John C. Castle

MICROARRAY ANALYSIS SHOWS THAT SOME MICRORNAS DOWNREGULATE LARGE NUMBERS OF TARGET MRNAS

MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression in plants and animals. To investigate the influence of miRNAs on transcript levels, we transfected miRNAs into human cells and used microarrays to examine changes in the messenger RNA profile. Here we show that delivering miR-124 causes the expression profile to shift towards that of brain, the organ in which miR-124 is preferentially expressed, whereas delivering miR-1 shifts the profile towards that of muscle, where miR-1 is preferentially expressed. In each case, about 100 messages were downregulated after 12u2009h. The 3′ untranslated regions of these messages had a significant propensity to pair to the 5′ region of the miRNA, as expected if many of these messages are the direct targets of the miRNAs. Our results suggest that metazoan miRNAs can reduce the levels of many of their target transcripts, not just the amount of protein deriving from these transcripts. Moreover, miR-1 and miR-124, and presumably other tissue-specific miRNAs, seem to downregulate a far greater number of targets than previously appreciated, thereby helping to define tissue-specific gene expression in humans.

Exploiting the Mutanome for Tumor Vaccination

Multiple genetic events and subsequent clonal evolution drive carcinogenesis, making disease elimination with single-targeted drugs difficult. The multiplicity of gene mutations derived from clonal heterogeneity therefore represents an ideal setting for multiepitope tumor vaccination. Here, we used next generation sequencing exome resequencing to identify 962 nonsynonymous somatic point mutations in B16F10 murine melanoma cells, with 563 of those mutations in expressed genes. Potential driver mutations occurred in classical tumor suppressor genes and genes involved in proto-oncogenic signaling pathways that control cell proliferation, adhesion, migration, and apoptosis. Aim1 and Trrap mutations known to be altered in human melanoma were included among those found. The immunogenicity and specificity of 50 validated mutations was determined by immunizing mice with long peptides encoding the mutated epitopes. One-third of these peptides were found to be immunogenic, with 60% in this group eliciting immune responses directed preferentially against the mutated sequence as compared with the wild-type sequence. In tumor transplant models, peptide immunization conferred in vivo tumor control in protective and therapeutic settings, thereby qualifying mutated epitopes that include single amino acid substitutions as effective vaccines. Together, our findings provide a comprehensive picture of the mutanome of B16F10 melanoma which is used widely in immunotherapy studies. In addition, they offer insight into the extent of the immunogenicity of nonsynonymous base substitution mutations. Lastly, they argue that the use of deep sequencing to systematically analyze immunogenicity mutations may pave the way for individualized immunotherapy of cancer patients.

Mutant MHC class II epitopes drive therapeutic immune responses to cancer

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient’s tumour possesses a unique set of mutations (‘the mutanome’) that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient’s individual tumour-specific mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient’s tumour with vaccines produced ‘just in time’.

RNA-Seq Atlas – A reference database for gene expression profiling in normal tissue by next generation sequencing

MOTIVATIONnNext-generation sequencing technology enables an entirely new perspective for clinical research and will speed up personalized medicine. In contrast to microarray-based approaches, RNA-Seq analysis provides a much more comprehensive and unbiased view of gene expression. Although the perspective is clear and the long-term success of this new technology obvious, bioinformatics resources making these data easily available especially to the biomedical research community are still evolving.nnnRESULTSnWe have generated RNA-Seq Atlas, a web-based repository of RNA-Seq gene expression profiles and query tools. The website offers open and easy access to RNA-Seq gene expression profiles and tools to both compare tissues and find genes with specific expression patterns. To enlarge the scope of the RNA-Seq Atlas, the data were linked to common functional and genetic databases, in particular offering information on the respective gene, signaling pathway analysis and evaluation of biological functions by means of gene ontologies. Additionally, data were linked to several microarray gene profiles, including BioGPS normal tissue profiles and NCI60 cancer cell line expression data. Our data search interface allows an integrative detailed comparison between our RNA-Seq data and the microarray information. This is the first database providing data mining tools and open access to large scale RNA-Seq expression profiles. Its applications will be versatile, as it will be beneficial in identifying tissue specific genes and expression profiles, comparison of gene expression profiles among diverse tissues, but also systems biology approaches linking tissue function to gene expression changes.nnnAVAILABILITY AND IMPLEMENTATIONnhttp://medicalgenomics.org/rna_seq_atlas.

Searching for seismic scattering off mantle interfaces between 800 km and 2000 km depth

[1]xa0We systematically examined seismic data from deep earthquakes in the western and northwestern Pacific, Tonga/Fiji, and South America for signs of sharp gradients in seismic properties that could represent either a mineralogical phase change or the boundary between geochemical reservoirs in the Earths lower mantle. This involved the stacking of short-period, vertical component teleseismic waveforms from the dense Pacific Northwest Seismic Network and searching for evidence of S-to-P conversions at sharp mantle structures between 800 and 2000 km depth. With these data, we estimate we should be able to detect seismic discontinuities with ΔVs > 2%, transition width <20 km, and limited local topography. Using data from over 45 earthquakes, we found no evidence of a horizontal global discontinuity, neither near 920 km nor near 1700 km, beneath the four convergent margins studied here. Consistent with previous results, we detected a deep structure with topography near 1600 km beneath the Marianas region. Because we observed the signal over a large geographical region beneath the Marianas (10°N to 20°N), it is likely a coherent local structural and not a point source. The absence of such a structure beneath the northwest Pacific, the Tonga/Fiji region, or South America, argues against a global feature near 1600 km depth, however. The lack of convincing and consistent scattering suggests that no composition boundary or thermal boundary layer exists between 800 to 2000 km depth beneath the convergent zones studied that has ΔVs < 2%, locally limited topography, and a transition interval <20 km.

HLA typing from RNA-Seq sequence reads

We present a method, seq2HLA, for obtaining an individuals human leukocyte antigen (HLA) class I and II type and expression using standard next generation sequencing RNA-Seq data. RNA-Seq reads are mapped against a reference database of HLA alleles, and HLA type, confidence score and locus-specific expression level are determined. We successfully applied seq2HLA to 50 individuals included in the HapMap project, yielding 100% specificity and 94% sensitivity at a P-value of 0.1 for two-digit HLA types. We determined HLA type and expression for previously un-typed Illumina Body Map tissues and a cohort of Korean patients with lung cancer. Because the algorithm uses standard RNA-Seq reads and requires no change to laboratory protocols, it can be used for both existing datasets and future studies, thus adding a new dimension for HLA typing and biomarker studies.

Topography of the 660‐km seismic discontinuity beneath Izu‐Bonin: Implications for tectonic history and slab deformation

We analyze the P wave codas of 65 paths from deep northwestern Pacific earthquakes recorded by arrays of stations in Germany, the western United States, India, and Turkmenistan. We identify a phase resulting from a near-source S-to-P conversion at a nearly horizontal discontinuity ranging in depth from 650 to 730 km, which we interpret as a thermally depressed spinel to perovskite and magnesiowustite phase transition. We migrate these data along with 39 more from Wicks and Richards [1993], accounting for three-dimensional ray bending by the sloping discontinuity, to produce a high-resolution topography map of the 660-km discontinuity in the Izu-Bonin region. Assuming an equilibrium phase transition, we interpret the discontinuity depth in terms of local temperatures. The slab, if defined by a thermal anomaly greater than −400°K, is only about 100 km thick near 28°N suggesting the slab is penetrating into the lower mantle with little or no advective thickening. Farther to the north, however, cold material appears spread out over a wide region, consistent with the slab having been laid down flat on the 660-km discontinuity as the trench retreated 2000 km eastward. Both the narrow slab to the south and the flat-lying slab to the north are consistent with recent high-resolution tomographic images. The depression to 745 km along the arc is consistent with a maximum thermal anomaly of about 1100°K. Along the entire arc, the depression occurs directly beneath the deepest earthquakes, even where seismicity is dipping at 45° and stops at 450 km depth, suggesting that the slab steepens to a vertical dip at the deepest seismicity. This change to a vertical orientation suggests that the slab loses strength temporarily through a physical process which causes the seismicity to increase dramatically and then abruptly cease.

The core^mantle boundary under the Gulf of Alaska: No ULVZ for shear waves

The Earth’s core^mantle boundary (CMB) marks the boundary between the hot, molten iron core and the silicate mantle and is a thermal, chemical, and flow boundary. Previous observations of very slow compressional wavespeeds suggest that thin ultra-low-velocity zones (ULVZs), possibly composed of a mixture of molten iron and silicates, exist at the base of the mantle. A molten or partially molten layer would cause a large shear wavespeed decrease; however this velocity drop has not been observed. Here, we use core reflected ScP phases to investigate the shear properties of ULVZs. These phases reveal at least two distinct regions: one region under Central America which is distinctly average (PREM and iasp91-like) and a second region under the entire Gulf of Alaska that produces large ScP reflections with amplitudes of up to 30 times larger than calculated with average Earth models. The large amplitudes suggest a combination of focusing by CMB topography, high shear wavespeeds at the bottom of the mantle, and low attenuation (high Q) along the ScP path; high shear wavespeeds and low attenuation are opposite from what would be expected from a shear wave ULVZ. A ULVZ in compressional wavespeeds (ultra-low Vp) has previously been observed in this region; however, in addition to the large amplitudes, the short-period ScP waveforms show no complexity that can be related to a ULVZ. Thus, either there is not a ULVZ under the Gulf of Alaska or, if it does exist, it is restricted to compressional wavespeed changes, precluding interpretation as partial melt but rather suggesting a chemical origin. fl 2000 Published by Elsevier Science B.V. All rights reserved.

A steeply dipping discontinuity in the lower mantle beneath Izu-Bonin

We analyze the coda of teleseismic P waves to deterministically map seismic scatterers in a 1000 km on-a-side cube beneath the Izu-Bonin trench by migrating and stacking waveforms. This method was applied to several hundred short-period vertical-component western United States seismometer recordings of 17 deep-focus Izu-Bonin earthquakes. Except for isolated arrivals, the midmantle appears devoid of sharp discontinuities. S-to-P conversions at the 660-km discontinuity generated the largest signals; the 410-km discontinuity occasionally generated signals. Horizontal discontinuities poorly explain other signals; however, S-to-P conversions generated at a nearly north-south trending, steeply dipping discontinuity at 30°N, 145°E, and 1000 km beneath and parallel to the Izu-Bonin trench explain most additional signals. Observed coherent S-to-P conversions of 2-s period waves limit the width of a gradient zone to <7 km. Compressional and shear tomographic models [Grand et al., 1997; Widiyantoro, 1997] image a fast velocity structure at a similar depth and orientation extending southward from the middle of the Izu-Bonin trench to the Mariana trench. We interpret the sharp scattering surface as a subducted crust and the tomographically imaged structure as the cold thermal anomaly associated with an ancient slab. We observe neither the deep scatterer nor high-seismic wavespeeds north of 32°N. This feature is as much as 800 km east of recent deep earthquakes near 30°N and is at a latitude where the slab appears to extend horizontally on the 660-km discontinuity to the west of the seismicity. We suggest that this deep slab fragment was carried north 500–1000 km owing to oblique subduction. In this scenario, it would be associated with the near vertical subduction at the Mariana subduction zone.

Immunomic, genomic and transcriptomic characterization of CT26 colorectal carcinoma

BackgroundTumor models are critical for our understanding of cancer and the development of cancer therapeutics. Here, we present an integrated map of the genome, transcriptome and immunome of an epithelial mouse tumor, the CT26 colon carcinoma cell line.ResultsWe found that Kras is homozygously mutated at p.G12D, Apc and Tp53 are not mutated, and Cdkn2a is homozygously deleted. Proliferation and stem-cell markers, including Top2a, Birc5 (Survivin), Cldn6 and Mki67, are highly expressed while differentiation and top-crypt markers Muc2, Ms4a8a (MS4A8B) and Epcam are not. Myc, Trp53 (tp53), Mdm2, Hif1a, and Nras are highly expressed while Egfr and Flt1 are not. MHC class I but not MHC class II is expressed. Several known cancer-testis antigens are expressed, including Atad2, Cep55, and Pbk. The highest expressed gene is a mutated form of the mouse tumor antigen gp70. Of the 1,688 non-synonymous point variations, 154 are both in expressed genes and in peptides predicted to bind MHC and thus potential targets for immunotherapy development. Based on its molecular signature, we predicted that CT26 is refractory to anti-EGFR mAbs and sensitive to MEK and MET inhibitors, as have been previously reported.ConclusionsCT26 cells share molecular features with aggressive, undifferentiated, refractory human colorectal carcinoma cells. As CT26 is one of the most extensively used syngeneic mouse tumor models, our data provide a map for the rationale design of mode-of-action studies for pre-clinical evaluation of targeted- and immunotherapies.