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Tumour‐promoting inflammation is an emerging hallmark of cancer that is increasingly recognised as a therapeutic target. As a constituent measure of inflammation, tumour‐infiltrating neutrophils (TINs) have been associated with inferior prognosis in several cancers. We analysed clinically annotated cohorts of clear cell renal cell carcinoma (ccRCC) to assess the presence of neutrophils within the tumour microenvironment as a function of outcome. We centrally reviewed ccRCC surgical resection and fine‐needle aspiration (FNA) specimens, including primary and metastatic sites, from three centres. TINs were scored based on the presence of neutrophils in resection and FNA specimens by two pathologists. TIN count was correlated with tumour characteristics including stage, WHO/ISUP grade, and immunohistochemistry (IHC). In parallel, we performed CIBERSORT analysis of the tumour microenvironment in a cohort of 516 ccRCCs from The Cancer Genome Atlas (TCGA). We included 102 ccRCC cases comprising 65 resection specimens (37 primary and 28 metastatic resection specimens) and 37 FNAs from primary lesions. High TINs were significantly associated with worse overall survival (p = 0.009) independent of tumour grade and stage. In ccRCCs sampled via FNA, all cases with high TINs had distant metastasis, whereas they were seen in only 19% of cases with low TINs (p = 0.0003). IHC analysis showed loss of E‐cadherin in viable tumour cells in areas with high TINs, and neutrophil activation was associated with elastase and citrullinated histone H3 expression (cit‐H3). In the TCGA cohort, neutrophilic markers were also associated with worse survival (p

Rhabdoid tumors (RTs) are frequent pediatric malignancies that are classified based on tumor localization: In the central nervous system, they are referred to as atypical teratoid/rhabdoid tumors (ATRTs) as compared to extra-cranial malignant RTs (MRTs). The common genetic hallmark of these tumors is the loss of SMARCB1 in 95% of all cases. Molecularly, RTs are heterogeneous. Previous studies reported molecular subgroups within RTs of the same anatomic compartment. However, biological similarities among RT subgroups of different sites, and molecular characteristics shared among them remained unknown. Investigating the similarity of RTs from different anatomic sites at a molecular level, we compared DNA methylation, gene expression, and H3K27ac profiles of 150 MRTs and 161 ATRTs generated by WGBS, 450K/850K-arrays, RNA-, and ChIP-Seq. Clustering of methylation data showed that a subset of MRTs (called Group1) clustered with the MYC-subgroup of ATRTs but not with SHH-and TYR-subgroups of ATRTs. Over-expression of MYC-ATRT representative genes, such as c-MYC and the non-coding regulatory RNA gene HOTAIR, was also observed in these MRTs. Transcription factor binding enrichment analyses displayed a high enrichment of transcription factors implicated in immune cell regulation such as GMEMB1/2, IRF5/8/9, and STAT1 in both ATRT-MYC and MRT. We therefore predicted the immune cell infiltration of RTs using the CIBERSORT and validated these in-silico analyses by immunohistochemistry (IHC) for CD3,CD8, CD68 and PD-L1. In line with the computational results, IHC demonstrated a high number of CD3+ and CD8+ in both MRT and ATRT-MYC, suggestive of abundant cytotoxic T-cell infiltration. Overall our analyses reveal a high degree of similarity between ATRT-MYC and (Group 1)- MRT not only at the epigenetic level, but also in the immune cell composition and underline the suitability of these tumors for a potential immunotherapy.

Extra-cranial malignant rhabdoid tumors (MRTs) and cranial atypical teratoid RTs (ATRTs) are heterogeneous pediatric cancers driven primarily by SMARCB1 loss. To understand the genome-wide molecular relationships between MRTs and ATRTs, we analyze multi-omics data from 140 MRTs and 161 ATRTs. We detect similarities between the MYC subgroup of ATRTs (ATRT-MYC) and extra-cranial MRTs, including global DNA hypomethylation and overexpression of HOX genes and genes involved in mesenchymal development, distinguishing them from other ATRT subgroups that express neural-like features. We identify five DNA methylation subgroups associated with anatomical sites and SMARCB1 mutation patterns. Groups 1, 3, and 4 exhibit cytotoxic T cell infiltration and expression of immune checkpoint regulators, consistent with a potential role for immunotherapy in rhabdoid tumor patients., In Brief Chun et al. report similarities between the MYC subgroup of cranial and extracranial rhabdoid tumors (RTs) at genetic, gene-expression, and epigenetic levels. They identify five DNA methylation subgroups of RTs across multiple organ sites, and some subgroups exhibit increased levels of immune cell infiltration and immune checkpoint expression., Graphical Abstract

Kulic et al. show that RBPJ, a transcriptional repressor of Notch, is frequently deleted in human cancers and can function as a tumor suppressor. Loss of RBPJ acts to derepress target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumor growth., Aberrant Notch activity is oncogenic in several malignancies, but it is unclear how expression or function of downstream elements in the Notch pathway affects tumor growth. Transcriptional regulation by Notch is dependent on interaction with the DNA-binding transcriptional repressor, RBPJ, and consequent derepression or activation of associated gene promoters. We show here that RBPJ is frequently depleted in human tumors. Depletion of RBPJ in human cancer cell lines xenografted into immunodeficient mice resulted in activation of canonical Notch target genes, and accelerated tumor growth secondary to reduced cell death. Global analysis of activated regions of the genome, as defined by differential acetylation of histone H4 (H4ac), revealed that the cell death pathway was significantly dysregulated in RBPJ-depleted tumors. Analysis of transcription factor binding data identified several transcriptional activators that bind promoters with differential H4ac in RBPJ-depleted cells. Functional studies demonstrated that NF-κB and MYC were essential for survival of RBPJ-depleted cells. Thus, loss of RBPJ derepresses target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumorigenesis.

Background Intellectual Disability (ID) is among the most common global disorders, yet etiology is unknown in ~30% of patients despite clinical assessment. Whole genome sequencing (WGS) is able to interrogate the entire genome, providing potential to diagnose idiopathic patients. Methods We conducted WGS on eight children with idiopathic ID and brain structural defects, and their normal parents; carrying out an extensive data analyses, using standard and discovery approaches. Results We verified de novo pathogenic single nucleotide variants (SNV) in ARID1B c.1595delG and PHF6 c.820C > T, potentially causative de novo two base indels in SQSTM1 c.115_116delinsTA and UPF1 c.1576_1577delinsA, and de novo SNVs in CACNB3 c.1289G > A, and SPRY4 c.508 T > A, of uncertain significance. We report results from a large secondary control study of 2081 exomes probing the pathogenicity of the above genes. We analyzed structural variation by four different algorithms including de novo genome assembly. We confirmed a likely contributory 165 kb de novo heterozygous 1q43 microdeletion missed by clinical microarray. The de novo assembly resulted in unmasking hidden genome instability that was missed by standard re-alignment based algorithms. We also interrogated regulatory sequence variation for known and hypothesized ID genes and present useful strategies for WGS data analyses for non-coding variation. Conclusion This study provides an extensive analysis of WGS in the context of ID, providing genetic and structural insights into ID and yielding diagnoses. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3671-0) contains supplementary material, which is available to authorized users.

Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (for example, VHL) and the maintenance of chromatin states (for example, PBRM1). We surveyed more than 400 tumours using different genomic platforms and identified 19 significantly mutated genes. The PI(3)K/AKT pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodelling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving downregulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, upregulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 (also known as MIR21) and GRB10. Remodelling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumour stage and severity and offers new views on the opportunities for disease treatment.

We performed an integrated genomic, transcriptomic and proteomic characterization of 373 endometrial carcinomas using array- and sequencing-based technologies. Uterine serous tumours and ~25% of high-grade endometrioid tumours had extensive copy number alterations, few DNA methylation changes, low oestrogen receptor/progesterone receptor levels, and frequent TP53 mutations. Most endometrioid tumours had few copy number alterations or TP53 mutations, but frequent mutations in PTEN, CTNNB1, PIK3CA, ARID1A and KRAS and novel mutations in the SWI/SNF chromatin remodelling complex gene ARID5B. A subset of endometrioid tumours that we identified had a markedly increased transversion mutation frequency and newly identified hotspot mutations in POLE. Our results classified endometrial cancers into four categories: POLE ultramutated, microsatellite instability hypermutated, copy-number low, and copy-number high. Uterine serous carcinomas share genomic features with ovarian serous and basal-like breast carcinomas. We demonstrated that the genomic features of endometrial carcinomas permit a reclassification that may affect post-surgical adjuvant treatment for women with aggressive tumours., National Institutes of Health (U.S.) (Grant 5U24CA143799-04), National Institutes of Health (U.S.) (Grant 5U24CA143835-04), National Institutes of Health (U.S.) (Grant 5U24CA143840-04), National Institutes of Health (U.S.) (Grant 5U24CA143843-04), National Institutes of Health (U.S.) (Grant 5U24CA143845-04), National Institutes of Health (U.S.) (Grant 5U24CA143848-04), National Institutes of Health (U.S.) (Grant 5U24CA143858-04), National Institutes of Health (U.S.) (Grant 5U24CA143866-04), National Institutes of Health (U.S.) (Grant 5U24CA143867-04), National Institutes of Health (U.S.) (Grant 5U24CA143882-04), National Institutes of Health (U.S.) (Grant 5U24CA143883-04), National Institutes of Health (U.S.) (Grant 5U24CA144025-04), National Institutes of Health (U.S.) (Grant U54HG003067-11), National Institutes of Health (U.S.) (Grant U54HG003079-10), National Institutes of Health (U.S.) (Grant U54HG003273-10)

SummaryLeukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the alternative splicing of self-renewal and survival genes. To elucidate splice-isoform regulators of human BC LSC maintenance, we performed whole-transcriptome RNA sequencing, splice-isoform-specific quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice-isoform expression in BC LSC maintenance and suggest that the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance.

Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1(E912A) mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28B upregulation. A small-molecule tool compound antagonizes ADAR1's effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling.

Background Dormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication. Methods To test these postulates, whole transcriptome RNA sequencing (RNA-seq), microarray, qRT-PCR, stromal co-culture, confocal fluorescence microscopic, nanoproteomic, serial transplantation and cell cycle analyses were performed on FACS purified normal, chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) phase CML progenitors with or without PF-04449913 treatment. Results Notably, RNA-seq analyses revealed that Hh pathway and cell cycle regulatory gene overexpression correlated with leukemic progression. While lentivirally enforced GLI2 expression enhanced leukemic progenitor dormancy in stromal co-cultures, this was not observed with a mutant GLI2 lacking a transactivation domain, suggesting that GLI2 expression prevented cell cycle transit. Selective SMO inhibition with PF-04449913 in humanized stromal co-cultures and LSC xenografts reduced downstream GLI2 protein and cell cycle regulatory gene expression. Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC. Conclusion In summary, while GLI2, forms part of a core HH pathway transcriptional regulatory network that promotes human myeloid leukemic progression and dormant LSC generation, selective inhibition with PF-04449913 reduces the dormant LSC burden thereby providing a strong rationale for clinical trials predicated on SMO inhibition in combination with TKIs or chemotherapeutic agents with the ultimate aim of obviating leukemic therapeutic resistance, persistence and progression. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0453-9) contains supplementary material, which is available to authorized users.

Cancer results from accumulated mutations in the genome. Sequencing is an accurate method to detect mutations. Second-generation sequencing technology, commonly referred to as next-generation sequencing technology, enables rapid, efficient and affordable DNA sequencing, and is transforming the scale and scope of cancer research. The technology is sufficiently flexible and affordable to allow sequencing of many cancer genomes, and thus facilitates both sequencing of samples from large patient cohorts and during disease progression in individual cancer patients. The high depths of redundant sequence coverage that can be obtained using some second-generation sequencing technologies, along with sequencing reads amplified from single DNA molecules, facilitate detection of subclones of cells in tumors. Large-scale genome sequencing of hundreds or even thousands of cancer samples is being conducted by several groups that aim to identify and characterize cancer driver mutations. Goals of such work, previously infeasible with Sanger sequencing instruments, are to use this information to improve cancer prognosis, diagnosis and therapeutic decision-making. The speed of data analysis is rate limiting, and investigators are struggling to accommodate and interpret the data deluge produced by second-generation technologies. In this chapter, we discuss cancer properties that are revealed by sequencing and the implication of such properties in experimental design and data interpretation. We describe past, current and upcoming sequencing technologies and the application of second-generation sequencing technologies in cancer genomics. Finally, we discuss the impact of second-generation sequencing technology in shaping personalized medicine.

The Cancer Genome Atlas (TCGA) Research Network has profiled and analyzed large numbers of human tumors to discover molecular aberrations at the DNA, RNA, protein and epigenetic levels. The resulting rich data provide a major opportunity to develop an integrated picture of commonalities, differences and emergent themes across tumor lineages. The Pan-Cancer initiative compares the first 12 tumor types profiled by TCGA. Analysis of the molecular aberrations and their functional roles across tumor types will teach us how to extend therapies effective in one cancer type to others with a similar genomic profile., National Cancer Institute (U.S.), National Human Genome Research Institute (U.S.)

Background Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. However, its role in malignant reprogramming of progenitors into self-renewing leukemia stem cells (LSCs) had not been established. Methods Whole transcriptome RNA sequencing (RNA-seq) was performed on FACS purified progenitors from normal, chronic phase and blast crisis chronic myeloid leukemia samples and analyzed using Cuff-links, GSEA and IPA software. Splice isoform specific qRT-PCR, confocal microscopy, lentiviral overexpression and shRNA knockdown experiments were performed according to published methods (Jamieson NEJM 2004; Geron et al Cancer Cell 2008; Goff et al Cell Stem Cell 2013). Results We performed LSC RNA-seq, lentiviral overexpression and knockdown and discovered that decreased expression of MBNL3, a repressor of an embryonic alternative splicing program and reprogramming, activated a pluripotency network and increased expression of a pro-survival isoform of CD44v3, which is more commonly expressed in human embryonic stem cells. This resulted in malignant reprogramming of progenitors in blast crisis CML endowing them with unbridled survival and self-renewal capacity. This is the first description of MBNL3 downregulation as a mechanism of reversion to an embryonic alternative splicing program, which elicits malignant progenitor reprogramming of progenitors into self-renewing leukemia stem cells. While isoform specific lentiviral CD44v3 overexpression enhanced chronic phase CML progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. In keeping with activation of a stem cell reprogramming network, CD44v3 upregulation was associated with increased expression of pluripotency transcription factors, including OCT4, SOX2 and b-catenin in addition to the pro-survival long isoforms of MCL1 and BCLX resulting in increased self-renewal and apoptosis resistance. Conclusion In summary, MBNL3 downregulation activates an embryonic alternative splicing program, typified by CD44v3 overexpression, and represents a novel mechanism governing LSC generation in malignant microenvironments. Reversal of malignant reprogramming by epigenetic modulation of embryonic alternative splicing or via monoclonal antibody targeting of CD44v3 splice isoform may represent a pivotal opportunity for selective BC LSC eradication. Disclosures Jamieson: Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.

To characterize somatic alterations in colorectal carcinoma, we conducted a genome-scale analysis of 276 samples, analysing exome sequence, DNA copy number, promoter methylation and messenger RNA and microRNA expression. A subset of these samples (97) underwent low-depth-of-coverage whole-genome sequencing. In total, 16% of colorectal carcinomas were found to be hypermutated: three-quarters of these had the expected high microsatellite instability, usually with hypermethylation and MLH1 silencing, and one-quarter had somatic mismatch-repair gene and polymerase ε (POLE) mutations. Excluding the hypermutated cancers, colon and rectum cancers were found to have considerably similar patterns of genomic alteration. Twenty-four genes were significantly mutated, and in addition to the expected APC, TP53, SMAD4, PIK3CA and KRAS mutations, we found frequent mutations in ARID1A, SOX9 and FAM123B. Recurrent copy-number alterations include potentially drug-targetable amplifications of ERBB2 and newly discovered amplification of IGF2. Recurrent chromosomal translocations include the fusion of NAV2 and WNT pathway member TCF7L1. Integrative analyses suggest new markers for aggressive colorectal carcinoma and an important role for MYC-directed transcriptional activation and repression., National Institutes of Health (U.S.) (Grant U24CA143799), National Institutes of Health (U.S.) (Grant U24CA143835), National Institutes of Health (U.S.) (Grant U24CA143840), National Institutes of Health (U.S.) (Grant U24CA143843), National Institutes of Health (U.S.) (Grant U24CA143845), National Institutes of Health (U.S.) (Grant U24CA143848), National Institutes of Health (U.S.) (Grant U24CA143858), National Institutes of Health (U.S.) (Grant U24CA143866), National Institutes of Health (U.S.) (Grant U24CA143867), National Institutes of Health (U.S.) (Grant U24CA143882), National Institutes of Health (U.S.) (Grant U24CA143883), National Institutes of Health (U.S.) (Grant U24CA144025), National Institutes of Health (U.S.) (Grant U54HG003067), National Institutes of Health (U.S.) (Grant U54HG003079), National Institutes of Health (U.S.) (Grant U54HG003273)

Background Members of the pine family (Pinaceae), especially species of spruce (Picea spp.) and pine (Pinus spp.), dominate many of the world's temperate and boreal forests. These conifer forests are of critical importance for global ecosystem stability and biodiversity. They also provide the majority of the world's wood and fiber supply and serve as a renewable resource for other industrial biomaterials. In contrast to angiosperms, functional and comparative genomics research on conifers, or other gymnosperms, is limited by the lack of a relevant reference genome sequence. Sequence-finished full-length (FL)cDNAs and large collections of expressed sequence tags (ESTs) are essential for gene discovery, functional genomics, and for future efforts of conifer genome annotation. Results As part of a conifer genomics program to characterize defense against insects and adaptation to local environments, and to discover genes for the production of biomaterials, we developed 20 standard, normalized or full-length enriched cDNA libraries from Sitka spruce (P. sitchensis), white spruce (P. glauca), and interior spruce (P. glauca-engelmannii complex). We sequenced and analyzed 206,875 3'- or 5'-end ESTs from these libraries, and developed a resource of 6,464 high-quality sequence-finished FLcDNAs from Sitka spruce. Clustering and assembly of 147,146 3'-end ESTs resulted in 19,941 contigs and 26,804 singletons, representing 46,745 putative unique transcripts (PUTs). The 6,464 FLcDNAs were all obtained from a single Sitka spruce genotype and represent 5,718 PUTs. Conclusion This paper provides detailed annotation and quality assessment of a large EST and FLcDNA resource for spruce. The 6,464 Sitka spruce FLcDNAs represent the third largest sequence-verified FLcDNA resource for any plant species, behind only rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), and the only substantial FLcDNA resource for a gymnosperm. Our emphasis on capturing FLcDNAs and ESTs from cDNA libraries representing herbivore-, wound- or elicitor-treated induced spruce tissues, along with incorporating normalization to capture rare transcripts, resulted in a rich resource for functional genomics and proteomics studies. Sequence comparisons against five plant genomes and the non-redundant GenBank protein database revealed that a substantial number of spruce transcripts have no obvious similarity to known angiosperm gene sequences. Opportunities for future applications of the sequence and clone resources for comparative and functional genomics are discussed.

Background The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. Results As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL)-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa × P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones for genes that were differentially expressed in poplar leaves attacked by forest tent caterpillars. Conclusion This study has generated a high-quality FLcDNA resource for poplar and the third largest FLcDNA collection published to date for any plant species. We successfully used the FLcDNA sequences to reassess gene prediction in the poplar genome sequence, perform comparative sequence annotation, and identify differentially expressed transcripts associated with defense against insects. The FLcDNA sequences will be essential to the ongoing curation and annotation of the poplar genome, in particular for targeting gaps in the current genome assembly and further improvement of gene predictions. The physical FLcDNA clones will serve as useful reagents for functional genomics research in areas such as analysis of gene functions in defense against insects and perennial growth. Sequences from this study have been deposited in NCBI GenBank under the accession numbers EF144175 to EF148838.

Genome size, a fundamental aspect of any organism, is subject to a variety of mutational and selection pressures. We investigated genome size evolution in haploid, diploid, and tetraploid initially isogenic lines of the yeast Saccharomyces cerevisiae. Over the course of ~1,800 generations of mitotic division, we observed convergence toward diploid DNA content in all replicate lines. This convergence was observed in both unstressful and stressful environments, although the rate of convergence was dependent on initial ploidy and evolutionary environment. Comparative genomic hybridization with microarrays revealed nearly euploid DNA content by the end of the experiment. As the vegetative life cycle of S. cerevisiae is predominantly diploid, this experiment provides evidence that genome size evolution is constrained, with selection favouring the genomic content typical of the yeast's evolutionary past., Synopsis Genome size is a fundamental aspect of all species and has the potential to influence a number of individual characteristics such as cell size, generation time, ecological tolerances, and reproductive traits. Although genome sizes range widely among species, the forces shaping the evolution of genome size are only poorly known. Here we provide the results of an ~1,800 generation evolution experiment using lines of the budding yeast S. cerevisiae with either one, two, or four copies of their genome (haploid, diploid and tetraploid, respectively). We found, surprisingly, that all haploid and tetraploid lines converged toward diploidy, the historical state of S. cerevisiae, by the end of the experiment. Further experiments suggest that entire sets of chromosomes were lost as genome size changed from tetraploid to diploid. Our results suggest that genome size is constrained by selection acting against changes from the historical genome size.

Introduction Malignant reprogramming, first described in chronic myeloid leukemia (CML), occurs upon activation of the Wnt/b-catenin pathway in granulocyte-macrophage progenitors (GMPs) that gain the capacity to self-renew and contribute to the emergence of BCR-ABL1 tyrosine kinase inhibitor (TKI) resistant blast crisis CML. Deregulation of the Wnt/b-catenin target gene, CD44, plays a vital role in leukemia stem cell (LSC) maintenance in the malignant microenvironment in mouse models of CML. However, extensive alternative mRNA splicing in humans results in expression of multiple CD44 isoforms, some of which have been implicated in cancer invasion and metastasis. In this study we investigated the role of CD44 splice variant expression on human blast crisis LSC maintenance in the malignant niche. Methods and Results CD44 Isoform Expression Analysis To investigate the splice isoform expression pattern of CD44, whole transcriptome RNA sequencing (RNA Seq; Illumina HiSeq 2000) was performed on FACS sorted chronic phase (CP; n=8) and blast crisis (BC; n=8) CML progenitors (CD34+CD38+Lin-) as well as their normal counterparts from cord blood (CB) (n=7) and adult peripheral blood (NPB; n=4). While whole gene expression analysis revealed upregulation of CD44 in blast crisis compared with chronic phase and normal progenitors, a plethora of CD44 transcript variants were also detected including variants 3, 4 (CD44s), 5, 6, 7, 8. Notably, RNA Seq isoform analysis detected a higher expression of CD44 transcript variant 3 in BC compared to CP and CB and NPB. Moreover, CD44 transcript variant 3 gene expression was highly expressed in undifferentied human embryonic stem cells (hESCs) while differentiated hESCs (embryoid bodies) had low expression, suggesting CD44 transcript variant 3 to be important for pluripotency. Lentiviral CD44 Variant 3 Overexpression To directly determine the impact of CD44 variant 3 expression on malignant reprogramming of CP progenitors into self-renewing LSC, we developed a lentiviral human CD44 variant 3 overexpression vector and transduced CP CML progenitors. Transduced CP progenitors harbored increased expression of migration specific markers, such as osteopontin and ICAM1, as well as an upregulation of the pro-survival long isoforms of BCL2 family members BCLX and MCL1, thereby enhancing survival and replating in hematopoietic progenitor assays. Moreover, hESCs transduced with CD44 transcript variant 3 showed upregulation of pro-survival BCL2 isoforms, enhanced proliferation and as well as maintenance of an undifferentiated state, suggesting that CD44 transcript variant 3 promotes pluripotency. Targeted Inhibition of CD44 variant 3 Expressing LSC Humanized RAG2-/-gc-/-mice engrafted with CD34+ BC CML patient samples showed a significant reduction of human progenitor cells post treatment with a clinical grade CD44 mAb, both alone and in combination with Dasatinib in all hematopoietic niches. Bone marrow and spleen samples from primary transplanted mice show a reduced gene expression level of CD44 and CD44 transcript variant 3 upon combination treatment of CD44 and Dasatinib. Most importantly, serial transplantation of progenitors treated with the CD44 mAb as well as in combination with Dasatinib revealed a significant reduction in LSC self-renewal capacity commensurate with a reduction in CD44 variant 3 expression. Conclusions Upregulation of an embryonic splice variant of CD44, variant 3, expands pluripotent stem cell populations and promotes malignant reprogramming of CML progenitors into self-renewing LSC. Treatment with a humanized CD44 specific mAb sensitizes CML LSC residing in malignant niches to Dasatinib. From these results CD44 mAb appears to be an excellent antibody for future combination clinical studies aimed at eradicating therapy resistant blast crisis LSC in CML. In addition, these observations strongly suggest that CD44 transcript variant 3 upregulation serves as a biomarker of progression from CP to BC as well as the generation of TKI resistant LSCs, with the potential of being a more specific target for future combination therapies. Disclosures No relevant conflicts of interest to declare.

Rhabdoid tumors (RT) of the kidney (RTK) are aggressive pediatric solid tumors that predominantly affect infants. There is no effective chemotherapy and the overall 4-year survival rate is 23%. RT has a characteristic loss of SMARCB1 function, found in >90% of the patients. SMARCB1 is a conserved core subunit of the SWI/SNF chromatin-remodeling complex, which in turn is responsible for proper chromatin assembly and dynamic regulation of gene expression. Previous studies showed a remarkable paucity of mutations in coding regions of genomes and a highly penetrant cancer susceptibility in a conditional knockout mouse model. These findings support the interpretation that SMARCB1 is a tumor suppressor whose inactivation is the primary driver in RT and that RT follows a tumorigenesis model in which cancer is driven by aberrant epigenetic regulation and gene expression instead of accumulation of somatic mutations. Characterizing interplays of mutations, gene expression and epigenetic regulation will be important in understanding RT development and biology. To achieve this goal, we will comprehensively characterize genetic and epigenetic aberrations in RT using HiSeq sequencing technology. Our research efforts include profiling whole genome, whole transcriptome, promoter methylation and histone modification in 40 primary RTK samples. Here, we report preliminary results from whole genome analyses. Using an amplification-free library construction method, we sequenced whole genomes of 40 RTK and matched normal cases to an average haploid coverage of 39.4X. The RTK genomes were mostly diploid, but we found 35 loci that are either recurrently focally amplified or deleted using GISTIC 2.0 at FDR ≤0.05. Using the Trans-ABySS de novo short-read assembler, we assembled the RT cases’ whole genomes and identified a total of 19 genes that were recurrently rearranged in 8 out of 40 cases. Eleven of the genes were either known tumor suppressors (e.g. CABIN1, BCR) or associated with developmental or neurodegenerative diseases (e.g. UPB1, SPECC1L). The genome-wide single nucleotide variant and indel analysis showed an average somatic mutation rate at 0.37 per Mb in RTK, comparable to the previous finding of 0.19 per Mb in AT/RT. Approximately 99% of the somatic mutations occurred in non-genic regions. SMARCB1 had homozygous loss of function in 83% of cases by somatic homozygous deletion, or heterozygous deletion or truncating point mutations followed by loss of heterozygosity. The remaining cases appeared to have at least 1 copy of the gene unaffected and analyses are ongoing to investigate the inactivation mechanism in these cases. Citation Format: Hye-Jung E. Chun, Kelsey Zhu, Jenny Q. Qian, Karen L. Mungall, Yussanne Ma, Yong-Jun Zhao, Andrew J. Mungall, Richard A. Moore, Jacquie E. Schein, Daniela S. Gerhard, Elizabeth J. Perlman, Marco A. Marra. Whole genome sequencing of rhabdoid tumors of the kidney. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3087. doi:10.1158/1538-7445.AM2014-3087

The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. While DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity may also be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole transcriptome sequencing of normal, chronic phase (CP) and serially transplantable blast crisis (BC) chronic myeloid leukemia (CML) progenitors revealed increased interferon-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the interferon responsive ADAR1 p150 isoform and a propensity for increased A-to-I RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promoted expression of the myeloid transcription factor PU.1 and induced malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a mis-spliced form of GSK3β implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of BC CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies. Citation Format: Qingfei Jiang, Leslie A. Crews, Christian L. Barrett, Angela Court-Recart, Daniel Goff, Anil Sadarangani, Jessica Rusert, Sheldon Morris, Lawrence Goldstein, Hye-Jung Chun, Marco Marra, Kelly Fraser, Kim-Hien Dao, Mark Minden, Catriona Jamieson. ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 247. doi:10.1158/1538-7445.AM2013-247

Abstract 910 In blast crisis transformation of CML (BC CML), the leukemia stem cells (LSC), via the acquisition of both enhanced survival and self-renewal capacity, become increasingly resistant to BCR-ABL targeted tyrosine kinase inhibition and thus often contribute to relapse after treatment, pointing to the need for alternative therapeutic strategies and a better understanding of the molecular mechanisms underlying disease progression. Janus kinase 2 (JAK2) plays an important role in BCR–ABL + cell survival and has profound effects on self-renewal and lineage commitment of normal and leukemic hematopoietic stem cells, through the activation of the transcription factor signal transducer and activator of transcription 5 (STAT5). To determine if JAK/STAT signaling pathway activation is related to CML progression, LSC from human Chronic Phase (CP CML) and BC CML samples were sorted using FACS Aria (Lin-CD34+CD38+) and analyzed using splice-isoform specific q-RT-PCR. Our results showed that, compared to CP CML, BC LSC harbor enhanced mRNA expression of BCR-ABL, JAK2 and STAT5A isoforms, confirming that progression of CP to BC, in CML LSC, is marked by activation of JAK/STAT pathway. Therefore, we investigated the response of BC CML LSC to a clinical grade JAK2 inhibitor, SAR302503 (Sanofi, Cambridge, MA) alone or in combination with a potent BCR-ABL inhibitor, dasatinib, in vivo. After two weeks of treatment, RAG2−/−gc−/− mice intrahepatic transplanted with BC LSC, showed a significant (p To test whether the combination therapy can impair self-renewal capacity of the BC CML LSC in vivo, we immunomagnetic bead selected CD34+ cells from BM and spleens of treated mice, and serially transplanted an equal number into secondary recipients. We observed a significant (p Validation studies, using nanoproteomic analysis, confirmed that LSC sorted cells from mice treated with SAR302503 had lower expression levels of p-JAK2 (Tyr 1007-08) and p-STAT5A (Tyr 694) compared with vehicle treated mice (51% and 64% of reduction, respectively), while no changes are observed for total JAK2 protein or B2M between both conditions. Full transcriptome sequencing and q-RT-PCR analysis, on sorted CML LSC from mice treated with SAR302503 in combination with dasatinib, confirmed that STAT5A specific isoforms decresed after treatment, suggesting JAK/STAT pathway could be used as biomarker of response and could explain the impairment of self-renewal in the combination therapy. Disclosures: No relevant conflicts of interest to declare.

Summary Malignant rhabdoid tumors (MRTs) are rare lethal tumors of childhood that most commonly occur in the kidney and brain. MRTs are driven by SMARCB1 loss, but the molecular consequences of SMARCB1 loss in extra-cranial tumors have not been comprehensively described and genomic resources for analyses of extra-cranial MRT are limited. To provide such data, we used whole-genome sequencing, whole-genome bisulfite sequencing, whole transcriptome (RNA-seq) and microRNA sequencing (miRNA-seq), and histone modification profiling to characterize extra-cranial MRTs. Our analyses revealed gene expression and methylation subgroups and focused on dysregulated pathways, including those involved in neural crest development.

We analysed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, messenger RNA arrays, microRNA sequencing and reverse-phase protein arrays. Our ability to integrate information across platforms provided key insights into previously defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity. Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at >10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the luminal A subtype. We identified two novel protein-expression-defined subgroups, possibly produced by stromal/microenvironmental elements, and integrated analyses identified specific signalling pathways dominant in each molecular subtype including a HER2/phosphorylated HER2/EGFR/phosphorylated EGFR signature within the HER2-enriched expression subtype. Comparison of basal-like breast tumours with high-grade serous ovarian tumours showed many molecular commonalities, indicating a related aetiology and similar therapeutic opportunities. The biological finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biological subtypes of breast cancer.