Isogenic Clones Research Articles

Cellular Repair of DNA-DNA Cross-Links Induced by 1,2,3,4-Diepoxybutane.

Xenobiotic-induced interstrand DNA–DNA cross-links (ICL) interfere with transcription and replication and can be converted to toxic DNA double strand breaks. In this work, we investigated cellular responses to 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) cross-links induced by 1,2,3,4-diepoxybutane (DEB). High pressure liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS) assays were used to quantify the formation and repair of bis-N7G-BD cross-links in wild-type Chinese hamster lung fibroblasts (V79) and the corresponding isogenic clones V-H1 and V-H4, deficient in the XPD and FANCA genes, respectively. Both V-H1 and V-H4 cells exhibited enhanced sensitivity to DEB-induced cell death and elevated bis-N7G-BD cross-links. However, relatively modest increases of bis-N7G-BD adduct levels in V-H4 clones did not correlate with their hypersensitivity to DEB. Further, bis-N7G-BD levels were not elevated in DEB-treated human clones with defects in the XPA or FANCD2 genes. Comet assays and γ-H2AX focus analyses conducted with hamster cells revealed that ICL removal was associated with chromosomal double strand break formation, and that these breaks persisted in V-H4 cells as compared to control cells. Our findings suggest that ICL repair in cells with defects in the Fanconi anemia repair pathway is associated with aberrant re-joining of repair-induced double strand breaks, potentially resulting in lethal chromosome rearrangements.

THE ROLE OF YERSINIA PESTIS RESIDENT PLASMIDS PMT1, PCD1, AND PPCP1 IN THE PRODUCTION OF LIPOPOLYSACCHARIDE EXTRACELLULAR FORM

Objective of the study is to investigate the role of resident plasmids pMT1, pCD1, and pPCP1 in the production of extracellular form of Yersinia pestis lipopolysaccharide (LPS). Materials and methods . The experiments have been performed using Y. pestis strain EV76 (pMT1, pCD1, pPCP1), carrying the whole plasmid set, as well as plasmid-free Y. pestis variant EV76 (pMT1 - , pCD1 - , pPCP1 - ), and isogenic clones, harbouring only one plasmid: Y. pestis EV76 (pMT1); Y. pestis EV76 (pCD1); Y. pestis EV76 (pPCP1). The presence of extracellular LPS in the incubation medium of Y. pestis EV76 cells has been confirmed by supernatant toxicity for laboratory animals and also by LAL-test reaction. Results and conclusions . It has been established that LPS extracellular form is produced by 37 °C Y. pestis EV76 cultures of the initial strain and its variants, carrying pMT1 or pPCP1 plasmid. Plasmid-free cultures and variant harbouring pCPP1 plasmid are deprived of such ability. The results of LAL-test has shown that the process of LPS separation from cell wall membrane into the environment is associated with translocation of proteins encoded by pMT1 and pCD1 plasmids and constitutes a natural form of existence of Y. pestis cells. The involvement of pCD1 plasmid in realization of the toxic potential of Y. pestis LPS has been established for the first time ever.

PECULIARITIES OF INTERACTION BETWEEN CITELLOPHILUS TESQUORUM ALTAICUS (IOFF, 1936) FLEA AND YERSINIA PESTIS WITH VARIOUS PLASMID COMPOSITION

Objective of the study is in vitro investigation of mutual relations between Citellophilus tesquorum altaicus and Yersinia pestis with various plasmid composition: influence of the strain on flea alimentary activity and mortality rate, frequency and dynamics of biofilm formation. Materials and methods . C. tesquorum altaicus were infected by three Yersinia pestis strains: virulent triple-plasmid I-3230 isolated in Mongolia, referential for the Tuva focus I-2638 carrying four plasmids (pYT, pYV, pYP, pTP 33) and also selected from it avirulent isogenic clone I-3480 that lost two plasmids (pYV, pYP). Peculiarities of interaction between fleas and Y. pestis strains were estimated through the lens of specimens with «conglomerates» and “blocks” for feeding, the period from infection prior to the beginning of conglomerates’ formation, alimentary activity, and mortality rate of the infected fleas. Results and conclusions . It was revealed that alimentary activity of the infected insects was higher than that of the control group, and the highest – in fleas infected with I-2638 strain. Greater numbers of dead fleas at feeding was noted in specimens inoculated with I-3230 strain. Predominant significance of I-2638 strain was established in C. tesquorum altaicus biofilm formation both as «conglomerates» and “blocks”. I-3480 strain also formed the conglomerates in fleas more actively than I-3230 lacking pTP33 plasmid. Thus, four-plasmid I-2638 strain surpassed triple-plasmid I-3230 and two-plasmid I-3480 strains in reference to all tested indicators except flea mortality rates. It may testify to co-adaptation of Y. pestis and C. tesquorum altaicus from the Tuva plague focus and to the possibility of a pTP33 functional role in enhancement of a biofilm formation in vivo.

Waldenstrom Macroglobulinemia Cells Modulate Mitochondrial Bioenergetics and Induce a Respiratory Hyper-Drive State upon Acquisition of Ibrutinib-Resistance

Background: Mitochondrial activity is critical to maintain a high cellular proliferation especially in drug-resistant clones. Ibrutinib-resistance (IR) in CLL is associated with highly aggressive clinical behavior, carrying a median survival of ~3 months. While these outcomes are not yet reported in WM, resistance to ibrutinib is foreseeable considering no WM patients achieve a complete remission with BTK inhibitors (reported series). The transition to a highly proliferative drive in the IR clone remains a biological dilemma and understanding this will allow rationale development of therapeutic strategies. It is established that increased cell proliferation is highly dependent on the functional capabilities of the mitochondria. Yet the respiratory proficiencies and adaptation of mitochondrial machinery in the IR state remains unknown. Here we interrogated mitochondrial function and its relevance in IR cells using our WM models.Materials: WM cell lines (BCWM.1, non-IR) and its isogenic IR clone (BCWM.1/IR; 20-fold resistant, without BTK or CXCR4WHIM-like mutations) were used. Mitochondrial respirometry was measured by Seahorse XF96 flux analyzer (Fig. 1a). Drug sensitivity and cytotoxicity were determined by Cell-Titer Glo cell viability assay. Whole exome sequencing (WES) was performed on an Illumina HiSeq2000.Results: IR WM cells were noted to be in G1 arrest with altered morphologic features and growth kinetics. Despite growth arrest, IR cells showed increased basal oxygen consumption rate (OCR) (>30% increase) vs. parental (non-IR) cells. Consistent with this we observed enhanced mitochondrial ATP production suggesting increased mitochondrial utilization by the IR clones. We then measured maximal respiratory potential of WM cells with FCCP pre-treatment, which evaluates cellular potential to respond to increased ATP demand. We observed upregulation of mitochondrial spare respiratory capacity in BCWM.1/IR cells vs. parental BCWM.1 cell. In addition, a >50% increase in non-mitochondrial oxygen consumption/respiration was also noted (Fig.1b, c). To identify potential genomic changes that could explain mitochondrial overdrive, WES of the non-IR and IR cells was performed. Interestingly, this analysis did not uncover any mutational alterations in genes directing cellular oxidative phosphorylation (NDUF, COX1, COQ7, SURF1, etc.), glycolysis (PDK1, HK2, PKM2, ALDOA, etc.) or ATP production (ATP5, ND1, UCP1 and others). We then asked if mitochondrial function impacts therapeutic potential of a specific drug (or a class of drug), focusing specifically on proteasome disruption. First, we observed that proteasome inhibition- independent of the site (20S vs. 19S), retained high cytotoxic potential, wherein IR cells showed exquisite sensitivity to proteasome disruption. Interestingly, VLX1570 (targets 19S proteasome) had a 2-fold lower IC50 in IR clones than its non-IR counterpart. Cell respirometry demonstrated that compared to parental cells, VLX1570 (500nM) treatment significantly (p<0.002) and rapidly (2hr) attenuated basal respiration in IR clones at concentrations half of those needed to achieve similar effects in BCWM.1 cells. A concomitant decrease in ATP production as well as spare respiratory capacity was observed at these time points/drug concentrations (significantly more evident than in BCWM.1 cells). While our focus was mitochondrial bioenergetics and its therapeutic relevance in drug-resistant WM, unexpectedly we observed for the first time, evidence that VLX1570 induces changes in mitochondrial bioenergetics as early as 2hrs leading to increased mitochondrial membrane permeability and cell apoptosis in 6-12hrs. Comparable observations were made with bortezomib (targeting 20S).Conclusions: This is the first report identifying the role of mitochondrial hyper-drive in WM cells with resistance to ibrutinib. Of note, enhancedmitochondrial bioenergetics in the IR state increased sensitivity to proteasome inhibition and this was independent of the site of disruption in the proteasome pathway. Although, WES findings alone were not sufficient in explaining mitochondrial overdrive noted in our system, ongoing analysis of various mitochondrial functions (Fig. 1d) at the gene expression, copy number and epigenetic level may elucidate the underlying biomechanics of mitochondrial disposition in IR WM cells. [Display omitted] DisclosuresAnsell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Linder:Vivolux, Ab: Other: Shareholder. Ailawadhi:Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy.

Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming.

The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.

Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions

Loss-of-function mutations in the BRCA1 and BRCA2 genes increase the risk of cancer. Owing to their function in homologous recombination repair, much research has focused on the unstable genomic phenotype of BRCA1/2 mutant cells manifest mainly as large-scale rearrangements. We used whole-genome sequencing of multiple isogenic chicken DT40 cell clones to precisely determine the consequences of BRCA1/2 loss on all types of genomic mutagenesis. Spontaneous base substitution mutation rates increased sevenfold upon the disruption of either BRCA1 or BRCA2, and the arising mutation spectra showed strong and specific correlation with a mutation signature associated with BRCA1/2 mutant tumours. To model endogenous alkylating damage, we determined the mutation spectrum caused by methyl methanesulfonate (MMS), and showed that MMS also induces more base substitution mutations in BRCA1/2-deficient cells. Spontaneously arising and MMS-induced insertion/deletion mutations and large rearrangements were also more common in BRCA1/2 mutant cells compared with the wild-type control. A difference in the short deletion phenotypes of BRCA1 and BRCA2 suggested distinct roles for the two proteins in the processing of DNA lesions, as BRCA2 mutants contained more short deletions, with a wider size distribution, which frequently showed microhomology near the breakpoints resembling repair by non-homologous end joining. An increased and prolonged gamma-H2AX signal in MMS-treated BRCA1/2 cells suggested an aberrant processing of stalled replication forks as the cause of increased mutagenesis. The high rate of base substitution mutagenesis demonstrated by our experiments is likely to significantly contribute to the oncogenic effect of the inactivation of BRCA1 or BRCA2.

Abstract 3591: Expression of 14-3-3 gamma stabilizes polyploidization in NSCLC cells

Abstract In normal lung tissue the expression levels of 14-3-3 gamma are very low and tightly regulated, however, in cancer it is evident that 14-3-3 gamma's expression patterns become deregulated and significantly elevated. In patients with advanced non-small cell lung cancer (NSCLC), increased expression of this isoform is associated with poorer survival and significantly correlates with lymph node and distant metastasis. These data prompted us to analyze TCGA's NSCLC gene expression dataset, and it is clear that 14-3-3 gamma's expression continues to increase in the progression from early to late stage cancers. Taken together, these data suggest that overexpression of 14-3-3 gamma is correlated with a more aggressive tumor phenotype, an observation also seen with breast and hepatocellular carcinoma. The focus of this study is to elucidate the mechanism(s) causing these more aggressive cancer phenotypes. We have previously shown that overexpressing 14-3-3 gamma in human lung cancer cells harboring very low levels of endogenous 14-3-3 gamma and no wildtype p53 results in a stable subpopulation of cells with polyploid DNA content. Interestingly, approximately 40% of lung adenocarcinomas present with hyper-triploid karyotypes, and even a higher percentage, 40-60%, have inactivation of p53. It is well established that polyploid tumors have the capacity to increase tumorigenicity by allowing resistance to conventional therapies and also permitting elevated tolerance to chromosomal instability (CIN). This leads us to hypothesize that in the absence of p53, NSCLC tumors overexpressing 14-3-3 gamma result in a polyploid population of cells that may be influencing the aggressiveness of the tumor. Further in vitro analysis of these 14-3-3 gamma induced tetraploid cells show that they have a prolonged mitosis with significantly more lagging chromosomes in anaphase than their diploid counterparts, indicating an increase in chromosomal instability (CIN). After isolating isogenic tetraploid clones with or without 14-3-3 gamma expression, the stability of the tetraploid cell state was assessed. Clones not expressing 14-3-3 gamma quickly reverted back to a pseudo-diploid state, whereas overexpression of 14-3-3 gamma significantly prolonged the tolerance of tetraploidy and eventually resulted in an aneuploid cell state. Our data suggests that overexpression of 14-3-3 gamma increases tumorigenicity through the stabilization of a polyploid cell state. Citation Format: Cecil J. Gomes, Michael Harman, Sara Centuori, Charles Wolgemuth, Jesse Martinez. Expression of 14-3-3 gamma stabilizes polyploidization in NSCLC cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3591.

Different metabolic responses to PI3K inhibition in NSCLC cells harboring wild-type and G12C mutant KRAS.

KRAS mutations in non-small-cell lung cancer (NSCLC) patients are considered a negative predictive factor and indicate poor response to anticancer treatments. KRAS mutations lead to activation of the PI3K/akt/mTOR pathway, whose inhibition remains a challenging clinical target. Since the PI3K/akt/mTOR pathway and KRAS oncogene mutations all have roles in cancer cell metabolism, we investigated whether the activity of PI3K/akt/mTOR inhibitors (BEZ235 and BKM120) in cells harboring different KRAS status is related to their metabolic effect. Isogenic NSCLC cell clones expressing wild-type (WT) and mutated (G12C) KRAS were used to determine the response to BEZ235 and BKM120. Metabolomics analysis indicated the impairment of glutamine in KRAS-G12C and serine metabolism in KRAS-WT, after pharmacological blockade of the PI3K signaling, although the net effect on cell growth, cell cycle distribution and caspase activation was similar. PI3K inhibitors caused autophagy in KRAS-WT, but not in KRAS-G12C, where there was a striking decrease in ammonia production, probably a consequence of glutamine metabolism impairment.These findings lay the grounds for more effective therapeutic combinations possibly distinguishing wild-type and mutated KRAS cancer cells in NSCLC, exploiting their different metabolic responses to PI3K/akt/mTOR inhibitors.

Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells

mtDNA sequence alterations are challenging to generate but desirable for basic studies and potential correction of mtDNA diseases. Here, we report a new method for transferring isolated mitochondria into somatic mammalian cells using a photothermal nanoblade, which bypasses endocytosis and cell fusion. The nanoblade rescued the pyrimidine auxotroph phenotype and respiration of ρ0 cells that lack mtDNA. Three stable isogenic nanoblade-rescued clones grown in uridine-free medium showed distinct bioenergetics profiles. Rescue lines 1 and 3 reestablished nucleus-encoded anapleurotic and catapleurotic enzyme gene expression patterns and had metabolite profiles similar to the parent cells from which the ρ0 recipient cells were derived. By contrast, rescue line 2 retained a ρ0 cell metabolic phenotype despite growth in uridine-free selection. The known influence of metabolite levels on cellular processes, including epigenome modifications and gene expression, suggests metabolite profiling can help assess the quality and function of mtDNA-modified cells.

Mutations in PIK3CA sensitize breast cancer cells to physiologic levels of aspirin.

A review of the literature finds that women diagnosed with breast cancer, who were on an aspirin regimen, experienced a decreased risk of distant metastases and death. Several recent studies have reported an improvement in overall survival in colorectal cancer patients who harbored mutations in the oncogene PIK3CA and received a daily aspirin regimen. Breast cancer patients on a daily aspirin regimen experienced decreased risk of distant metastases and death. PIK3CA is the most frequently mutated oncogene in breast cancer, occurring in up to 45 % of all breast cancers. In order to determine if mutations in PIK3CA sensitized breast cancers to aspirin treatment, we employed the use of isogenic cellular clones of the non-tumorigenic, breast epithelial cell line MCF-10A that harbored mutations in either PIK3CA or KRAS or both. We report that mutations in both PIK3CA and KRAS are required for the greatest aspirin sensitivity in breast cancer, and that the GSK3β protein was hyperphosphorylated in aspirin-treated double knockin cells, but not in other clones/treatments. A more modest effect was observed with single mutant PIK3CA, but not KRAS alone. These observations were further confirmed in a panel of breast cancer cell lines. Our findings provide the first evidence that mutations in PIK3CA sensitize breast cancer cells to aspirin.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-016-3729-8) contains supplementary material, which is available to authorized users.

TALENs-mediated gene disruption of FLT3 in leukemia cells: Using genome-editing approach for exploring the molecular basis of gene abnormality.

Novel analytic tools are needed to elucidate the molecular basis of leukemia-relevant gene mutations in the post-genome era. We generated isogenic leukemia cell clones in which the FLT3 gene was disrupted in a single allele using TALENs. Isogenic clones with mono-allelic disrupted FLT3 were compared to an isogenic wild-type control clone and parental leukemia cells for transcriptional expression, downstream FLT3 signaling and proliferation capacity. The global gene expression profiles of mutant K562 clones and corresponding wild-type controls were compared using RNA-seq. The transcriptional levels and the ligand-dependent autophosphorylation of FLT3 were decreased in the mutant clones. TALENs-mediated FLT3 haplo-insufficiency impaired cell proliferation and colony formation in vitro. These inhibitory effects were maintained in vivo, improving the survival of NOD/SCID mice transplanted with mutant K562 clones. Cluster analysis revealed that the gene expression pattern of isogenic clones was determined by the FLT3 mutant status rather than the deviation among individual isogenic clones. Differentially expressed genes between the mutant and wild-type clones revealed an activation of nonsense-mediated decay pathway in mutant K562 clones as well as an inhibited FLT3 signaling. Our data support that this genome-editing approach is a robust and generally applicable platform to explore the molecular bases of gene mutations.

Base excision repair-mediated resistance to cisplatin in KRAS(G12C) mutant NSCLC cells.

KRAS mutations in NSCLC are supposed to indicate a poor prognosis and poor response to anticancer treatments but this feature lacks a mechanistic basis so far. In tumors, KRAS was found to be mutated mostly at codons 12 and 13 and a pool of mutations differing in the base alteration and the amino acid substitution have been described. The different KRAS mutations may differently impact on cancerogenesis and drug sensitivity. On this basis, we hypothesized that a different KRAS mutational status in NSCLC patients determines a different profile in the tumor response to treatments. In this paper, isogenic NSCLC cell clones expressing mutated forms of KRAS were used to determine the response to cisplatin, the main drug used in the clinic against NSCLC. Cells expressing the KRAS(G12C) mutation were found to be less sensitive to treatment both in vitro and in vivo. Systematic analysis of drug uptake, DNA adduct formation and DNA damage responses implicated in cisplatin adducts removal revealed that the KRAS(G12C) mutation might be particular because it stimulates Base Excision Repair to rapidly remove platinum from DNA even before the formation of cross-links. The presented results suggest a different pattern of sensitivity/resistance to cisplatin depending on the KRAS mutational status and these data might provide proof of principle for further investigations on the role of the KRAS status as a predictor of NSCLC response.

Identification of genotoxic compounds using isogenic DNA repair deficient DT40 cell lines on a quantitative high throughput screening platform.

DNA repair pathways play a critical role in maintaining cellular homeostasis by repairing DNA damage induced by endogenous processes and xenobiotics, including environmental chemicals. Induction of DNA damage may lead to genomic instability, disruption of cellular homeostasis and potentially tumours. Isogenic chicken DT40 B-lymphocyte cell lines deficient in DNA repair pathways can be used to identify genotoxic compounds and aid in characterising the nature of the induced DNA damage. As part of the US Tox21 program, we previously optimised several different DT40 isogenic clones on a high-throughput screening platform and confirmed the utility of this approach for detecting genotoxicants by measuring differential cytotoxicity in wild-type and DNA repair-deficient clones following chemical exposure. In the study reported here, we screened the Tox21 10K compound library against two isogenic DNA repair-deficient DT40 cell lines (KU70 (-/-) /RAD54 (-/-) and REV3 (-/-) ) and the wild-type cell line using a cell viability assay that measures intracellular adenosine triphosphate levels. KU70 and RAD54 are genes associated with DNA double-strand break repair processes, and REV3 is associated with translesion DNA synthesis pathways. Active compounds identified in the primary screening included many well-known genotoxicants (e.g. adriamycin, melphalan) and several compounds previously untested for genotoxicity. A subset of compounds was further evaluated by assessing their ability to induce micronuclei and phosphorylated H2AX. Using this comprehensive approach, three compounds with previously undefined genotoxicity-2-oxiranemethanamine, AD-67 and tetraphenylolethane glycidyl ether-were identified as genotoxic. These results demonstrate the utility of this approach for identifying and prioritising compounds that may damage DNA.

Abstract 1084: An EMT-like gene expression profile differentiates helical from kinase domain PIK3CA mutations

Abstract A recent study of the mutation landscape of &amp;gt;3000 cancers across 12 major cancer types from the Cancer Genome Atlas (TCGA) program revealed PIK3CA as the second most commonly mutated gene, occurring at &amp;gt;10% frequency across most cancer types, with mutations occurring mostly in the PIK3CA kinase (39% overall, 52% for breast cancer) and helical (45% overall, 39% for breast cancer) domains and their most frequent hotspot mutations being H1047R and E545K, respectively. Limited preclinical evidence has suggested that mutations affecting the kinase and helical domains can produce different phenotypic consequences; and our recent analysis of the TCGA data indicates that helical domain mutations, relative to kinase domain mutations, are preferentially associated with pathway/PARADIGM activity features enriched for cell motility mechanisms (e.g. RHO GTPases) and loss of gap junctions, suggesting that helical domain mutations might serve to enhance cell invasiveness and metastatic motility. To validate the functional differences predicted by these two classes of PIK3CA mutations, we turned to the non-malignant breast epithelial cell line MCF10A bearing targeted pathogenic knock-ins of H1047R or E545K at the PIK3CA locus; and we compared RNA-seq expression profiles from the wildtype (wt) and knockin isogenic clones. RSEM was used to estimate gene and isoform abundance; and EBseq was used to perform differential expression analysis. In a three-group comparison, over 2500 differentially expressed genes were identified as differentiating wt cells and the PIK3CA mutated MCF10A clones, with the greatest transcriptional changes (2043 genes) observed between either kinase or helical domain mutations and wt cells. Functional enrichment analysis (DAVID) revealed enrichment of 17 pathway categories among these 2043 genes, representing 3 major functional groups, the most prominent being protein translation. Only 409 genes were differentially expressed across all three groups, suggesting the transcriptome effect differentiating E545K from H2047R bearing MCF10A clones was less pronounced. In a direct comparison of the kinase and helical domain clones, &amp;gt;900 differentially expressed genes were identified that showed enrichment in 64 pathways representing altered cell morphology, motility, apoptosis and phosphorylation, but without enrichment in protein translation function. In particular, MCF10A E545K clones showed significant loss of expression in 19 tight junction-associated genes linked to oncogenic EMT (e.g. OCLN, CLDN-7, TJP1, MYL9). Thus, consistent with our pathway conclusions drawn from the TCGA clinical dataset, helical domain mutations appear to confer an EMT-like expression phenotype beyond the effects of both helical and kinases domain mutations on protein translation. Citation Format: Christina Yau, Joe Garay, Stephen Benz, Joe Gray, Ben Park, Christopher C. Benz. An EMT-like gene expression profile differentiates helical from kinase domain PIK3CA mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1084. doi:10.1158/1538-7445.AM2015-1084

Fitness of artemisinin-resistant Plasmodium falciparum in vitro.

Drug resistance confers a fitness advantage to parasites exposed to frequent drug pressure, yet these mutations also may incur a fitness cost. We assessed fitness advantages and costs of artemisinin resistance in Plasmodium falciparum in vitro to understand how drug resistance will spread and evolve in a competitive environment. Genotyping of SNPs, drug susceptibility assays and copy number determination were used to assess the impact of artemisinin resistance on parasite fitness. An artemisinin-resistant clone (C9) selected in vitro from an isogenic parental clone (D6) was used to conduct competitive growth studies to assess fitness of artemisinin resistance. The resistant and susceptible clones were mixed or grown alone in the presence and absence of drug pressure (dihydroartemisinin or pyrimethamine) to quantify the rate at which artemisinin resistance was gained or lost. We experimentally demonstrate for the first time that artemisinin resistance provides a fitness advantage that is selected for with infrequent exposure to drug, but is lost in the absence of exposure to artemisinin drugs. The best correlations with artemisinin resistance were decreased in vitro drug susceptibility to artemisinin derivatives, increased copy number of Pf3D7_1030100 and an SNP in Pf3D7_0307600. An SNP conferring an E208K mutation in the kelch gene (Pf3D7_1343700) was not associated with resistance. Furthermore, we observed second-cycle ring-stage dormancy induced by pyrimethamine, suggesting that dormancy is a fitness trait that provides an advantage for survival from antimalarial drug stress. Artemisinin-resistant P. falciparum have a fitness advantage to survive and predominate in the population even in the face of infrequent exposure to artemisinin drugs.

Inoculation of Transgenic Resistant Potato by Phytophthora infestans Affects Host Plant Choice of a Generalist Moth.

Pathogen attack and the plant’s response to this attack affect herbivore oviposition preference and larval performance. Introduction of major resistance genes against Phytophthora infestans (Rpi-genes), the cause of the devastating late blight disease, from wild Solanum species into potato changes the plant-pathogen interaction dynamics completely, but little is known about the effects on non-target organisms. Thus, we examined the effect of P. infestans itself and introduction of an Rpi-gene into the crop on host plant preference of the generalist insect herbivore, Spodoptera littoralis (Lepidoptera: Noctuidae). In two choice bioassays, S. littoralis preferred to oviposit on P. infestans-inoculated plants of both the susceptible potato (cv. Desiree) and an isogenic resistant clone (A01-22: cv. Desiree transformed with Rpi-blb1), when compared to uninoculated plants of the same genotype. Both cv. Desiree and clone A01-22 were equally preferred for oviposition by S. littoralis when uninoculated plants were used, while cv. Desiree received more eggs compared to the resistant clone when both were inoculated with the pathogen. No significant difference in larval and pupal weight was found between S. littoralis larvae reared on leaves of the susceptible potato plants inoculated or uninoculated with P. infestans. Thus, the herbivore’s host plant preference in this system was not directly associated with larval performance. The results indicate that the Rpi-blb1 based resistance in itself does not influence insect behavior, but that herbivore oviposition preference is affected by a change in the plant-microbe interaction.

Generation of CCR5-defective CD34 cells from ZFN-driven stop codon-integrated mesenchymal stem cell clones.

BackgroundsHomozygous 32-bp deletion of the chemokine receptor 5 gene (CCR5) is associated with resistance to human immunodeficiency virus (HIV) infection, while heterozygosity delays HIV progression. Bone marrow transplantation (BMT) from a 32/32 donor has been shown to cure an HIV-infected patient. However, the rarity of this mutation and the safety risks associated with current BMT protocols are the major obstacles to this treatment. Zinc finger nuclease (ZFN) targeting is a powerful method for achieving genomic disruption at specific DNA sites of interest.ResultsTaking advantage of the self-renewal and plasticity properties of stem cells, in this study, we successfully generated isogenic and six-cell clones of bone marrow-derived mesenchymal stem cells that carry the stop codon of the CCR5 gene by using a ZFN-mediated homology-directed repair technique. These cells were expandable for more than 5 passages, and thus show potential to serve as an individual’s cell factory. When Oct4 was overexpressed, the mutated cells robustly converted to CD34+ progenitor cells.ConclusionWe here reported the novel approach on generation of patients own CD34 cells from high fidelity ZFN-mediated HDR MSC clones. We believe that our approach will be beneficial in future HIV treatment.

In vitro passage selects for Chlamydia muridarum with enhanced infectivity in cultured cells but attenuated pathogenicity in mouse upper genital tract.

Although modern Chlamydia muridarum has been passaged for decades, there are no reports on the consequences of serial passage with strong selection pressure on its fitness. In order to explore the potential for Pasteurian selection to induce genomic and phenotypic perturbations to C. muridarum, a starter population was passaged in cultured cells for 28 generations without standard infection assistance. The resultant population, designated CMG28, displays markedly reduced in vitro dependence on centrifugation for infection and low incidence and severity of upper genital tract pathology following intravaginal inoculation into mice compared to the parental C. muridarum population, CMG0. Deep sequencing of CMG0 and CMG28 revealed novel protein variants in the hypothetical genes TC0237 (Q117E) and TC0668 (G322R). In vitro attachment assays of isogenic plaque clone pairs with mutations in either TC0237 and TC0668 or only TC0237 reveal that TC0237(Q117E) is solely responsible for enhanced adherence to host cells. Paradoxically, double mutants, but not TC0237(Q117E) single mutants, display severely attenuated in vivo pathogenicity. These findings implicate TC0237 and TC0668 as novel genetic factors involved in chlamydial attachment and pathogenicity, respectively, and show that serial passage under selection pressure remains an effective tool for studying Chlamydia pathogenicity.

Semi-solid tumor model in Xenopus laevis/gilli cloned tadpoles for intravital study of neovascularization, immune cells and melanophore infiltration

Tumors have the ability to grow as a self-sustaining entity within the body. This autonomy is in part accomplished by the tumor cells ability to induce the formation of new blood vessels (angiogenesis) and by controlling cell trafficking inside the tumor mass. These abilities greatly reduce the efficacy of many cancer therapies and pose challenges for the development of more effective cancer treatments. Hence, there is a need for animal models suitable for direct microscopy observation of blood vessel formation and cell trafficking, especially during early stages of tumor establishment. Here, we have developed a reliable and cost effective tumor model system in tadpoles of the amphibian Xenopus laevis. Tadpoles are ideally suited for direct microscopy observation because of their small size and transparency. Using the thymic lymphoid tumor line 15/0 derived from, and transplantable into, the X. laevis/gilli isogenic clone LG-15, we have adapted a system that consists in transplanting 15/0 tumor cells embedded into rat collagen under the dorsal skin of LG-15 tadpole recipients. This system recapitulates many facets of mammalian tumorigenesis and permits real time visualization of the active formation of the tumor microenvironment induced by 15/0 tumor cells including neovascularization, collagen rearrangements as well as infiltration of immune cells and melanophores.

Abstract 4342: Crosstalk between initiating cells with different metabolism in a murine model of malignant glioma

Abstract Background The metabolic preference of malignant glioma for glycolysis as an energy source is a potential therapeutic target. As a result of the cellular heterogeneity of these tumors, however, the relation between glycolytic preference, tumor formation, and tumor cell clonogenicity has remained unknown. To address this issue, we analyzed the metabolic profiles of isogenic glioma-initiating cells (GICs) in a mouse model. Methods GICs were established by overexpression of H-RasV12 in Ink4a/Arf-null neural stem cells. Subpopulations of these cells were obtained by single-cell cloning, and clones differing in extracellular acidification potential were assessed for metabolic characteristics by quantification of intra- and extracellular metabolites. Tumorigenicity was assessed by implantation of 100 cells of each subpopulation into the forebrain of wild-type mice. Tumors were examined for pathological features of glioma and expression of glycolytic enzymes. Results Malignant transformation of neural stem cells resulted in a shift in metabolism characterized by a significant increase in glucose uptake and lactic acid production. Clonal populations of GICs also manifested pronounced differences in their metabolic profiles. Certain GICs consumed more glucose and produced more lactate, while others had higher oxygen consumption. These differences were reflected in the levels of intracellular metabolites and they were paralleled by a differential expression of glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2. GIC clones with different metabolic profiles had the same level of tumorigenic ability and the tumors formed by all types of GICs displayed the histopathological features of glioblastoma. However, the differential expression of the glycolytic enzymes was also evident in the tumors formed by each of the clones. Implantation of a mix of GICs with different metabolic profiles showed that clones with higher glycolytic ability were the major component of the tumor mass and that they provided a scaffold for the less glycolytic clones, supporting their expansion. Conclusions The metabolic characteristics of glioma cells appear early during malignant transformation and persist until the late stages of tumor formation. Even isogenic clones may be heterogeneous in terms of metabolic features, however, and this heterogeneity may play a role in tumor cell proliferation and survival. Our results suggest that a more detailed understanding of the metabolic profile of malignant gliomas is imperative for their effective therapeutic targeting. Citation Format: Oltea Sampetrean, Isako Saga, Shunsuke Shibao, Jun Okubo, Satoru Osuka, Nobuyuki Onishi, Hideyuki Saya. Crosstalk between initiating cells with different metabolism in a murine model of malignant glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4342. doi:10.1158/1538-7445.AM2014-4342