Selective pressures of platinum compounds shape the evolution of therapy-related myeloid neoplasms.

Therapy-related myeloid neoplasms (t-MN) arise as a complication of chemo- and/or radiotherapy. Although t-MN can occur both in adult and childhood cancer survivors, the mechanisms driving therapy-related leukemogenesis likely vary across different ages. Chemotherapy is thought to induce driver mutations in children, whereas in adults pre-existing mutant clones are selected by the exposure. However, selective pressures induced by chemotherapy early in life are less well studied. Here, we use single-cell whole genome sequencing and phylogenetic inference to show that the founding cell of t-MN in children starts expanding after cessation of platinum exposure. In patients with Li-Fraumeni syndrome, characterized by a germline TP53 mutation, we find that the t-MN already expands during treatment, suggesting that platinum-induced growth inhibition is TP53-dependent. Our results demonstrate that germline aberrations can interact with treatment exposures in inducing t-MN, which is important for the development of more targeted, patient-specific treatment regimens and follow-up.

Improved detection of colibactin-induced mutations by genotoxic E. coli in organoids and colorectal cancer.

Co-culture of intestinal organoids with a colibactin-producing pks+E. coli strain (EcC) revealed mutational signatures also found in colorectal cancer (CRC). E. coli Nissle 1917 (EcN) remains a commonly used probiotic, despite harboring the pks operon and inducing double strand DNA breaks. We determine the mutagenicity of EcN and three CRC-derived pks+E. coli strains with an analytical framework based on sequence characteristic of colibactin-induced mutations. All strains, including EcN, display varying levels of mutagenic activity. Furthermore, a machine learning approach attributing individual mutations to colibactin reveals that patients with colibactin-induced mutations are diagnosed at a younger age and that colibactin can induce a specific APC mutation. These approaches allow the sensitive detection of colibactin-induced mutations in ∼12% of CRC genomes and even in whole exome sequencing data, representing a crucial step toward pinpointing the mutagenic activity of distinct pks+E. coli strains.

Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox.

Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole-genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole-genome sequencing (WGS) but still generate hundreds of artifacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolbox (PTATO), to accurately detect single base substitutions, insertions-deletions (indels), and structural variants in PTA-based WGS data. PTATO includes a machine learning approach and filtering based on recurrence to distinguish PTA artifacts from true mutations with high sensitivity (up to 90%), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that hematopoietic stem cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS, have normal somatic single base substitution burdens but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy.

Elevated Mutational Age in Blood of Children Treated for Cancer Contributes to Therapy-Related Myeloid Neoplasms.

Childhood cancer survivors are confronted with various chronic health conditions like therapy-related malignancies. However, it is unclear how exposure to chemotherapy contributes to the mutation burden and clonal composition of healthy tissues early in life. Here, we studied mutation accumulation in hematopoietic stem and progenitor cells (HSPC) before and after cancer treatment of 24 children. Of these children, 19 developed therapy-related myeloid neoplasms (t-MN). Posttreatment HSPCs had an average mutation burden increase comparable to what treatment-naïve cells accumulate during 16 years of life, with excesses up to 80 years. In most children, these additional mutations were induced by clock-like processes, which are also active during healthy aging. Other patients harbored mutations that could be directly attributed to treatments like platinum-based drugs and thiopurines. Using phylogenetic inference, we demonstrate that most t-MN in children originate after the start of treatment and that leukemic clones become dominant during or directly after chemotherapy exposure. SIGNIFICANCE: Our study shows that chemotherapy increases the mutation burden of normal blood cells in cancer survivors. Only few drugs damage the DNA directly, whereas in most patients, chemotherapy-induced mutations are caused by processes similar to those present during normal aging. This article is highlighted in the In This Issue feature, p. 1825.

MutationalPatterns: the one stop shop for the analysis of mutational processes.

BACKGROUND: The collective of somatic mutations in a genome represents a record of mutational processes that have been operative in a cell. These processes can be investigated by extracting relevant mutational patterns from sequencing data. RESULTS: Here, we present the next version of MutationalPatterns, an R/Bioconductor package, which allows in-depth mutational analysis of catalogues of single and double base substitutions as well as small insertions and deletions. Major features of the package include the possibility to perform regional mutation spectra analyses and the possibility to detect strand asymmetry phenomena, such as lesion segregation. On top of this, the package also contains functions to determine how likely it is that a signature can cause damaging mutations (i.e., mutations that affect protein function). This updated package supports stricter signature refitting on known signatures in order to prevent overfitting. Using simulated mutation matrices containing varied signature contributions, we showed that reliable refitting can be achieved even when only 50 mutations are present per signature. Additionally, we incorporated bootstrapped signature refitting to assess the robustness of the signature analyses. Finally, we applied the package on genome mutation data of cell lines in which we deleted specific DNA repair processes and on large cancer datasets, to show how the package can be used to generate novel biological insights. CONCLUSIONS: This novel version of MutationalPatterns allows for more comprehensive analyses and visualization of mutational patterns in order to study the underlying processes. Ultimately, in-depth mutational analyses may contribute to improved biological insights in mechanisms of mutation accumulation as well as aid cancer diagnostics. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns .

Human induced pluripotent stem cells display a similar mutation burden as embryonic pluripotent cells in vivo.

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, but genetic instability is a major concern. Embryonic pluripotent cells also accumulate mutations during early development, but how this relates to the mutation burden in iPSCs remains unknown. Here, we directly compared the mutation burden of cultured iPSCs with their isogenic embryonic cells during human embryogenesis. We generated developmental lineage trees of human fetuses by phylogenetic inference from somatic mutations in the genomes of multiple stem cells, which were derived from different germ layers. Using this approach, we characterized the mutations acquired pre-gastrulation and found a rate of 1.65 mutations per cell division. When cultured in hypoxic conditions, iPSCs generated from fetal stem cells of the assessed fetuses displayed a similar mutation rate and spectrum. Our results show that iPSCs maintain a genomic integrity during culture at a similar degree as their pluripotent counterparts do in vivo.

Mutation signatures of pediatric acute myeloid leukemia and normal blood progenitors associated with differential patient outcomes.

Acquisition of oncogenic mutations with age is believed to be rate limiting for carcinogenesis. However, the incidence of leukemia in children is higher than in young adults. Here we compare somatic mutations across pediatric acute myeloid leukemia (pAML) patient-matched leukemic blasts and hematopoietic stem and progenitor cells (HSPCs), as well as HSPCs from age-matched healthy donors. HSPCs in the leukemic bone marrow have limited genetic relatedness and share few somatic mutations with the cell-of-origin of the malignant blasts, suggesting polyclonal hematopoiesis in pAML patients. Compared to normal HSPCs, a subset of pAML cases harbored more somatic mutations and a distinct composition of mutational process signatures. We hypothesize these cases might have arisen from a more committed progenitor. This subset had better outcomes than pAML cases with mutation burden comparable to age-matched healthy HSPCs. Our study provides insights into the etiology and patient stratification of pAML.

Antiviral treatment causes a unique mutational signature in cancers of transplantation recipients.

Genetic instability is a major concern for successful application of stem cells in regenerative medicine. However, the mutational consequences of the most applied stem cell therapy in humans, hematopoietic stem cell transplantation (HSCT), remain unknown. Here we characterized the mutation burden of hematopoietic stem and progenitor cells (HSPCs) of human HSCT recipients and their donors using whole-genome sequencing. We demonstrate that the majority of transplanted HSPCs did not display altered mutation accumulation. However, in some HSCT recipients, we identified multiple HSPCs with an increased mutation burden after transplantation. This increase could be attributed to a unique mutational signature caused by the antiviral drug ganciclovir. Using a machine learning approach, we detected this signature in cancer genomes of individuals who received HSCT or solid organ transplantation earlier in life. Antiviral treatment with nucleoside analogs can cause enhanced mutagenicity in transplant recipients, which may ultimately contribute to therapy-related carcinogenesis.

Mutation accumulation and developmental lineages in normal and Down syndrome human fetal haematopoiesis.

Children show a higher incidence of leukemia compared to young adolescents, yet their cells have less age-related (oncogenic) somatic mutations. Newborns with Down syndrome have an even higher risk of developing leukemia, which is thought to be driven by mutations that accumulate during fetal development. To characterize mutation accumulation in individual stem and progenitor cells of Down syndrome and karyotypically normal fetuses, we clonally expanded single cells and performed whole-genome sequencing. We found a higher mutation rate in haematopoietic stem and progenitor cells during fetal development compared to the post-infant rate. In fetal trisomy 21 cells the number of somatic mutations is even further increased, which was already apparent during the first cell divisions of embryogenesis before gastrulation. The number and types of mutations in fetal trisomy 21 haematopoietic stem and progenitor cells were similar to those in Down syndrome-associated myeloid preleukemia and could be attributed to mutational processes that were active during normal fetal haematopoiesis. Finally, we found that the contribution of early embryonic cells to human fetal tissues can vary considerably between individuals. The increased mutation rates found in this study, may contribute to the increased risk of leukemia early during life and the higher incidence of leukemia in Down syndrome.

Somatic Mutations Reveal Lineage Relationships and Age-Related Mutagenesis in Human Hematopoiesis.

Mutation accumulation during life can contribute to hematopoietic dysfunction; however, the underlying dynamics are unknown. Somatic mutations in blood progenitors can provide insight into the rate and processes underlying this accumulation, as well as the developmental lineage tree and stem cell division numbers. Here, we catalog mutations in the genomes of human-bone-marrow-derived and umbilical-cord-blood-derived hematopoietic stem and progenitor cells (HSPCs). We find that mutations accumulate gradually during life with approximately 14 base substitutions per year. The majority of mutations were acquired after birth and could be explained by the constant activity of various endogenous mutagenic processes, which also explains the mutation load in acute myeloid leukemia (AML). Using these mutations, we construct a developmental lineage tree of human hematopoiesis, revealing a polyclonal architecture and providing evidence that developmental clones exhibit multipotency. Our approach highlights features of human native hematopoiesis and its implications for leukemogenesis.

Systemic miRNA-7 delivery inhibits tumor angiogenesis and growth in murine xenograft glioblastoma.

Tumor-angiogenesis is the multi-factorial process of sprouting of endothelial cells (EC) into micro-vessels to provide tumor cells with nutrients and oxygen. To explore miRNAs as therapeutic angiogenesis-inhibitors, we performed a functional screen to identify miRNAs that are able to decrease EC viability. We identified miRNA-7 (miR-7) as a potent negative regulator of angiogenesis. Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration. Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib. Local administration of miR-7 in an in vivo murine neuroblastoma tumor model significantly inhibited angiogenesis and tumor growth. Finally, systemic administration of miR-7 using a novel integrin-targeted biodegradable polymeric nanoparticles that targets both EC and tumor cells, strongly reduced angiogenesis and tumor proliferation in mice with human glioblastoma xenografts. Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7. Our study provides a comprehensive validation of miR-7 as novel anti-angiogenic therapeutic miRNA that can be systemically delivered to both EC and tumor cells and offers promise for miR-7 as novel anti-tumor therapeutic.

Correction: A Functional Screen Identifies Specific MicroRNAs Capable of Inhibiting Human Melanoma Cell Viability.

[This corrects the article DOI: 10.1371/journal.pone.0043569.].

A functional screen identifies specific microRNAs capable of inhibiting human melanoma cell viability.

Malignant melanoma is an aggressive form of skin cancer with poor prognosis. Despite improvements in awareness and prevention of this disease, its incidence is rapidly increasing. MicroRNAs (miRNAs) are a class of small RNA molecules that regulate cellular processes by repressing messenger RNAs (mRNAs) with partially complementary target sites. Several miRNAs have already been shown to attenuate cancer phenotypes, by limiting proliferation, invasiveness, tumor angiogenesis, and stemness. Here, we employed a genome-scale lentiviral human miRNA expression library to systematically survey which miRNAs are able to decrease A375 melanoma cell viability. We highlight the strongest inhibitors of melanoma cell proliferation, including the miR-15/16, miR-141/200a and miR-96/182 families of miRNAs and miR-203. Ectopic expression of these miRNAs resulted in long-term inhibition of melanoma cell expansion, both in vitro and in vivo. We show specifically miR-16, miR-497, miR-96 and miR-182 are efficient effectors when introduced as synthetic miRNAs in several melanoma cell lines. Our study provides a comprehensive interrogation of miRNAs that interfere with melanoma cell proliferation and viability, and offers a selection of miRNAs that are especially promising candidates for application in melanoma therapy.

Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals.

BACKGROUND: The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive. RESULTS: As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR) system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background. Indeed, Msh6 knockout rats were found to be more sensitive to ENU treatment and the germ line mutation rate was boosted more than two-fold to 1 mutation per 585 kb. In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by approximately 20%, resulting in an overall increase in efficiency of approximately 2.5 fold. The improved effectiveness was demonstrated by high throughput mutation discovery in 70 Mb of sequence in a set of only 310 mutant F1 rats. This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes. CONCLUSION: Taken together, we show that the use of a MMR-deficient background considerably improves ENU-driven target-selected mutagenesis in the rat, thereby reducing animal use as well as screening costs. The use of a mismatch repair-deficient genetic background for improving mutagenesis and target-selected knockout efficiency is in principle applicable to any organism of interest.

Adaptations in pre- and postsynaptic 5-HT1A receptor function and cocaine supersensitivity in serotonin transporter knockout rats.

RATIONALE: While individual differences in vulnerability to psychostimulants have been largely attributed to dopaminergic neurotransmission, the role of serotonin is not fully understood. OBJECTIVES: To study the rewarding and motivational properties of cocaine in the serotonin transporter knockout (SERT-/-) rat and the involvement of compensatory changes in 5-HT1A receptor function are the objectives of the study. MATERIALS AND METHODS: The SERT-/- rat was tested for cocaine-induced locomotor activity, cocaine-induced conditioned place preference, and intravenous cocaine self-administration. In addition, the function and expression of 5-HT1A receptors was assessed using telemetry and autoradiography, respectively, and the effect of 5-HT1A receptor ligands on cocaine's psychomotor effects were studied. RESULTS: Cocaine-induced hyperactivity and conditioned place preference, as well as intravenous cocaine self-administration were enhanced in SERT-/- rats. Furthermore, SERT-/- rats displayed a reduced hypothermic response to the 5-HT1A receptor agonist 8-OHDPAT. S-15535, a selective somatodendritic 5-HT1A receptor agonist, reduced stress-induced hyperthermia (SIH) in wild-type controls (SERT+/+), while it increased SIH in SERT-/- rats. As 5-HT1A receptor binding was reduced in selective brain regions, these thermal responses may be indicative for desensitized 5-HT1A receptors. We further found that both 8-OHDPAT and S-15535 pretreatment increased low-dose cocaine-induced locomotor activity in SERT-/- rats, but not SERT+/+ rats. At a high cocaine dose, only SERT+/+ animals responded to 8-OHDPAT and S-15535. CONCLUSION: These data indicate that SERT-/- -associated 5-HT1A receptor adaptations facilitate low-dose cocaine effects and attenuate high-dose cocaine effects in cocaine supersensitive animals. The role of postsynaptic and somatodendritic 5-HT1A receptors is discussed.

Distribution and functional impact of DNA copy number variation in the rat.

The abundance and dynamics of copy number variants (CNVs) in mammalian genomes poses new challenges in the identification of their impact on natural and disease phenotypes. We used computational and experimental methods to catalog CNVs in rat and found that they share important functional characteristics with those in human. In addition, 113 one-to-one orthologous genes overlap CNVs in both human and rat, 80 of which are implicated in human disease. CNVs are nonrandomly distributed throughout the genome. Chromosome 18 is a cold spot for CNVs as well as evolutionary rearrangements and segmental duplications, suggesting stringent selective mechanisms underlying CNV genesis or maintenance. By exploiting gene expression data available for rat recombinant inbred lines, we established the functional relationship of CNVs underlying 22 expression quantitative trait loci. These characteristics make the rat an excellent model for studying phenotypic effects of structural variation in relation to human complex traits and disease.

Lack of DNA mismatch repair protein MSH6 in the rat results in hereditary non-polyposis colorectal cancer-like tumorigenesis.

To understand genetic instability in relation to tumorigenesis, experimental animal models have proven very useful. The DNA mismatch repair (MMR) machinery safeguards genomic integrity by repairing mismatches, insertion or deletion loops and responding to genotoxic agents. Here, we describe the functional characterization of a novel rat mutant model in which the MMR gene Msh6 has been genetically inactivated by N-ethyl-N-nitrosourea-driven target-selected mutagenesis. This model shows a robust mutator phenotype that is reflected by microsatellite instability and an increased germ line point mutation frequency. Consequently, these rats develop a spectrum of tumors with a high similarity to atypical hereditary non-polyposis colorectal cancer in humans. The MSH6 knockout rat complements existing models for studying genetic instable tumorigenesis as it provides experimental opportunities that are not available or suboptimal in current models.

A genome-wide SNP panel for mapping and association studies in the rat.

BACKGROUND: The laboratory rat (Rattus norvegicus) is an important model for human disease, and is extensively used for studying complex traits for example in the physiological and pharmacological fields. To facilitate genetic studies like QTL mapping, genetic makers that can be easily typed, like SNPs, are essential. RESULTS: A genome-wide set of 820 SNP assays was designed for the KASPar genotyping platform, which uses a technique based on allele specific oligo extension and energy transfer-based detection. SNPs were chosen to be equally spread along all chromosomes except Y and to be polymorphic between Brown Norway and SS or Wistar rat strains based on data from the rat HapMap EU project. This panel was tested on 38 rats of 34 different strains and 3 wild rats to determine the level of polymorphism and to generate a phylogenetic network to show their genetic relationships. As a proof of principle we used this panel to map an obesity trait in Zucker rats and confirmed significant linkage (LOD 122) to chromosome 5: 119-129 Mb, where the leptin receptor gene (Lepr) is located (chr5: 122 Mb). CONCLUSION: We provide a fast and cost-effective platform for genome-wide SNP typing, which can be used for first-pass genetic mapping and association studies in a wide variety of rat strains.

Many novel mammalian microRNA candidates identified by extensive cloning and RAKE analysis.

MicroRNAs are 20- to 23-nucleotide RNA molecules that can regulate gene expression. Currently > 400 microRNAs have been experimentally identified in mammalian genomes, whereas estimates go up to 1000 and beyond. Here we show that many more mammalian microRNAs exist. We discovered novel microRNA candidates using two approaches: testing of computationally predicted microRNAs by a modified microarray-based detection system, and cloning and sequencing of large numbers of small RNAs from different human and mouse tissues. Together these efforts experimentally identified 348 novel mouse and 81 novel human microRNA candidate genes. Most novel microRNAs candidates are not conserved beyond mammals, and ~10% are taxon-specific. Our analyses indicate that the entire microRNA repertoire is not remotely exhausted.

Haplotype block structure is conserved across mammals.

Genetic variation in genomes is organized in haplotype blocks, and species-specific block structure is defined by differential contribution of population history effects in combination with mutation and recombination events. Haplotype maps characterize the common patterns of linkage disequilibrium in populations and have important applications in the design and interpretation of genetic experiments. Although evolutionary processes are known to drive the selection of individual polymorphisms, their effect on haplotype block structure dynamics has not been shown. Here, we present a high-resolution haplotype map for a 5-megabase genomic region in the rat and compare it with the orthologous human and mouse segments. Although the size and fine structure of haplotype blocks are species dependent, there is a significant interspecies overlap in structure and a tendency for blocks to encompass complete genes. Extending these findings to the complete human genome using haplotype map phase I data reveals that linkage disequilibrium values are significantly higher for equally spaced positions in genic regions, including promoters, as compared to intergenic regions, indicating that a selective mechanism exists to maintain combinations of alleles within potentially interacting coding and regulatory regions. Although this characteristic may complicate the identification of causal polymorphisms underlying phenotypic traits, conservation of haplotype structure may be employed for the identification and characterization of functionally important genomic regions.