Overexpressed TP73 induces apoptosis in medulloblastoma

Abstract Background Medulloblastoma is the most common malignant brain tumor of childhood. Children who relapse usually die of their disease, which reflects resistance to radiation and/or chemotherapy. Improvements in outcome require a better understanding of the molecular basis of medulloblastoma growth and treatment response. TP73 is a member of the TP53 tumor suppressor gene family that has been found to be overexpressed in a variety of tumors and mediates apoptotic responses to genotoxic stress. In this study, we assessed expression of TP73 RNA species in patient tumor specimens and in medulloblastoma cell lines, and manipulated expression of full-length TAp73 and amino-terminal truncated ΔNp73 to assess their effects on growth. Methods We analyzed medulloblastoma samples from thirty-four pediatric patients and the established medulloblastoma cell lines, Daoy and D283MED, for expression of TP73 RNA including the full-length transcript and the 5'-terminal variants that encode the ΔNp73 isoform, as well as TP53 RNA using quantitative real time-RTPCR. Protein expression of TAp73 and ΔNp73 was quantitated with immunoblotting methods. Clinical outcome was analyzed based on TP73 RNA and p53 protein expression. To determine effects of overexpression or knock-down of TAp73 and ΔNp73 on cell cycle and apoptosis, we analyzed transiently transfected medulloblastoma cell lines with flow cytometric and TUNEL methods. Results Patient medulloblastoma samples and cell lines expressed full-length and 5'-terminal variant TP73 RNA species in 100-fold excess compared to non-neoplastic brain controls. Western immunoblot analysis confirmed their elevated levels of TAp73 and amino-terminal truncated ΔNp73 proteins. Kaplan-Meier analysis revealed trends toward favorable overall and progression-free survival of patients whose tumors display TAp73 RNA overexpression. Overexpression of TAp73 or ΔNp73 induced apoptosis under basal growth conditions in vitro and sensitized them to cell death in response to chemotherapeutic agents. Conclusion These results indicate that primary medulloblastomas express significant levels of TP73 isoforms, and suggest that they can modulate the survival and genotoxic responsiveness of medulloblastomas cells

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Conversion of AFLP markers to high-throughput markers in a complex polyploid, sugarcane

The application of DNA markers linked to traits of commercial value in sugarcane may increase the efficiency of sugarcane breeding. The majority of markers generated for quantitative trait locus mapping in sugarcane have been single sequence repeats or AFLPs (amplified fragment length polymorphisms). Since AFLP markers are not adapted for large-scale implementation in plant breeding, our objective was to assess the feasibility of converting AFLP markers to fast, cheap and reliable PCR-based assays in a complex polyploid, sugarcane. Three AFLP markers were selected on the basis of an association to resistance to the fungal pathogen Ustilago scitaminea, the causal agent of smut in sugarcane. We developed an approach which enabled the identification of polymorphisms in these AFLP markers. Towards this goal, we employed GenomeWalking and 454 sequencing to isolate sequences adjacent to the linked AFLP markers and identify SNP (single nucleotide polymorphisms) haplotypes present in the homo(eo)logous chromosomes of sugarcane. One AFLP marker was converted to a cleavage amplified polymorphic sequence marker, another to a SCAR (sequence characteristered amplified region) marker and the final AFLP marker to a SNP PCR-based assay. However, validation of each of the markers in 240 genotypes resulted in 99, 90 and 60% correspondence with the original AFLP marker. These experiments indicate that even in a complex polyploid such as sugarcane, polymorphisms identified by AFLP can be converted to high-throughput marker systems, but due to the complexity this would only be carried out for high-value markers. In some cases, the polymorphisms identified are not transferable to more sequence-specific PCR applications

Development of a high-throughput, low-cost SNP genotyping panel for sugarcane breeding

SUGARCANE (SACCHARUM SPP.) IS one of the world's most important economic crops, grown for its sugar and biofuel production. Investigating genomic sequence variation is critical for identifying alleles contributing to important agronomic traits. The development and delivery of varieties that are higher yielding and disease resistant is one of the main research goals in sugarcane breeding. To achieve this, sugarcane breeding is focusing on genetic improvement programs assisted by single nucleotide polymorphisms (SNP) molecular markers. SNPs are now the molecular marker of choice in animal and crop breeding programs around the world because very large numbers of SNPs per genotype can be accurately screened for, and are amenable to high-throughput screening. However, a particular difficulty when working at the DNA level with sugarcane is its highly complex and polyploid genome. The development of SNP markers in sugarcane can overcome the current limitations as large numbers throughout the genome can be easily screened across many genotypes. Recently, an Affymetrix® Axiom® 45K SNP chip was developed for sugarcane and has dramatically improved the construction of high-density linkage maps and identification of target QTLs for agronomic traits in sugarcane. Marker-assisted selection (MAS) in plant breeding requires markers that are tightly linked to genes of interest and are cost-effective. The use of high-density SNP chips, such as the Axiom® sugarcane SNP chip, for MAS is still price-prohibitive in sugarcane breeding. The objective of this study was to develop a low-density, low-cost SNP panel that could be used for selection of disease resistant clones in the breeding program. Initially, we used the 45K sugarcane SNP chip to identify new markers, which were found to be linked to resistance to smut across different genetic backgrounds. A comparison was then made of these SNP markers converted to two different SNP marker technologies, the LGC® KASPTM assay and the Fluidigm® SNPTypeTM assay, for the development of a high-throughput, low-cost SNP marker panel for disease resistance in sugarcane. We discuss aspects that should be considered during the design of these SNP genotyping arrays, including the importance of validation of SNP markers in diverse genetic backgrounds, costs of marker implementation and decisions on where to use markers in the breeding program. These results offer promise for making selection during the breeding process more rapid, accurate and less expensive and could result in the faster delivery of new disease resistant varieties to the sugarcane industry

Comparative mapping in the Poaceae family reveals translocations in the complex polyploid genome of sugarcane

Background: The understanding of sugarcane genetics has lagged behind that of other members of the Poaceae family such as wheat, rice, barley and sorghum mainly due to the complexity, size and polyploidization of the genome. We have used the genetic map of a sugarcane cultivar to generate a consensus genetic map to increase genome coverage for comparison to the sorghum genome. We have utilized the recently developed sugarcane DArT array to increase the marker density within the genetic map. The sequence of these DArT markers plus SNP and EST-SSR markers was then used to form a bridge to the sorghum genomic sequence by BLAST alignment to start to unravel the complex genomic architecture of sugarcane. Results: Comparative mapping revealed that certain sugarcane chromosomes show greater levels of synteny to sorghum than others. On a macrosyntenic level a good collinearity was observed between sugarcane and sorghum for 4 of the 8 homology groups (HGs). These 4 HGs were syntenic to four sorghum chromosomes with from 98% to 100% of these chromosomes covered by these linked markers. Four major chromosome rearrangements were identified between the other four sugarcane HGs and sorghum, two of which were condensations of chromosomes reducing the basic chromosome number of sugarcane from x=10 to x=8. This macro level of synteny was transferred to other members within the Poaceae family such as maize to uncover the important evolutionary relationships that exist between sugarcane and these species. Conclusions: Comparative mapping of sugarcane to the sorghum genome has revealed new information on the genome structure of sugarcane which will help guide identification of important genes for use in sugarcane breeding. Furthermore of the four major chromosome rearrangements identified in this study, three were common to maize providing some evidence that chromosome reduction from a common paleo-ancestor of both maize and sugarcane was driven by the same translocation events seen in both species

Analysis of the resistance mechanisms in sugarcane during <i>Sporisorium scitamineum</i> infection using RNA-seq and microscopy

<div><p>Smut caused by biotrophic fungus <i>Sporisorium scitamineum</i> is a major disease of cultivated sugarcane that can cause considerable yield losses. It has been suggested in literature that there are at least two types of resistance mechanisms in sugarcane plants: an external resistance, due to chemical or physical barriers in the sugarcane bud, and an internal resistance governed by the interaction of plant and fungus within the plant tissue. Detailed molecular studies interrogating these two different resistance mechanisms in sugarcane are scarce. Here, we use light microscopy and global expression profiling with RNA-seq to investigate these mechanisms in sugarcane cultivar CP74-2005, a cultivar that possibly possesses both internal and external defence mechanisms. A total of 861 differentially expressed genes (DEGs) were identified in a comparison between infected and non-infected buds at 48 hours post-inoculation (hpi), with 457 (53%) genes successfully annotated using BLAST2GO software. This includes genes involved in the phenylpropanoid pathway, cell wall biosynthesis, plant hormone signal transduction and disease resistance genes. Finally, the expression of 13 DEGs with putative roles in <i>S</i>. <i>scitamineum</i> resistance were confirmed by quantitative real-time reverse transcription PCR (qRT-PCR) analysis, and the results were consistent with the RNA-seq data. These results highlight that the early sugarcane response to <i>S</i>. <i>scitamineum</i> infection is complex and many of the disease response genes are attenuated in sugarcane cultivar CP74-2005, while others, like genes involved in the phenylpropanoid pathway, are induced. This may point to the role of the different disease resistance mechanisms that operate in cultivars such as CP74-2005, whereby the early response is dominated by external mechanisms and then as the infection progresses, the internal mechanisms are switched on. Identification of genes underlying resistance in sugarcane will increase our knowledge of the sugarcane-<i>S</i>. <i>scitamineum</i> interaction and facilitate the introgression of new resistance genes into commercial sugarcane cultivars.</p></div

A comprehensive genetic map of sugarcane that provides enhanced map coverage and integrates high-throughput Diversity Array Technology (DArT) markers

Background: Sugarcane genetic mapping has lagged behind other crops due to its complex autopolyploid genome structure. Modern sugarcane cultivars have from 110-120 chromosomes and are in general interspecific hybrids between two species with different basic chromosome numbers: Saccharum officinarum (2n = 80) with a basic chromosome number of 10 and S. spontaneum (2n = 40-128) with a basic chromosome number of 8. The first maps that were constructed utilised the single dose (SD) markers generated using RFLP, more recent maps generated using AFLP and SSRs provided at most 60% genome coverage. Diversity Array Technology (DArT) markers are high throughput allowing greater numbers of markers to be generated

Assessment of <i>de novo</i> transcriptome assembly in BUSCO notation.

<p>Assessment of <i>de novo</i> transcriptome assembly in BUSCO notation.</p

Analysis of the resistance mechanisms in sugarcane during <i>Sporisorium scitamineum</i> infection using RNA-seq and microscopy - Fig 1

<p>Colonization of sugarcane bud sections by <i>S</i>. <i>scitamineum</i>, <b>a</b>: bud section of Q117, 4 weeks following dip inoculation with 1.5 x 10⁶ mixed sporidia showing extensive hyphal growth, <b>b</b>: Intercellular colonisation in the meristematic cells from Q208 bud section following wound inoculation with <i>S</i>. <i>scitamineum</i>, <b>c</b>: Intracellular colonisation (arrow) in the meristematic cells from QN80-3425 bud section, <b>d</b>: Clumping of fungal hyphae (arrow) in bud section of CP74-2005 following wound inoculation with <i>S</i>. <i>scitamineum</i>, <b>e</b>: Non-detection of fungal hyphae in bud section of Q99 following 4 weeks after inoculation with 1.5 x 10⁶ mixed sporidia. Bar = 40 μm.</p