Mitochondrial Dysfunction and Polo-Like Kinases

mitochondriaThe observation that cancer cells have a high rate of glycolysis, even in the presence of oxygen, was first made by Otto Warburg in 1924. Warburg assumed that the reason for this was a mitochondrial malfunction in cancer cells that drove a dependency on anaerobic glycolysis to generate ATP. Subsequently, it has been shown that the Warburg Effect is in operation even in cancer cells with intact mitochondria. It has been hypothesised that cancer cells exploit the less efficient glycolytic route to ATP to meet the high energy requirements of rapid proliferation and/or to provide a defence against the highly oxidative environment in which they survive. Indeed, increased glucose metabolism provides cancer cells with high levels of antioxidants, ATP and metabolites for growth. Down-regulation of mitochondrial activity further protects cancer cells from apoptosis. Understanding of the mechanisms underlying the Warburg Effect is still far from complete (recently reviewed), but the pathways remain an intense area of study to identify new approaches to cancer chemotherapy.

Scientists at the Translational Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health and Kyoto Prefectural University of Medicine, Kyoto have now reported that ablation of mitochondrial respiration markedly increases expression of Polo-like kinase 2 (PLK2). In these cells, PLK2 is required for in vitro growth and the PLK2-mediated phosphorylation of Ser-137 of Polo-like kinase 1 (PLK1) is sufficient for survival. The researchers also showed that, in vivo, knockdown of PLK2 in an isogenic human cell line with a modest defect in mitochondrial respiration eliminated xenograft formation, indicating that PLK2 activity is necessary for growth of cells with compromised respiration.

The study is reported in the August 11th early edition of Proceedings of the National Academy of Sciences.

Resveratrol’s Anti-inflammatory Effect Linked to Sphingosine Kinase

red wineSince the early 1990s, when it was first suggested that the presence of the anti-oxidant, resveratrol, could explain the cardioprotective effects of red wine, the health benefits attributed to this compound have grown and grown. Based largely on studies in simple organisms and in rodents, resveratrol has been credited with cardioprotective and neuroprotective properties, as well as with anti-diabetic, anti-cancer, anti-inflammatory, and anti-ageing powers. Resveratrol has been shown to interact with numerous biochemical pathways: the anti-ageing properties of resveratrol have been linked to activation of sirtuins, and researchers at the University of Glasgow and the University of Singapore have now identified a pathway involved in its anti-inflammatory effects.

Pretreatment of human or mouse neutrophils with resveratrol was found to significantly block oxidative burst, leukocyte migration, degranulation, and inflammatory cytokine production caused by the inflammatory mediator, C5 anaphylatoxin. The anti-inflammatory activity of resveratrol was attributed to its ability to inhibit sphingosine kinase (IC50 ~20µM) and so prevent activation of phospholipase D. Resveratrol also blocked ERK1/2 phosphorylation independently of sphingosine kinase. In further studies, prior injection of resveratrol reduced the inflammatory response of mice to challenge with C5 anaphylatoxin. The authors suggest that inhibition of sphingosine kinase by resveratrol could provide an effective treatment for systemic sepsis, appendicitis, and peritonitis, which are currently very difficult to treat. The study is published in the August print issue of FASEB Journal.

Given the profusion of biochemical targets that are being uncovered for resveratrol, it will be very interesting to see whether analogues that are being optimised specifically for one pathway retain the full activity of the parent compound in selected animal studies.

New Hope for Damaged Hearts

Scars of HeartA number of organs, including the heart, have limited regenerative powers, but US scientists have now shown that fully differentiated cardiac muscle cells can be induced to proliferate and regenerate. Writing in the journal Cell, they show that the growth factor neuregulin 1, which plays a role in early development of the heart and nervous system, induces mononucleated, but not binucleated, cardiomyocytes to divide in vitro by acting on the receptor tyrosine kinase, ErbB4. In mice, genetic inactivation of ErbB4 was shown to reduce cardiomyocyte proliferation, whereas increasing ErbB4 expression enhanced proliferation. Following heart attack in adult mice, daily intraperitoneal injection of neuregulin 1 for 12 weeks led to regeneration of the heart muscle and improved function. Unlike the control mice, the treated animals showed reduced signs of heart failure such as left-ventricular dilation and cardiac hypertrophy. If the neuregulin/ErbB4 signalling pathway plays the same role in human heart muscle, stimulating proliferation of differentiated cardiomyocytes by activation of this pathway may provide an alternative to stem cell therapy to regenerate damaged heart muscle in patients with heart failure or children with congenital heart defects. Since ErbB receptor tyrosine kinases and neuregulins have oncogenic potential and may cause proliferation of other tissues, a full safety assessment would be needed before any clinical studies.

Neuregulin 1 has previously been associated with susceptibility to schizophrenia and has also been shown to protect neurones following stroke.

Host Kinases Linked to HCV Infection

geodesic domeGlobally, around 200 million people are infected with Hepatitis C virus (HCV). Infection generally leads to chronic liver disease, albeit over a period of decades, which can lead to cirrhosis, hepatocellular carcinoma and liver failure. Current treatment, a combination of pegylated interferon-α and ribavirin, is only effective in 50-85% of patients, dependent on viral genotype. Not surprisingly, much effort is being expended to identify new therapeutic interventions.

Study of the viral lifecycle has been difficult in the past since serum-derived HCV (sHCV) replicates poorly in primary human hepatocytes and hepatoma cells in vitro. Surrogate systems have been developed, however, that have enabled reproduction of all steps of the HCV replication cycle, including cell entry (recently reviewed).

Viral cell entry is still incompletely understood, but a new study from researchers at the University of Rennes 1 has identified host kinases involved in HCV infectivity. Using a small-interfering RNA (siRNA) library, the scientists have shown that knock-down of phosphatidylinositol 4-kinase type III-α (PI4KIIIα) prevents infection by either HCV pseudoparticles (HCVpp) or by cell-culture grown JFH-1-based HCV (HCVcc). A second PI4K-family member, PI4KIIIβ, also influenced cellular susceptibility to HCVpp infection and the ability of the cells to sustain HCV replication. These kinases may therefore represent new targets for HCV therapeutics.

The study is published in the FASEB Journal.

Targeting ‘Normal’ Proteins to Kill Cancer Cells

For the most part, cancer therapy has been aimed at exploiting pathways that are present in cancer cells and not in normal cells but two studies published in the May 29th issue of the journal Cell suggest potential for an alternative approach. arrowBlocking the activity of oncogenic protein kinases – either with antibodies or small molecules – has become an important field of interest in cancer research but, despite the prevalence of RAS mutations in human tumours, inhibition of oncogenic RAS has not been realised as a therapeutic strategy. As an alternative to directly targeting RAS, US researchers have now identified ‘normal’ genes that are needed for cell survival in the presence of mutant, but not wild-type, KRAS.

To identify genes that are essential for survival only in the context of mutant KRAS, the researchers used a short hairpin RNA (shRNA) library to carry out high-throughput loss-of-function RNA interference (RNAi) screens in cancer cell lines as well as in normal cells. Dozens of potential drug targets were identified including serine/threonine kinase 33 (STK33) and mitotic polo-like kinase 1 (PLK1).

Patients with KRAS tumours are more likely to survive if they also have reduced expression of genes in the PLK1 pathway, suggesting that PLK1 inhibitors may have the potential to prolong survival. Although not required by normal cells, STK33 was found to be essential for the survival of cancer cells, irrespective of their tissue of origin, again suggesting therapeutic potential for inhibitors.

As well as identifying new targets for which it may be possible to develop therapeutically useful inhibitors, the two studies demonstrate the potential of RNAi screens to discover functional dependencies between oncogenes and normal genes in cancer cells. Targeting proteins which are essential for the survival of cancer cells, but not normal cells, could lead to a substantial therapeutic window, especially if only partial knock-down is needed to kill cancer cells.

IKKβ Linked to Huntington’s Disease

George HuntingtonThe neurodegenerative disorder, Huntington’s Disease (HD, Huntington’s Chorea), is caused by mutations in the gene for the protein Huntingtin (Htt). Mutant Htt (mHtt) results when the number of trinucleotide repeats, in this case the CAG sequence encoding glutamine, exceeds a threshold value. Typically, HD affects patients when the number of repeats is greater than 35.

Although the mechanisms by which mHtt causes the disease are poorly understood, proteolysis of the mutant protein is key and neurotoxicity is attributed to the cleaved N-terminal fragments of mHtt. Scientists at the California Institute of Technology and the Wallenberg Neuroscience Center, Lund, Sweden have now reported that the pro-inflammatory kinase, IKKβ , can regulate mHtt proteolysis in response to neuronal DNA damage.

The team demonstrated that DNA damage in neurons induced by etoposide or γ-irradiation results in cleavage of both wild-type and mutant Htt and that the proteolysis requires IKKβ. Elevation of IKKα, inhibition of IKKβ expression or inhibition of IKKβ catalytic activity all suppressed proteolysis and increased neuronal resistance to DNA damage.

Since elevated neuronal DNA damage is observed in HD patients and animal models of HD, inhibition of IKKβ activity may represent a treatment option for the disease. The study is published in the journal PLoS one.

Just Another Kinase Structure?

crystal structure of the kinase domain of JAK-1The Janus family of protein tyrosine kinases is comprised of four members: JAK-1, JAK-2, JAK-3 and Tyk-2. These kinases provide membrane proximal signalling through association with type 1 and type 2 cytokine receptors, phosphorylating and activating Signal Transducers and Activators of Transcription proteins (STATs) in response to cytokine binding. Expression of JAK-3 is predominantly restricted to haematopoietic cells, whilst the other family members are ubiquitously expressed. Inhibitors of these kinases have received interest since aberrant JAK activity has been associated with a variety of hematopoietic malignancies, cardiovascular diseases and immune-related disorders. A number of clinical studies are in progress to evaluate JAK inhibitors in transplantation, myelofibrosis, polycythaemia vera and essential thrombocythaemia.

Crystal structures of the kinase domains of JAK-2 and JAK-3 have previously been solved, enabling structure-based design of inhibitors. Now collaborators at Cytopia Research and Monash University have published the high-resolution crystal structure of the JAK-1 kinase domain. Whilst the ATP-binding sites of protein kinases are highly conserved, particularly amongst family members, the researchers have identified subtle differences surrounding the JAK1 and JAK2 ATP-binding sites. There is no doubt that developing JAK-1 or JAK-2 selective inhibitors (at the ATP-site) will be challenging, but the new data at least suggest that it could be possible. Whether it is necessary or desirable to achieve such selectivity is, as yet, unclear.

Full details are published in the Journal of Molecular Biology.

GSK-3 Inhibitors Help Maintain Pluripotency

stem cellsAlthough the use of embryonic stem cells is controversial and hotly debated from both sides, many researchers believe that these cells offer the promise of revolutionary treatments for a wide variety of diseases and injuries, including spinal cord injuries and degenerative diseases.

Embryonic stem cells are derived from the inner cell mass of an early stage embryo and are pluripotent, meaning that they are able to differentiate into any of the more than 200 cell types that make up the human body. For their full potential to be realised, it is important to maintain this pluripotency whilst growing them in cell culture. Glycogen synthase kinase 3 (GSK-3) had previously been implicated as a regulator of both self-renewal and differentiation, and a study published in the journal Chemistry and Biology now clarifies the role of GSK-3 in murine embryonic stem cell development by showing that inhibitors of GSK-3 enhance self-renewal in the presence of serum and leukaemia inhibitory factor. The authors propose that, by inhibiting GSK-3 activity in the appropriate cell culture environment, it will be possible to more easily obtain large numbers of undifferentiated, pluripotent, cells for medical use. Once the inhibitor is removed from the cell culture medium, it is possible to induce the stem cells to differentiate into the chosen type of specialised cells.
gsk-3 inhibitor

Role for CDK5 in Adult Neurogenesis

For many years, it was been believed that we were born with all the brain cells we would ever have, and that new ones could not be formed. More recently, it has been shown that the adult human brain creates new neurons, a process known as neurogenesis. The most active area of neurogenesis is the hippocampus, an area of the brain important for learning and memory. Although the exact role of the newly created neurons is uncertain, to play a part in the acquisition and storage of memories they must locate themselves correctly and be able to communicate with pre-existing circuits in the brain. A new study published in PLoS Biology shows that cyclin-dependent kinase 5 (CDK5) plays a key role in this integration process. The scientists used retroviruses to alter the activity of CDK5, and found that more than 50% of CDK5-deficient cells failed to migrate to the right position in the brain or make the correct connections with surrounding neurons. Instead, the CDK5-deficient cells put out processes in the wrong direction and formed connections with the wrong cells. hippocampusCells that fail to integrate correctly could interfere with normal information processing. By increasing understanding of the factors needed for newly formed neurons to become properly integrated into brain, the study may suggest ways to increase the success of neural transplantation to treat brain injuries or diseases such as Parkinson’s disease.

Specificity or Polypharmacology?

As a target class, kinases have received considerable attention over the last 20 years. There are now 8 kinase inhibitors on the market, approved for use in a number of cancers, with many more in clinical development. Targeting of the catalytic ATP-binding site has proved the most fruitful in drug discovery, but the conserved nature of the site has presented challenges to identification of specific inhibitors. In fact all of the approved compounds inhibit more than one tyrosine kinase, although they maintain selectivity over the serine/threonine and phosphoinositide (PI) kinase classes. This multiple target activity has, however, proven advantageous in an oncology setting.

co-crystal structuresA collaborative group of researchers has recently reported the identification of dual inhibitors of tyrosine and PI kinases. Their rationale for searching for such compounds was the knowledge that reactivation of PI3 kinase signalling is a common mechanism of resistance to tyrosine kinase inhibitors. Furthermore, preclinical studies combining tyrosine and PI kinase inhibitors have demonstrated efficacy. Despite the sequence dissimilarity between tyrosine and PI kinases, the scientists were able to identify a number of molecules with novel selectivity profiles. In addition, one compound was discovered with unexpected specificity for mTOR compared to other members of the PI kinase family. Full details are reported in Nature Chemical Biology.