Landscape of Targeted Anti-Cancer Drug Synergies in Melanoma Identifies a Novel BRAF-VEGFR/PDGFR Combination Treatment

A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year. Thus, there is a pressing need for identification of combinations of targeted agents which induce more complete responses and prevent disease progression. We describe the results of a combination screen of an unprecedented scale in mammalian cells performed using a collection of targeted, clinically tractable agents across a large panel of melanoma cell lines. We find that even the most synergistic drug pairs are effective only in a discrete number of cell lines, underlying a strong context dependency for synergy, with strong, widespread synergies often corresponding to non-specific or off-target drug effects such as multidrug resistance protein 1 (MDR1) transporter inhibition. We identified drugs sensitizing cell lines that are BRAF^V600E mutant but intrinsically resistant to BRAF inhibitor PLX4720, including the vascular endothelial growth factor receptor/kinase insert domain receptor (VEGFR/KDR) and platelet derived growth factor receptor (PDGFR) family inhibitor cediranib. The combination of cediranib and PLX4720 induced apoptosis in vitro and tumor regression in animal models. This synergistic interaction is likely due to engagement of multiple receptor tyrosine kinases (RTKs), demonstrating the potential of drug- rather than gene-specific combination discovery approaches. Patients with elevated biopsy KDR expression showed decreased progression free survival in trials of mitogen-activated protein kinase (MAPK) kinase pathway inhibitors. Thus, high-throughput unbiased screening of targeted drug combinations, with appropriate library selection and mechanistic follow-up, can yield clinically-actionable drug combinations.     

NDUFA4 Mutations Underlie Dysfunction of a Cytochrome c Oxidase Subunit Linked to Human Neurological Disease

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Engineering a high-affinity anti-IL-15 antibody: crystal structure reveals an α-helix in VH CDR3 as key component of paratope

Interleukin (IL) 15 is an inflammatory cytokine that plays an essential role in the activation, proliferation, and maintenance of specific natural killer cell and T-cell populations, and has been implicated as a mediator of inflammatory diseases. An anti-IL-15 antibody that blocked IL-15-dependent cellular responses was isolated by phage display and optimised via mutagenesis of the third complementarity-determining regions (CDRs) of variable heavy (VH) and variable light chains. Entire repertoires of improved variants were recombined with each other to explore the maximum potential sequence space. DISC0280, the most potent antibody isolated using this comprehensive strategy, exhibits a 228-fold increase in affinity and a striking 40,000-fold increase in cellular potency compared to its parent. Such a wholesale recombination strategy therefore represents a useful method for exploiting synergistic potency gains as part of future antibody engineering efforts. The crystal structure of DISC0280 Fab (fragment antigen binding), in complex with human IL-15, was determined in order to map the structural epitope and paratope. The most remarkable feature revealed lies within the paratope and is a novel six-amino-acid α-helix that sits within the VH CDR3 loop at the center of the antigen binding site. This is the first report to describe an α-helix as a principal component of a naturally derived VH CDR3 following affinity maturation.     

Mitochondrial ND5 gene variation associated with encephalomyopathy and mitochondrial ATP consumption

Mitochondrial encephalomyopathy and lactic acidosis with strokelike episodes (MELAS) is a severe young onset stroke disorder without effective treatment. We have identified a MELAS patient harboring a 13528A-->G mitochondrial DNA (mtDNA) mutation in the Complex I ND5 gene. This mutation was homoplasmic in mtDNA from patient muscle and nearly homoplasmic (99.9%) in blood. Fibroblasts from the patient exhibited decreased mitochondrial membrane potential (Deltapsim) and increased lactate production, consistent with impaired mitochondrial function. Transfer of patient mtDNA to a new nuclear background using transmitochondrial cybrid fusions confirmed the pathogenicity of the 13528A-->G mutation; Complex I-linked respiration and Deltapsim were both significantly reduced in patient mtDNA cybrids compared with controls. Inhibition of the adenine nucleotide translocase or the F1F0-ATPase with bongkrekic acid or oligomycin caused a loss of potential in patient mtDNA cybrid mitochondria, indicating a requirement for glycolytically generated ATP to maintain Deltapsim. This was confirmed by inhibition of glycolysis with 2-deoxy-D-glucose, which caused depletion of ATP and mitochondrial depolarization in patient mtDNA cybrids. These data suggest that in response to impaired respiration due to the mtDNA mutation, mitochondria consume ATP to maintain Deltapsim, representing a potential pathophysiological mechanism in human mitochondrial disease.     

Some Observations upon Biochemical Causes of Ataxia and a New Disease Entity Ubiquinone, CoQ10 Deficiency

Some hereditary ataxias are treatable and the insight required for this has come from an in depth knowledge of the phenotypes and clinical biochemistry of the conditions. This has required both fundamental and translational clinical research. Prof John Blass was fortunate to begin his career at what we can now recognise as a golden era for such studies and he worked upon two important conditions; Refsum’s disease and Friedreich’s ataxia. More recently the mitochondrial encephalomyopathies have been described and similar investigative work has been undertaken upon them. Ubiquinone, CoQ₁₀, deficiency is the most recently recognised encephalomyopathy and is itself treatable. Though rare, it is becoming increasingly recognised and patients are benefiting from the same scholarly approach to its’ investigation as was afforded Refsums’ disease and Friedreich’s ataxia. A dedication to Professor John P. Blass.     

Physiological shifts in the microbial community drive changes in enzyme activity in a perennial agroecosystem

Perennial agroecosystems have the potential to promote plant–microbial linkages by increasing the quantity of root carbon entering the soil. However, an understanding of how perennial cropping systems affect microbial communities remains incomplete. The objective of this study was to determine the potential for a fertilized perennial bioenergy cropping system to impact microbial growth and enzyme activity. Three times throughout the growing season we examined the activity of four enzymes involved in decomposition (ß-glucosidase, ß-xylosidase, cellobiohydrolase, and N-acetyl glucosaminidase) in replicated plots of an annual (corn) and perennial-based (switchgrass) cropping system. We also took simultaneous measurements of microbial biomass and potential rates of microbial respiration and net N mineralization. Microbial biomass was unaffected by cropping system. Mid-summer, however, we observed increases in enzyme activity and potential microbial respiration in the perennial system that were independent of microbial biomass, likely in response to labile carbon inputs. Further, we observed lower net N mineralization, higher microbial biomass nitrogen and higher activity of nitrogen liberating enzymes, which are indicative of a community with high nitrogen demands. Overall, our research demonstrates that perennial agroecosystems can affect the physiological capacity of the microbial community, yielding communities with greater nitrogen retention and greater rates of decomposition as a result of allocation of resources towards enzyme production and nitrogen mining. These results can inform biogeochemical models with respect to the importance of temporally dynamic changes in carbon and nitrogen availability and microbial carbon use efficiency as drivers of enzyme production.