutic potential. Here, we discuss opportunities offered by biologicals for this purpose.     

Selective susceptibility to nanosecond pulsed electric field (nsPEF) across different human cell types

Tumor ablation by nanosecond pulsed electric fields (nsPEF) is an emerging therapeutic modality. We compared nsPEF cytotoxicity for human cell lines of cancerous (IMR-32, Hep G2, HT-1080, and HPAF-II) and non-cancerous origin (BJ and MRC-5) under strictly controlled and identical conditions. Adherent cells were uniformly treated by 300-ns PEF (0–2000 pulses, 1.8 kV/cm, 50 Hz) on indium tin oxide-covered glass coverslips, using the same media and serum. Cell survival plotted against the number of pulses displayed three distinct regions (initial resistivity, logarithmic survival decline, and residual resistivity) for all tested cell types, but with differences in LD₅₀ spanning as much as nearly 80-fold. The non-cancerous cells were less sensitive than IMR-32 neuroblastoma cells but more vulnerable than the other cancers tested. The cytotoxic efficiency showed no apparent correlation with cell or nuclear size, cell morphology, metabolism level, or the extent of membrane disruption by nsPEF. Increasing pulse duration to 9 µs (0.75 kV/cm, 5 Hz) produced a different selectivity pattern, suggesting that manipulation of PEF parameters can, at least for certain cancers, overcome their resistance to nsPEF ablation. Identifying mechanisms and cell markers of differential nsPEF susceptibility will critically contribute to the proper choice and outcome of nsPEF ablation therapies.     

Opposing effects on the cell cycle of T lymphocytes by Fbxo7 via Cdk6 and p27

G₁ phase cell cycle proteins, such as cyclin-dependent kinase 6 (Cdk6) and its activating partners, the D-type cyclins, are important regulators of T-cell development and function. An F-box protein, called F-box only protein 7 (Fbxo7), acts as a cell cycle regulator by enhancing cyclin D-Cdk6 complex formation and stabilising levels of p27, a cyclin-dependent kinase inhibitor. We generated a murine model of reduced Fbxo7 expression to test its physiological role in multiple tissues and found that these mice displayed a pronounced thymic hypoplasia. Further analysis revealed that Fbxo7 differentially affected proliferation and apoptosis of thymocytes at various stages of differentiation in the thymus and also mature T-cell function and proliferation in the periphery. Paradoxically, Fbxo7-deficient immature thymocytes failed to undergo expansion in the thymus due to a lack of Cdk6 activity, while mature T cells showed enhanced proliferative capacity upon T-cell receptor engagement due to reduced p27 levels. Our studies reveal differential cell cycle regulation by Fbxo7 at different stages in T-cell development.     

Advances in anti-viral immune defence: revealing the importance of the IFN JAK/STAT pathway

Interferon-alpha (IFN-α) is a potent anti-viral cytokine, critical to the host immune response against viruses. IFN-α is first produced upon viral detection by pathogen recognition receptors. Following its expression, IFN-α embarks upon a complex downstream signalling cascade called the JAK/STAT pathway. This signalling pathway results in the expression of hundreds of effector genes known as interferon stimulated genes (ISGs). These genes are the basis for an elaborate effector mechanism and ultimately, the clearance of viral infection. ISGs mark an elegant mechanism of anti-viral host defence that warrants renewed research focus in our global efforts to treat existing and emerging viruses. By understanding the mechanistic role of individual ISGs we anticipate the discovery of a new “treasure trove” of anti-viral mediators that may pave the way for more effective, targeted and less toxic anti-viral therapies. Therefore, with the aim of highlighting the value of the innate type 1 IFN response in our battle against viral infection, this review outlines both historic and recent advances in understanding the IFN-α JAK/STAT pathway, with a focus on new research discoveries relating to specific ISGs and their potential role in curing existing and future emergent viral infections.     

Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD

The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.     

Disruption of calcitonin gene-related peptide signaling accelerates muscle denervation and dampens cytotoxic neuroinflammation in SOD1 mutant mice

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Neuronal vacuolization and glial activation are pathologic hallmarks in the superoxide dismutase 1 (SOD1) mouse model of ALS. Previously, we found the neuropeptide calcitonin gene-related peptide (CGRP) associated with vacuolization and astrogliosis in the spinal cord of these mice. We now show that CGRP abundance positively correlated with the severity of astrogliosis, but not vacuolization, in several motor and non-motor areas throughout the brain. SOD1 mice harboring a genetic depletion of the βCGRP isoform showed reduced CGRP immunoreactivity associated with vacuolization, while motor functions, body weight, survival, and astrogliosis were not altered. When CGRP signaling was completely disrupted through genetic depletion of the CGRP receptor component, receptor activity-modifying protein 1 (RAMP1), hind limb muscle denervation, and loss of muscle performance were accelerated, while body weight and survival were not affected. Dampened neuroinflammation, i.e., reduced levels of astrogliosis in the brain stem already in the pre-symptomatic disease stage, and reduced microgliosis and lymphocyte infiltrations during the late disease phase were additional neuropathology features in these mice. On the molecular level, mRNA expression levels of brain-derived neurotrophic factor (BDNF) and those of the anti-inflammatory cytokine interleukin 6 (IL-6) were elevated, while those of several pro-inflammatory cytokines found reduced in the brain stem of RAMP1-deficient SOD1 mice at disease end stage. Our results thus identify an important, possibly dual role of CGRP in ALS pathogenesis.     

Immunological properties of oxygen-transport proteins: hemoglobin,hemocyanin and hemerythrin

It is now well documented that peptides with enhanced or alternative functionality (termed cryptides) can be liberated from larger, and sometimes inactive, proteins. A primary example of this phenomenon is the oxygen-transport protein hemoglobin. Aside from respiration, hemoglobin and hemoglobin-derived peptides have been associated with immune modulation, hematopoiesis, signal transduction and microbicidal activities in metazoans. Likewise, the functional equivalents to hemoglobin in invertebrates, namely hemocyanin and hemerythrin, act as potent immune effectors under certain physiological conditions. The purpose of this review is to evaluate the true extent of oxygen-transport protein dynamics in innate immunity, and to impress upon the reader the multi-functionality of these ancient proteins on the basis of their structures. In this context, erythrocyte–pathogen antibiosis and the immune competences of various erythroid cells are compared across diverse taxa.     

Intracellular localization of DR5 and related regulatory pathways as a mechanism of resistance to TRAIL in cancer

TNF-related apoptosis-inducing ligand (TRAIL) is a prominent cytokine capable of inducing apoptosis. It can bind to five different cognate receptors, through which diverse intracellular pathways can be activated. TRAIL’s ability to preferentially kill transformed cells makes it a promising potential weapon for targeted tumor therapy. However, recognition of several resistance mechanisms to TRAIL-induced apoptosis has indicated that a thorough understanding of the details of TRAIL biology is still essential before this weapon can be confidently unleashed. Critical to this aim is revealing the functions and regulation mechanisms of TRAIL’s potent death receptor DR5. Although expression and signaling mechanisms of DR5 have been extensively studied, other aspects, such as its subcellular localization, non-signaling functions, and regulation of its membrane transport, have only recently attracted attention. Here, we discuss different aspects of TRAIL/DR5 biology, with a particular emphasis on the factors that seem to influence the cell surface expression pattern of DR5, along with factors that lead to its nuclear localization. Disturbance of this balance apparently affects the sensitivity of cancer cells to TRAIL-mediated apoptosis, thus constituting an eligible target for potential new therapeutic agents.     

Endothelial cell apoptosis in angiogenesis and vessel regression

Blood vessel regression is an essential process for ensuring blood vessel networks function at optimal efficiency and for matching blood supply to the metabolic needs of tissues as they change over time. Angiogenesis is the major mechanism by which new blood vessels are produced, but the vessel growth associated with angiogenesis must be complemented by remodeling and maturation events including the removal of redundant vessel segments and cells to fashion the newly forming vasculature into an efficient, hierarchical network. This review will summarize recent findings on the role that endothelial cell apoptosis plays in vascular remodeling during angiogenesis and in vessel regression more generally.