In vivo interrogation of regulatory genomes reveals extensive quasi-insufficiency in cancer evolution.

In contrast to mono- or biallelic loss of tumor-suppressor function, effects of discrete gene dysregulations, as caused by non-coding (epi)genome alterations, are poorly understood. Here, by perturbing the regulatory genome in mice, we uncover pervasive roles of subtle gene expression variation in cancer evolution. Genome-wide screens characterizing 1,450 tumors revealed that such quasi-insufficiency is extensive across entities and displays diverse context dependencies, such as distinct cell-of-origin associations in T-ALL subtypes. We compile catalogs of non-coding regions linked to quasi-insufficiency, show their enrichment with human cancer risk variants, and provide functional insights by engineering regulatory alterations in mice. As such, kilo-/megabase deletions in a Bcl11b-linked non-coding region triggered aggressive malignancies, with allele-specific tumor spectra reflecting gradual gene dysregulations through modular and cell-type-specific enhancer activities. Our study constitutes a first survey toward a systems-level understanding of quasi-insufficiency in cancer and gives multifaceted insights into tumor evolution and the tissue-specific effects of non-coding mutations.

The EurOPDX Data Portal: an open platform for patient-derived cancer xenograft data sharing and visualization.

BACKGROUND: Patient-derived xenografts (PDX) mice models play an important role in preclinical trials and personalized medicine. Sharing data on the models is highly valuable for numerous reasons - ethical, economical, research cross validation etc. The EurOPDX Consortium was established 8 years ago to share such information and avoid duplicating efforts in developing new PDX mice models and unify approaches to support preclinical research. EurOPDX Data Portal is the unified data sharing platform adopted by the Consortium. MAIN BODY: In this paper we describe the main features of the EurOPDX Data Portal ( https://dataportal.europdx.eu/ ), its architecture and possible utilization by researchers who look for PDX mice models for their research. The Portal offers a catalogue of European models accessible on a cooperative basis. The models are searchable by metadata, and a detailed view provides molecular profiles (gene expression, mutation, copy number alteration) and treatment studies. The Portal displays the data in multiple tools (PDX Finder, cBioPortal, and GenomeCruzer in future), which are populated from a common database displaying strictly mutually consistent views. (SHORT) CONCLUSION: EurOPDX Data Portal is an entry point to the EurOPDX Research Infrastructure offering PDX mice models for collaborative research, (meta)data describing their features and deep molecular data analysis according to users' interests.

PDX Finder: A portal for patient-derived tumor xenograft model discovery.

Patient-derived tumor xenograft (PDX) mouse models are a versatile oncology research platform for studying tumor biology and for testing chemotherapeutic approaches tailored to genomic characteristics of individual patients' tumors. PDX models are generated and distributed by a diverse group of academic labs, multi-institution consortia and contract research organizations. The distributed nature of PDX repositories and the use of different metadata standards for describing model characteristics presents a significant challenge to identifying PDX models relevant to specific cancer research questions. The Jackson Laboratory and EMBL-EBI are addressing these challenges by co-developing PDX Finder, a comprehensive open global catalog of PDX models and their associated datasets. Within PDX Finder, model attributes are harmonized and integrated using a previously developed community minimal information standard to support consistent searching across the originating resources. Links to repositories are provided from the PDX Finder search results to facilitate model acquisition and/or collaboration. The PDX Finder resource currently contains information for 1985 PDX models of diverse cancers including those from large resources such as the Patient-Derived Models Repository, PDXNet and EurOPDX. Individuals or organizations that generate and distribute PDXs are invited to increase the 'findability' of their models by participating in the PDX Finder initiative at www.pdxfinder.org.

PDX-MI: Minimal Information for Patient-Derived Tumor Xenograft Models.

Patient-derived tumor xenograft (PDX) mouse models have emerged as an important oncology research platform to study tumor evolution, mechanisms of drug response and resistance, and tailoring chemotherapeutic approaches for individual patients. The lack of robust standards for reporting on PDX models has hampered the ability of researchers to find relevant PDX models and associated data. Here we present the PDX models minimal information standard (PDX-MI) for reporting on the generation, quality assurance, and use of PDX models. PDX-MI defines the minimal information for describing the clinical attributes of a patient's tumor, the processes of implantation and passaging of tumors in a host mouse strain, quality assurance methods, and the use of PDX models in cancer research. Adherence to PDX-MI standards will facilitate accurate search results for oncology models and their associated data across distributed repository databases and promote reproducibility in research studies using these models. Cancer Res; 77(21); e62-66. ©2017 AACR.

Molecular synergy underlies the co-occurrence patterns and phenotype of NPM1-mutant acute myeloid leukemia.

NPM1 mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with FLT3 internal tandem duplications (ITD) or, less commonly, NRAS or KRAS mutations. Co-occurrence of mutant NPM1 with FLT3-ITD carries a significantly worse prognosis than NPM1-RAS combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis show that compound Npm1cA/+;NrasG12D/+ or Npm1cA;Flt3ITD share a number of features: Hox gene overexpression, enhanced self-renewal, expansion of hematopoietic progenitors, and myeloid differentiation bias. However, Npm1cA;Flt3ITD mutants displayed significantly higher peripheral leukocyte counts, early depletion of common lymphoid progenitors, and a monocytic bias in comparison with the granulocytic bias in Npm1cA/+;NrasG12D/+ mutants. Underlying this was a striking molecular synergy manifested as a dramatically altered gene expression profile in Npm1cA;Flt3ITD , but not Npm1cA/+;NrasG12D/+ , progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with Npm1cA/+;NrasG12D/+ During AML evolution, both models acquired additional copies of the mutant Flt3 or Nras alleles, but only Npm1cA/+;NrasG12D/+ mice showed acquisition of other human AML mutations, including IDH1 R132Q. We also find, using primary Cas9-expressing AMLs, that Hoxa genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with NPM1c/FLT3-ITD vs NPM1/NRAS-G12D-mutant AML and functionally confirm the role of HOXA genes in NPM1c-driven AML.

Identifiers for the 21st century: How to design, provision, and reuse persistent identifiers to maximize utility and impact of life science data.

In many disciplines, data are highly decentralized across thousands of online databases (repositories, registries, and knowledgebases). Wringing value from such databases depends on the discipline of data science and on the humble bricks and mortar that make integration possible; identifiers are a core component of this integration infrastructure. Drawing on our experience and on work by other groups, we outline 10 lessons we have learned about the identifier qualities and best practices that facilitate large-scale data integration. Specifically, we propose actions that identifier practitioners (database providers) should take in the design, provision and reuse of identifiers. We also outline the important considerations for those referencing identifiers in various circumstances, including by authors and data generators. While the importance and relevance of each lesson will vary by context, there is a need for increased awareness about how to avoid and manage common identifier problems, especially those related to persistence and web-accessibility/resolvability. We focus strongly on web-based identifiers in the life sciences; however, the principles are broadly relevant to other disciplines.

Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium.

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.

Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells.

The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50-70 de novo single nucleotide variants (SNVs) between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs), their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer.

A mouse informatics platform for phenotypic and translational discovery.

The International Mouse Phenotyping Consortium (IMPC) is providing the world's first functional catalogue of a mammalian genome by characterising a knockout mouse strain for every gene. A robust and highly structured informatics platform has been developed to systematically collate, analyse and disseminate the data produced by the IMPC. As the first phase of the project, in which 5000 new knockout strains are being broadly phenotyped, nears completion, the informatics platform is extending and adapting to support the increasing volume and complexity of the data produced as well as addressing a large volume of users and emerging user groups. An intuitive interface helps researchers explore IMPC data by giving overviews and the ability to find and visualise data that support a phenotype assertion. Dedicated disease pages allow researchers to find new mouse models of human diseases, and novel viewers provide high-resolution images of embryonic and adult dysmorphologies. With each monthly release, the informatics platform will continue to evolve to support the increased data volume and to maintain its position as the primary route of access to IMPC data and as an invaluable resource for clinical and non-clinical researchers.

Chromosome instability induced by Mps1 and p53 mutation generates aggressive lymphomas exhibiting aneuploidy-induced stress.

Aneuploidy is a hallmark of human solid cancers that arises from errors in mitosis and results in gain and loss of oncogenes and tumor suppressors. Aneuploidy poses a growth disadvantage for cells grown in vitro, suggesting that cancer cells adapt to this burden. To understand better the consequences of aneuploidy in a rapidly proliferating adult tissue, we engineered a mouse in which chromosome instability was selectively induced in T cells. A flanked by Lox mutation was introduced into the monopolar spindle 1 (Mps1) spindle-assembly checkpoint gene so that Cre-mediated recombination would create a truncated protein (Mps1(DK)) that retained the kinase domain but lacked the kinetochore-binding domain and thereby weakened the checkpoint. In a sensitized p53(+/-) background we observed that Mps1(DK/DK) mice suffered from rapid-onset acute lymphoblastic lymphoma. The tumors were highly aneuploid and exhibited a metabolic burden similar to that previously characterized in aneuploid yeast and cultured cells. The tumors nonetheless grew rapidly and were lethal within 3-4 mo after birth.

Histone deacetylase 1 and 2 are essential for normal T-cell development and genomic stability in mice.

Histone deacetylase 1 and 2 (HDAC1/2) regulate chromatin structure as the catalytic core of the Sin3A, NuRD and CoREST co-repressor complexes. To better understand the key pathways regulated by HDAC1/2 in the adaptive immune system and inform their exploitation as drug targets, we have generated mice with a T-cell specific deletion. Loss of either HDAC1 or HDAC2 alone has little effect, while dual inactivation results in a 5-fold reduction in thymocyte cellularity, accompanied by developmental arrest at the double-negative to double-positive transition. Transcriptome analysis revealed 892 misregulated genes in Hdac1/2 knock-out thymocytes, including down-regulation of LAT, Themis and Itk, key components of the T-cell receptor (TCR) signaling pathway. Down-regulation of these genes suggests a model in which HDAC1/2 deficiency results in defective propagation of TCR signaling, thus blocking development. Furthermore, mice with reduced HDAC1/2 activity (Hdac1 deleted and a single Hdac2 allele) develop a lethal pathology by 3-months of age, caused by neoplastic transformation of immature T cells in the thymus. Tumor cells become aneuploid, express increased levels of c-Myc and show elevated levels of the DNA damage marker, γH2AX. These data demonstrate a crucial role for HDAC1/2 in T-cell development and the maintenance of genomic stability.

The piggyBac transposon displays local and distant reintegration preferences and can cause mutations at noncanonical integration sites.

The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome.

PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice.

Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability.

Analysis of Pax6 contiguous gene deletions in the mouse, Mus musculus, identifies regions distinct from Pax6 responsible for extreme small-eye and belly-spotting phenotypes.

In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca(+2)-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.

Extensive genomic copy number variation in embryonic stem cells.

Recent analysis of the human and mouse genomes has revealed that highly identical duplicated elements account for >5% of the sequence content. These elements vary in copy number between individuals. Copy number variations (CNVs) contribute significantly to genetic differences among individuals and are increasingly recognized as a causal factor in human diseases with different etiologies. In inbred mouse strains, CNVs have been fixed by inbreeding, but they are highly variable among strains. Within strains, de novo germ-line CNVs can occur, leading to interindividual variation. By analyzing the genome of clonal isolates of mouse ES cells derived from common parental lines, we have uncovered extensive and recurrent CNVs. This variation arises during mitosis and can be cotransmitted into the mouse germ line along with engineered alleles, contributing to genetic variability. The frequency and extent of these genomic changes in ES cells suggests that all somatic tissues in individuals will be mosaics composed of variants of the zygotic genome. Human ES (hES) cells and derived somatic lineages may be similarly affected, challenging the concept of a stable somatic genome.

Segmental trisomy of chromosome 17: a mouse model of human aneuploidy syndromes.

Triplication of whole autosomes or large autosomal segments is detrimental to the development of a mammalian embryo. The trisomy of human chromosome (Chr) 21, known as Down's syndrome, is regularly associated with mental retardation and a variable set of other developmental anomalies. Several mouse models of Down's syndrome, triplicating 33-104 genes of Chr16, were designed in an attempt to analyze the contribution of specific orthologous genes to particular developmental features. However, a recent study challenged the concept of dosage-sensitive genes as a primary cause of an abnormal phenotype. To distinguish between the specific effects of dosage-sensitive genes and nonspecific effects of a large number of arbitrary genes, we revisited the mouse Ts43H/Ph segmental trisomy. It encompasses >310 known genes triplicated within the proximal 30 megabases (Mb) of Chr17. We refined the distal border of the trisomic segment to the interval bounded by bacterial artificial chromosomes RP23-277B13 (location 29.0 Mb) and Cbs gene (location 30.2 Mb). The Ts43H mice, viable on a mixed genetic background, exhibited spatial learning deficits analogous to those observed in Ts65Dn mice with unrelated trisomy. Quantitative analysis of the brain expression of 20 genes inside the trisomic interval and 12 genes lying outside on Chr17 revealed 1.2-fold average increase of mRNA steady-state levels of triplicated genes and 0.9-fold average down-regulation of genes beyond the border of trisomy. We propose that systemic comparisons of unrelated segmental trisomies, such as Ts65Dn and Ts43H, will elucidate the pathways leading from the triplicated sequences to the complex developmental traits.

Aurora B -TACC1 protein complex in cytokinesis.

Taxins are a family of centrosomal proteins important for the regulation of mitosis and microtubule dynamics. Cytokinesis, the last step of M phase, is essential for chromosomal integrity and cell division. It is highly regulated and involves a reorganization of microtubules and actin filaments. We show here that TACC1 localizes diffusely to the midzone spindle in anaphase and strongly to the midbody during cytokinesis, indicating a possible involvement of this protein in the exit of M phase. TACC1 also relocalizes to the nucleolus in interphase. We demonstrate that TACC1 and the mitotic kinase Aurora B belong to the same complex during cytokinesis. We further show that Aurora B knocked down by RNA-mediated interference prevents the formation of the midbody - and consequently affects TACC1 localization at this site - and leads to abnormal cell division and multinucleated cells.

TACC1-chTOG-Aurora A protein complex in breast cancer.

The three human TACC (transforming acidic coiled-coil) genes encode a family of proteins with poorly defined functions that are suspected to play a role in oncogenesis. A Xenopus TACC homolog called Maskin is involved in translational control, while Drosophila D-TACC interacts with the microtubule-associated protein MSPS (Mini SPindleS) to ensure proper dynamics of spindle pole microtubules during cell division. We have delineated here the interactions of TACC1 with four proteins, namely the microtubule-associated chTOG (colonic and hepatic tumor-overexpressed gene) protein (ortholog of Drosophila MSPS), the adaptor protein TRAP (tudor repeat associator with PCTAIRE2), the mitotic serine/threonine kinase Aurora A and the mRNA regulator LSM7 (Like-Sm protein 7). To measure the relevance of the TACC1-associated complex in human cancer we have examined the expression of the three TACC, chTOG and Aurora A in breast cancer using immunohistochemistry on tissue microarrays. We show that expressions of TACC1, TACC2, TACC3 and Aurora A are significantly correlated and downregulated in a subset of breast tumors. Using siRNAs, we further show that depletion of chTOG and, to a lesser extent of TACC1, perturbates cell division. We propose that TACC proteins, which we also named 'Taxins', control mRNA translation and cell division in conjunction with microtubule organization and in association with chTOG and Aurora A, and that these complexes and cell processes may be affected during mammary gland oncogenesis.

Loss of heterozygosity at microsatellite markers from region p11-21 of chromosome 8 in microdissected breast tumor but not in peritumoral cells.

Alterations of chromosomal region 8p11-21 are very frequent in human cancers, and especially in breast cancer; yet, most of the genes involved have not been identified. We performed laser capture microdissection in a series of 52 consecutive breast tumor samples to obtain pure tumor cells without surrounding normal breast. To determine genomic subregions in which some of the cancer genes may be located, we conducted a search for loss of heterozygosity (LOH) at 13 microsatellite markers from this region. Two-thirds of the tumors showed LOH at least at one marker. Microdissection of pure tumor samples was helpful to precisely define four LOH subregions. No LOH was observed in the corresponding peritumoral tissues. We studied by immunohistochemistry (IHC) on tissue-microarrays the expression in the same tumors, of the protein product of three potential tumor genes lying close to or within the subregions of LOH. In most samples, the TACC1 gene product was downregulated in tumor cells as compared to normal cells. Our results show that the centromeric portion of chromosome arm 8p is frequently altered in breast tumor cells.

Distinct and complementary information provided by use of tissue and DNA microarrays in the study of breast tumor markers.

Emerging high-throughput screening technologies are rapidly providing opportunities to identify new diagnostic and prognostic markers and new therapeutic targets in human cancer. Currently, cDNA arrays allow the quantitative measurement of thousands of mRNA expression levels simultaneously. Validation of this tool in hospital settings can be done on large series of archival paraffin-embedded tumor samples using the new technique of tissue microarray. On a series of 55 clinically and pathologically homogeneous breast tumors, we compared for 15 molecules with a proven or suspected role in breast cancer, the mRNA expression levels measured by cDNA array analysis with protein expression levels obtained using tumor tissue microarrays. The validity of cDNA array and tissue microarray data were first verified by comparison with quantitative reverse transcriptase-polymerase chain reaction measurements and immunohistochemistry on full tissue sections, respectively. We found a good correlation between cDNA and tissue array analyses in one-third of the 15 molecules, and no correlation in the remaining two-thirds. Furthermore, protein but not RNA levels may have prognostic value; this was the case for MUC1 protein, which was studied further using a tissue microarray containing approximately 600 tumor samples. For THBS1 the opposite was observed because only RNA levels had prognostic value. Thus, differences extended to clinical prognostic information obtained by the two methods underlining their complementarity and the need for a global molecular analysis of tumors at both the RNA and protein levels.