Systematic genomic and translational efficiency studies of uveal melanoma

To further our understanding of the somatic genetic basis of uveal melanoma, we sequenced the protein-coding regions of 52 primary tumors and 3 liver metastases together with paired normal DNA. Known recurrent mutations were identified in GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. The role of mutated EIF1AX was tested using loss of function approaches including viability and translational efficiency assays. Knockdown of both wild type and mutant EIF1AX was lethal to uveal melanoma cells. We probed the function of N-terminal tail EIF1AX mutations by performing RNA sequencing of polysome-associated transcripts in cells expressing endogenous wild type or mutant EIF1AX. Ribosome occupancy of the global translational apparatus was sensitive to suppression of wild type but not mutant EIF1AX. Together, these studies suggest that cells expressing mutant EIF1AX may exhibit aberrant translational regulation, which may provide clonal selective advantage in the subset of uveal melanoma that harbors this mutation

Sooty Mangabey Genome Sequence Provides Insight into AIDS Resistance in a Natural SIV Host

In contrast to infections with human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques, SIV infection of a natural host, sooty mangabeys (Cercocebus atys), is non-pathogenic despite high viraemia. Here we sequenced and assembled the genome of a captive sooty mangabey. We conducted genome-wide comparative analyses of transcript assemblies from C. atys and AIDS-susceptible species, such as humans and macaques, to identify candidates for host genetic factors that influence susceptibility. We identified several immune-related genes in the genome of C. atys that show substantial sequence divergence from macaques or humans. One of these sequence divergences, a C-terminal frameshift in the toll-like receptor-4 (TLR4) gene of C. atys, is associated with a blunted in vitro response to TLR-4 ligands. In addition, we found a major structural change in exons 3-4 of the immune-regulatory protein intercellular adhesion molecule 2 (ICAM-2); expression of this variant leads to reduced cell surface expression of ICAM-2. These data provide a resource for comparative genomic studies of HIV and/or SIV pathogenesis and may help to elucidate the mechanisms by which SIV-infected sooty mangabeys avoid AIDS

Paired exome analysis of Barrett's esophagus and adenocarcinoma.

Barrett's esophagus is thought to progress to esophageal adenocarcinoma (EAC) through a stepwise progression with loss of CDKN2A followed by TP53 inactivation and aneuploidy. Here we present whole-exome sequencing from 25 pairs of EAC and Barrett's esophagus and from 5 patients whose Barrett's esophagus and tumor were extensively sampled. Our analysis showed that oncogene amplification typically occurred as a late event and that TP53 mutations often occurred early in Barrett's esophagus progression, including in non-dysplastic epithelium. Reanalysis of additional EAC exome data showed that the majority (62.5%) of EACs emerged following genome doubling and that tumors with genomic doubling had different patterns of genomic alterations, with more frequent oncogenic amplification and less frequent inactivation of tumor suppressors, including CDKN2A. These data suggest that many EACs emerge not through the gradual accumulation of tumor-suppressor alterations but rather through a more direct path whereby a TP53-mutant cell undergoes genome doubling, followed by the acquisition of oncogenic amplifications

Paired Exome Analysis of Barrett’s Esophagus and Adenocarcinoma

Barrett’s esophagus, is thought to progress to esophageal adenocarcinoma (EAC) through a step-wise progression with loss of CDKN2A followed by p53 inactivation and aneuploidy. Here, we present whole exome sequencing from 25 pairs of EAC and Barrett’s and five patients whose Barrett’s and tumor were extensively sampled. Our analysis revealed that oncogene amplification typically occurred as a late event and that TP53 mutations often occur early in Barrett’s progression, including in non-dysplastic epithelium. Reanalysis of additional EAC exome data revealed that the majority (62.5%) of EACs emerged following genome doubling and that tumors with genomic doubling had different patterns of genomic alterations with more frequent oncogenic amplifications and less frequent inactivation of tumor suppressors, including CDKN2A. These data suggest that many EACs emerge not through gradual accumulation of tumor suppressor alterations but rather through a more direct path whereby a TP53-mutant cell undergoes genome doubling, followed by acquisition of oncogenic amplifications

Prospective derivation of a living organoid biobank of colorectal cancer patients

In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely recapitulate several properties of the original tumor. The spectrum of genetic changes within the "living biobank" agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell-line- and xenograft-based drug studies, and allow personalized therapy design. PAPERCLIP

Somatic mutations in primary and metastatic uveal melanoma.

<p><b>(A)</b> The number of synonymous and nonsynonymous mutations per megabase of DNA sequence for 52 samples, arranged in columns. <b>(B)</b> Mutations in recurrently mutated genes are color-coded and ordered by significance. <b>(C)</b> Boxplots represent the distributions of allelic fractions observed per sample where the thick line represents 25-75^th percentile, and thin line 5-95^th. <b>(D)</b> The percentage of tumor cells (CCF) harboring a given mutation in the primary tumor in comparison to a metastatic liver sample from the same patient (UM45). <b>(E)</b> As in (D), but comparing a pre-treatment metastatic tumor sample to a post-treatment metastasis (Trio 2).</p

EIF1AX-regulated growth and translation in uveal melanoma.

<p><b>(A)</b> Distribution of <i>EIF1AX</i> mutations observed in cohort of 52 uveal melanomas in comparison to other cancer types (as reported by <a href="http://www.tumorportal.org" target="_blank">http://www.tumorportal.org</a>). <b>(B)</b> <i>EIF1AX</i> wild type (WT) or mutant (MUT) uveal melanoma cells were infected with <i>EIF1AX</i> or control shRNAs and cell viability was determined after 6 days using MTS. Percent growth is relative to shLuc-expressing cells. Error bars represent SD of mean from 3 independent experiments. <b>(C)</b> Immunoblot analysis of EIF1AX protein levels in shRNA-expressing cells. <b>(D)</b> Polysome profiles of cell lines expressing shRNAs against <i>EIF1AX</i> and <i>Luciferase</i>.</p

Decreased EIF1AX expression impairs translation of protein synthesis machinery in wildtype, but not mutated setting.

<p><b>(A)</b> Principal component analysis depicts 4 color-coded clusters of 141 genes. <b>(B)</b> The trend in translational efficiency is depicted for each cluster in cells expressing control shRNAs (CN) or <i>EIF1AX</i> shRNAs (KD). Each line represents a different gene. Ribosomal protein genes are highlighted in red. Translational efficiency was calculated as polysome CPM / total CPM. <b>(C)</b> Boxplots demonstrate the distribution of the translational efficiencies of 78 ribosomal proteins in cells as in (B).</p