Trichodesmium increase under high carbon dioxide (CO2) levels in short-term studies due to physiological plasticity. However, its long-term adaptive responses to ongoing anthropogenic CO2 increases are unknown. Here we show that experimental evolution under extended selection at projected future elevated CO2 levels results in irreversible, large increases in nitrogen fixation and growth rates, even after being moved back to lower present day CO2 levels for hundreds of generations. This represents an unprecedented microbial evolutionary response, as reproductive fitness increases acquired in the selection environment are maintained after returning to the ancestral environment. Constitutive rate increases are accompanied by irreversible shifts in diel nitrogen fixation patterns, and increased activity of a potentially regulatory DNA methyltransferase enzyme. High CO2-selected cell lines also exhibit increased phosphorus-limited growth rates, suggesting a potential advantage for this keystone organism in a more nutrient-limited, acidified future ocean.Nitrogen fixation rates of the globally distributed, biogeochemically important marine cyanobacterium  The long-term response of marine cyanobacteria to increased anthropogenic CO2 are not known. Here, Hutchins et al. show that Trichodesmium exposed to long-term selection at elevated CO2 display irreversible increases in nitrogen fixation and growth rates, even after returning to present day conditions. Enhancement of Trichodesmium N2 fixation rates by up to 50% at projected year 2100 CO2 atmospheric levels (∼800 p.p.m.) relative to present day concentrations (∼400 p.p.m.) points to the possibility that new nitrogen inputs from this cyanobacterium could greatly increase as a result of future anthropogenic CO2 emissions79102 fertilization effect on marine N2 fixation would fundamentally change the nitrogen cycle of the ocean2-fixing cyanobacteria offers a more nuanced viewpoint, whereby particular strains, species and perhaps clades appear to be optimized for growth and N2 fixation under either low or high CO2 conditions. This observation of taxon-specific CO2 niche specialization suggests that marine N2-fixing cyanobacteria may have undergone differential selection by the many spatial and temporal CO2 fluctuations they have experienced during their long evolutionary historyA number of recent studies have demonstrated that carbon dioxide  or 750 p.p.m. , and then evaluate changes in their reproductive fitness using growth rate measurements, as well as changes in their physiological rates of N2 fixation. Every replicate cell line selected under expected future high-CO2 conditions exhibits elevated N2 fixation and growth rates that are constitutively expressed, in that they remain fixed at high levels despite reciprocal transfers back to long-term growth under lower current CO2 concentrations. This surprising microbial evolutionary response is characterized by apparently permanent changes in diel periodicity of N2 fixation and increased expression of a DNA-methylating enzyme with a possible regulatory function, and persists even when growth rates are limited by the nutrient phosphorus. Trichodesmium reacts to projected anthropogenic CO2 increases not only through transitory physiological plasticity responses, but also in wholly unexpected adaptive ways that could have large consequences for nitrogen and carbon biogeochemical cycling in the future ocean.To address this issue, here we employ classic experimental evolution methods that provide insights into the fundamental principles of microbial adaptive responses13141516172 fixation rates when stock cultures maintained at 380 p.p.m. CO2  were transferred to 750 p.p.m. CO2  for 2 weeks , as expected based on the previous short-term experiments cited above. At the end of the 4.5 year selection period, N2 fixation rates of the six 380- and 750-selected cell lines were not significantly different from those of the ancestral lineage at these same respective CO2 levels before selection . N2 fixation rates of the 750-selected cell lines were still 43% higher than those of the 380-selected cultures . This is consistent with the responses of the ancestral cell line . These same six 750-selected to 380 switch cell lines were then maintained at the reciprocal CO2 level of 380 p.p.m. over the subsequent ∼2 years, along with the long-term 750-selected and 380-selected cell lines maintained at their respective selection CO2 levels. N2 fixation rates of the 750-selected cultures switched back to 380 p.p.m. for 2 years (>350 generations) remained virtually identical to those of the 750-selected cell lines maintained continuously at 750 p.p.m., with both still ∼43% higher relative to rates of the 380-adapted cell lines maintained at 380 p.p.m. . Thus, every cell line selected at 750 p.p.m. CO2 was unable to reduce their rates when moved back to the ancestral 380 p.p.m. CO2 level, with N2 fixation physiology instead becoming ‘stuck in the fast lane'.Unexpectedly though, the 750-selected cell lines returned to 380 p.p.m. CO2 fixation rates. 750-selected to 380 switch cultures maintained at 380 p.p.m. for 2 years grew at the same rates as the 750-selected cell lines maintained continuously at 750 p.p.m.; both grew up to ∼44% faster than the 380-selected cultures maintained at 380 p.p.m. . Thus as with N2 fixation rates, the growth rates of all six 750-selected cell lines became constitutively elevated relative to CO2 concentration following 4.5 years of selection at high CO2, with the same universally increased growth rates persisting regardless of whether they were grown at 380 or 750 p.p.m.Cell-specific growth rates of all six replicate cell lines responded in the same way as N2 fixation in the selected cell lines offers possible insights into the physiological mechanisms behind this unexpected adaptive response to prolonged growth under high CO2. Trichodesmium fixes nitrogen only during daylight hours, with rates often peaking somewhere near mid-photoperiod2 fixation rates occurring between 3 and 5 h into the photoperiod . Methyltransferase activity levels were elevated to varying degrees in all six 750-selected to 380 switch cell lines, but were below detection or nearly so in every one of the 380-selected and 750-selected cell lines . Phosphorus-limited growth rates of both sets of cell lines were lower than phosphorus-replete rates, as expected . In phosphorus-replete cultures, the 750-selected cell lines grown at 750 p.p.m. had growth rates that were much higher than those of the 380-selected cell lines growing at 380 p.p.m. , again as expected from previous results .Phosphorus is one of the key limiting nutrients for N results . NotablyTrichodesmium growth rates, a proxy for microbial reproductive fitness132, but then remained unchanged despite the subsequent ∼850 generations of selection at this elevated CO2 level. Thus, beyond the previously documented rapid initial physiological plasticity response, long-term selection by high CO2 did not lead to further measurable fitness increases in the selection environment. However, these same 750-selected cell lines exhibited a 44% fitness increase when transplanted back to the ancestral CO2 condition of 380 p.p.m., relative to the 380-selected and ancestral cell lines. This surprising adaptive response–in which short-term plastic growth rate increases become fixed during extended selection in a novel environment, resulting in permanent large fitness increases even in the ancestral environment–appears to be unique in the microbial experimental evolution literature132 selection environment, and some but not all high CO2-selected cell lines exhibited fitness decreases when switched back to ambient CO2 concentrationsTrichodesmium cell lines, in another study measuring Escherichia coli long-term evolutionary adaptation to several temperature regimes all cell lines improved fitness relative to their ancestors in the selection environment. However, these fitness increases occurred only following thousands of generations of exposure, and replicate E. coli cell lines demonstrated considerable heterogeneity in whether fitness was decreased or unchanged at ancestral temperatures; none exhibited fitness increases in the ancestral environmentTrichodesmium results differ markedly from these prior results, in that within a few hundred generations all six biological replicates of our high CO2-selected cell lines evolved a homogeneous, correlated phenotype. This phenotype consisted of permanent retention of fitness increases originally acquired due to a physiological plasticity response, and that conferred increased fitness even in the ancestral environment.Changes in evolutionary fitness often show tradeoffs in relative fitness across different environments13142 ocean. Rapidly growing, high CO2-selected Trichodesmium might appear to possess a potential competitive advantage over other primary producers; however, if faster growth rates are universally advantageous, it is remarkable that our 380-selected cell lines did not also acquire the same adaptation that each of our 750-selected cell lines did. Indeed, it is questionable why wild Trichodesmium populations growing at current CO2 levels have not adapted to grow at similarly high rates. It is evident that there must be tradeoffs involved with maintaining constitutively elevated growth and N2 fixation rates that make them intrinsically maladaptive at present day CO2 concentrations.Regardless of the ultimate mechanisms, our findings could have major ecological and evolutionary implications for these biogeochemically critical cyanobacteria in the future high-CO2-selected Trichodesmium may enjoy a competitive advantage in the future more stratified and nutrient-limited ocean, and that this advantage is also likely to persist, even if they are subsequently returned to lower CO2 conditions. However, since limitation by iron and simultaneous co-limitation by both iron and phosphorus are also ecologically important12Trichodesmium62 as well.Although the nature of these tradeoffs is currently unknown, one potential disadvantage with elevated growth rates is that they result in increased cellular demand for limiting resources such as nutrients that are in short supply over large parts of the ocean345626282 levels controlled artificially by bubbling, which maintained partial pressures within ±<5% of our target values of 380 and 750 p.p.m. (Methods section). In the oligotrophic ocean where Trichodesmium grows, CO2 concentrations in contemporary surface waters vary over a similar range around current atmospheric levels of ∼400 p.p.m.  were maintained in a modified Aquil medium35142 effects during acclimated, steady-state growth. The cultures were kept optically thin to avoid self-shading, nutrient limitation and perturbations of targeted CO2 levels. Each bottle was diluted individually based on the growth rate calculated for that bottle7811in vivo chlorophyll fluorescence measurements with a Turner 10 AU fluorometer for semi-daily dilution calculations in real time during the experiments. Comparisons between cell counts and in vivo-based growth rates revealed no significant differences between the two methods of assessing biomass changes.Cultures of 2 mixtures (Praxair) to maintain stable CO2 concentrations of 380 p.p.m. or 750 p.p.m. CO2 for ∼4.5 years. At this time, a set of short-term 2 week CO2 reciprocal transfer incubation experiments was performed using the long-term cultures. These two treatments consisted of switching 380 p.p.m. CO2-conditioned cell lines to 750 p.p.m. CO2, and 750 p.p.m. CO2-conditioned cultures to 380 p.p.m. CO2 (referred to as switch cultures). The switch incubations were performed under experimental conditions and dilution frequencies identical to those of the long-term cultures.Experimental evolution cultures were continuously bubbled with prepared air/CO2 levels. During this subsequent 2 year period, all culturing protocols for the switch and long-term selected cultures remained the same as outlined above. The classical method to determine fitness changes in microbial experimental evolution experiments is competition of selected cell lines against the ancestral strain1314Trichodesmium, though, since it is not amenable to cryopreservation. Consequently, we used the best available measurable indicator of relative fitness, specific growth rates, to assess adaptive changes, with the caveat that this proxy may not fully capture all fitness changes in our CO2-selected Trichodesmium cell lines. For all experiments, significant differences between the six replicates in each treatment were tested using one-way Anova followed by student's t-test.Following the 2 week reciprocal transfers, the six 750 to 380 switch cell lines were then maintained for a further 2 years at 380 p.p.m., in parallel with the 750-selected and 380-selected cell lines maintained at their relative selection COTrichodesmium cell lines were used in experiments assessing the responses of cell-specific growth rates to phosphorous limitation. Medium was prepared for P-replete cultures as described above for experimental cultures, and was prepared in the same way except the phosphate concentration was reduced to 0.5 μM for P-limited cultures. Steady-state growth rates of the semi-continuous cultures were measured after 2 months of growth at 26 °C in the P-limited medium, and results are reported as the means and averages of the six replicate cell lines for each treatment. P limitation cultures were maintained in nutrient-limited exponential growth by transferring them every 2 days using semi-continuous culturing methods, so cultures were never allowed to deplete medium nutrients or reach stationary phaseThe six replicate long-term 750-selected and 380-selected 2H2:mol N2 reduced) was used to convert rates of ethylene production (C2H2 reduction) to N2 fixation. Assays were initiated by adding 2 ml of C2H2 to the headspace of 28-ml serum vials containing 10 ml of culture. A measure of 100 μl headspace was removed to measure ethylene production at 2–3 h intervals over the entire 12-h light period. Samples were gently agitated to equilibrate gas concentrations between the headspace and culture samples following injection of acetylene and before measuring ethylene concentrations7811Nitrogen fixation rates were measured using the acetylene reduction assay with a Shimadzu gas chromatograph GC-8a  equipped with a flame ionization detector. A theoretical ratio of 3:1  with a combined glass electrode. The metre was calibrated with National Bureau of Standards buffer solutions of pH 4, 7 and 10. Dissolved inorganic carbon was measured with CO2 coulometry . The partial pressure of CO2 in the samples was calculated from the measured pH and dissolved inorganic carbon values using CO2SYS software782 equilibrium was closely controlled using continuous bubbling, photosynthesis and respiration had minimal effects on the seawater carbonate system and measured pCO2 was always within ∼5% of the two target values.To ensure that target selection COg  for 20 min at RT. The supernatants were removed and concentrated by membrane centrifugation to ∼300 μl in 6 ml, 5 K molecular weight cutoff Vivaspin units . Each sample was precipitated with cold 50% methanol (MeOH) 50% acetone 0.5 mM HCl for 3 days at –20 °C, centrifuged at 14,100g for 30 m at 4 °C, decanted and dried by vacuum concentration (Thermo Savant Speedvac) for 10 min or until dry. Pellets were resuspended in 1% SDS extraction buffer and left at RT for 1 h to completely dissolve. Total protein was quantified  with BSA as a standard.For protein extraction, 1.5 ml of 1% SDS extraction buffer  was added to unfolded 25 mm filter samples. Each sample was incubated at room temperature (RT) for 15 min, heated at 95 °C for 10 min, and shaken at RT, 350 r.p.m. for 1 h. The protein extract was decanted and placed in a new tube and centrifuged at 14,1002O) and incubated at 350 r.p.m., RT for 2 h. Liquid was then removed, gel cut up into 1 mm cubes and then added back to tubes containing 1 ml of 50:50 acetonitrile:25 mM ammonium bicarbonate (ambic) incubated for 1 h, 350 r.p.m. at RT. Liquid was removed and replaced with fresh 50:50 acetonitrile:ambic and incubated at 16 °C 350 r.p.m. overnight. The above step was repeated for 1 h the following morning. Gel pieces were then dehydrated twice in 800 μl of acetonitrile for 10 min at RT and dried for 10 min in a ThermoSavant DNA110 speedvac after removing solvent. 600 μl of 10 mM dithiothreitol in 25 mM ambic was added to reduce proteins incubating at 56 °C, 350 r.p.m. for 1 h. Unabsorbed dithiothreitol solution was then removed with volume measured. Gel pieces were washed with 25 mM ambic and 600 μl of 55 mM iodacetamide was added to alkylate proteins at RT, 350 r.p.m. for 1 h. Gel cubes were then washed with 1 ml ambic for 20 min, 350 r.p.m. at RT. Acetonitrile dehydrations and speedvac drying were repeated as above. Trypsin (Promega #V5280) was added in appropriate volume of 25 mM ambic to rehydrate and submerse gel pieces at a concentration of 1:20 μg trypsin:protein. Proteins were digested overnight at 350 r.p.m. 37 °C. Unabsorbed solution was removed and transferred to a new tube. 50 μl of peptide extraction buffer  was added to gels, incubated for 20 min at RT then centrifuged at 14,100g for 2 min. Supernatant was collected and combined with unabsorbed solution. The above peptide extraction step was repeated combining all supernatants. Combined protein extracts were centrifuged at 14,100g for 20 min, supernatants transferred into a new tube and dehydrated down to ∼10–20 μl in the speedvac. Concentrated peptides were then diluted in 2% acetonitrile 0.1% formic acid in water for storage until analysis. All water used in the tube gel digestion protocol was LC/MS grade, and all plastic microtubes were ethanol rinsed and dried before use.For protein digestion, extracted proteins were purified from SDS detergent, reduced, alkylated and trypsin digested while embedded within a polyacrylamide tube gel−1 flow rate.For MS global proteome analyses, chromatography was performed using a Michrom Advance nanoflow LC and autosampler (Michrom Bioresources) and a 100 μm inner diameter 15 cm long capillary column with a pulled tip (packed in-lab with MAGIC C18AQ 200 Å pore size 3 μm particle size from Michrom Bioresources). Samples were first loaded on a 200 μm I.D. 1 cm long trap  and washed with 50 μl of 2% acetonitrile and 0.1% formic acid in water. The trap was then switched in-line with the 15 cm column and eluted with a non-linear gradient of 5–35% solvent B (0.1% formic acid in acetonitrile) balanced with solvent A (0.1% formic acid in water) at 500 nl minm/z.Electrospray ionization was performed with a Thermo Flex ion source in positive ion mode at 1,400 V. Eluting peptides were analysed on a Thermo Fusion mass spectrometer with MS1 scans at an Orbitrap resolution of 60 K, 350–1,800 m/z scan range, 2.0e5 automatic gain control target, and a maximum injection time of 35 ms. MS2 scans were analysed on the linear ion trap in topN data dependent mode at a cycle time of 3 s using normal scan rate and range with a maximum injection time of 75 ms and a 0.7 m/z isolation window. Charge states of 2–7 were analysed with a dynamic exclusion of 15 s with a mass tolerance of 10 p.p.m. Monoisotopic precursor selection was used, and a user-defined lock mass of 445.12003 Protein identifications were made using the SEQUEST peptide mapping algorithm within Proteome Discoverer and the Peptide Prophet algorithm within Scaffold 3.0  using 99% protein and 95% peptide confidence levels, allowing one minimum peptide per protein, resulting in a 1.1% false discovery rate for proteins and a 0.01% false discovery rate for peptides resulting in 1,499 identified proteins.Changes in relative abundance of proteins between samples were determined using label-free spectral count enumeration within Scaffold. Spectral counts compare a specific protein's abundance between treatments, rather than against other proteins, because the sensitivity of spectral counts can vary between proteins depending on the number of tryptic peptides within the sequence and their chemical characteristics. These problems do not affect comparisons of a protein with itself between treatments. Spectral counts were normalized within each experimental treatment to the total number of spectra collected to correct for small variations in the number of spectra between samples sets; under stable and consistent MS conditions this results in a very small difference3940Cultures were rapidly and gently filtered during the middle of the photoperiod onto 5 μm polycarbonate filters (Whatman), washed off of the filters with 2 ml of 50 mM (pH=7) Tris-HCl into PowerPlant Bead tubes from the PowerPlant Pro DNA Isolation Kit (#13200-100), and put on ice. The tubes were vortexed for 2 min , and technical replicates were made for each biological replicate. Protein concentrations were quantified using the Pierce BCA Protein Assay Kit (#23225), and the (Epigentek) Epiquick DNMT Activity/Inhibition Assay Ultra Kit (Fluorometric) (#P-3010) was used to determine DNA methylation activity according the manufacturer's instructions.How to cite this article: Hutchins, D.A. et al. Irreversibly increased nitrogen fixation in Trichodesmium experimentally adapted to elevated CO2. Nat. Commun. 6:8155 doi: 10.1038/ncomms9155 (2015).


His 12201T>C mutation. The destabilization of a highly conserved base-pairing (5A-68U) by the m.12201T>C mutation alters structure and function of tRNAHis. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (ρo) cells, we showed ∼70% decrease in the steady-state level of tRNAHis in mutant cybrids, compared with control cybrids. The mutation changed the conformation of tRNAHis, as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. However, ∼60% increase in aminoacylated level of tRNAHis was observed in mutant cells. The failure in tRNAHis metabolism was responsible for the variable reductions in seven mtDNA-encoded polypeptides in mutant cells, ranging from 37 to 81%, with the average of ∼46% reduction, as compared with those of control cells. The impaired mitochondrial translation caused defects in respiratory capacity in mutant cells. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cells. The data provide the evidence for a mitochondrial tRNAHis mutation leading to deafness.In this report, we investigated the molecular genetic mechanism underlying the deafness-associated mitochondrial tRNA Leu(UUR) gene (Ser(UCN) 7445A>G, 7472insC, 7505T>C, 7510T>C and 7511T>C, tRNAHis 12201T>C and tRNAIle 4295A>G mutations (Ser(UCN) precursor (Ile (Ser(UCN), thereby altering the tRNA metabolisms , and non-syndromic deafness . Mutations in mitochondrial DNA (mtDNA) are one of the important causes of syndromic and non-syndromic deafness . In partUR) gene . The nonutations . These mutations . The m.7recursor , while tsor  (Life Technologies) (containing 4.5 mg of glucose and 0.11 mg pyruvate/ml), supplemented with 100 μg of BrdU/ml and 5% FBS. The mtDNA-less ρ°206 cell line, derived from 143B.TK−  of the Chinese family carrying the m.12201T>C mutation and one genetically unrelated Chinese control individual belonging to the same mtDNA haplogroup Z3 but lacking the mutation (H7)  were grown in RPMI 1640 medium with 10% fetal bovine serum (FBS). The bromodeoxyuridine (BrdU) resistant 143B.TKImmortalized lymphoblastoid cell lines derived from one affected member of the Chinese family (IV-11) and one Chinese control individual (H7) were used for the generation of cybrid cell lines. Transformation by cytoplasts of mtDNA less ρ°206 cells was performed as described elsewhere .His gene was carried out as described in the supplemental materials. The quantification of mtDNA copy numbers from different cybrids was performed by slot blot hybridization as detailed elsewhere , as described previously , tRNAGly and 5S RNA were described as elsewhere  from mitochondria isolated from the cybrid cell lines  10% polyacrylamide–7 M urea gel to separate the charged and uncharged tRNA as detailed elsewhere ,26. The 1 and CO2 (Abcam), rabbit anti-ND4 and CYTB (Santa Cruz), and mouse anti-actin (Beyotime). Peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as a secondary antibody and protein signals were detected using the ECL system (CWBIO).Twenty micrograms of proteins obtained from lysed cells were denatured and loaded on sodium dodecyl sulfate polyacrylamide gels. Afterward, the proteins were transferred to polyvinylidene difluoride (PVDF) membrane and subjected to Western blotting. Membranes were blocked in Tris-Buffered Saline and Tween20 (TBST)  containing 5% (w/v) milk, then incubated with the corresponding primary and secondary antibodies. The primary antibodies used for this experiment were the rabbit anti-ND1, ND5 and A6, mouse anti-CO2 were determined under basal condition and the addition of oligomycin (1.5 μM), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (0.5 μM), rotenone (1 μM) and antimycin A (1 μM), as detailed elsewhere , as detailed elsewhere ,28. XF96lsewhere ,28.® Luminescent Cell Viability Assay kit (Promega) was used for ATP assay according to the manufacturer's instructions  for 30 min at 37°C, 5% CO2 prior to staining with JC-10 dye. The fluorescent intensities for both J-aggregates and monomeric forms of JC-10 were measured at Ex/Em = 490/530 and 490/590 nm with a microplate reader .Mitochondrial membrane potential was assessed with JC-10 Assay Kit-Microplate (Abcam) following general manufacturer's recommendations with some modifications ,31. In b6 cells of each cell line were harvested, resuspended in PBS supplemented with 100 μM of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and then incubated at 37°C for 20 min. After washing with PBS twice, cells were resuspended in PBS in the presence of 2 mM freshly prepared H2O2 and 2% FBS and then incubated at room temperature for another 45 min. Cells were further washed with PBS and resuspended with 1 ml of PBS with 0.5% paraformaldehyde. Samples with or without H2O2 stimulation were analyzed by BD-LSR II flow cytometer system , with an excitation at 488 nm and emission at 529 nm. Ten thousand events were analyzed in each sample.ROS measurements were performed following the procedures detailed elsewhere ,32. Briet-test contained in the Microsoft-Excel program. Unless indicated otherwise, a P-value <0.05 was considered statistically significant.Statistical analysis was carried out using the Student's unpaired, two-tailed − cell line (The lymphoblastoid cells derived from one affected subject (IV-11) and one control individual (H7) were enucleated, and subsequently fused to a large excess of mtDNA-less human ρ°206 cells, derived from the 143B.TKell line . The cybell line ,20. BetwHis, we subjected mitochondrial RNAs from cybrids to northern blots and hybridized them with DIG-labeled oligodeoxynucleotide probes for tRNAHis, tRNAThr, tRNALys, tRNALeu(CUN) and tRNAGly as well as a nucleus-encoded mitochondrial 5S RNA. As shown in Figure His in three mutant cell lines were markedly decreased, compared with those in three control cybrid cell lines. For comparison, the average level of each tRNA in control or mutant cell lines was normalized to the average levels in the same cell line for reference 5S RNA. As shown in Figure His in three cell lines were 31.8% (P = 0.0002) of three controls cell lines after normalization to 5S RNA. However, the average steady-state levels of tRNAThr, tRNALys, tRNALeu(CUN) and tRNAGly in three mutant cell lines were 84.2, 99.7, 109.7 and 96.3%, respectively of those in three cell lines after normalization to 5S RNA.To further examine whether the m.12201T>C mutation affects the stability of tRNAHis, tRNAThr, tRNALys, tRNALeu(CUN) and tRNAGly in control and mutant cell lines were examined by the use of electrophoresis in an acid polyacrylamide/urea gel system to separate uncharged tRNA species from the corresponding charged tRNA, electroblotting and hybridizing with above tRNA probes  and tRNAGly between the cell lines carrying the m.12201T>C mutation and cell lines lacking this mutation. Notably, the efficiencies of aminoacylated tRNAHis in these mutant cell lines reflected 62% increase, ranged from 30 to 86%, relative to the average control values (P = 0.0257). However, the levels of aminoacylation in tRNAThr, tRNALys, tRNALeu(CUN) and tRNAGly in mutant cell lines were comparable with those in the control cell lines.The aminoacylation capacities of tRNAA probes . As showc oxidase; p.MT-ND1, p.MT-ND4 and p.MT-ND5, subunits 1, 4 and 5 of NADH dehydrogenase; p.MT-A6, subunit 6 of the H+-ATPase; p.MT-CYTB, apocytochrome b were decreased in three mutant cell lines, as compared with those of three control cell lines. As shown in Figure P = 0.0007). Notably, the average levels of p.MT-ND1, p.MT-ND4, p.MT-ND5, p.MT-CO1, p.MT-CO2, p.MT-A6 and p.MT-CYTB in the mutant cells were 34, 64, 60, 43, 19, 61 and 63% of the average values of control cells, respectively. However, the levels of synthesis of polypeptides in mutants relative to that in controls did not correlate with either the number of codons or proportion of histidine residues . This result was in contrast to previous studies with the deafness-associated m.7445A>G mutation in the precursor of tRNASer(UCN) gene  gene .Figure P = 0.0021) relative to the mean value measured in the control cell lines. To investigate which of the enzyme complexes of the respiratory chain was affected in the mutant cell lines, OCR were measured after the sequential addition of oligomycin (inhibit the ATP synthase), FCCP , rotenone (to inhibit complex I) and antimycin A (to inhibit complex III). The difference between the basal OCR and the drug-insensitive OCR yields the amount of ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity and non-mitochondrial OCR. As shown in Figure P = 0.0005, 0.1109, 0.0005, 0.0562 and 0.3084), respectively.To evaluate if the m.12201T>C mutation alters cellular bioenergetics, we examined the oxygen consumption rates (OCR) of cell lines derived from three mutant cell lines carrying the m.12201T>C mutation and three control cell lines. As shown in Figure d-glucose with pyruvate , ranged from 54 to 86%, with an average of 69% relative to the mean value measured in the control cell lines (P = 0.0379).The capacity of oxidative phosphorylation in mutant and wild-type cells was examined by measuring the levels of cellular and mitochondrial ATP using a luciferin/luciferase assay. Populations of cells were incubated in the media in the presence of glucose, and 2-deoxy-pyruvate . As showΨm) changes were measured in three mutant and three control cell lines using a fluorescence probe JC-10 assay system. The ratio of fluorescence intensities Ex/Em = 490/590 and 490/530 nm (FL590/FL530) were recorded to delineate the ΔΨm level of each sample. The relative ratios of FL590/FL530 geometric mean between mutant and control cell lines were calculated to represent the level of ΔΨm As shown in Figure Ψm in the mutant cell lines carrying m.12201T>C mutation ranged from 14.2 and 21.4%, with an average 17.7% (P < 0.0001) of the mean value measured in the control cell lines. In contrast, the levels of ΔΨm in mutant cells in the presence of CCCP were comparable with those measured in the control cell lines (P = 0.4748).The mitochondrial membrane potential  of the mean value measured in the control cell lines.The levels of the ROS generation in the vital cells derived from three mutant cell lines carrying the m.12201T>C mutation and three control cell lines lacking the mutation were measured with flow cytometry under normal and Hmulation ,32. GeomHis gene. The mutation is localized at a highly conserved nucleotide (68U) to form a base-pairing (5A-68U) on the acceptor stem of the tRNAHis  may contribute to the increasing aminoacylation level of tRNAHis  .In the present study, we investigated the pathogenetic mechanism of the deafness-associated m.12201T>C mutation in the tRNA tRNAHis . This nu tRNAHis ,34. The  tRNAHis . It was mutation . Howevery effect ,37. Alte tRNAHis . A failuLeu(UUR) ,39. In tmutation . These rmutation ,18,39. IHis then leads to the impairment of mitochondrial protein synthesis. In the present study, a markedly decreased level of mitochondrial proteins (an average decrease of ∼46%) was observed in mutant cybrid cell lines, as compared to the average levels in control cell lines. The reduced level of variable mitochondrial proteins detected in cybrids using a Western blot analysis was comparable with the reduced rate of mitochondrial protein synthesis observed in lymphoblastoid cell lines (Ser(UCN)  7445A>G, 7472insC and 7511T>C mutations (A shortage or improper aminoacylation of the tRNAll lines . In mutaSer(UCN)  or the MLys gene . The imputations ,18,41.His 12201T>C mutation results in decreased efficiency of the mitochondrial ATP synthesis. In this study, 31% drop in mitochondrial ATP production in cybrid carrying the m.12201T>C mutation was much lower than those in cells carrying MERRF-associated tRNALys 8344A>G and MELAS-associated tRNALeu(UUR) 3243A>G mutations (The respiratory deficiency caused by the tRNAutations ,43. Alteutations . As a reutations . In thisutations ,46. The utations . The haiutations . This woHis 12201T>C mutation. The m.12201T>C mutation alters the secondary structure and function of tRNA. A failure in tRNA metabolism impaired mitochondrial translation and respiration. As a result, this respiratory deficiency reduced mitochondrial ATP production and membrane potentials. Consequently, the mutation leads to the increasing production of oxidative reactive species and subsequent hearing loss. Thus, our findings may provide the new insights into the understanding of pathophysiology of maternally inherited hearing loss.In summary, our findings convincingly demonstrate the pathogenic mechanism leading to an impaired oxidative phosphorylation in cybrid cell lines carrying the deafness-associated tRNASupplementary Data are available at NAR Online.


Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with poor overall survival. New therapeutic strategies that target specific molecular lesions driving advanced disease are needed. Herein we demonstrate the utility of the chicken chorioallantoic membrane (CAM) assay for in vivo human HNSCC tumor growth and metastasis and the tumor suppressive effects of a new chemotherapeutic agent.We tested anti-metastatic effects of a WNT pathway inhibitor, WNT974 , which targets porcupine (PORCN) the palmityl-transferase that is essential for secretion of Wnt proteins. CAM assays were performed with 8 HNSCC cell lines: UM-SCC-1, UM-SCC-10A, UM-SCC-10B, UM-SCC-11A, UM-SCC-14A UM-SCC-17A, UM-SCC-17B, UM-SCC-25, and UM-SCC-34.PORCN inhibitor WNT974 reduced metastatic spread of UM-SCC cells, especially in UM-SCCs with Notch1 deficiency.UM-SCC-1 (University of Michigan Squamous Cell Carcinoma cell line) CAM xenografts contain CD44+ and ALDH+ cancer stem cell (CSC) proportions similar to UM-SCC-1 mouse xenografts supporting the applicability of the CAM assay for study of CSCs. Inhibition of WNT signaling by the Our data demonstrate decreased tumor growth and metastases in tumors from cell lines that showed in vitro responses to WNT974, providing evidence that this agent may have a role in future HNSCC therapy. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common form of cancer worldwide . DespiteThe Wnt signaling pathway is an appealing target, as this developmental pathway has been implicated in a large number of human cancers, with recent evidence for an oncogenic role in HNSCC . DeregulAXIN2, as well as reduced Wnt-dependent phosphorylation of LRP6. Of the 96 HNSCC cell lines analyzed, 31 demonstrated a pharmacodymanic (PD) AXIN2 mRNA reduction response and were thus considered as cell lines that were responsive to treatment with WNT974. Interestingly, there was an enriched rate of response to WNT974 among head and neck cancer cell lines with Notch1 loss-of-function (LOF) mutations [A small molecule inhibitor of the Wnt-pathway, LGK974, was recently described . The druutations .NOTCH1 LOF mutations were found in 10–15 % of tumors and abnormalities of the Notch pathway in 66 % of patients [Like Wnt, Notch is also a developmental pathway gene that has recently been implicated in HNSCC tumorigenesis –17. Whilpatients . The Notpatients , 20 withpatients . Consequpatients –24. HereThe aim of this study was to determine the feasibility of the chicken chorioallantoic membrane assay for assessing in vivo tumor response to a novel WNT pathway inhibitor, WNT974, in human head and neck cancer cell lines.The UM-SCC-1, -10A, -10B, -11A, 14A -17A, -17B, -25, and -34 cell lines were derived in our laboratory from human head and neck tumor explants taken during surgical resection from patients treated at the University of Michigan.The cell line donor-patients gave written informed consent for the use of their tissue to create cell lines in studies reviewed and approved by the University of Michigan Medical School (Ann Arbor) IRBMED institutional review board.The cell lines have been carefully characterized in our laboratory for HNSCC characteristics and each has been genotyped at a minimum of 10 microsatellite markers  to confirm their unique origin . Cell liThe use of fertilized chicken eggs is exempt from vertebrate animal use approval.2 window was drawn on the egg shell overlying the most vascularized area of each viable embryo. Two small holes were then bored into the egg shell, one in the center of the window and the other at the apex of the egg, overlying a naturally occurring air pocket. A rubber pipette bulb was used to suction a small amount of air out of this apical pocket, causing the chorioallantoic membrane to drop downward, away from the vent hole in the drawn window. The window was then opened using a Dremel 1100-N/25 7.2-Volt Stylus Lithium-Ion Cordless Rota Drill (Robert Bosch Tool Company) and was covered with a piece of clear adhesive tape to protect the embryo and prevent dehydration. This window served as the site for subsequent cancer cell inoculation in our preliminary experiment, and cancer cell and drug inoculation in our WNT974 treatment experiment .Fertilized white leghorn chicken eggs were obtained from Charles River Labs . The use of fertilized chicken eggs is exempt from vertebrate animal use approval. The eggs were kept in an incubator at 99.5 degrees Fahrenheit at a humidity range of 45–55 %. Eight days following arrival, the embryos were assessed for viability, performed by trans-illumination of the egg in a dark room to identify the presence of an embryo and surrounding blood vessels. A 1 cmSeven days after the administration of cancer cells, the chick eggs were opened with sterile scissors, and primary tumors were dissected out and weighed. The chick embryos were then dissected, removing the lungs and livers which were immediately placed on ice in labeled 10 mL conical containers. The samples were stored in the -80 degree C freezer until DNA extraction.Chick embryo livers and lungs were thawed, rinsed with 5 mL PBS and homogenized with a handheld homogenizer  using a sterile tip for each sample. DNA extraction was performed using the QIAgen DNeasy Blood & Tissue Kit (QIAGEN Group), following the manufacturer’s specifications. Purified DNA was quantified using a spectrophotometer, adjusted to a concentration of 0.2ug/uL, and stored at -20 degrees C. Quantitative PCR (qPCR) was performed on the DNA samples using primers specific for a human Alu sequence. Alu sequences are primate-specific, and thus their detection in chick organs represents disseminated human cells, or cancer metastases. The copy threshold, or CT, values of the liver and lung specimens from the eight HNSCC cell lines were compared to the CT values of the negative controls of the respective organ. Student T-tests were used to compare the CT values of both organs of each cell line to the respective negative control.2 plastic flasks, trypsinized, counted, and resuspended in a mixture of 10 % matrigel (BD Biosciences) and 90 % DMEM for a total volume of 30uL per eggs such that each egg received 2.5 million cancer cells. The prepared chick eggs were removed from the incubator, and the cancer cell suspension was laid on top of the chorioallantoic membrane using a 100uL pipette tips. For each cell line, five eggs were each inoculated. An additional ten eggs received a 30uL suspension of 10 % matrigel and 90 % DMEM with no cancer cells to serve as treatment, embryo viability, and specificity controls.The initial experiments were designed to demonstrate feasibility of the CAM assay for use with HNSCC cancer cells using eight cell lines ..The cells were grown in 150 cmUM-SCC-1 cancer cells were administered to an additional ten eggs for cancer stem cell analysis. Seven days following cancer cell administration, primary tumors were dissected from eight viable embryos and placed on ice in 15 mL tubes filled with DMEM media. Tumor tissue was minced and digested in DMEM/F12 (Gibco) with 1x collagenase/hyaluronidase (Stem Cell Technologies). After two hours of digestion, the mixture was strained through a 40 μm sieve and the cells were counted before being prepared for flow cytometry. CD44 expression was detected using an APC-conjugated antibody (BD Pharmingen). Aldehyde dehydrogenase (ALDH) expression was detected using the ALDEFLUOR kit (StemCell Technologies). The aldefluor substrate freely diffuses into cells and reacts with human aldehyde dehydrogenase enzyme, producing a fluorescent reaction product that is proportional to the enzymatic activity. This reaction does not occur in the chicken host cells.Tumor cells from the primary tumors of the eight viable chick embryos were pooled together to gather sufficient cells for an adequate assessment of CSCs in the tumors grown on the CAM. Fluorescence-activated cell sorting gates were established for ALDH expression using the inhibited control (DEAB) along the fluorescein isothiocyanate (FITC) channel with excitation and emission wavelengths of approximately 495 nm/521 nm. For CD44 expression, cell sorting gates were established using the APC-conjugated isotype control along the allophycocyanin (APC) channel with excitation and emission wavelengths of approximately 650 nm/660 nm.Notch1 mutations , and both exhibited a pharmacodynamic (PD) change in AXIN2 mRNA expression in response to WNT974 during the in vitro analysis performed by GNF. A third cell line, UM-SCC-1, containing wildtype NOTCH1, also showed a PD response to WNT974. The fourth line, UM-SCC-14A, also wildtype for NOTCH1, was a PD non-responder in vitro. The CAM assay was prepared as described above. For each cell line, 2.5 million cells were administered to each of 32 eggs (performed in separate assays per cell line due to limited incubator space). Of these, half served as treatment eggs and the other half were vehicle control eggs. The treatment arm received 5 μL of 1 μM WNT974 once daily, every other day starting the day of cancer cell inoculation for a total of four doses. The control arm received 5uL DMSO every other day for a total of four doses. There were also 5 manipulation-only control eggs per cell line, which were injected with 30 μL of 10 % matrigel and 90 % DMEM but no cancer cells. Eggs were opened seven days following cancer cell administration, all identifiable primary tumors dissected and weighed, the lung and liver tissues harvested and DNA was purified as described above.To test the effect of Wnt pathway inhibition on tumor growth and distant metastasis we first performed a dose-finding assay with WNT974 to assess for potential toxicity of this pathway inhibitor on chick embryos. Three drug concentrations were tested, 0.1 μM, 0.31 μM, and 1 μM. μM. For each drug concentration, five chick eggs were dosed every other day for a total of four doses. An additional five eggs were treated with 5uL DMSO (the WNT974 vehicle) to control for vehicle toxicity to the embryos. This assay showed minimal toxicity of these drug concentrations on the chick embryos, with all five embryos given 1 μM WNT974 remaining viable and phenotypically normal through harvesting. Thus 1 μM was selected as the treatment dose. Four UM-SCC cell lines were selected to test the effect of WNT974 on tumor growth on the CAM and on distant metastases to liver and lung. Two cell lines, UM-SCC-11A and -25, were shown via deep exome sequencing to have a priori power calculation was performed. Given a Type I error rate of 0.05 and power of approximately 85 %, it was determined that a sample size of at least 9 tumors in each treatment group was needed to detect a treatment effect size of 1.5 standard deviations in magnitude. For each of the four cell lines, tumor weights for the treatment and non-treatment groups were recorded and compared using two-tailed T-tests. Given the need to run the assay twice for each cell line due to space limitations, an ANOVA model for each cell line was used to evaluate treatment effect controlling for assay batch as a main effect and as potential effect modifier in the model parameterization. These analyses revealed no significant batch effect in any of the four cell lines, thus allowing pooling of the data from both batches. The qPCR cycle threshold (CT) values for both the liver and lung specimens in the treatment group for each cell line were compared to the CT values of the corresponding organ and cell line using a two-tailed T-test, generating a P-value for both the liver and lungs in each cell line. P-values <0.05 were considered statistically significant.An . This demonstrated that all eight of the cell lines metastasized to the livers of the developing chicks. qPCR was also performed on DNA isolated from the chick lungs. However, only one negative control was included in the qPRC analysis due to the initial poor egg viability, and therefore these data have not been included in Fig. Five chick embryos were implanted with cancer cells for each of the eight UMSCC cell lines tested in this study. For each cell line, the number of viable embryos examined for tumor growth is given following the cell line: UM-SCC-1: 5, UM-SCC-10A: 3, UM-SCC-10B: 4, UM-SCC-11A: 5, UM-SCC-17A: 3, UM-SCC-17B: 5, UM-SCC-25: 5, and UM-SCC-34: 4 embryos. Of the viable embryos, a subset was randomly chosen for primary tumor dissection and weighing. Tumor weights ranged from 27.4 to 82.4 mg with consistency within cell lines  and less consistency between cell lines (up to a fourfold difference in tumor weights). To assess the metastatic ability of these cell lines in this assay, quantitative PCR performed on DNA isolated from the livers of xenograft bearing animals and negative control specimens are shown in Fig. 1high  cells Fig. . Of the pulation ) and 81.NOTCH1 mutation status and in vitro pharmacodynamic response to WNT974: UM-SCC-11A , UM-SCC-25 , UM-SCC-1 , and UM-SCC-14A . Tumor weight analysis shown in Fig. p = 1.0 × 10-7), UM-SCC-1 (p = 0.002), and UM-SCC-25 (p = 0.0076), the three cell lines that previously showed in vitro pharmacodynamic responses to WNT974 [p = 2.5 × 10-5) and UM-SCC-25 (p = 0.02) (Fig. After establishing the CAM model using several of the genetically characterized UM-SCC cell lines , we soughigh and ALDHhigh cells at similar rates to those observed in murine model HNSCC tumors. Our ability to detect CSCs on tumor cells grown in the CAM assay provides opportunities for use of this model in the study of HNSCC CSC populations, behavior, and response to drug therapies. The CAM model has several advantages as an adjunct to murine xenografts [We and others have used the CAM assay for a variety of studies , 26, 27.nografts . The CAMIn addition to evaluating CAM assays as a model of therapeutic response, we explored the interaction between the Wnt and Notch signaling pathways in HNSCC, specifically in the context of the anti-cancer effects of Wnt pathway inhibition in tumors with different types of Notch pathway mutation. Given the previously proposed inhibitory effect of the Notch pathway on Wnt signaling , 20, it HNSCC cell line xenograft growth, cancer stem cell distribution, metastasis and therapeutic response can be effectively assessed in CAM assays in a manner consistent with mouse xenograft assays. This allows for an expeditious means to screen the efficacy of new therapeutic agents across a large number of head and neck tumors such that more time consuming mouse xenograft models can be focused on the precision medicine protocols most likely to advance to clinical trials. Therefore, we expect that implementation of this model will significantly reduce the pre-clinical phase of drug development timelines.


MDM4 mRNA/protein levels or TP53 status. Alkaline/neutral comet and γH2AX immunofluorescence assays revealed that the cytotoxic effects of XI-006 could not be attributed to the induction of DNA damage. RNA expression analysis revealed that the mechanism of action of XI-006 could be accredited to the inhibition of cell division and cycle regulators such as KIF20A and GPSM2. Finally, potent synergy between XI-006 and olaparib (PARP inhibitor) were observed due to the down-regulation of Mre11. Our findings suggest that XI-006 represents a novel therapeutic intervention for the treatment of Ewing sarcoma.There is an imperious need for the development of novel therapeutics for the treatment of Ewing sarcoma, the second most prevalent solid bone tumour observed in children and young adolescents. Recently, a 4-nitrobenzofuroxan derivative, XI-006 (NSC207895) was shown to diminish MDM4 promoter activity in breast cancer cell lines. As amplification of MDM4 is frequently observed in sarcomas, this study examined the therapeutic potential of XI-006 for the treatment of Ewing and osteosarcoma. XI-006 treatment of Ewing and osteosarcoma cell lines (n = 11) resulted in rapid and potent apoptosis at low micro-molar concentrations specifically in Ewing sarcoma cell lines (48 hr IC50 0.099–1.61 μM). Unexpectedly, apoptotic response was not dependent on  Sarcomas are a group of rare malignancies that affect approximately 200,000 individuals worldwide each yearMDM4 (Mouse Double Minute 4), a structural homologue of MDM2, thought to promote tumourigenesis via its ability to inhibit the tumour suppressor function of TP53MDM4 has been documented across a wide spectrum of tumours including cutaneous melanoma (68.5%)710Cancer is a multifaceted process that can arise due to the activation of proto-oncogenes and/or inactivation of tumour suppressor genes. The development of novel anti-cancer agents, specifically those focused on targeting oncogene addiction has undergone a dramatic renaissance over the past decade. One particular oncogene which has gained significant interest is melanoma 8.5%6 , bin vitro, uptake was attenuated in the presence of serum, and complete regression of xenograft tumours was not achieved4MDM4 mRNA and protein expression and cell viability in MDM4 amplified breast cancer cell linesOwing to the prevalence of MDM4 genomic amplification/mRNA overexpression in human cancers, several strategies aimed at inhibiting the oncogenic activity of MDM4 have been explored. Although a selective MDM4 small-molecule inhibitor does not currently exist, the first reported p53-MDM4 antagonist, SJ-172550, did exhibit cytotoxicity in retinoblastoma cellsin vitro. Specifically, treatment of Ewing sarcoma cell lines resulted in potent apoptosis that was remarkably not dependent on MDM4 mRNA or protein levels or TP53 status.To our knowledge, no studies have hitherto directly addressed whether repression of MDM4 activity can represent a novel therapeutic strategy for the treatment of sarcomas. In particular, as MDM4 amplification is a characteristic of both Ewing and osteosarcoma, this study has examined the biological effects of XI-006 both as a single agent and in combination with standard chemotherapeutic agents and olaparib (PARP inhibitor) in a comprehensive panel of Ewing and osteosarcoma cell lines MDM4 mRNA expression was recently shown not to correlate with protein expression in freshly isolated human melanomasMDM4 mRNA overexpression was not observed in our previous cohort of 24 sarcoma tissuesp < 0.0001)  .MDM4 was associated with statistically significant increased MDM4 protein expression in high-grade ovarian carcinomashsa-miR-191, which only recognised the 3’UTR of the MDM4-C allele resulting in decreased MDM4 mRNA and protein expression. To determine whether SNP34091 regulates MDM4 protein expression in sarcomas, the 3’UTR of MDM4 was sequenced. Genotypes were as follows, 17 (47.2%) were homozygous for the wild-type allele (A/A), 12 (33.3%) were heterozygous (A/C) and 7 (19.4%) were homozygous for SNP34091 (C/C) (P = 0.521) (AA vs CC: P = 0.624)  located 32 nucleotides downstream of the stop codon in the 3’UTR of 91 (C/C) . Presenc= 0.624) .MDM4 is frequently observed in Ewing and osteosarcomasP = 0.008) to achieve an IC50, range of 3.60–>10 μM and 2.14–5.41 μM, 24 and 48 hr treatment respectively  staining . In the MDM4 mRNA and protein levels in cell lines with varying MDM4 genomicMDM4 mRNA expression was observed in U20S cells  following XI-006 treatment; however U20S cells were the least sensitive cell line to XI-006 , the least sensitive Ewing sarcoma cell line. Consistent with these findings, a dose-dependent reduction in MDM4 protein levels following XI-006 treatment was only observed in U20S and WE-68 cells, but not TC252 cells  (P = 0.05), equating to a 30.5% reduction in activity (P = 0.491) (As XI-006 was shown to decrease MDM4 expression in breast cancer cell lineso XI-006 . Althoug52 cells . We next 0.0005) . To confactivity . No effe= 0.491) . As X1-0TP53 target genes implicated in apoptosis , cell cycle arrest (CDKN1A) and p53 regulation (MDM2). Wild-type TP53 Ewing  and osteosarcoma  cell lines were treated with XI-006 , with target gene expression assessed through real-time qPCR assays . In contrast, induction of these target genes was significantly lower in all osteosarcoma cell lines, in particular BAX and CDKN1A, where no induction was observed for all time points and XI-006 concentrations.We next examined whether XI-006 can induce expression of R assays . XI-006 TP53 target genes, the cytotoxic effects of XI-006 were shown to be TP53 independent, as XI-006 sensitivity was not correlated with TP53 status (P = 0.190)   or relative viability at any concentration tested was observed, endorsing the p53-independent cytotoxic effects of XI-006.To determine whether induction of p53 target genes specifically in Ewing sarcoma cells could be attributed to activation and stabilisation of p53, p53 protein levels following XI-006 treatment were examined. Activation and stabilisation of p53 protein levels was only observed in TC252 and WE-68 Ewing sarcoma cell lines but not U20S cells following 4 hrs of XI-006 treatment . Further= 0.190) . Indeed,= 0.190) . To furt0–10 μM) . No signPrevious reports proposed that XI-006 activates the DNA damage response pathway leading to a delay in cell cycle progressionataxia telangiectasia mutated) is critical for sensing and co-ordinating repair of DNA DSBs. To further confirm that the cytotoxic effects of XI-006 was not due to DNA damage at low concentrations, Ewing cells  were pre-treated with the ATM inhibitor KU-55933, before the addition of XI-006 (0.02–5 μM). A significant reduction in both ATM and ATR mRNA expression (82.0% and 87.5% respectively) was observed following monotherapy KU-55933 treatment (5 μM) (The serine/threonine protein kinase ATM (t (5 μM) . No signt (5 μM) .EWS-FLI1, the hallmark gene fusion of Ewing sarcoma, at ThrEWS-FLI1 fusion, we examined the role p38α/p38β MAPKs in XI-006 sensitivity. TC252 and RD-ES cells were pre-treated with the p38α/p38β MAPK inhibitor BIRB 796  before the addition of XI-006 (0–2.5 μM) for 24 and 48 hrs. BIRB 796 was previously shown to inhibit p38α/p38β MAPKs specifically at 0.1 μM, p38y/p38δ MAPKs at 1 μM and completely suppress the activation and activity of all JNKs at 10 μMPrevious chemogenomic profiling studies suggested that XI-006 imparts its cytotoxic effect through the activation of the DNA-damage-response pathway. Phosphorylation of the N-terminal domain of Finally, alkaline comet assays were employed to investigate whether XI-006 induces single-strand break DNA damage. Following 4 hrs of XI-006 treatment, a significant increase in comet tail and length was only observed at concentrations exceeding 2 μM . Collect2 = 0.722) was observed between XI-006 and vincristine IC50 values in all Ewing sarcoma cell lines, suggesting that these two agents may have a similar mechanism of action  was assessed in five Ewing sarcoma cell lines . Modest synergistic combination indexes (CI < 1) were observed in 4/5 cell lines for actinomycin and doxorubicin and 3/5 cell lines tested for etoposide (CI range 0.753–0.989) over multiple chemotherapeutic doses . Althougf action .poly-ADP-ribose polymerase), which subsequently further promotes transcriptional activation by EWS-FLIEWS-FLI positive Ewing sarcoma cell lines. TC252, RD-ES and WE-68 cells were pre-treated with olaparib prior to addition of XI-006 (0–5 μM). Potent synergy between these two agents was observed in all cell lines tested and across multiple XI-006 concentrations, 24 and 48 hrs post XI-006 treatment. A maximum 51.9% increase in apoptosis was observed in RD-ES cells when these two agents were combined (48 hr XI-006 treatment) (The EWS-FLI fusion protein has been shown to drive expression of PARP1 (eatment) . Olaparimeiotic recombination 11) for end processing to facilitate replication restart and recombination repairMre11 mRNA expression levels following XI-006 treatment was assessed. A decrease in Mre11 mRNA expression was observed across all cell lines tested, with a maximum 50.1% reduction observed in STA-ET-1 cells (8 hr treatment) (Mre11 expression in RD-ES pre-treated with olaparib (1 μM) before the addition of XI-006 was assessed. Olaparib treatment alone resulted in a significant 25% decrease in Mre11 mRNA expression  . As PARPpression . Co-treaP < 0.05, Bonferroni correction) repressed  or induced  following non DNA-damaging XI-006 treatment (0.5 μM)  were repressed on average by 51.2% and 41.3% respectively across all cell lines following XI-006 treatment  . Real-tireatment .UTRN, HEG1, FLOT1, EDIL3) were identified and validated through real-time qPCR analysis  which undergo apoptosis following XI-006 treatment, and repressed in osteosarcoma cell lines (average 31.9% decrease) (P = 0.025) which undergo cell-cycle arrest following XI-006 treatment.We next sought to identify genes that were differentially expressed between Ewing and osteosarcoma cell lines following XI-006 treatment. Four genes  in the pathogenesis, maintenance, and chemo-resistance of human cancer, there are currently no selective MDM4 antagonists undergoing clinical trial evaluation. As MDM4 gene amplification is a characteristic of both Ewing and osteosarcomaMDM4 mRNA or protein levels, and reduction of MDM4 mRNA and protein levels were only observed in the least sensitive Ewing sarcoma and osteosarcoma cell lines at high XI-006 concentrations (>1 μM) that also induced DNA damage  in the absence of both observable DNA damage and effect on MDM4 expression levels , Fig. 2.A damage  and 4. IThe introduction of systemic chemotherapy in the 1960’s greatly improved survival rates for patients with localised Ewing sarcomaEWS-FLI1, the hallmark translocation of Ewing sarcoma, and sensitivity to the olaparib. Indeed, FLI1 expression levels in Ewing sarcoma cell lines were highly correlated with olaparib sensitivityEWS-FLI1 fusion acted in a positive feedback loop to maintain the expression of PARP1 which was required for EWS-FLI–mediated transcriptionEWS-FLI1 was potentiated by PARP1 inhibition in vitro. As PARP1 inhibitors have exhibited promising activity in early clinical trialsPARP-1 is a member of the base excision repair pathway that sensors and modulates the spatial and temporal organization of single-strand break repairPrevious chemogenomic profiling studies suggested that XI-006 (referred to as NSC-207895) activates the DNA-damage-response pathway through an indirect mechanism leading to a significant delay in replication and cell cycle progression38UTRN (Utrophin) was significantly upregulated in Ewing sarcoma cell lines but repressed in osteosarcoma cell lines (P = 0.025). The GTPase UTRN also known as Drip1, mediates outer mitochondrial membrane fission and is essential for the normal progression of several mitochondria dependent apoptosis pathwaysUTRN is recruited from the cytosol to the mitochondrial outer membrane, where it colocalizes with Bax at fission sites and mediates the release of apoptotic regulatory proteins including cytochrome c prior to caspase activation43FLOT1 (flotillin) was also significantly repressed in osteosarcoma cell lines (P = 0.044) compared to Ewing. The flotillin family of proteins have been implicated in numerous cellular processes such as actin-cytoskeleton reorganization, endocytosis, adhesion and transduction of cellular signalsFLOT1 has been shown to significantly impair cell proliferation and tumourigenicity of breast and esophageal squamous cell carcinoma cells in vitro and in vivo through the Akt/FOXO3a pathways46FLOT1 induced G1-S-phase arrest of breast cancer cells due to upregulated expression of the CDK inhibitors p21Cip1 and p27Kip1. Following XI-006 treatment, both UTRN and FLOT1 were strongly repressed in osteosarcoma cell lines (31.9% and 33.8% decrease in mRNA expression respectively) but not in Ewing cell lines. This may be the basis of the finding that XI-006 induces apoptosis in Ewing sarcoma cell lines but cell cycle arrest in osteosarcoma cell lines , have been implicated in cell proliferation. KIF20A belongs to the family of kinesin microtubule-dependent motor proteins, which are required for bipolar spindle assembly, chromosome alignment, chromosome segregation, and cytokinesis512 = 0.722)  , a widelin vivo.In summary, our findings demonstrate that XI-006 is a promising new potential therapeutic for the treatment of Ewing sarcoma as it induced potent p53-independent apoptosis at non-DNA damaging concentrations specifically in Ewing sarcoma cell lines. Notably, strong synergy was observed with olaparib, a PARP inhibitor that is gaining significant interest for the treatment of solid cancers. As such, our preclinical findings warrant further pharmacokinetic and pharmacodynamic investigations of XI-006 Ewing sarcoma cell lines were cultured as previously describedXI-006 and BIRB 796 were purchased from Merck Millipore, KU-55933 and olaparib (AZD2281) were purchased from Selleck. Vincristine sulfate (Hospira), doxorubicin HCI (Pfizer), actinomycin D/Cosmegen (Lundbeck) and etoposide  were supplied by M.P Brown .National Health and Medical Research (NHMRC) approved guidelines.Chemotherapy/radiotherapy naïve tumour specimens were collected from thirty-six patients with sarcoma  undergoing surgical resection/core biopsy at three clinical institutions; Royal Adelaide Hospital (RAH), Calvary Wakefield Hospital, and St Andrew’s Hospital between 2010 and 2013. Patient consent was obtained for accrual of surgically excised tissue. Study was approved by the Royal Adelaide Hospital Human Ethics Committee (RAH Protocol #100505). The different morphological subtypes were represented by nine undifferentiated pleormorphic sarcomas, eight liposarcomas , six leiomyosarcomas, five myxofibrosarcomas, two osteosarcomas, two synovial and one Ewing sarcoma, angiosarcoma, chondrosarcoma and radiation induced sarcoma. Twenty patients were previously describedMDM4 immunohistochemical protocol was adapted fromDNA from sarcoma tissues was isolated using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions. The 3’UTR region of MDM4 was amplified using the following primer pair4 cells/well and treated with XI-006 alone or in combination with chemotherapeutics agents. For inhibitor studies, cells were pre-treated with olaparib, KU-55933 or BIRB 796 for 2 hrs prior to the addition of XI-006. Following treatment for 24 and/or 48 hrs, cells were centrifuged at 2500 rpm for 5 mins, washed in phosphate buffered saline (PBS) and stained with 7-amino-actinomycin-D solution  for 10 mins at room temperature.For viability assays, cell were seeded in 96-well micro-titer plates at a density of 3 × 10For cell cycle analysis, XI-006 and vehicle control treated cells were permeabilized with cold 70% ethanol overnight, and stained with a solution containing 50 μg/ml propidium iodide , 0.05% Triton-X and 100 μg/ml RNase A at 37 °C for 40 mins. Cell viability and DNA content was determined through the use of a FACS Calibur flow cytometer (Becton-Dickinson Immunocytometry Systems) with cell cycle profiles and viability analyzed using FLOWJO software (V7.6.5).4) were seeded overnight in 24-well plates (triplicate wells per treatment) and transfected with 200 ng of pCM-luci-MDM4 or p21-pro-Luc reporter constructs and 25 ng of pRL-TK plasmid (Promega) using Lipofectamine LTX (Invitrogen) according to the manufacturers’ instructions. pCM-Luci-MDM4 was kindly supplied by C. Yan (GRU Cancer Center). Cells were treated with vehicle control or XI-006 for 6 hrs with Dual-luciferase reporter assays (Promega) performed according to the manufacturer’s instructions.U20S cells . Real-time qPCR reactions were performed using iTaq Universal Sybr Green Supermix (BIORAD) and processed on a CFX Real-Time PCR detection system (BIORAD). Cycling parameters were as follows: 95 °C for 3 mins, followed by 45 cycles of denaturation at 95 °C for 10 secs, annealing at 59–63 °C for 15 secs, and extension at 72 °C for 30 secs. Relative target mRNA expression was determined using the ΔCT method from triplicate reactions, with the levels of gene expression normalized to the relative average Ct value of Peptidylprolyl Isomerase-G (PPIG). Primer sequences and annealing temperatures are listed in Total RNA was extracted using RNeasy mini kit (Qiagen), using on-column RNase-free DNase digestion according to the manufacturer’s instructions. cDNA was synthesized by reverse transcribing 600 ng of total RNA using random primers (Promega) and Moloney murine leukemia virus reverse transcriptase  were seeded on microscopes slides and treated with XI-006  or vehicle control (DMSO) for 4 hrs. Cells were fixed with 10% neutral-buffered formalin, washed with PBS and blocked with 5% bovine albumin serum with 0.3% Triton-X 100 (Sigma-Aldrich). Slides were washed in PBS (3 × 5 min), then incubated with 1 μg/ml biotinylated mouse anti-human anti-phospho-histone H2AX (ser139)  overnight at 4 °C. After further washing, samples were incubated with 5 μg/ml streptavidin-Alexa Fluor 488 (Life Technologies) followed by counterstaining with 1 μg/ml DAPI. Slides were examined using an Olympus IX71 microscope (x40 magnification) with CellSens Standard (v1.6) software. Images were analysed using ImageJ (v1.45) .TC252 and U20S cells (1.5 × 105 cells were plated in 6 well plates and treated with XI-006 or vehicle control for 4 or 20 hrs. Lysed cells were subjected to electrophoresis for 40 mins at 30 V (300 mA) at 4 °C. Cells were stained with 2.5 μg/ml PI for 15 mins and visualised with an Olympus IX71 microscope (x20 magnification) with CellSens Standard (v1.6) software. Tail length and moment were assessed using AutoComet software (TriTek) from a minimum of 60 cells.Neutral and alkaline comet assays were performed with the Trevigen CometAssay kit according to the manufacturer’s protocol. Briefly, 3.5 × 10Ewing and osteosarcoma cell lines (n = 11) were treated with XI-006 (0.5 μM) or vehicle control for 4 hrs. One μg of RNA was used for polyA selection and library construction with NEBNext UltraT RNA Library Prep Kits for Illumina sequencing, according to the manufacturer’s instructions (E7530 Version 2). The mRNA library size was validated with the Agilent BioAnalyzer on High Sensitivity chips, with yield determined with a Life Technologies Qubit 2.0 Fluorometer. The mRNA libraries were pooled and sequenced across five lanes of an Illumina HiSeq 2500 flowcell (1 × 50 bp reads) at the Australian Cancer Research Foundation Cancer Genomics Facility . Reads were trimmed for the NEB single end adapter “AGATCGGAAGAGCACACGTCTGAACTCCAG TCAC” with Cutadapt v1.3, requiring a minimum overlap of 5, allowing a 20% error rate and discarding trimmed sequences shorter than 18 bases. Reads were mapped to the UCSC hg19 genome and GTF annotations with Tophat 2.0.9 using default parameters. Gene counts were performed with HTSeq-count v0.6.1p1 using gene_id as the GTF feature ID.P values were calculated using Student t test using Graph Pad Prism Version 6.Combination Index (CI) values were used to determine synergy between XI-006 and cytotoxic agents. A CI value of <1, =1 and >1 indicates synergistic, additive and antagonistic effects respectivelyHow to cite this article: Pishas, K. I. et al. XI-006 induces potent p53-independent apoptosis in Ewing sarcoma. Sci. Rep. 5, 11465; doi: 10.1038/srep11465 (2015).


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.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 BRAFV600E mutant melanoma patients receiving the BRAF inhibitor vemurafenib [de novo resistance to vemurafenib in the context BRAFV600E [de novo and acquired resistance but predicting drug combination activity from single agents is not yet feasible in part because only relatively small datasets of combination exist. Candidate-based discovery of combination drug targets such as sequencing tumors for additional driver somatic mutations [3) project, we screened a large collection of melanoma cell lines across several thousand combinations of targeted inhibitors. Melanoma was selected in light of the availability of a large number of cell lines harboring a common mutated oncogene (BRAFV600E) and a validated targeted therapy.Although response rates within genetically-selected subpopulations of solid tumor cancer patients can be high, such as 60–80% among urafenib , few paturafenib , 3. Receurafenib –7, pointutations , 10, whiutations . To undeTo gain insight into the landscape of clinically relevant synergistic combinations targeted agents in cancer, we assembled a library of 108 compounds. Since we were interested in finding drug combinations with potential for clinical translation and for which mechanism of action would be tractable, we selected well-characterized oncology drugs approved by the Food and Drug Administration (FDA) or in late clinical trials; two-thirds of these agents have been in clinical use . We thenInitial analysis of the synergy values showed that a large number of combinations demonstrated > 40% Bliss independence in more than one cell line, including several drugs with broad lethality and synergy when paired with any other drug across the cell line collection . HoweverA number of drugs induced increased cell death as measured by cPARP, including ABT263 and the broad kinase inhibitor midostaurin . Fewer dbona fide synergistic interactions. Among these included MK1775, a WEE-1 inhibitor, and AZD7762, a CHK1/2 inhibitor , when compared to sensitive lines such as UACC62 and SkMel28 (IC50 < 500nM)  . Many of< 500nM) . We used< 500nM) . Among t< 500nM) , with a < 500nM) , but not< 500nM) . Long-te< 500nM) . The com< 500nM) . While s< 500nM) . In cont< 500nM) .Cediranib is a potent and selective inhibitor of the PDGFR and VEGFR family of receptor tyrosine kinases in clinical trials, used primarily as an anti-angiogenesis agent . While Pin vitro and clinical studies [Activation or re-activation of major pathways downstream of RTKs such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)–AKT pathways previously has been implicated in resistance to targeted therapies including vemurafenib in both  studies , 27–32.  studies , this pain vivo. In two xenograft models with the resistant lines ISTMel1 and RPMI7951, we observed a strong interaction between the inhibitors on tumor progression  may be in vivo in several non-melanoma tumor types [V600E mutant but PLX4720-resistant cells. There is, however, growing recognition of a cell-autonomous role for VEGF receptors in tumor cells [V600E mutation and elevated KDR/PDGFRα/β expression. Furthermore, we found synergistic interaction between cediranib and PLX4720 in non-melanoma BRAFV600E mutant cell lines with expression of KDR/PDGFRα/β family members  data through the C-Bio portal  did not or types , 41; theor cells . Indeed, members , suggest2. “WM” lines were obtained from cryopreserved collections at the Wistar Institute, courtesy of M. Herlyn. “CBRC” lines were derived from biopsies of MGH melanoma patients, as described [http://www.cancerRxgene.org).Cells were grown in RPMI or DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin and 2 mM L-glutamine, and maintained at 37°C in a humidified atmosphere at 5% COescribed . All werescribed , all CMTescribed , as follCompounds for screening were purchased from the commercial vendors as listed in Approximately 250 cells per well for each cell line were seeded in each well of 1,536-well microtiter plates (PerkinElmer) and incubated overnight. Combinatorial drug library was prepared as 300X stocks in 384 well plate and 20nL pin-transferred to 1,536-well plates. Plates were incubated for 72 hours and fixed in 4% formaldehyde, washed in PBS containing 0.1% Triton, and incubated overnight with antibodies (1:500) to cleaved PARP (above). The plates were then washed twice, incubated with Alexa 647 secondary antibodies  and DAPI 2–16 hours, and washed with PBST. Plates were then imaged using the Cellworx high-throughput microscope (Applied Precision Inc.) and counting nuclei and cells with cPARP staining with the Multi-Wavelength Cell Scoring module of the MetaExpress image analysis software (Molecular Devices). Z’ values were between 0.8–0.9 for most cell lines.R toolbox ‘ezyfit’:For every cell-line in the collection, we performed a dual regression fit  on the relationship between cell counts (% viability) and apoptosis (% cPARP) levels, using the Matlabτ’, within exponential curve fitting approach: evaluate ‘a’ and ‘τ’, within f(x) = a * x + τ.linear fitting approach: evaluate ‘a’ and ‘f(x)) and their fitted values , JNK2 (Dharmacon #L-003505-00-0005), JNK3 (Dharmacon #L-004324-00-0005), EGFR (Dharmacon L-003114-00-0005), HER2 (Dharmacon #L-003126-00-0005), KDR (Dharmacon #L-003148-00-0005), PDGFRα, PDGFRβ (Dharmacon #L-003163-00-0005), MDR1 (Dharmacon #L-003868-00-0005), GSK3β (Dharmacon #L-003010-00-0005) or control non-targeting siRNA (Dharmacon #D-001810-10-20) or individual siRNAs targeting KDR  were transfected at final concentration of 10nM (single gene knockdown) or 20nM  using the lipidoid delivery agent C12-113-B as previously described . LipidoiAll secondary viability assays were performed using the CellTiter Glo assay (Promega) after seeding 3,000 cells per plate, and the following day adding the indicated drugs for 96 hours. For long-term cell proliferation assays, cells were seeded into 12-well plates and cultured both in presence of drugs or controls as indicated, and stained by crystal violet staining.actB or hTBP mRNA as indicated. Primers were as follows, all 5’ to 3’ orientation: hTBP (forward), AACAACAGCCTGCCACCTTA, (reverse), GCCATAAGGCATCATTGGAC; actB (forward) CTGGAACGGTGAAGGTGACA, (reverse), AAGGGACTTCCTGTAACAATGCA; EGFR (forward), AGGCACGAGTAACAAGCTCAC, (reverse), ATGAGGACATAACCAGCCACC; HER2 (forward), GCTCATCGCTCACAACCAAGT, (reverse), ACAGGGGTGGTATTGTTCAGC; JNK1 (forward), TGTGTGGAATCAAGCACCTTC, (reverse), AGGCGTCATCATAAAACTCGTTC; JNK2 (forward), GAAACTAAGCCGTCCTTTTCAGA, (reverse), TCCAGCTCCATGTGAATAACCT; JNK3 (forward), CAAATGTTGTGTGGCATTAAGCA, (reverse), TCAATGTGCAATCAGACTTGACT; GSK3β (forward) CCACTGTTGTCACCTTGCTG, (reverse), GAGTGATCATGTCAGGGCG; KDR (forward), GGCCCAATAATCAGAGTGGCA, (reverse), CCAGTGTCATTTCCGATCACTTT; PDGFRα (forward), TGGCAGTACCCCATGTCTGAA, (reverse), CCAAGACCGTCACAAAAAGGC; PDGFRβ (forward), AGCACCTTCGTTCTGACCTG, (reverse), TATTCTCCCGTGTCTAGCCCA; MDR1 (forward), TTGCTGCTTACATTCAGGTTTCA, (reverse), AGCCTATCTCCTGTCGCATTA; MRP1 (forward), CGACATGACCGAGGCTACATT, (reverse), AGCAGACGATCCACAGCAAAA; BCRP (forward), ACGAACGGATTAACAGGGTCA, (reverse), CTCCAGACACACCACGGAT.mRNA was extracted from cell lines using the RNeasy kit (Qiagen) and homogenized using the Qiashredder kit (Qiagen). Total mRNA was used for subsequent reverse transcription using the Kapa SYBR FAST qPCR master mix (Kapa Biosystems). Expression was normalized to 2O2. Secondary antibody Dako EnVision anti-rabbit  was applied for 30 minutes. Slides were developed with DAB+ (Dako K3468) for 10 min, and counterstained 1 min with hematoxylin (Vector H-3401), prior to dehydration and mounting. Slides were imaged on an Olympus BX51 microscope with Olympus DP25 camera using Olympus WHN10X-H/22 oculars, Olympus UPlan FL N -20x/0.50 and -40x/0.50 objectives, an Olympus DP25 camera, and images acquired using Olympus DP2-TWAIN software and Adobe Photoshop 7.0. Slides were scored for intensity and distribution of KDR and PDGFRβ by a dermatopathologist blinded to clinical outcome. Scoring of staining (by A. P.) and bio statistical analysis was doubled-blinded.For western blotting, whole cell lysates were collected in Radio-Immunoprecipitation Assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors (Roche). Centrifuged supernatants normalized for protein content by bicinchoninic acid (BCA) protocol (Pierce). Equal amounts of protein were resolved by electrophoresis on 4–15% or 10–20% gradient gels and transferred to polyvinylidene difluoride membranes. IRDye 800CW (Rockland) or Alexa 680-conjugated secondary antibodies were used, followed by imaging on the LiCor Odyssey (Li-Cor Biosciences). For immunohistochemistry, 4 μM sections of formalin-fixed, paraffin-embedded specimens were heated at 60°C, deparaffinized in xylene, and hydrated in a series of ethanol dilutions. Epitope retrieval was by microwaving  in 10 mM Tris-EDTA buffer pH 9.0. Slides were blocked 10 minutes in 3% BSA in TBST . Primary antibodies were as follows: KDR, 1:100 in 3% BSA in TBST, clone 55B11 ; PDGFRβ, 1:100 in 3% BSA in TBST, clone 28E1 . Slides underwent 10 min peroxidase block in 3% H7 ISTMel1 or RPMI7951 cells were injected subcutaneously into the flanks of female nu/nu (ISTMel1) or NSG (RPMI7951) female mice aged to 7–8 weeks. Six to eight animals were used per experiment as per standard procedures. After tumors reached 100–150 mm3 in size, animals were assigned to treatment groups by simple randomization and given ad libitum mouse chow containing 2% PLX4720 by weight or control chow acquired from Plexxikon Inc. Cediranib (6mg/kg) was administered daily by oral gavage 5 days per week. Tumor volume was calculated by the formula ½ x (length x width2); investigator was not blinded to treatment group. All studies and procedures involving animal subjects were approved by the Institutional Animal Care and Use Committees of Massachusetts General Hospital and Dana-Farber Harvard Cancer Center and were conducted strictly in accordance with the approved animal handling protocol.For mouse xenotransplant experiments, 1 × 10De-identified biopsies of patients for generation of short-term cultures or for immunohistochemical staining to KDR or PDGFRβ were obtained after informed consent on Dana-Farber/Harvard Cancer Center-approved protocols 11–181 and 02–017. All samples were analyzed via hematoxylin and eosin (H&E) staining to confirm that viable tumor was present.t-test (two-sided) of patient samples was implemented after confirming normal distribution and equal variance by F test. To identify significantly synergistic combinations with PLX4720 specific to the PLX4720-resistant cell lines, we performed SAM analysis on both the original and modeled combination data. Heat maps, hierarchical clustering, and SAM analysis were implemented using the MeV platform [Cell culture growth data were modeled using a nonlinear sigmoid regression curve using GraphPad Prism 6 for Mac (GraphPad). All statistical tests excluding those explicitly discussed above were implemented using GraphPad. For animal ANOVA tests, the data were log transformed prior to analysis to equalize variance by Bartlett’s test; Tukey’s test was used to account for multiple comparisons. Student’s platform .S1 Figx axis) and the normalized and log-transformed percent of cPARP positive nuclei (y axis). Shown are the data for the low (left) and standard (right) library concentration. In general, single drugs that result in reduced viability also show an increase in cPARP percentage. Insets show population of single drugs resulting in relatively normal viability, some of which also show increase cPARP staining. (B) As in (A) but shown are results for all combinations across all melanoma cells lines. Also demonstrated in insets are specific population of drug-drug-cell line triads with relatively normal viability but increased cPARP staining.(A) Single drug results across all melanoma cell lines, plotted as the relationship between the normalized cell count ((TIF)Click here for additional data file.S2 Fign = 4). (B) CHIR265, an inhibitor of BRAF and other kinases, showed a synergistic interaction with ABT263, a BCL2 family inhibitor, at high doses of CHIR265; this effect was confirmed (below) in UACC62 cells. Error bars represent s.d. of measurement replicates (n = 3). (C) BI78D3, a JNK family inhibitor, showed strong synergy with TZDZ8, a GSK3β inhibitor, across multiple melanoma cell lines. This interaction was confirmed in secondary experiments (below) in A375 cells. Error bars represent s.d. of measurement replicates (n = 8). (D) siRNA knockdown of TZDZ8 target GSK3β  or BI78D3 targets JNK1, JNK2, or JNK3  showed no synergy with 500nM BI78D3 or 5μM TZDZ8, respectively. Error bars represent s.d. of measurement replicates. RT-PCR confirming siRNA-mediated target knockdown is shown at right. Expression is normalized to ActB. Error bars represent s.d. of measurement replicates (n = 2). (E) No synergy was observed between 5μM TZDZ8 and a variety of other JNK inhibitors, including CC401 (n = 2), SP600125 (n = 3), and TCS JNK5a (n = 2). No synergy was observed between BI78D3 and other GSK3β inhibitors, including 1μM CHIR99021 (n = 4) and 1μM SB216763 (n = 3). Error bars represent s.d. of measurement replicates. (F) Synergistic interaction was seen between TZDZ8 and 5μM BI78D3 across a range of non-melanoma cells, including BxPc3 pancreatic cell line (n = 2), DU145 prostate cell line (n = 2), MCF7 breast cancer cell line (n = 2), and normal human fetal melanocytes (n = 2). Summary of maximum Bliss measurements for each cell line is shown on bottom right. Error bars represent s.d. of measurement replicates.(A) AZD7762, a CHK1/2 inhibitor, displayed broad synergistic effects with MK1775, a WEE1 inhibitor, across multiple melanoma cell lines. This effect was confirmed in secondary experiments, below, showing synergistic interaction in A375 cells. Error bars represent s.d. of measurement replicates ((TIF)Click here for additional data file.S3 Fign = 7), SkMel30 , and IPC298 . Error bars represent s.d. of measurement replicates. (C) Significant synergy was also seen in the primary screen across multiple melanoma cell lines between vincristine and erlotinib. This synergy was confirmed in secondary experiments in A375 cells (right). Error bars represent s.d. of measurement replicates (n = 3). (D) siRNA-mediated knockdown of lapatinib targets EGFR and HER2 demonstrated no synergy with 2nM vincristine either alone  or in combination . Error bars represent s.d. of measurement replicates. Target knockdown was confirmed by RT-PCR measurement and normalized to hTBP or ActB (below). Error bars represent s.d. of measurement replicates (n = 4). (E) Canonical MDR inhibitor verapamil (5μM) showed significant synergy with vincristine in A375 cells. Error bars represent s.d. of measurement replicates (n = 7). (F) Although not statistically significant, a general trend was observed between increased MDR1 mRNA expression [MDR1, relative to hTBP control. Error bars represent s.d. of measurement replicates (n = 3). Also shown in the blot is basal MDR1 protein in WM451Lu cells, which is decreased compared to A375, correlating to decreased mRNA expression. (H) Western blotting confirmed over-expression of HA-tagged MDR1 in WM451Lu cells, relative to GFP control over-expression, and GAPDH loading control. (I) Synergistic interaction was seen between vincristine and 5μM lapatinib across a range of non-melanoma rapidly proliferating cells, including BxPc3 pancreatic cell line (n = 3), DU145 prostate cell line (n = 4), HeLa cervical cancer cell line (n = 3), MCF7 breast cancer cell line (n = 2), and PANC1 pancreatic cell line (n = 3). Much less synergy was seen in normal, more quiescent cells such as human fetal melanocytes (n = 4) and normal human fibroblasts (n = 2). Summary of maximum Bliss measurements for each cell line is shown on bottom right. Error bars represent s.d. of measurement replicates.(A) Isobologram demonstrating significant synergy between vincristine and lapatinib, as shown in A375 cells. Combination Index was, on average 0.37, with minimum of 0.085. (B) Confirmation of synergy between vincristine and 5μM lapatinib in multiple other melanoma cell lines, including UACC62 Click here for additional data file.S4 Fig(A) Hierarchical clustering of the Bliss synergy scores for all combinations with PLX4720, at both library concentrations (left), and the corresponding relative cell count values for each PLX4720-drug-cell line triad (right). (B) Top results of SAM analysis of combinations with PLX4720 specifically demonstrating synergy in the PLX4720-resistant cell lines.(TIF)Click here for additional data file.S5 Fign = 2–3). (C) Cediranib induces a synergistic decrease in cell growth when in triple combination with PLX4720 and selumetinib (100nM). Error bars represent s.d. of measurement replicates (n = 2). (D) Cell cycle analysis by flow cytometry with propidium iodide. PLX4720 causes less G1 arrest in ISTMel1 cells compared to UACC62 cells. Addition of cediranib has no significant effect on cell cycle in either cell line. Data shown is average of three independent experiments. (E) No significant synergy was seen between PLX4720 and the canonical MDR inhibitor verapamil. Error bars represent s.d. of measurement replicates (n = 2).(A) Isobologram demonstrating significant synergy between PLX4720 and cediranib, as shown in ISTMel1 cells. The Combination Index was 0.35, with a minimum of 0.01. (B) Synergy (Bliss ~30–40%) in ISTMel1 and RPMI7951 cells between cediranib and PLX4720 (top) and between cediranib and selumetinib . Error bars represent s.d. of measurement replicates ((TIF)Click here for additional data file.S6 Fign = 2). (B) Expression data for cediranib targets KDR and PDGFRα/β from the Center for Molecular Therapeutics(CMT)/Sanger database across melanoma cell lines screened in this study. Higher expression was seen in resistant lines ISTMel1 and RPMI7951. (C) Expression of KDR across the entire CMT/Sanger database of cell lines, showing melanoma lines have the highest expression of KDR compared to other tissues types.(A) Summary table (top) of maximum Bliss synergy scores for interaction between the given RTK inhibitor and PLX4720 in ISTMel1 and RPMI7951 cells. Strongest synergies were observed with cediranib and tivozanib across the KDR/PDGFR class of targets, compared to more specific or broader spectrum or less potent inhibitors. Results for tivozanib , crenolanib (PDGFRα/β inhibitor), and axitinib (KDR inhibitor) are shown below for ISTMel1 and RPMI7951 cell lines. Error bars represent s.d. of measurement replicates ((TIF)Click here for additional data file.S7 FighTBP in ISTMel1 cells following siRNA treatment to pooled KDR siRNA or two individual siRNAs. Knockdown efficiency correlated with synergy with PLX4720. Error bars represent s.d. of measurement replicates (n = 3). (B) RT-PCR results of PDGFRα, PDGFRβ, and KDR expression normalized to hTBP in ISTMel1 (left) and RPMI7951 (right) cells following siRNA treatment. Total siRNA concentration was normalized across single, double, and triple knockdown, using siRNA control reagent. Error bars represent s.d. of measurement replicates (n = 2).(A) RT-PCR results of KDR expression normalized to (TIF)Click here for additional data file.S8 Fign = 2). (B) Western blotting to dpERK and pS6K following single or combined treatment with PLX4720 and cediranib in PLX4720 resistant ISTMel1 and PLX4720-sensitive UACC62. Despite intrinsic resistance to PLX4720, ISTMel1 showed near complete suppression of ERK activation after PLX4720, as in UACC62 cells. Combined cediranib and PLX4720 treatment caused further suppression of pS6K in ISTMel1 cells after 24 hours. (C) Akt pathway phospho-antibody array  results in resistant ISTMel1 and RPMI7951 cells and PLX4720 sensitive UACC62 cells, after 24 hours of treatment. ISTMel1 and RPMI7951 cells showed decreased pathway activation at multiple levels following combined treatment of cediranib and PLX4720. (D) Combined treatment of ISTMel1 and RPMI7951 cells with PLX4720 and various inhibitors of the mTor/Akt pathway showed only modest synergy (summary table below for maximum Bliss scores), suggesting the more pronounced synergy with cediranib is due to suppression of additional pathways. Error bars represent s.d. of measurement replicates (n = 2). (E) Western blotting to Akt pathway marker phospho S6K showed the pathway inhibitors had variable but expected effects on suppression of S6K activation but no correlation between pathway suppression and synergy with PLX4720.(A) No synergistic interaction was seen between MEK inhibition by selumetinib and PLX4720 in ISTMel1 (left) or RPMI7951 (right) cell lines. Error bars represent s.d. of measurement replicates ((TIF)Click here for additional data file.S9 Figin vivo. Results are shown from lysates of ISTMel1 tumors after 72 hours of treatment. PLX4720-impregnated chow resulted in suppression of dpERK in tumors. Although Akt pathway suppression was seen in cell culture, significant suppression was not seen in tumors. Cediranib suppressed PDGFRα phosphorylation. Due to low expression levels, immunoprecipitation was performed to observe on-target suppression of pPDGFRβ and pKDR in tumors, detected by phospho-tyrosine antibodies following RTK immunoprecipitation. (C) Spider plots of all tumors from individual animals in the in vivo experiments using ISTMel1 or RPMI7951 cells for xenografts. (D) Animal weight during in vivo xenograft experiments showed no significant effects of single or combined cediranib and PLX4720 treatment. Error bars represent s.d. of measurement replicates (n = 7–9). (E) Synergistic interaction between PLX4720 and cediranib was observed in multiple non-melanoma BRAFV600E-mutant cell lines, including NMC-G1 glioma (average Bliss synergy 31%), SW872 liposarcoma (average Bliss synergy 30%), and SW982 (average Bliss synergy 53%) sarcoma. No synergy was observed in the BRAFV600E colon cancer cell lines COLO201 and WiDr. Error bars represent s.d. of measurement replicates (n = 2–3).(A) Interaction between PLX4720 and several Wnt pathway modulators including the β-catenin inhibitor FH535, GSK3β inhibitor TZDZ-8, and Wnt pathway inducer Wnti. (B) On-target pathway activity of PLX4720 and cediranib (TIF)Click here for additional data file.S1 TableListing of 108 drugs used in building the combinatorial library, including current stage of clinical development, putative primary targets, and standard and low concentrations used.(XLSX)Click here for additional data file.S2 TableListing of 36 cell lines used in the combination screen, including genotype of major oncogenes and key data from analysis of the vincristine-lapatinib and PLX4720-cediranib combinations discussed in detail in the manuscript.(XLSX)Click here for additional data file.S3 TableFull dataset of effects of drug combinations and individual drugs across all cell lines. Cell count data are presented as percent of DMSO control for single and combination drug effects. For combination data, values for Bliss synergy  are also given. Cleaved PARP values are given as percent of DMSO control percent of nuclei positive for cleaved PARP; positive Bliss synergy indicates synergistic induction of cleaved PARP.(XLSX)Click here for additional data file.S4 TablePatients with metastatic melanoma containing BRAFV600E mutation (confirmed by genotyping) were enrolled on clinical trials for treatment with a BRAF inhibitor (BRAFi), or combined BRAF inhibition and MEK inhibition (BRAFi + MEKi). Patient, age, site of disease, treatment, maximal response, and duration of response are reported. SD = stable disease, PR = partial response, CR = complete response, sc = subcutaneous, n = nodal. lu = lung, li = liver, br = brain, b = bone.(XLSX)Click here for additional data file.