Parental Clones Research Articles

The role of transgenerational effects in adaptation of clonal offspring of white clover (Trifolium repens) to drought and herbivory

Environmentally induced transgenerational effects can increase success of offspring and thereby be adaptive if offspring experience conditions similar to the parental environment. The ecological and evolutionary significance of these effects in plants have been considered overwhelmingly in the context of sexual generations. We investigated whether drought stress and jasmonic acid, a key hormone involved in induction of plant defenses against herbivores, applied in the parental generation, trigger transgenerational effects in clonal offspring of Trifolium repens and whether these effects are adaptive. We found that drought stress experienced by parents significantly affected phenotypes of offspring ramets. Offspring ramets were bigger if they were produced in the parental water regime (control/drought). Repeated application of jasmonic acid to parents increased the subsequent growth of offspring ramets produced by stolons after they were disconnected from the parental clone. However, these offspring ramets experienced similar herbivory by the generalist Spodoptera littoralis caterpillar as did control offspring ramets, indicating that this jasmonic acid application in the parental generation did not result in a transgenerational effect comprising increased herbivory resistance. We conclude that, overall, environmental interaction in the parental generation can trigger transgenerational effects in clonal plants and some of these effects can be adaptive. Moreover, transgenerational effects in clonal plants that significantly influence their growth and behavior can ultimately affect the evolutionary trajectories of clonal populations.

Essential roles of the interaction between cancer cell-derived chemokine, CCL4, and intra-bone CCR5-expressing fibroblasts in breast cancer bone metastasis

From a murine breast cancer cell line, 4T1, we established a subclone, 4T1.3, which consistently metastasizes to bone upon its injection into the mammary fat pad. 4T1.3 clone exhibited similar proliferation rate and migration capacity as the parental clone. However, the intra-bone injection of 4T1.3 clone caused larger tumors than that of the parental cells, accompanied with increases in fibroblast, but not osteoclast or osteoblast numbers. 4T1.3 clone displayed an enhanced expression of a chemokine, CCL4, but not its specific receptor, CCR5. CCL4 shRNA-transfection of 4T1.3 clone had few effects on its in vitro properties, but reduced the tumorigenicity arising from the intra-bone injection. Moreover, intra-bone injection of 4T1.3 clone caused smaller tumors in mice deficient in CCR5 or those receiving CCR5 antagonist than in wild-type mice. The reduced tumor formation was associated with attenuated accumulation of CCR5-positive fibroblasts expressing connective tissue growth factor (CTGF)/CCN2. Tumor cell-derived CCL4 could induce fibroblasts to express CTGF/CCN2, which could support 4T1.3 clone proliferation under hypoxic culture conditions. Thus, the CCL4-CCR5 axis can contribute to breast cancer metastasis to bone by mediating the interaction between cancer cells and fibroblasts in bone cavity.

Genetic analysis of resistance to Alternaria leaf petiole and stem blight of sweetpotato in Uganda

Alternaria blight (Alternaria spp.) is an important sweetpotato disease in Uganda causing yield losses of over 50 % in susceptible genotypes. In Uganda, Alternaria bataticola and Alternaria alternata are the major species with A. bataticola the more aggressive of the two. The most effective control measure for this disease is the use of resistant genotypes. This study was conducted to determine the inheritance of resistance to Alternaria blight and the general and specific combining abilities of the available germplasm. Sixteen parental clones varying in reaction to Alternaria blight were crossed using the North Carolina II mating scheme. Due to incompatibility of some parents, two sets of compatible parents were formed. Differences among the families for Alternaria blight severity were significant while general combining ability (GCA) and specific combining ability (SCA) mean squares were highly significant (P < 0.001) for the disease with GCA sum of squares (SS) being more predominant at 67.4 % of the treatment SS for Set 1 and the SCA SS predominant at 54.0 % of the treatment SS for Set 2. This indicated that both additive and non-additive effects are important in controlling this trait. Some parents with high, negative GCA effects produced families with undesirable SCA effects and the reverse was also true. This implies that the best parents should not be chosen on GCA alone but also on SCA of their best crosses. The wide range in the area under disease progress curve for the families indicated that it was possible to select for highly resistant genotypes.

Development and Integration of an SSR-Based Molecular Identity Database into Sugarcane Breeding Program

Sugarcane breeding is very difficult and it takes 12 to 14 years to develop a new cultivar for commercial production. This is because sugarcane varieties are highly polyploid, inter-specific hybrids with 100 to 130 chromosomes that may vary across geographical areas. Other obstacles/constraints include the small size of flowers that may not synchronize but may self-pollinate, difficulty in distinguishing hybrids from self progenies, extreme (G × E) interactive effect, and potential variety mis-identification during vegetative propagation and varietal exchange. To help cane breeders circumvent these constraints, a simple sequence repeats (SSR)-based molecular identity database has been developed at the United States Department of Agriculture-Agricultural Research Service, Sugarcane Research Unit in Houma, LA. Since 2005, approximately 2000 molecular identities have been constructed for clones of sugarcane and related Saccharum species that cover geographical areas including Argentina, Australia, Bangladesh, China, Colombia, India, Mexico, Pakistan, South Africa, Thailand, USA (Louisiana, Florida, Texas, and Hawaii), and Venezuela. The molecular identity database is updated annually and has been utilized to: (1) provide molecular descriptors to newly registered cultivars; (2) identify in a timely fashion any mislabeled or unidentifiable clones from cross parents and field evaluation plots; (3) develop de novo clones of energy cane with S. spontaneum cytoplasm; (4) provide clone-specific fingerprint information for assessing cross quality and paternity of polycross; (5) determine genetic relatedness of parental clones; (6) select F1 hybrids from (elite × wild) or (wild × elite) crosses; and (7) investigate the inheritance of SSR markers in sugarcane. The integration of the molecular identity database into the sugarcane breeding program may improve the overall efficacy of cultivar development and commercialization.

CHO cells knocked out for TSC2 display an improved productivity of antibodies under fed batch conditions.

The kinase mTOR operates in two cellular complexes, mTORC1 and mTORC2. mTORC1 adjusts metabolic activity according to external growth conditions and nutrients availability. When conditions are prosperous, mTOR facilitates protein and lipid biosyntheses and inhibits autophagy, while under metabolic constraints, however, its attenuation induces a catabolic program, energy preservation and autophagy. CHO is a key cell line for manufacturing of biologics owing to its remarkable ability to grow to high densities and maintain protein production and secretion for extended times. While high mTOR activity has been associated with high productivity in CHO cells, its inhibition by rapamycin has also been documented to augment productivity via promotion of viability. Here using CRISPR/Cas9 editing we engineered CHO cells to enforce high mTORC1 activity by knocking-out TSC2, a major mTOR inhibitory protein, or PTEN, a phosphatase that attenuates the PI3K/AKT/mTOR pathway. Only TSC2-deleted cells exhibited a constitutive activation of mTORC1 under fed batch conditions. Cells grew larger in size, synthesized more proteins and displayed an over twofold elevation in their specific productivity. While peak viable cell density was compromised, overall titers increased to an extent dependent upon the parental clone. Our data underscore manipulation of TSC as a strategy to improve performance of CHO cell in bioreactors. Biotechnol. Bioeng. 2016;113: 1942-1952. © 2016 Wiley Periodicals, Inc.

Efficient expression of single chain variable fragment antibody against paclitaxel using the Bombyx mori nucleopolyhedrovirus bacmid DNA system and its characterizations.

A single chain variable fragment (scFv), the smallest unit of functional recombinant antibody, is an attractive format of recombinant antibodies for various applications due to its small fragment and possibility of genetic engineering. Hybridoma clone 3A3 secreting anti-paclitaxel monoclonal antibody was used to construct genes encoding its variable domains of heavy (VH) and light (VL) chains. The VH and VL domains were linked to be the PT-scFv3A3 using flexible peptide linker in a format of VH-(GGGGS)5-VL. The PT-scFv3A3 was primarily expressed using the pET28a(+) vector in the Escherichia coli system, and was then further expressed by using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Interestingly, the reactivity of PT-scFv3A3 expressed in the hemolymph of B. mori using the BmNPV bacmid DNA system was much higher than that expressed in the E. coli system. Using indirect competitive enzyme-linked immunosorbent assay (icELISA), the PT-scFv3A3 (B. mori) reacted not only with immobilized paclitaxel, but also with free paclitaxel in a concentration-dependent manner, with the linear range of free paclitaxel between 0.156 and 5.00µg/ml. The PT-scFv3A3 (B. mori) exhibited less cross-reactivity (%) than its parental MAb clone 3A3 against paclitaxel-related compounds, including docetaxel (31.1%), 7-xylosyltaxol (22.1%), baccatin III (<0.68%), 10-deacetylbaccatin III (<0.68%), 1-hydroxybaccatin I (<0.68%), and 1-acetoxy-5-deacetylbaccatin I (<0.68%). With the exception of cephalomannine, the cross-reactivity was slightly increased to 8.50%. The BmNPV bacmid DNA system was a highly efficient expression system of active PT-scFv3A3, which is applicable for PT-scFv3A3-based immunoassay of paclitaxel. In addition, the PT-scFv3A3 can be applied to evaluate its neutralizing property of paclitaxel or docetaxel toxicity.

An assessment of genetic relationship in tea clones based on important chemical compound and yield potential

Assembling of new excellence high yiel­ding and good quality tea clones was started from identification accession as gene sources, crossing design of parents, selection mother bushes and propagated vegetatively. Crossing beetwen two parent clones which near or remote genetic relationship in tea will failed. Assessment of genetic relationship clone accession was done using secondary datas from description of first and second tea clone gene­ration which cultivated in Indonesia. Data of yield potentials and main chemical com­pound of quality among others catechine, gallat cate­chine, catechine gallo and cafeine were ana­lysed using biplot and cluster analysis. The results showed that no correllation beetwen main chemical compound of quality and yield potentials. Biplot with two dimensions could be explained 92,5% variation of five characters and showed that yield has the highest and catechine the smallest variances. The first gene­ration of tea clones was separated to second at the 46,79% similarity levels. At the 15,60% similarity levels, there were six clusters. GMB 1, GMB 2, GMB 6, GMB 9, and GMB 10 clones were in one cluster was caused of they had high of yield and catechine. GMB 4 and GMB 5 had similarity on medium yields, GMB 7 and GMB 11 had similarity on medium catechine and the highest yield, and GMB 3 and GMB 8 had similarity on low catechine. PS 1 with the lowest catechine gallo was separated to the others. The sixth cluster were SA 40, Kiara 8, SA 35, RB 3, and Cin 143 were characterized by low catechine and yields.Â

Integrated Multiregional Analysis Proposing a New Model of Colorectal Cancer Evolution.

Understanding intratumor heterogeneity is clinically important because it could cause therapeutic failure by fostering evolutionary adaptation. To this end, we profiled the genome and epigenome in multiple regions within each of nine colorectal tumors. Extensive intertumor heterogeneity is observed, from which we inferred the evolutionary history of the tumors. First, clonally shared alterations appeared, in which C>T transitions at CpG site and CpG island hypermethylation were relatively enriched. Correlation between mutation counts and patients’ ages suggests that the early-acquired alterations resulted from aging. In the late phase, a parental clone was branched into numerous subclones. Known driver alterations were observed frequently in the early-acquired alterations, but rarely in the late-acquired alterations. Consistently, our computational simulation of the branching evolution suggests that extensive intratumor heterogeneity could be generated by neutral evolution. Collectively, we propose a new model of colorectal cancer evolution, which is useful for understanding and confronting this heterogeneous disease.

Generation of a neutralization-resistant CCR5 tropic simian/human immunodeficiency virus (SHIV-MK38) molecular clone, a derivative of SHIV-89.6.

Previously, we reported that a new genetically diverse CCR5 (R5) tropic simian/human immunodeficiency virus (SHIV-MK38) adapted to rhesus monkeys became more neutralization resistant to SHIV-infected plasma than did the parental SHIV-KS661 clone. Here, to clarify the significance of the neutralization-resistant phenotype of SHIV in a macaque model, we initially investigated the precise neutralization phenotype of the SHIVs, including SHIV-MK38 molecular clones, using SHIV-MK38-infected plasma, a pooled plasma of human immunodeficiency virus (HIV)-infected individuals, soluble CD4 and anti-HIV-1 neutralizing mAbs, the epitopes of which were known. The results show that SHIV-KS661 had tier 1 neutralization sensitivity, but monkey-adapted R5 tropic SHIV-MK38 acquired neutralization resistance similar to that of tier 2 or 3 as a clone virus. Sequence analysis of the env gene suggested that the neutralization-resistant phenotype of SHIV-MK38 was acquired by conformational changes in Env associated with the net charge and potential N-linked glycosylation sites. To examine the relationship between neutralization phenotype and stably persistent infection in monkeys, we performed in vivo rectal inoculation experiments using a SHIV-MK38 molecular clone. The results showed that one of three rhesus monkeys exhibited durable infection with a plasma viral load of 105 copies ml- 1 despite the high antibody responses that occurred in the host. Whilst further improvements are required in the development of a challenge virus, it will be useful to generate a neutralization-resistant R5 tropic molecular clone of the SHIV-89.6 lineage commonly used for vaccine development - a result that can be used to explore the foundation of AIDS pathogenesis.

Overexpression of G6PDH does not affect the behavior of HEK-293 clones stably expressing interferon-α2b

HEK293 cells are gaining in interest for the production of recombinant proteins. However, further understanding and engineering of cell metabolism are still needed to improve protein titers. The importance of G6PDH has already been studied regarding redox balance, and a correlation has recently been established between the oxidative pentose phosphate pathway (PPP) and antibody peak production. In this work, HEK293 cells stably expressing interferon-α2b, a parental clone, and a further engineered clone expressing the cytosolic yeast pyruvate-carboxylase (PYC) were transiently transfected to overexpress glucose-6-phosphate dehydrogenase (G6PDH) and increase fluxes through the PPP. The aim of the study was thus to evaluate the effect of overexpressing G6PDH on the “pull” effect brought by the PYC phenotype. Results indicate that the cell metabolism is, however, highly robust, showing a highly regulated PPP damping the potential effects of overexpressing G6PDH. A metabolomic study also clearly demonstrates, by metabolites profiling, that the PYC clone has a highly robust and more efficient metabolic efficiency, compared to its parental clone.

Quantitative Assessment of Circulating Clonal IG-VDJ Sequences in Plasma of Follicular Lymphoma at Diagnosis Is Highly Predictive of Progression Free Survival (PFS)

Background:Recent advances in next-generation sequencing (NGS) techniques enable the quantitation of circulating tumor DNA (ctDNA) encoding the clonal rearranged V(D)J immunoglobulin (IG) receptor gene sequence. The aims of this study were 1) To evaluate the clonal heterogeneity of follicular lymphoma (FL) and its variations between tumor and plasma at diagnosis in patients (pts) included in PRIMA trial, and 2) To assess the prognostic value of the level of ctDNA at diagnosis on PFS (median follow up of 6 years).Methods:Using the NGS-based immunosequencing method (Adaptive Biotechnologies, South San Francisco, CA), the lymphoma clonotype was first established in the tumor biopsy using locus-specific primer sets for IGH-V, IGH-D and IGK rearrangements. Clonotypes regarded as originating from the lymphoma clone were present at a frequency greater than 5%. The lymphoma-derived sequences identified in the biopsy sample were then used as targets to assess the presence of ctDNA in plasma samples. The quantity and frequency of the ctDNA in plasma is calculated relative to the total number of reads in the sample and defined as lymphoma clonotype molecules per million diploid genomes. For 34 FL pts from the PRIMA trial, both tumor biopsy and plasma samples at diagnosis were available.Results:One tumor clonotype or more could be detected in 29 pts (85%) in the tumor diagnostic sample (DNA isolated from fresh frozen tissue). Indeed, with the IGH-V assay, between 1 to 3 different clonotypes were identified in 23 pts. With the IGH-D assay, 1 clonotype was detected in 2 pts. With the IGK assay, 1 to 2 different clonotypes were detected in 17 pts. The tumor clonotypes were detected in diagnostic plasma samples in 25 out of the 29 pts (83%). Moreover, in 14 pts we detected clones in the plasma samples that are related (with point mutations in the V(D)J sequence) to the highest frequency index clone identified in the tumor sample. In half of the cases, we observed a different repartition of the subclonal populations between tumor and plasma samples at diagnosis: for these cases the highest frequency clone in the tumor was not the highest frequency clone in the plasma.Among the 24 pts with an IGH clonotype (IGH-V or IGH-D assay), ctDNA could be detected in 19 cases. We could not find a correlation between peripheral blood dissemination and the presence of a stereotyped sequence or an N-glycosylation motif within the CDR3 region of IGH clonotypes. Interestingly, in one case, two different related clones were detected at high frequency in tumor sample and the putative parental clone, not detected in the tumor sample, was detected in plasma sample as the highest frequency clone.The level of ctDNA ranged from 0 to 345,000/million diploid genomes in the 29 plasma samples (median 39,720). The presence of ctDNA was correlated with bone marrow involvement (p=.005), but not with FLIPI score, LDH level, anemia, bulky disease, presence of circulating lymphoma cells (detected by morphology or flow examination), Ann Arbor stage or β2microglobulin level. The absolute level of ctDNA in diagnostic samples did not correlate with any of the clinical characteristics. We assessed the prognostic value of the level of ctDNA at diagnosis. Pts (14/29) with higher levels of ctDNA at diagnosis (>40,000/million diploid genomes) had shorter PFS than patients with lower levels of ctDNA (median 15.7 m vs NR, p=.027, Fig 1). In an exploratory multivariate Cox regression model including FLIPI score, bulky disease and β2-microglobuline level, high ctDNA level was the only factor significantly associated with a worse PFS (HR 4.2, p=.039). In the observation arm, pts with high ctDNA levels had a median PFS of only 11.6 months vs NR (p=.01). Whereas in the maintenance arm, the prognostic value of elevated ctDNA levels was erased (p=.6).Conclusion:Using the NGS-based immunosequencing method, we were able to show a variation in clonal heterogeneity between tumor biopsy and plasma samples obtained at diagnosis. The poor prognostic value of elevated ctDNA levels in plasma at diagnosis appeared higher than other clinical parameters in a multivariate cox regression model. These findings need to be validated in larger cohorts. [Display omitted] DisclosuresCarlton:Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Faham:adaptive biotech: Employment, Other: stockholders. Salles:Roche: Honoraria, Research Funding.

Abstract C59: Role of MTH1 (NUTD1) in cancer cell survival

Abstract Human mutT homolog MTH1 (also known as NUDT1) is a purine nucleoside triphosphatase which hydrolyses oxidised nucleotides (8-oxo-dGTP and 2-OH-dATP) into mono-phosphate forms to prevent these damaged bases from being incorporated into DNA. Recent studies have suggested a key role of MTH1 in the survival of cancer cells. It was hypothesized that in cancer cells with high levels of reactive oxygen species (ROS), small molecule inhibition or loss of MTH1 would lead to accumulation of oxidised nucleotides in DNA, increased genome instability and loss of cell viability. In order to validate MTH1 as a potential cancer therapeutic target we have developed additional potent small molecule inhibitors of MTH1 and generated siRNA knock-down and CRISPR-mediated knock-out cell lines. Using these tools we evaluated the impact of either MTH1 inhibition or loss of expression on cell viability, proliferation, induction of DNA damage and cell cycle arrest across multiple cell lines in vitro. While we have confirmed that the previous study compounds (TH588, TH287, (S)-crizotinib) are potent inhibitors of MTH1 and have broad cancer cell line growth inhibition activity, we were unable to demonstrate a corresponding induction of γH2AX, 53BP1 or phospho-ATM DNA damage response (DDR). In addition none of our MTH1 inhibitors were able to show significant growth inhibitory activity across cell lines panels or induce a DDR despite being able to potently engage MTH1 in cells as evidenced by cell thermal stability assay (CETSA). Due to the lack of DNA damage induction and inconsistent anti-proliferative activity between MTH1 inhibitors, we investigated the effects of loss of protein expression using additional, independent siRNA and CRISPR knockout cell lines. Despite potent and long lasting siRNA knockdown of MTH1 protein expression in U2OS cells, we did not observe significant differences in cell viability or induction in DNA damage when compared to control cell lines. Consistent with these findings we were also able to generate SW480 clones with complete knockout of all MTH1 alleles and this did not impair growth rates when compared to parental cells. Finally, to probe the specificity of MTH1 inhibitors we assessed their ability to impact the growth of these cells which do not express MTH1. We did not observe a differential response in U2OS cells with or without MTH1 siRNA or in the SW480 parental versus MTH1 knockout clones. Here we show through the use of novel small-molecule MTH1 inhibitors, with further experiments with RNAi-mediated knockdown of MTH1 and the ability to generate stable CRISPR-mediated MTH1 knockouts, that the cellular effects previously ascribed to MTH1 inhibition may be driven through alternative mechanisms. In summary, our results do not support an essential role for MTH1 in cancer cell survival. Citation Format: Husam Alwan, Kay Eckersley, Louise Goodwin, Alan Lau, David Jones, Jason Kettle, Willem M. Nissink, Jon Read, James S. Scott, Benjamin J.M. Taylor, Graeme E. Walker, Kevin M. Foote. Role of MTH1 (NUTD1) in cancer cell survival. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C59.

Abstract C97: Beyond intratumoural steroidogenesis: abiraterone resistance mediated by AR variants and glucocorticoid receptor signalling

Abstract Introduction Castration resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signalling, driven by adrenal precursors and potentially de novo steroid synthesis in other organ tissues including prostate. Abiraterone, an inhibitor of the steroidogenic enzyme CYP17A1 and the AR has been demonstrated to prolong survival of CRPC patients. In this study, we created a co-culture model using human prostate and adrenal tumours to study abiraterone resistance. Materials and Methods Human androgen-dependent PC (VCaP) and CPRC clones were cultured with substrates for de novo androgen synthesis or with adrenal androgens, or cultured with human adrenal cells (H295R) and treated with either the CYP17A1 inhibitor abiraterone or the antiandrogen MDV3100. Male mice bearing VCaP tumours and human adrenal H295R xenografts were castrated and treated with placebo or abiraterone. Tumour response was assessed by tumour growth, PSA release, steroid quantitation by (LC/MS-MS), immunohistochemistry and mRNA expression analysis of steroidogenic enzymes and nuclear receptors. Results In vitro, physiological levels of adrenal androgen precursors DHEA and androstenedione induced cell growth in parental and CRPC VCaP sub clones, whereas precursor steroids pregnenolone and progesterone for de novo synthesis did not. In a co-culture model, abiraterone blocked H295R-induced growth of VCaP cells. Likewise, in vivo, H295R tumours stimulated castration-resistant VCaP growth. This stimulative effect was inhibited by abiraterone, reducing - but not fully blocking - growth and PSA production. In the absence of H295R tissue, VCaP xenografts grew slow but became castration resistant nonetheless. In contrast to the observed effects on VCaP growing in castrate animals bearing H295R tumours, abiraterone was unable to inhibit the slow VCaP growth and low PSA production in castrate mice without H295R xenografts. LC/MS-MS analysis of plasma and tumour tissue could not confirm increased de novo production of androgens. Castrate and abiraterone-resistant VCaP tumours were characterised by increased levels of AR, AR variants and glucocorticoid receptor (GR) expression, resulting in equal AR target gene expression levels. Conclusions Our data indicate that AR ligand dependent regrowth of CRPC is predominantly supported via adrenal steroid production. Abiraterone resistant disease of VCaP relies on AR overexpression, expression of ligand independent AR variants and GR signalling. Citation Format: Jan Matthijs Moll, Johannes Hofland, Wilma Teubel, Corrina M.A. de Ridder, Anne E. Taylor, Ralph Graeser, Wiebke Arlt, Guido W. Jenster, Wytske M. van Weerden. Beyond intratumoural steroidogenesis: abiraterone resistance mediated by AR variants and glucocorticoid receptor signalling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C97.

Abstract B88: Mechanisms of resistance to the Hsp90 inhibitor ganetespib in triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC), which accounts for 10-17% of all breast cancers, is challenging to treat due to its heterogeneity and the absence of identified targeted therapies. Heat shock protein 90 kDa (Hsp90) is a molecular chaperone protein essential for regulating the stability and activity of several client proteins required for oncogenesis, and selective Hsp90 inhibitors have shown anti-cancer activity in multiple cancer models including TNBC. Ganetespib, a second generation Hsp90 inhibitor, has shown promising clinical activity in TNBC patients but responders develop acquired resistance. Here, we generated an in vitro model of ganetespib-resistant TNBC cells and investigated the mechanisms of resistance to ganetespib in TNBC using a 96-well based small molecule screen. Methods: Ganetespib treatment sensitivity in a panel of seven TNBC cell lines (HS578T, HCC1143, HCC1937, MDA-MB-468, MDA-MB-231, MDA-MB-453 and BT20) was assessed using a resazurin-based cell proliferation assay. HS578T cells were incubated in a high concentration of ganetespib (&amp;gt;IC50) to generate ganetespib-resistant clones. The effects on Hsp90 oncogenic client proteins (AKT and EGFR) and Hsp70 were determined by western blotting. Cross resistance to other Hsp90 inhibitors was also assessed. 96-well based small molecule screen with a library of 326 bioactive small molecule inhibitors was performed on parental HS578T and ganetespib-resistant clones in the absence and presence of ganetespib. Resazurin-based cell proliferation assay was used as an end point. Positive hits were identified as log10(percentage of proliferation in parental/resistant) ≥ 2SD. Results: TNBC cell lines showed differential sensitivity to ganetespib (IC50 range: 4.75 to 20.16 nM), with HS578T being the most sensitive and HCC1143 the most resistant. Independent ganetespib-resistant HS578T clones (CR2 and CR3) were successfully generated by incubating parental HS578T cells in ganetespib (30 nM) for 42 days. IC50 values for the ganetespib-resistant clones (CR2: 15.57 ± 1.90 nM and CR3: 20.28 ± 2.75 nM) were 3 to 4-fold higher than the parental HS578T cells (IC50: 4.79 ± 0.32 nM). The client proteins were not further down-regulated after ganetespib treatment in the ganetespib-resistant clones compared to the parental HS578T cells. Resistant clones also showed cross resistance to 1st generation (17-AAG) and 2nd generation (NVP-AUY922) Hsp90 inhibitors. The small molecule screen was reproducible as the independent runs and duplicates strongly correlated (R2 &amp;gt;0.7). The ganetespib-resistant CR3 clones showed selective sensitivity to 11/326 compounds (Z score ≥ 2), only in the presence of ganetespib when compared to the parental HS578T cells, most notable being LY2784544, a JAK2 inhibitor. Conclusion: Ganetespib-resistant HS578T cells provide a promising model to study the resistance mechanisms to ganetespib in TNBC. Our current data suggest that TNBC cells are responsive to JAK2 inhibition in the presence of ganetespib despite prior acquisition of resistant to ganetespib. Activation of a JAK2/STAT3 survival pathway may be a potential resistance mechanism to Hsp90 inhibition and combined Hsp90 and JAK2 inhibition could potentially be a useful therapeutic strategy for TNBC. Citation Format: Nuramalina H. Mumin, Sivan Bokobza, Fiona Cahill, Neele Drobnitzky, Yanyan Jiang, Luiza Madia Lourenco, Aoife Vaughan, Anika Weber, Anthony Kong, Anderson Ryan. Mechanisms of resistance to the Hsp90 inhibitor ganetespib in triple negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B88.

Novel candidate genes AuxRP and Hsp90 influence the chip color of potato tubers.

Potato (Solanum tuberosum L.) tubers exhibit significant variation in reducing sugar content directly after harvest, cold storage and reconditioning. Here, we performed QTL analysis for chip color, which is strongly influenced by reducing sugar content, in a diploid potato mapping population. Two QTL on chromosomes I and VI were detected for chip color after harvest and reconditioning. Only one region on chromosome VI was linked with cold-induced sweetening. Using the RT-PCR technique, we showed differential expression of the auxin-regulated protein (AuxRP) gene. The AuxRP transcript was presented in light chip color parental clone DG 97-952 and the RNA progeny of the bulk sample consisting of light chip color phenotypes after cold storage. This amplicon was absent in dark chip parental clone DG 08-26/39 and the RNA bulk sample of dark chip progeny. Genetic variation of AuxRP explained up to 16.6 and 15.2 % of the phenotypic variance after harvest and 3 months of storage at 4 °C, respectively. Using an alternative approach, the RDA-cDNA method was used to recognize 25 gene sequences, of which 11 could be assigned to potato chromosome VI. One of these genes, Heat-shock protein 90 (Hsp90), demonstrated higher mRNA and protein expression in RT-qPCR and western blotting assays in the dark chip color progeny bulk sample compared with the light chip color progeny bulk sample. Our study, for the first time, suggests that the AuxRP and Hsp90 genes are novel candidate genes capable of influencing the chip color of potato tubers.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-015-0415-1) contains supplementary material, which is available to authorized users.

Estimation of narrow sense heritability of powdery mildew resistance in pseudo F2 (F1) population of flowering dogwoods (cornus Florida L)

Powdery mildew disease caused by Erysiphe (sect. Microsphaera) pulchra a (Cook & Peck, Braun & Takamatsu) is a devastating disease in flowering dogwoods (Cornus Florida) throughout southeastern USA where flowering dogwood is widely grown. Host resistance is the most sustainable and economical way for controlling this disease, but resistance breeding has been slow and genetic information on powdery mildew resistance is unavailable. In this study, pseudo F2 populations derived from crosses between selected resistant (R) and susceptible (S) plants (RxR, RxS and SxR), were evaluated for disease severity at two locations in three years, McMinnville in 2005 and 2012, and McMinnville and Nashville, TN, in 2013. Disease reaction in progeny from reciprocal crosses was similar indicating that there was no cytoplasmic inheritance of resistance genes. Disease severity of progenies showed continuous distributions, suggesting that powdery mildew resistance is inherited quantitatively. High narrow sense heritability (0.74 in 2005, 0.84 in 2012 and 0.90 in 2013) and broad sense heritability (0.9) in parental clones were observed. This information will be useful in breeding programs for dogwood improvement.

Multiple region whole-exome sequencing reveals dramatically evolving intratumor genomic heterogeneity in esophageal squamous cell carcinoma.

Cancer is a disease of genome instability and genomic alterations; now, genomic heterogeneity is rapidly emerging as a defining feature of cancer, both within and between tumors. Motivation for our pilot study of tumor heterogeneity in esophageal squamous cell carcinoma (ESCC) is that it is not well studied, but the highest incidences of esophageal cancers are found in China and ESCC is the most common type. We profiled the mutations and changes in copy number that were identified by whole-exome sequencing and array-based comparative genomic hybridization in multiple regions within an ESCC from two patients. The average mutational heterogeneity rate was 90% in all regions of the individual tumors in each patient; most somatic point mutations were nonsynonymous substitutions, small Indels occurred in untranslated regions of genes, and copy number alterations varied among multiple regions of a tumor. Independent Sanger sequencing technology confirmed selected gene mutations with more than 88% concordance. Phylogenetic analysis of the somatic mutation frequency demonstrated that multiple, genomically heterogeneous divergent clones evolve and co-exist within a primary ESCC and metastatic subclones result from the dispersal and adaptation of an initially non-metastatic parental clone. Therefore, a single-region sampling will not reflect the evolving architecture of a genomically heterogeneous landscape of mutations in ESCC tumors and the divergent complexity of this genomic heterogeneity among patients will complicate any promise of a simple genetic or epigenetic diagnostic signature in ESCC. We conclude that any potential for informative biomarker discovery in ESCC and targeted personalized therapies will require a deeper understanding of the functional biology of the ontogeny and phylogeny of the tumor heterogeneity.

Simultaneous selection for nutritive value and agronomic traits in timothy (Phleum pratense L.)

Improvement of forage nutritive value through breeding should lead to enhanced livestock productivity but often results in reduced agronomic performance. This study investigated the potential for simultaneous improvement of nutritive and agronomic traits in timothy (Phleum pratense L.). Two unrelated groups were used as the base populations: group A focused on agronomic traits and group N focused on three nutritive traits (low-digestible fiber, water-soluble carbohydrate content, and the ratio of low-digestible fiber to organic cell wall content). Although the two base populations contained few genotypes with both high nutritive and agronomic performance compared with the standard cultivar ‘Kiritappu’, the Cycle 1 populations selected for the traits simultaneously included promising genotypes. The two strains improved via two cycles of selection were superior to the Cycle 1 populations and ‘Kiritappu’ based on a comprehensive analysis of the targeted traits in a yield trial test. However, the nutritive traits of the strain derived from group A were more similar to the Cycle 1 population and ‘Kiritappu’ unlike in the successful case of group N. This may be attributable to the more similar phenotypic distribution of the strain A parental clones to ‘Kiritappu’ compared with that of group N for the nutritive traits. The strain derived from both groups showed high nutritive value with the best yield of all tested genotypes, suggesting the possibility of heterosis for yield. These results indicate that application of recurrent selection simultaneously for nutritive and agronomic traits is likely to provide effective improvement without detriment to important traits.

Assessing 62 Chinese Fir (Cunninghamia lanceolata) Breeding Parents in a 12-Year Grafted Clone Test

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is one of the major commercial conifer species in China. The present study concentrated on the assessment of growth, wood property traits, and strobili number in a 12-year grafted clone test of 62 Chinese fir breeding parents, aiming to describe the variation and correlations between these traits and to identify parent clones with the highest potential for future breeding. The results indicate that all of the growth (height, diameter at breast height, stem volume, crown-width) and wood property (wood basic density and hygroscopicity) traits varied significantly (p &lt; 0.01) among clones, with coefficients of variation ranging from 7.6% to 30.6%. Furthermore, these traits consistently had a moderate to high (0.39–0.87) repeatability estimate (broad-sense heritability). Remarkable clonal differences were also observed for the production of male and female strobili. Phenotypic correlations among growth traits were strong (p &lt; 0.01) and positive. Significantly negative correlations (p &lt; 0.01 or 0.05) were found between wood basic density and growth (except for height) and hygroscopicity. The production of male and female strobili appeared to be significantly (p &lt; 0.01) positively correlated with each other. A notable number of faster-growing parent clones were identified (n = 30); 11 of these had higher density wood with an average realized gain of 10.5% in diameter, and a 5.4% gain in wood basic density. When selection was made for growth and strobili, 10 faster-growing parent clones with medium to high production of female strobili were identified.

Abstract 5467: BRCA1 N-terminal-deficient proteins provide PARP inhibitor and platinum resistance

Abstract Introduction: Tumors harboring BRCA1 mutations initially respond well to platinum and PARP inhibitor therapy; however, resistance invariably arises and is a major clinical problem. The BRCA1 185delAG allele is a common founder mutation located close to the protein translation start site, thought to produce a short peptide devoid of function. Experimental procedures: In this study, we utilized the SUM1315MO2 breast cancer cell line that harbors a BRCA1 185delAG mutation to study mechanisms of PARP inhibitor and platinum resistance. SUM1315MO2 cells were cultured in the presence of increasing concentrations of the PARP inhibitor rucaparib or cisplatin until rucaparib resistant (RR) and cisplatin resistant (CR) clones emerged. Results: DNA sequencing revealed that no BRCA1 gene reversion mutations were present in resistant cells. We next measured BRCA1 protein levels by Western blotting with antibodies specific for the N- and C-terminal domains of BRCA1. As a control, the wild-type BRCA1 protein expressed in MDA-MB-231 cells was detectable with both N- and C-terminal antibodies. In contrast, BRCA1 protein was undetectable in both SUM1315MO2 parental and resistant clones using the N-terminal specific antibody. However, the C-terminal specific antibody identified a more quickly migrating band of low abundance in parental cells, but with elevated expression in both RR and CR clones. We inferred that this protein was devoid of the extreme N-terminal RING domain-containing region that mediates interaction with BARD1. To investigate the functionality of the N-terminal truncated BRCA1 protein (Nt-BRCA1), we measured BRCA1 and RAD51 irradiation-induced focus formation by immunofluorescence. RR and CR clones demonstrated 2.25 - 2.75-fold increase (P = 0.024) in the number of cells with BRCA1 foci compared to parental cells. Similarly, we detected a 2 - 2.85-fold increase (P = 0.0325) in RAD51 foci formation in resistant clones compared to parental cells. Additionally, BRCA1 siRNA treated cells were 12- and 9-fold more sensitive to rucaparib compared to scrambled siRNA-treated control cells (P = 0.0002). Similarly, CR-1 cells treated with BRCA1 siRNAs were 1.56- and 1.7-fold more sensitive to cisplatin (P = 0.0346) compared to scrambled siRNA treated cells. Furthermore, N-terminal truncated BRCA1 proteins were detectable in a primary tumor from a germline BRCA1185delAG mutation carrier. Conclusions: Taken together, these results provide evidence for a novel, mutation location-dependent mechanism of PARP inhibitor and platinum resistance. Citation Format: Yifan Wang, Andrea Bernhardy, Emmanuelle Nicolas, Ryan Winters, Kathy Cai, Kelly Duncan, James Duncan, Maria Harrell, Elizabeth Swisher, Neil Johnson. BRCA1 N-terminal-deficient proteins provide PARP inhibitor and platinum resistance. [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 5467. doi:10.1158/1538-7445.AM2015-5467