Ruth S. Kirk

Gatekeeper Mutations Mediate Resistance to BRAF-Targeted Therapies

Gatekeeper mutations in BRAF confer drug resistance. Getting Back on Target The identification of clinically useful small-molecule inhibitors of dysregulated proteins that trigger a cascade of detrimental downstream effects during the process of tumorigenesis requires a highly precise understanding of the specific residues responsible for the damage. However, although this information can elucidate promising candidate targets, clinical data have revealed that the utility of anticancer drugs within tumors of similar origin and mutational status is variable. It has been postulated that the haphazard effects of such drugs are the result of off-targeting effects—that is, the ability of a drug to inhibit tumor growth in a manner less specific than that which it was optimally designed for. In an era that now seeks to further personalize medicine, these off-targeting effects reveal how distant we remain from truly achieving an individualized cancer cocktail. In melanoma, the failure of the FDA-approved drug sorafenib to consistently curb disease in individuals with tumors that harbor cancer-driving mutations in BRAF—the overactive kinase presumed to be the target of the drug on the basis of its efficacy against CRAF—raised doubts as to the utility of BRAF as true melanoma target, despite its more successful application as a liver and kidney cancer therapeutic. Now, Marais and colleagues have designed a system by which to tease apart whether BRAF-mediated therapeutics such as sorafenib and PLX4720 inhibit tumor growth by homing in on the gatekeeper mutations that cause aberrant signaling in BRAF or by other nonspecific tumor-inhibiting mechanisms. Their compelling results reveal that BRAF is indeed a tractable target for melanoma, and while a plethora of tractable cancer targets are continuously being resolved, the need for proper preclinical validation—where the effects can easily be discerned as being mechanistic or off-target—is paramount to developing the right drug for the specific target. BRAF is a serine-threonine–specific protein kinase that is mutated in 2% of human cancers. Oncogenic BRAF is a validated therapeutic target that constitutively activates mitogen-activated protein kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) signaling, driving tumor cell proliferation and survival. Drugs designed to target BRAF have been developed, but it is difficult to prove that they mediate their antitumor effects by inhibiting BRAF rather than by working through off-target effects. We generated drug-resistant versions of oncogenic BRAF by mutating the gatekeeper residue. Signaling by the mutant proteins was resistant to the small-molecule inhibitor sorafenib, but sorafenib still inhibited the growth of tumors driven by the mutant protein. In contrast, both BRAF signaling and tumor growth were resistant to another RAF drug, PLX4720. These data provide unequivocal evidence that sorafenib mediates its antitumor effects in a manner that is independent of its ability to target oncogenic BRAF, whereas PLX4720 inhibits tumor growth by targeting oncogenic BRAF directly.

An evaluation of pollutant induced changes in the gills of rainbow trout using scanning electron microscopy

Abstract The effects of phenol, copper and ammonia on the surface ultrastructure of the gills of rainbow trout were determined by scanning electron microscopy (SEM). Although the gills exhibited some common pollutant‐induced changes, certain types of pathological response were pollutant‐specific. Phenol caused destruction of epithelial layers down to supporting cartilage. Copper damage was characterized by lamellar fusion, swelling in the tips of the filaments and formation of epithelial swellings and ridges. Ammonia caused the development of distinctive circular depressions and pits in the epithelium. These results suggest that SEM of fish gills should be evaluated further as a diagnostic tool in water pollution investigations.

Survival and transmission of Anguillicola crassus Kuwahara, Niimi & Itagaki, 1974 (Nematoda) in seawater eels.

The study aims were to quantify the survivorship of Anguillicola crassus in eels in seawater and investigate transmission in estuarine (50% seawater) and marine (100% seawater) simulated conditions. Most A. crassus were able to survive and reproduce in 50% and 100% seawater eels for up to 6 months and therefore could survive during the spawning migration of eels to the Sargasso Sea. Up to 10% of the parasite metapopulation were damaged after long-term exposure to 50% or 100% seawater. Transmission of A. crassus was completed in 50% and 100% seawater in the laboratory, although infection levels in 100% seawater hosts were always lower. Transmission in estuaries is ecologically possible since the copepod Eurytemora affinis is susceptible to infection and is the dominant autochthonous species in most tidal estuaries in the Northern hemisphere. Transmission at sea is unlikely due to lack of suitable intermediate hosts. The data support the suggestion that dissemination of A. crassus has been facilitated by natural movement of eels in coastal waters. It is possible that A. crassus could impair the success of the eel spawning migration to the Sargasso Sea, although there is no direct evidence to support this.

The life-cycle and morphology of Sanguinicola inermis Plehn, 1905 (Digenea: Sanguinicolidae)

The life-cycle and morphology of Sanguinicola inermis in laboratory infected common carp Cyprinus carpio and Lymnaea peregra at 20°C are described. Furcocercous cercariae develop from two generations of sporocysts within four to five weeks and directly infect a cyprinid definitive host. Post-penetration larvae migrate to the blood circulatory system of the fish and mature into adults which release eggs into the blood by 30 days post-exposure. Eggs reaching the gills mature and liberate miracidia within seven days. The miracidia emigrate through host tissue to the water, where they locate and penetrate a snail host. A detailed description of the developmental stages is given and the surface structure of the cercaria, the oral muscular organ of the adult and the vitelline membrane of the egg are described for the first time.

Nitric oxide production by Biomphalaria glabrata haemocytes: effects of Schistosoma mansoni ESPs and regulation through the extracellular signal-regulated kinase pathway

BackgroundSchistosoma mansoni uses Biomphalaria glabrata as an intermediate host during its complex life cycle. In the snail, the parasite initially transforms from a miracidium into a mother sporocyst and during this process excretory-secretory products (ESPs) are released. Nitric oxide (NO) and its reactive intermediates play an important role in host defence responses against pathogens. This study therefore aimed to determine the effects of S. mansoni ESPs on NO production in defence cells (haemocytes) from schistosome-susceptible and schistosome-resistant B. glabrata strains. As S. mansoni ESPs have previously been shown to inhibit extracellular signal-regulated kinase (ERK) phosphorylation (activation) in haemocytes from susceptible, but not resistant, B. glabrata the regulation of NO output by ERK in these cells was also investigated.ResultsHaemocytes from resistant snails challenged with S. mansoni ESPs (20 μg/ml) over 5 h displayed an increase in NO production that was 3.3 times greater than that observed for unchallenged haemocytes; lower concentrations of ESPs (0.1–10 μg/ml) did not significantly increase NO output. In contrast, haemocytes from susceptible snails showed no significant change in NO output following challenge with ESPs at any concentration used (0.1–20 μg/ml). Western blotting revealed that U0126 (1 μM or 10 μM) blocked the phosphorylation (activation) status of ERK in haemocytes from both snail strains. Inhibition of ERK signalling by U0126 attenuated considerably intracellular NO production in haemocytes from both susceptible and resistant B. glabrata strains, identifying ERK as a key regulator of NO output in these cells.ConclusionS. mansoni ESPs differentially influence intracellular NO levels in susceptible and resistant B. glabrata haemocytes, possibly through modulation of the ERK signalling pathway. Such effects might facilitate survival of S. mansoni in its intermediate host.

Novel hinge binder improves activity and pharmacokinetic properties of BRAF inhibitors.

Mutated BRAF serine/threonine kinase is implicated in several types of cancer, with particularly high frequency in melanoma and colorectal carcinoma. We recently reported on the development of BRAF inhibitors based on a tripartite A-B-C system featuring an imidazo[4,5]pyridin-2-one group hinge binder. Here we present the design, synthesis, and optimization of a new series of inhibitors with a different A-B-C system that has been modified by the introduction of a range of novel hinge binders (A ring). The optimization of the hinge binding moiety has enabled the development of compounds with low nanomolar potencies in both BRAF inhibition and cellular assays. These compounds display optimal pharmacokinetic properties that warrant further in vivo investigations.

Disruption of ERK signalling in Biomphalaria glabrata defence cells by Schistosoma mansoni: Implications for parasite survival in the snail host

Biomphalaria glabrata is an intermediate snail host for the human blood fluke Schistosoma mansoni. To survive in B. glabrata, S. mansoni must suppress the snails haemocyte-mediated defence response; the molecular mechanisms by which this is achieved remain largely unknown. We report here that S. mansoni excretory-secretory products (ESPs) attenuate phosphorylation of extracellular signal-regulated kinase (ERK) in haemocytes from a B. glabrata strain susceptible to S. mansoni. Whole S. mansoni sporocysts also impair ERK signalling in these cells. In striking contrast, ERK signalling in haemocytes from a B. glabrata strain refractory to schistosome infection is unaffected by ESPs or sporocysts. Effects of ESPs on ERK are similar in the presence or absence of snail plasma, thus ESPs seem to affect haemocytes directly. These findings reveal novel schistosome interference mechanisms; as ERK regulates various haemocyte defence reactions, we propose that disruption of ERK signalling in haemocytes facilitates S. mansoni survival within susceptible B. glabrata.

The laboratory maintenance of Sanguinicola inermis Plehn, 1905 (Digenea : Sanguinicolidae)

Methodology is described for the laboratory maintenance of the life-cycle of Sanguinicola inermis, using common carp (Cyprinus carpio) as the definitive host and Lymnaea peregra as the intermediate host. Lymnaea auricularia was also infected but is not considered to be a suitable laboratory host. Tench (Tinca tinca) were susceptible to infection at high cercarial doses but subsequent infection rates were low. Goldfish (Carassius auratus) and Lymnaea stagnalis were refractory to laboratory infection.

A Novel, Selective and Efficacious Nanomolar Pyridopyrazinone Inhibitor of V600EBRAF

Oncogenic BRAF is a critical driver of proliferation and survival and is thus a validated therapeutic target in cancer. We have developed a potent inhibitor, termed 1t (CCT239065), of the mutant protein kinase, (V600E)BRAF. 1t inhibits signaling downstream of (V600E)BRAF in cancer cells, blocking DNA synthesis, and inhibiting proliferation. Importantly, we show that 1t is considerably more selective for mutated BRAF cancer cell lines compared with wild-type BRAF lines. The inhibitor is well tolerated in mice and exhibits excellent oral bioavailability (F = 71%). Suppression of (V600E)BRAF-mediated signaling in human tumor xenografts was observed following oral administration of a single dose of 1t. As expected, the growth rate in vivo of a wild-type BRAF human tumor xenograft model is unaffected by inhibitor 1t. In contrast, 1t elicits significant therapeutic responses in mutant BRAF-driven human melanoma xenografts.

Differences in the Gene Expression Profiles of Haemocytes from Schistosome-Susceptible and -Resistant Biomphalaria glabrata Exposed to Schistosoma mansoni Excretory-Secretory Products

During its life cycle, the helminth parasite Schistosoma mansoni uses the freshwater snail Biomphalaria glabrata as an intermediate host to reproduce asexually generating cercariae for infection of the human definitive host. Following invasion of the snail, the parasite develops from a miracidium to a mother sporocyst and releases excretory-secretory products (ESPs) that likely influence the outcome of host infection. To better understand molecular interactions between these ESPs and the host snail defence system, we determined gene expression profiles of haemocytes from S. mansoni-resistant or -susceptible strains of B. glabrata exposed in vitro to S. mansoni ESPs (20 μg/ml) for 1 h, using a 5K B. glabrata cDNA microarray. Ninety-eight genes were found differentially expressed between haemocytes from the two snail strains, 57 resistant specific and 41 susceptible specific, 60 of which had no known homologue in GenBank. Known differentially expressed resistant-snail genes included the nuclear factor kappa B subunit Relish, elongation factor 1α, 40S ribosomal protein S9, and matrilin; known susceptible-snail specific genes included cathepsins D and L, and theromacin. Comparative analysis with other gene expression studies revealed 38 of the 98 identified genes to be uniquely differentially expressed in haemocytes in the presence of ESPs, thus identifying for the first time schistosome ESPs as important molecules that influence global snail host-defence cell gene expression profiles. Such immunomodulation may benefit the schistosome, enabling its survival and successful development in the snail host.