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Enhanced transcriptome maps from multiple mouse tissues reveal evolutionary constraint in gene expression.

Pervouchine DD, Djebali S, Breschi A, Davis CA, Barja PP, Dobin A, Tanzer A, Lagarde J, Zaleski C, See LH, Fastuca M, Drenkow J, Wang H, Bussotti G, Pei B, Balasubramanian S, Monlong J, Harmanci A, Gerstein M, Beer MA, Notredame C, Guigó R, Gingeras TR

Nat Commun
6 5903

2015

Abstract Illumina Summary

Abstract

Mice have been a long-standing model for human biology and disease. Here we characterize, by RNA sequencing, the transcriptional profiles of a large and heterogeneous collection of mouse tissues, augmenting the mouse transcriptome with thousands of novel transcript candidates. Comparison with transcriptome profiles in human cell lines reveals substantial conservation of transcriptional programmes, and uncovers a distinct class of genes with levels of expression that have been constrained early in vertebrate evolution. This core set of genes captures a substantial fraction of the transcriptional output of mammalian cells, and participates in basic functional and structural housekeeping processes common to all cell types. Perturbation of these constrained genes is associated with significant phenotypes including embryonic lethality and cancer. Evolutionary constraint in gene expression levels is not reflected in the conservation of the genomic sequences, but is associated with conserved epigenetic marking, as well as with characteristic post-transcriptional regulatory programme, in which sub-cellular localization and alternative splicing play comparatively large roles.

Illumina Summary

Mice are a good model organism for studying the progression of cancer and genetic diseases in humans. In this study a comprehensive map of transcriptional profiles was generated across 30 mouse tissue cell lines and 18 human cell lines using Illumina RNA sequencing. In this novel data set the authors identified thousands of novel transcript candidates in the mouse transcriptome. In comparison with transcriptome profiles in human cell lines, the mouse transcriptome across tissues shows a substantial conservation of transcriptional programmes.

Integrated genome and transcriptome sequencing of the same cell.

Dey SS, Kester L, Spanjaard B, Bienko M, van Oudenaarden A

Nat Biotechnol
33 285-9

2015

Abstract Illumina Summary

Abstract

Single-cell genomics and single-cell transcriptomics have emerged as powerful tools to study the biology of single cells at a genome-wide scale. However, a major challenge is to sequence both genomic DNA and mRNA from the same cell, which would allow direct comparison of genomic variation and transcriptome heterogeneity. We describe a quasilinear amplification strategy to quantify genomic DNA and mRNA from the same cell without physically separating the nucleic acids before amplification. We show that the efficiency of our integrated approach is similar to existing methods for single-cell sequencing of either genomic DNA or mRNA. Further, we find that genes with high cell-to-cell variability in transcript numbers generally have lower genomic copy numbers, and vice versa, suggesting that copy number variations may drive variability in gene expression among individual cells. Applications of our integrated sequencing approach could range from gaining insights into cancer evolution and heterogeneity to understanding the transcriptional consequences of copy number variations in healthy and diseased tissues.

Illumina Summary

Single-cell genomics and single-cell transcriptomics have emerged as powerful tools to study the biology of single cells at a genome-wide scale. In this breakthrough paper, the authors demonstrate how they extracted both DNA and mRNA from the same cell to perform complete transcriptome and genome analysis. Using a quasilinear amplification strategy and Illumina HiSeq for sequencing, they show that the efficiency of their approach is similar to existing methods for single-cell sequencing of either genomic DNA or mRNA. In addition, they found that genes with high cell-to-cell variability in transcript numbers generally have lower genomic copy numbers, and vice versa.

A genome-wide association study of marginal zone lymphoma shows association to the HLA region.

Vijai J, Wang Z, Berndt SI, Skibola CF, Slager SL, de Sanjose S, Melbye M, Glimelius B, Bracci PM, Conde L, Birmann BM, Wang SS, Brooks-Wilson AR, Lan Q, de Bakker PI, Vermeulen RC, Portlock C, Ansell SM, Link BK, Riby J, North KE, Gu J, Hjalgrim H, Cozen W, Becker N, Teras LR, Spinelli JJ, Turner J, Zhang Y, Purdue MP, Giles GG, Kelly RS, Zeleniuch-Jacquotte A, Ennas MG, Monnereau A, Bertrand KA, Albanes D, Lightfoot T, Yeager M, Chung CC, Burdett L, Hutchinson A, Lawrence C, Montalvan R, Liang L, Huang J, Ma B, Villano DJ, Maria A, Corines M, Thomas T, Novak AJ, Dogan A, Liebow M, Thompson CA, Witzig TE, Habermann TM, Weiner GJ, Smith MT, Holly EA, Jackson RD, Tinker LF, Ye Y, Adami HO, Smedby KE, De Roos AJ, Hartge P, Morton LM, Severson RK, Benavente Y, Boffetta P, Brennan P, Foretova L, Maynadie M, McKay J, Staines A, Diver WR, Vajdic CM, Armstrong BK, Kricker A, Zheng T, Holford TR, Severi G, Vineis P, Ferri GM, Ricco R, Miligi L, Clavel J, Giovannucci E, Kraft P, Virtamo J, Smi

Nat Commun
6 5751

2015

Abstract Illumina Summary

Abstract

Marginal zone lymphoma (MZL) is the third most common subtype of B-cell non-Hodgkin lymphoma. Here we perform a two-stage GWAS of 1,281 MZL cases and 7,127 controls of European ancestry and identify two independent loci near BTNL2 (rs9461741, P=3.95 × 10(-15)) and HLA-B (rs2922994, P=2.43 × 10(-9)) in the HLA region significantly associated with MZL risk. This is the first evidence that genetic variation in the major histocompatibility complex influences MZL susceptibility.

Illumina Summary

Marginal zone lymphoma (MZL) is the third most common subtype of B-cell non-Hodgkin lymphoma. In this two-stage genome-wide association study (GWAS) using Illumina HumanOmni genotyping arrays, the authors identified two independent SNPs within the HLA region that are robustly associated with the risk of MZL. However, the HLA region appears to be commonlyassociated with multiple major subtypes, and the authors suggest further studies are needed to identify biological mechanisms underlying these relationships.

Blocking PGE2-induced tumour repopulation abrogates bladder cancer chemoresistance.

Kurtova AV, Xiao J, Mo Q, Pazhanisamy S, Krasnow R, Lerner SP, Chen F, Roh TT, Lay E, Ho PL, Chan KS

Nature
517 209-13

2015

Abstract Illumina Summary

Abstract

Cytotoxic chemotherapy is effective in debulking tumour masses initially; however, in some patients tumours become progressively unresponsive after multiple treatment cycles. Previous studies have demonstrated that cancer stem cells (CSCs) are selectively enriched after chemotherapy through enhanced survival. Here we reveal a new mechanism by which bladder CSCs actively contribute to therapeutic resistance via an unexpected proliferative response to repopulate residual tumours between chemotherapy cycles, using human bladder cancer xenografts. Further analyses demonstrate the recruitment of a quiescent label-retaining pool of CSCs into cell division in response to chemotherapy-induced damages, similar to mobilization of normal stem cells during wound repair. While chemotherapy effectively induces apoptosis, associated prostaglandin E2 (PGE2) release paradoxically promotes neighbouring CSC repopulation. This repopulation can be abrogated by a PGE2-neutralizing antibody and celecoxib drug-mediated blockade of PGE2 signalling. In vivo administration of the cyclooxygenase-2 (COX2) inhibitor celecoxib effectively abolishes a PGE2- and COX2-mediated wound response gene signature, and attenuates progressive manifestation of chemoresistance in xenograft tumours, including primary xenografts derived from a patient who was resistant to chemotherapy. Collectively, these findings uncover a new underlying mechanism that models the progressive development of clinical chemoresistance, and implicate an adjunctive therapy to enhance chemotherapeutic response of bladder urothelial carcinomas by abrogating early tumour repopulation.

Illumina Summary

Cytotoxic chemotherapy remains the standard of care for many advanced carcinomas. However, in some patients tumour become progressively unresponsive to treatment. In this study, cancer stem cells were studied for their contribution to therapeutic resistance in a human bladder cancer xenograft model. The authors used RNA sequencing on Illumina HiSeq2500 to characterize the expression patterns before, during and after chemotherapy. The xenografts which were derived from a patient who was resistant to chemotherapy exhibited progressive chemoresistance.

Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing.

Yadav M, Jhunjhunwala S, Phung QT, Lupardus P, Tanguay J, Bumbaca S, Franci C, Cheung TK, Fritsche J, Weinschenk T, Modrusan Z, Mellman I, Lill JR, Delamarre L

Nature
515 572-6

2014

Abstract Illumina Summary

Abstract

Human tumours typically harbour a remarkable number of somatic mutations. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as 'non-self' neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, ~13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide-MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.

Illumina Summary

AlthoughCD8 T cells can recognize tumour cells andmediate tumour regression following immunotherapy10, the antigens driving effective anti-tumour CD8 T-cell responses remain largely unknown. This study used a combination of Illumina whole-exome and transcriptome sequencing with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. More than 1,300 amino acid changes were identified using this approach; 13% of those were predicted to bind MHCI and a small fraction of which were confirmed by mass spectrometry. The authors demonstrated how vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses.

PD-1 blockade induces responses by inhibiting adaptive immune resistance.

Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, Chmielowski B, Spasic M, Henry G, Ciobanu V, West AN, Carmona M, Kivork C, Seja E, Cherry G, Gutierrez AJ, Grogan TR, Mateus C, Tomasic G, Glaspy JA, Emerson RO, Robins H, Pierce RH, Elashoff DA, Robert C, Ribas A

Nature
515 568-71

2014

Abstract Illumina Summary

Abstract

Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types. One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8(+) T cells (termed adaptive immune resistance). Here we show that pre-existing CD8(+) T cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analysed samples from 46 patients with metastatic melanoma obtained before and during anti-PD-1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next-generation sequencing for T-cell antigen receptors (TCRs). In serially sampled tumours, patients responding to treatment showed proliferation of intratumoral CD8(+) T cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8-, PD-1- and PD-L1-expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumour regression after therapeutic PD-1 blockade requires pre-existing CD8(+) T cells that are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance..

Illumina Summary

The process of expression of PD-L1 in response to cytokines has been termed adaptive immune resistance6 and represents a mechanism by which cancer cells attempt to protect themselves from immune-cell-mediated killing. This study sought to determine whether pre-existing tumour associated CD8(+) T cells are a factor in determining clinical response to PD-1 blocking therapy. Using Illumina HiSeq sequencing, the authors characterized and quantified the T-cell receptor clonality in serially sampled tumours. They found that patients responding to treatment showed proliferation of intratumoral CD8(+) T cells, directly correlating with radiographic reduction in tumour size.

Spectrum of diverse genomic alterations define non-clear cell renal carcinoma subtypes.

Durinck S, Stawiski EW, Pavía-Jiménez A, Modrusan Z, Kapur P, Jaiswal BS, Zhang N, Toffessi-Tcheuyap V, Nguyen TT, Pahuja KB, Chen YJ, Saleem S, Chaudhuri S, Heldens S, Jackson M, Peña-Llopis S, Guillory J, Toy K, Ha C, Harris CJ, Holloman E, Hill HM, Stinson J, Rivers CS, Janakiraman V, Wang W, Kinch LN, Grishin NV, Haverty PM, Chow B, Gehring JS, Reeder J, Pau G, Wu TD, Margulis V, Lotan Y, Sagalowsky A, Pedrosa I, de Sauvage FJ, Brugarolas J, Seshagiri S

Nat Genet

2014

Abstract Illumina Summary

Abstract

To further understand the molecular distinctions between kidney cancer subtypes, we analyzed exome, transcriptome and copy number alteration data from 167 primary human tumors that included renal oncocytomas and non-clear cell renal cell carcinomas (nccRCCs), consisting of papillary (pRCC), chromophobe (chRCC) and translocation (tRCC) subtypes. We identified ten significantly mutated genes in pRCC, including MET, NF2, SLC5A3, PNKD and CPQ. MET mutations occurred in 15% (10/65) of pRCC samples and included previously unreported recurrent activating mutations. In chRCC, we found TP53, PTEN, FAAH2, PDHB, PDXDC1 and ZNF765 to be significantly mutated. Gene expression analysis identified a five-gene set that enabled the molecular classification of chRCC, renal oncocytoma and pRCC. Using RNA sequencing, we identified previously unreported gene fusions, including ACTG1-MITF fusion. Ectopic expression of the ACTG1-MITF fusion led to cellular transformation and induced the expression of downstream target genes. Finally, we observed upregulation of the anti-apoptotic factor BIRC7 in MiTF-high RCC tumors, suggesting a potential therapeutic role for BIRC7 inhibitors.

Illumina Summary

This study examined the molecular distinctions between kidney cancer subtypes, by analyzing exome, transcriptome and copy number alteration data from 167 primary human tumors that included renal oncocytomas and non-clear cell renal cell carcinomas (nccRCCs). Using high throughput sequencing on Illumina HiSeq2000 in combination with genotyping data from the Illumina HumanOmni2.5 array all results were validated and evaluated for mutational significance, copy number and gene fusions. The authors identified previously unreported gene fusions, including ACTG1-MITF fusion.

MicroRNA silencing for cancer therapy targeted to the tumour microenvironment.

Cheng CJ, Bahal R, Babar IA, Pincus Z, Barrera F, Liu C, Svoronos A, Braddock DT, Glazer PM, Engelman DM, Saltzman WM, Slack FJ

Nature

2014

Abstract Illumina Summary

Abstract

MicroRNAs are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. As such, microRNAs are critical cogs in numerous biological processes, and dysregulated microRNA expression is correlated with many human diseases. Certain microRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumours that depend on these microRNAs are said to display oncomiR addiction. Some of the most effective anticancer therapies target oncogenes such as EGFR and HER2; similarly, inhibition of oncomiRs using antisense oligomers (that is, antimiRs) is an evolving therapeutic strategy. However, the in vivo efficacy of current antimiR technologies is hindered by physiological and cellular barriers to delivery into targeted cells. Here we introduce a novel antimiR delivery platform that targets the acidic tumour microenvironment, evades systemic clearance by the liver, and facilitates cell entry via a non-endocytic pathway. We find that the attachment of peptide nucleic acid antimiRs to a peptide with a low pH-induced transmembrane structure (pHLIP) produces a novel construct that could target the tumour microenvironment, transport antimiRs across plasma membranes under acidic conditions such as those found in solid tumours (pH approximately 6), and effectively inhibit the miR-155 oncomiR in a mouse model of lymphoma. This study introduces a new model for using antimiRs as anti-cancer drugs, which can have broad impacts on the field of targeted drug delivery.

Illumina Summary

MicroRNAs are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. In this study, the authors present a novel method for delivery of antisense oligomers for oncogenic microRNAs (antimiRs). Using Illumina TruSeq sequencing, the authors validate the antimiR delivery platform as an approach to target the acidic tumour microenvironment and facilitates cell entry via a non-endocytic pathway, effectively inhibiting the miR-155 oncomiR in a mouse model of lymphoma.

IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo.

Venkatanarayan A, Raulji P, Norton W, Chakravarti D, Coarfa C, Su X, Sandur SK, Ramirez MS, Lee J, Kingsley CV, Sananikone EF, Rajapakshe K, Naff K, Parker-Thornburg J, Bankson JA, Tsai KY, Gunaratne PH, Flores ER

Nature

2014

Abstract Illumina Summary

Abstract

TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas the ?N isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes that promote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ?N isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the ß cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.

Illumina Summary

P53 is commonly altered in human cancer, and p53 reactivation suppresses tumours in vivo in mice. In this study the authors examine how to reactivate p53 as a therapeutic strategy by manipulating the p53 family members p63 and p73. Creating delta-Np73 and delta-Np63 knockout mice and validating by Illumina sequencing, the authors showed that the deletion of the delta-N isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours. This approach presents a novel strategy to target p53-deficient cancers.

Convergent loss of PTEN leads to clinical resistance to a PI(3)Kα inhibitor.

Juric D, Castel P, Griffith M, Griffith OL, Won HH, Ellis H, Ebbesen SH, Ainscough BJ, Ramu A, Iyer G, Shah RH, Huynh T, Mino-Kenudson M, Sgroi D, Isakoff S, Thabet A, Elamine L, Solit DB, Lowe SW, Quadt C, Peters M, Derti A, Schegel R, Huang A, Mardis ER, Berger MF, Baselga J, Scaltriti M

Nature

2014

Abstract Illumina Summary

Abstract

Broad and deep tumour genome sequencing has shed new light on tumour heterogeneity and provided important insights into the evolution of metastases arising from different clones. There is an additional layer of complexity, in that tumour evolution may be influenced by selective pressure provided by therapy, in a similar fashion to that occurring in infectious diseases. Here we studied tumour genomic evolution in a patient (index patient) with metastatic breast cancer bearing an activating PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha, PI(3)Ka) mutation. The patient was treated with the PI(3)Ka inhibitor BYL719, which achieved a lasting clinical response, but the patient eventually became resistant to this drug (emergence of lung metastases) and died shortly thereafter. A rapid autopsy was performed and material from a total of 14 metastatic sites was collected and sequenced. All metastatic lesions, when compared to the pre-treatment tumour, had a copy loss of PTEN (phosphatase and tensin homolog) and those lesions that became refractory to BYL719 had additional and different PTEN genetic alterations, resulting in the loss of PTEN expression. To put these results in context, we examined six other patients also treated with BYL719. Acquired bi-allelic loss of PTEN was found in one of these patients, whereas in two others PIK3CA mutations present in the primary tumour were no longer detected at the time of progression. To characterize our findings functionally, we examined the effects of PTEN knockdown in several preclinical models (both in cell lines intrinsically sensitive to BYL719 and in PTEN-null xenografts derived from our index patient), which we found resulted in resistance to BYL719, whereas simultaneous PI(3)K p110ß blockade reverted this resistance phenotype. We conclude that parallel genetic evolution of separate metastatic sites with different PTEN genomic alterations leads to a convergent PTEN-null phenotype resistant to PI(3)Ka inhibition.

Illumina Summary

Tumour evolution may be influenced by selective pressure provided by therapy, in a similar fashion to that occurring in infectious diseases acquiring resistance. In this study the tumour genomic evolution of a patient with metastatic breast cancer was studied via whole-exome sequencing on the Illumina HiSeq2500 platform. The patient was treated with the PI(3)Ka, which achieved a lasting clinical response, but the patient eventually became resistant to this drug and died. The analysis of the metastatic cancer genome revealed copy loss of PTEN and lesions refractory to treatment had additional and different PTEN alterations resulting in loss of PTEN expression.