Susanne Horn

TERT Promoter Mutations in Familial and Sporadic Melanoma.

Promoter Mutations and Cancer Cancer genome sequencing projects have highlighted the pathogenic role of recurrent mutations within the protein-coding regions of genes. Now, two studies suggest that the scope of mutations in human tumors extends to gene regulatory regions. In a study of 70 melanomas, Huang et al. (p. 957, published online 24 January) found that 71% harbored one of two specific mutations in the promoter region of TERT, the gene coding for the catalytic subunit of telomerase, the enzyme that caps chromosome ends. Independently, Horn et al. (p. 959, published online 24 January) identified a disease-segregating germline mutation in the TERT promoter in a family predisposed to melanoma and found additional TERT promoter mutations in a high percentage of sporadic melanomas and melanoma cell lines. The mutations in both studies generated new binding sites for specific transcription factors and, in reporter assays, caused an increase in transcription. A large fraction of human melanomas harbor mutations in sequences that regulate the expression of telomerase. Cutaneous melanoma occurs in both familial and sporadic forms. We investigated a melanoma-prone family through linkage analysis and high-throughput sequencing and identified a disease-segregating germline mutation in the promoter of the telomerase reverse transcriptase (TERT) gene, which encodes the catalytic subunit of telomerase. The mutation creates a new binding motif for Ets transcription factors and ternary complex factors (TCFs) near the transcription start and, in reporter gene assays, caused up to twofold increase in transcription. We then screened the TERT promoter in sporadic melanoma and observed recurrent ultraviolet signature somatic mutations in 125 of 168 (74%) of human cell lines derived from metastatic melanomas, 45 of 53 corresponding metastatic tumor tissues (85%), and 25 of 77 (33%) primary melanomas. The majority of those mutations occurred at two positions in the TERT promoter and also generated binding motifs for Ets/TCF transcription factors.

Mitochondrial genomes reveal slow rates of molecular evolution and the timing of speciation in beavers (Castor), one of the largest rodent species.

Background Beavers are one of the largest and ecologically most distinct rodent species. Little is known about their evolution and even their closest phylogenetic relatives have not yet been identified with certainty. Similarly, little is known about the timing of divergence events within the genus Castor. Methodology/Principal Findings We sequenced complete mitochondrial genomes from both extant beaver species and used these sequences to place beavers in the phylogenetic tree of rodents and date their divergence from other rodents as well as the divergence events within the genus Castor. Our analyses support the phylogenetic position of beavers as a sister lineage to the scaly tailed squirrel Anomalurus within the mouse related clade. Molecular dating places the divergence time of the lineages leading to beavers and Anomalurus as early as around 54 million years ago (mya). The living beaver species, Castor canadensis from North America and Castor fiber from Eurasia, although similar in appearance, appear to have diverged from a common ancestor more than seven mya. This result is consistent with the hypothesis that a migration of Castor from Eurasia to North America as early as 7.5 mya could have initiated their speciation. We date the common ancestor of the extant Eurasian beaver relict populations to around 210,000 years ago, much earlier than previously thought. Finally, the substitution rate of Castor mitochondrial DNA is considerably lower than that of other rodents. We found evidence that this is correlated with the longer life span of beavers compared to other rodents. Conclusions/Significance A phylogenetic analysis of mitochondrial genome sequences suggests a sister-group relationship between Castor and Anomalurus, and allows molecular dating of species divergence in congruence with paleontological data. The implementation of a relaxed molecular clock enabled us to estimate mitochondrial substitution rates and to evaluate the effect of life history traits on it.

Genetic Evolution of T-cell Resistance in the Course of Melanoma Progression

Purpose: CD8+ T lymphocytes can kill autologous melanoma cells, but their activity is impaired when poorly immunogenic tumor phenotypes evolve in the course of disease progression. Here, we analyzed three consecutive melanoma lesions obtained within one year of developing stage IV disease for their recognition by autologous T cells. Experimental Design: One skin (Ma-Mel-48a) and two lymph node (Ma-Mel-48b, Ma-Mel-48c) metastases were analyzed for T-cell infiltration. Melanoma cell lines established from the respective lesions were characterized, determining the T-cell–stimulatory capacity, expression of surface molecules involved in T-cell activation, and specific genetic alterations affecting the tumor–T-cell interaction. Results: Metastases Ma-Mel-48a and Ma-Mel-48b, in contrast with Ma-Mel-48c, were infiltrated by T cells. The T-cell–stimulatory capacity was found to be strong for Ma-Mel-48a, lower for Ma-Mel-48b, and completely abrogated for Ma-Mel-48c cells. The latter proved to be HLA class I–negative due to an inactivating mutation in one allele of the beta-2-microglobulin (B2M) gene and concomitant loss of the other allele by a deletion on chromosome 15q. The same deletion was already present in Ma-Mel-48a and Ma-Mel-48b cells, pointing to an early acquired genetic event predisposing to development of β2m deficiency. Notably, the same chronology of genetic alterations was also observed in a second β2m-deficient melanoma model. Conclusion: Our study reveals a progressive loss in melanoma immunogenicity during the course of metastatic disease. The genetic evolvement of T-cell resistance suggests screening tumors for genetic alterations affecting immunogenicity could be clinically relevant in terms of predicting patient responses to T-cell–based immunotherapy. Clin Cancer Res; 20(24); 6593–604. ©2014 AACR.

Genomic analysis of hepatitis B virus reveals antigen state and genotype as sources of evolutionary rate variation.

Hepatitis B virus (HBV) genomes are small, semi-double-stranded DNA circular genomes that contain alternating overlapping reading frames and replicate through an RNA intermediary phase. This complex biology has presented a challenge to estimating an evolutionary rate for HBV, leading to difficulties resolving the evolutionary and epidemiological history of the virus. Here, we re-examine rates of HBV evolution using a novel data set of 112 within-host, transmission history (pedigree) and among-host genomes isolated over 20 years from the indigenous peoples of the South Pacific, combined with 313 previously published HBV genomes. We employ Bayesian phylogenetic approaches to examine several potential causes and consequences of evolutionary rate variation in HBV. Our results reveal rate variation both between genotypes and across the genome, as well as strikingly slower rates when genomes are sampled in the Hepatitis B e antigen positive state, compared to the e antigen negative state. This Hepatitis B e antigen rate variation was found to be largely attributable to changes during the course of infection in the preCore and Core genes and their regulatory elements.

Thermal boundary layer equation for turbulent Rayleigh-Bénard convection

We report a new thermal boundary layer equation for turbulent Rayleigh-Bénard convection for Prandtl number Pr>1 that takes into account the effect of turbulent fluctuations. These fluctuations are neglected in existing equations, which are based on steady-state and laminar assumptions. Using this new equation, we derive analytically the mean temperature profiles in two limits: (a) Pr≳1 and (b) Pr≫1. These two theoretical predictions are in excellent agreement with the results of our direct numerical simulations for Pr=4.38 (water) and Pr=2547.9 (glycerol), respectively.

Acquired IFNγ resistance impairs anti-tumor immunity and gives rise to T-cell-resistant melanoma lesions

Melanoma treatment has been revolutionized by antibody-based immunotherapies. IFNγ secretion by CD8+ T cells is critical for therapy efficacy having anti-proliferative and pro-apoptotic effects on tumour cells. Our study demonstrates a genetic evolution of IFNγ resistance in different melanoma patient models. Chromosomal alterations and subsequent inactivating mutations in genes of the IFNγ signalling cascade, most often JAK1 or JAK2, protect melanoma cells from anti-tumour IFNγ activity. JAK1/2 mutants further evolve into T-cell-resistant HLA class I-negative lesions with genes involved in antigen presentation silenced and no longer inducible by IFNγ. Allelic JAK1/2 losses predisposing to IFNγ resistance development are frequent in melanoma. Subclones harbouring inactivating mutations emerge under various immunotherapies but are also detectable in pre-treatment biopsies. Our data demonstrate that JAK1/2 deficiency protects melanoma from anti-tumour IFNγ activity and results in T-cell-resistant HLA class I-negative lesions. Screening for mechanisms of IFNγ resistance should be considered in therapeutic decision-making.

Rotating non-Oberbeck–Boussinesq Rayleigh–Bénard convection in water

Rotating Rayleigh–Benard convection in water is studied in direct numerical simulations, where the temperature dependence of the viscosity, the thermal conductivity, and the density within the buoyancy term is taken into account. In all simulations, the arithmetic mean of the lowest and highest temperature in the system equals 40 °C, corresponding to a Prandtl number of Pr = 4.38. In the non-rotational case, the Rayleigh number Ra ranges from 107 to 1.16 × 109 and temperature differences Δ up to 70 K are considered, whereas in the rotational case the inverse Rossby number range from 0.07 ⩽ 1/Ro ⩽ 14.1 is studied for Δ = 40 K with the focus on Ra = 108. The non-Oberbeck–Boussinesq (NOB) effects in water are reflected in an up to 5.5 K enhancement of the center temperature and in an up to 5% reduction of the Nusselt number. The top thermal and viscous boundary layer thicknesses increase and the bottom ones decrease, while the sum of the corresponding top and bottom thicknesses remains as in the classical Oberbeck–Boussinesq (OB) case. Rotation applied to NOB thermal convection reduces the central temperature enhancement. Under NOB conditions the top (bottom) thermal and viscous boundary layers become equal for a slightly larger (smaller) inverse Rossby number than in the OB case. Furthermore, for rapid rotation the thermal bottom boundary layers become thicker than the top ones. The Nusselt number normalized by that in the non-rotating case depends similarly on 1/Ro  in both, the NOB and the OB cases. The deviation between the Nusselt number under OB and NOB conditions is minimal when the thermal and viscous boundary layers are equal.

Melanoma lesions independently acquire t-cell resistance during metastatic latency

Melanoma often recurs after a latency period of several years, presenting a T cell-edited phenotype that reflects a role for CD8(+) T cells in maintaining metastatic latency. Here, we report an investigation of a patient with multiple recurrent lesions, where poorly immunogenic melanoma phenotypes were found to evolve in the presence of autologous tumor antigen-specific CD8(+) T cells. Melanoma cells from two of three late recurrent metastases, developing within a 6-year latency period, lacked HLA class I expression. CD8(+) T cell-resistant, HLA class I-negative tumor cells became clinically apparent 1.5 and 6 years into stage IV disease. Genome profiling by SNP arrays revealed that HLA class I loss in both metastases originated from a shared chromosome 15q alteration and independently acquired focal B2M gene deletions. A third HLA class I haplotype-deficient lesion developed in year 3 of stage IV disease that acquired resistance toward dominant CD8(+) T-cell clonotypes targeting stage III tumor cells. At an early stage, melanoma cells showed a dedifferentiated c-Jun(high)/MITF(low) phenotype, possibly associated with immunosuppression, which contrasted with a c-Jun(low)/MITF(high) phenotype of T cell-edited tumor cells derived from late metastases. In summary, our work shows how tumor recurrences after long-term latency evolve toward T-cell resistance by independent genetic events, as a means for immune escape and immunotherapeutic resistance. Cancer Res; 76(15); 4347-58. ©2016 AACR.

Toroidal and poloidal energy in rotating Rayleigh–Bénard convection

We consider rotating Rayleigh–Benard convection of a fluid with a Prandtl number of Pr = 0.8 in a cylindrical cell with an aspect ratio Γ = 1/2. Direct numerical simulations were performed for the Rayleigh number range 10 6 Ra 6 10 and the inverse Rossby number range 0 6 1/Ro 6 20. We propose a method to capture regime transitions based on the decomposition of the velocity field into toroidal and poloidal parts. We identify four different regimes. First, a buoyancy dominated regime occurring as long as the toroidal energy etor is not affected by rotation and remains equal to that in the non-rotating case, e0tor. Second, a rotation influenced regime, starting at rotation rates where etor > e 0 tor and ending at a critical inverse Rossby number 1/Rocr that is determined by the balance of the toroidal and poloidal energy, etor = epol. Third, a rotation dominated regime, where the toroidal energy etor is larger than both, epol and e 0 tor. Fourth, a geostrophic turbulence regime for high rotation rates where the toroidal energy drops below the value of nonrotating convection.

Ancient mitochondrial DNA and the genetic history of Eurasian beaver (Castor fiber) in Europe

After centuries of human hunting, the Eurasian beaver Castor fiber had disappeared from most of its original range by the end of the 19th century. The surviving relict populations are characterized by both low genetic diversity and strong phylogeographical structure. However, it remains unclear whether these attributes are the result of a human‐induced, late Holocene bottleneck or already existed prior to this reduction in range. To investigate genetic diversity in Eurasian beaver populations during the Holocene, we obtained mitochondrial control region DNA sequences from 48 ancient beaver samples and added 152 modern sequences from GenBank. Phylogeographical analyses of the data indicate a differentiation of European beaver populations into three mitochondrial clades. The two main clades occur in western and eastern Europe, respectively, with an early Holocene contact zone in eastern Europe near a present‐day contact zone. A divergent and previously unknown clade of beavers from the Danube Basin survived until at least 6000 years ago, but went extinct during the transition to modern times. Finally, we identify a recent decline in effective population size of Eurasian beavers, with a stronger bottleneck signal in the western than in the eastern clade. Our results suggest that the low genetic diversity and the strong phylogeographical structure in recent beavers are artefacts of human hunting‐associated population reductions. While beaver populations have been growing rapidly since the late 19th century, genetic diversity within modern beaver populations remains considerably reduced compared to what was present prior to the period of human hunting and habitat reduction.