Prognostic Relevance and In Vitro Targeting of Concomitant PTEN and p16 Deficiency in Chordomas.

Chordomas are rare bone tumors arising along the spine. Due to high resistance towards chemotherapy, surgical resection-often followed by radiation therapy-is currently the gold standard of treatment. So far, targeted systemic therapies have not been approved. The most frequent molecular alterations include the loss of PTEN and CDKN2A (encoding p16), being associated with poor prognoses in chordoma patients. Specific inhibitors of the PI3K/AKT/mTOR pathway as well as CDK4/6 have shown antitumor activity in preclinical studies and have recently been under investigation in phase II clinical trials; however, the clinical impacts and therapeutic consequences of concomitant PTEN and p16 deficiency have not yet been investigated in chordomas. In a cohort of 43 chordoma patients, 16% of the cases were immunohistochemically negative for both markers. The simultaneous loss of PTEN and p16 was associated with a higher KI-67 index, a tendency to metastasize, and significantly shorter overall survival. Additionally, 30% of chordoma cell lines (n = 19) were PTEN-/p16-negative. Treating these chordoma cells with palbociclib (CDK4/6 inhibitor), rapamycin (mTOR inhibitor) or the pan-PI3K inhibitor buparlisib significantly reduced cell viability. Synergistic effects were observed when combining palbociclib with rapamycin. In conclusion, we show that patients with PTEN-/p16-negative chordomas have poor prognoses and provide strong preclinical evidence that these patients might benefit from a Palbociclib/rapamycin combination treatment.

A Reference Genome Sequence for the European Silver Fir (Abies alba Mill.): A Community-Generated Genomic Resource.

Silver fir (Abies alba Mill.) is a keystone conifer of European montane forest ecosystems that has experienced large fluctuations in population size during during the Quaternary and, more recently, due to land-use change. To forecast the species' future distribution and survival, it is important to investigate the genetic basis of adaptation to environmental change, notably to extreme events. For this purpose, we here provide a first draft genome assembly and annotation of the silver fir genome, established through a community-based initiative. DNA obtained from haploid megagametophyte and diploid needle tissue was used to construct and sequence Illumina paired-end and mate-pair libraries, respectively, to high depth. The assembled A. alba genome sequence accounted for over 37 million scaffolds corresponding to 18.16 Gb, with a scaffold N50 of 14,051 bp. Despite the fragmented nature of the assembly, a total of 50,757 full-length genes were functionally annotated in the nuclear genome. The chloroplast genome was also assembled into a single scaffold (120,908 bp) that shows a high collinearity with both the A. koreana and A. sibirica complete chloroplast genomes. This first genome assembly of silver fir is an important genomic resource that is now publicly available in support of a new generation of research. By genome-enabling this important conifer, this resource will open the gate for new research and more precise genetic monitoring of European silver fir forests.

Environmental effects on fine-scale spatial genetic structure in four Alpine keystone forest tree species.

Genetic responses to environmental changes take place at different spatial scales. While the effect of environment on the distribution of species' genetic diversity at large geographical scales has been the focus of several recent studies, its potential effects on genetic structure at local scales are understudied. Environmental effects on fine-scale spatial genetic structure (FSGS) were investigated in four Alpine conifer species (five to eight populations per species) from the eastern Italian Alps. Significant FSGS was found for 11 of 25 populations. Interestingly, we found no significant differences in FSGS across species but great variation among populations within species, highlighting the importance of local environmental factors. Interannual variability in spring temperature had a small but significant effect on FSGS of Larix decidua, probably related to species-specific life history traits. For Abies alba, Picea abies and Pinus cembra, linear models identified spring precipitation as a potentially relevant climate factor associated with differences in FSGS across populations; however, models had low explanatory power and were strongly influenced by a P. cembra outlier population from a very dry site. Overall, the direction of the identified effects is according to expectations, with drier and more variable environments increasing FSGS. Underlying mechanisms may include climate-related changes in the variance of reproductive success and/or environmental selection of specific families. This study provides new insights on potential changes in local genetic structure of four Alpine conifers in the face of environmental changes, suggesting that new climates, through altering FSGS, may also have relevant impacts on plant microevolution.

Increased fire frequency promotes stronger spatial genetic structure and natural selection at regional and local scales in Pinus halepensis Mill.

Background and Aims: The recurrence of wildfires is predicted to increase due to global climate change, resulting in severe impacts on biodiversity and ecosystem functioning. Recurrent fires can drive plant adaptation and reduce genetic diversity; however, the underlying population genetic processes have not been studied in detail. In this study, the neutral and adaptive evolutionary effects of contrasting fire regimes were examined in the keystone tree species Pinus halepensis Mill. (Aleppo pine), a fire-adapted conifer. The genetic diversity, demographic history and spatial genetic structure were assessed at local (within-population) and regional scales for populations exposed to different crown fire frequencies. Methods: Eight natural P. halepensis stands were sampled in the east of the Iberian Peninsula, five of them in a region exposed to frequent crown fires (HiFi) and three of them in an adjacent region with a low frequency of crown fires (LoFi). Samples were genotyped at nine neutral simple sequence repeats (SSRs) and at 251 single nucleotide polymorphisms (SNPs) from coding regions, some of them potentially important for fire adaptation. Key Results: Fire regime had no effects on genetic diversity or demographic history. Three high-differentiation outlier SNPs were identified between HiFi and LoFi stands, suggesting fire-related selection at the regional scale. At the local scale, fine-scale spatial genetic structure (SGS) was overall weak as expected for a wind-pollinated and wind-dispersed tree species. HiFi stands displayed a stronger SGS than LoFi stands at SNPs, which probably reflected the simultaneous post-fire recruitment of co-dispersed related seeds. SNPs with exceptionally strong SGS, a proxy for microenvironmental selection, were only reliably identified under the HiFi regime. Conclusions: An increasing fire frequency as predicted due to global change can promote increased SGS with stronger family structures and alter natural selection in P. halepensis and in plants with similar life history traits.

Environmental versus geographical determinants of genetic structure in two subalpine conifers.

Alpine ecosystems are facing rapid human-induced environmental changes, and so more knowledge about tree adaptive potential is needed. This study investigated the relative role of isolation by distance (IBD) versus isolation by adaptation (IBA) in explaining population genetic structure in Abies alba and Larix decidua, based on 231 and 233 single nucleotide polymorphisms (SNPs) sampled across 36 and 22 natural populations, respectively, in the Alps and Apennines. Genetic structure was investigated for both geographical and environmental groups, using analysis of molecular variance (AMOVA). For each species, nine environmental groups were defined using climate variables selected from a multiple factor analysis. Complementary methods were applied to identify outliers based on these groups, and to test for IBD versus IBA. AMOVA showed weak but significant genetic structure for both species, with higher values in L. decidua. Among the potential outliers detected, up to two loci were found for geographical groups and up to seven for environmental groups. A stronger effect of IBD than IBA was found in both species; nevertheless, once spatial effects had been removed, temperature and soil in A. alba, and precipitation in both species, were relevant factors explaining genetic structure. Based on our findings, in the Alpine region, genetic structure seems to be affected by both geographical isolation and environmental gradients, creating opportunities for local adaptation.

Micro- and macro-geographic scale effect on the molecular imprint of selection and adaptation in Norway spruce.

Forest tree species of temperate and boreal regions have undergone a long history of demographic changes and evolutionary adaptations. The main objective of this study was to detect signals of selection in Norway spruce (Picea abies [L.] Karst), at different sampling-scales and to investigate, accounting for population structure, the effect of environment on species genetic diversity. A total of 384 single nucleotide polymorphisms (SNPs) representing 290 genes were genotyped at two geographic scales: across 12 populations distributed along two altitudinal-transects in the Alps (micro-geographic scale), and across 27 populations belonging to the range of Norway spruce in central and south-east Europe (macro-geographic scale). At the macrogeographic scale, principal component analysis combined with Bayesian clustering revealed three major clusters, corresponding to the main areas of southern spruce occurrence, i.e. the Alps, Carpathians, and Hercynia. The populations along the altitudinal transects were not differentiated. To assess the role of selection in structuring genetic variation, we applied a Bayesian and coalescent-based F(ST)-outlier method and tested for correlations between allele frequencies and climatic variables using regression analyses. At the macro-geographic scale, the F(ST)-outlier methods detected together 11 F(ST)-outliers. Six outliers were detected when the same analyses were carried out taking into account the genetic structure. Regression analyses with population structure correction resulted in the identification of two (micro-geographic scale) and 38 SNPs (macro-geographic scale) significantly correlated with temperature and/or precipitation. Six of these loci overlapped with F(ST)-outliers, among them two loci encoding an enzyme involved in riboflavin biosynthesis and a sucrose synthase. The results of this study indicate a strong relationship between genetic and environmental variation at both geographic scales. It also suggests that an integrative approach combining different outlier detection methods and population sampling at different geographic scales is useful to identify loci potentially involved in adaptation.

Contrasting patterns of nucleotide diversity for four conifers of Alpine European forests.

A candidate gene approach was used to identify levels of nucleotide diversity and to identify genes departing from neutral expectations in coniferous species of the Alpine European forest. Twelve samples were collected from four species that dominate montane and subalpine forests throughout Europe: Abies alba Mill, Larix decidua Mill, Pinus cembra L., and Pinus mugo Turra. A total of 800 genes, originally resequenced in Pinus taeda L., were resequenced across 12 independent trees for each of the four species. Genes were assigned to two categories, candidate and control, defined through homology-based searches to Arabidopsis. Estimates of nucleotide diversity per site varied greatly between polymorphic candidate genes (range: 0.0004-0.1295) and among species (range: 0.0024-0.0082), but were within the previously established ranges for conifers. Tests of neutrality using stringent significance thresholds, performed under the standard neutral model, revealed one to seven outlier loci for each species. Some of these outliers encode proteins that are involved with plant stress responses and form the basis for further evolutionary enquiries.