Molecular Genetics of Pre-B Acute Lymphoblastic Leukemia Sister Cell Lines during Disease Progression

For many years, immortalized tumor cell lines have been used as reliable tools to understand the function of oncogenes and tumor suppressor genes. Today, we know that tumors can comprise subclones with common and with subclone-specific genetic alterations. We sequenced DNA and RNA of sequential sister cell lines obtained from patients with pre-B acute lymphoblastic leukemia at different phases of the disease. All five pairs of cell lines carry alterations that are typical for this disease: loss of tumor suppressors (CDKN2A, CDKN2B), expression of fusion genes (ETV6-RUNX1, BCR-ABL1, MEF2D-BCL9) or of genes targeted by point mutations (KRAS A146T, NRAS G12C, PAX5 R38H). MEF2D-BCL9 and PAX R38H mutations in cell lines have hitherto been undescribed, suggesting that YCUB-4 (MEF2D-BCL9), PC-53 (PAX R38H) and their sister cell lines will be useful models to elucidate the function of these genes. All aberrations mentioned above occur in both sister cell lines, demonstrating that the sisters derive from a common ancestor. However, we also found mutations that are specific for one sister cell line only, pointing to individual subclones of the primary tumor as originating cells. Our data show that sequential sister cell lines can be used to study the clonal development of tumors and to elucidate the function of common and clone-specific mutations.

RGB-Marking to Identify Patterns of Selection and Neutral Evolution in Human Osteosarcoma Models

Osteosarcoma (OS) is a highly aggressive tumor characterized by malignant cells producing pathologic bone; the disease presents a natural tendency to metastasize. Genetic studies indicate that the OS genome is extremely complex, presenting signs of macro-evolution, and linear and branched patterns of clonal development. However, those studies were based on the phylogenetic reconstruction of next-generation sequencing (NGS) data, which present important limitations. Thus, testing clonal evolution in experimental models could be useful for validating this hypothesis. In the present study, lentiviral LeGO-vectors were employed to generate colorimetric red, green, blue (RGB)-marking in murine, canine, and human OS. With this strategy, we studied tumor heterogeneity and the clonal dynamics occurring in vivo in immunodeficient NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ (NSG) mice. Based on colorimetric label, tumor clonal composition was analyzed by confocal microscopy, flow cytometry, and different types of supervised and unsupervised clonal analyses. With this approach, we observed a consistent reduction in the clonal composition of RGB-marked tumors and identified evident clonal selection at the first passage in immunodeficient mice. Furthermore, we also demonstrated that OS could follow a neutral model of growth, where the disease is defined by the coexistence of different tumor sub-clones. Our study demonstrates the importance of rigorous testing of the selective forces in commonly used experimental models.

Heterogeneous flow environments mediate defector exclusion during yeast floc formation

<p>Flocculation of Saccharomyces cerevisiae is cooperative phenotype that offers protection against various external stresses. It is modulated by cell-surface proteins, called flocculins, that bind with sugar residues of neighboring cells, effectively increasing cell-cell adhesion. These flocculins are predominantly encoded by the FLO1, which is considered as a ‘green beard’ gene as it governs both the cooperative phenotype and kin recognition of other cooperators. Conversely, defecting cells, lacking FLO1 expression, can still be incorporated in the flocs and ‘cheat’ on the cooperative benefits.</p> <p>In this work, we investigate various pathways involved in floc formation and specifically their effect on the exclusion of defecting cells. Initially, we experimentally confirmed the heterophilic binding mode of FLO1 and consequently the potential to ‘cheat’. These measured cell mechanical properties were adopted to calibrate the parameters of an individual cell-based model of interacting yeast cells. Using this model, we investigate exclusion of defecting cells for the two mechanisms of group formation; clonal development (staying together) and shear-driven aggregation (coming together). Our results indicate the need for a multi-stage mechanism and selection for the macroscopic flocs with high degree of defector exclusion, as observed experimentally. The stages in this process are (i) nucleation of clusters with FLO1+ rich cores, either due to aggregation or growth, (ii) high shear flow conditions causing erosion of flo1- cells from clusters and exclusion of aggregates due to remodeling, (iii) fast clustering of small but dense flocs of FLO1+ cells in low shear conditions or due to gravitational sedimentation.</p>

Role of oncoviruses in oncogenesis

The author describes DNA oncoviruses and RNA oncoviruses, their ways of infiltrating the host’s cells, and the possibilities of neoplastic transformation of cells by these microorganisms. The role of protooncogenesis and oncogenesis in both humans and animals is discussed. The transformation of cells by viruses is normally insufficient for oncogenesis; the cells also need to gain “immortality,” which usually requires 4-5 genetic changes (the so-called clonal development of cells), (Fig. 1). Oncoviruses remove suppressor growth factors while enhancing the effects that stimulate cell growth through e.g. hormones, cytokines, or transcription activators. In addition, the author discusses the role of the optimization principle in neogenesis.

Multicellular group formation in Saccharomyces cerevisiae

Understanding how and why cells cooperate to form multicellular organisms is a central aim of evolutionary biology. Multicellular groups can form through clonal development (where daughter cells stick to mother cells after division) or by aggregation (where cells aggregate to form groups). These different ways of forming groups directly affect relatedness between individual cells, which in turn can influence the degree of cooperation and conflict within the multicellular group. It is hard to study the evolution of multicellularity by focusing only on obligately multicellular organisms, like complex animals and plants, because the factors that favour multicellular cooperation cannot be disentangled, as cells cannot survive and reproduce independently. We support the use of Saccharomyces cerevisiae as an ideal model for studying the very first stages of the evolution of multicellularity. This is because it can form multicellular groups both clonally and through aggregation and uses a family of proteins called ‘flocculins’ that determine the way in which groups form, making it particularly amenable to laboratory experiments. We briefly review current knowledge about multicellularity in S. cerevisiae and then propose a framework for making predictions about the evolution of multicellular phenotypes in yeast based on social evolution theory. We finish by explaining how S. cerevisiae is a particularly useful experimental model for the analysis of open questions concerning multicellularity.

IgEvolution: clonal analysis of antibody repertoires

Constructing antibody repertoires is an important error-correcting step in analyzing immunosequencing datasets that is important for reconstructing evolutionary (clonal) development of antibodies. However, the state-of-the-art repertoire construction tools typically miss low-abundance antibodies that often represent internal nodes in clonal trees and are crucially important for clonal tree reconstruction. Thus, although repertoire construction is a prerequisite for follow up clonal tree reconstruction, the existing repertoire reconstruction algorithms are not well suited for this task. Since clonal analysis has the potential to reveal errors in the constructed repertoires and contribute to constructing more accurate repertoires, we advocate a tree-guided construction of antibody repertoires that combines error correction and clonal reconstruction as interconnected (rather than independent) tasks. We developed the IgEvolution algorithm for simultaneous repertoire and clonal tree reconstruction and applied it for analyzing multiple immunosequencing datasets representing antigen-specific immune responses. We demonstrate that analysis of clonal trees reveals highly mutable positions that correlate with antigen-binding sites and light-chain contacts in crystallized antibody-antigen complexes. We further demonstrate that this analysis leads to a new approach for identifying complementarity determining regions (CDRs) in antibodies.

The Potential Role of Quorum Sensing in Clonal Growth and Subsequent Expansion of Bone Marrow Stromal Cell Strains in Culture

Clonal development (clonogenicity) is an inherent property of a subset of postnatal bone marrow (BM) adherent stromal mesenchymal stem cells (MSCs) from which a multipotent progeny develops in culture. Our data suggest that clonogenicity and BM-MSC expansion are two distinct biological events. This hypothesis is based on the following observations: (1) the beginning of clonal growth is a property strictly dependent on serum and independent of the social context, (2) the expansion of individual clone is influenced by events deriving from a social context during initial growth, (3) clonogenic cells grown in a social context in presence of serum can emancipate themselves to generate a secondary different progeny, and (4) the ability of socially generated clones to develop an inherent potential for further growth suggests that quorum sensing may operate in BM-MSC cultures and determine the potential growth of clonal strains.

Cdkn2a (Arf) loss drives NF1-associated atypical neurofibroma and malignant transformation

Plexiform neurofibroma (PN) tumors are a hallmark manifestation of neurofibromatosis type 1 (NF1) that arise in the Schwann cell (SC) lineage. NF1 is a common heritable cancer predisposition syndrome caused by germline mutations in the NF1 tumor suppressor, which encodes a GTPase-activating protein called neurofibromin that negatively regulates Ras proteins. Whereas most PN are clinically indolent, a subset progress to atypical neurofibromatous neoplasms of uncertain biologic potential (ANNUBP) and/or to malignant peripheral nerve sheath tumors (MPNSTs). In small clinical series, loss of 9p21.3, which includes the CDKN2A locus, has been associated with the genesis of ANNUBP. Here we show that the Cdkn2a alternate reading frame (Arf) serves as a gatekeeper tumor suppressor in mice that prevents PN progression by inducing senescence-mediated growth arrest in aberrantly proliferating Nf1−/− SC. Conditional ablation of Nf1 and Arf in the neural crest-derived SC lineage allows escape from senescence, resulting in tumors that accurately phenocopy human ANNUBP and progress to MPNST with high penetrance. This animal model will serve as a platform to study the clonal development of ANNUBP and MPNST and to identify new therapies to treat existing tumors and to prevent disease progression.

Leo Sachs. 14 October 1924—12 December 2013

Leo Sachs was a worldwide renowned scientist for his major contributions in several fields. In the mid 1950s he showed that amniocentesis could be used for prenatal diagnosis of sex and blood group antigens. He then focused on various aspects of normal development and cancer, including the control of normal haematopoiesis and leukaemia, carcinogenesis in vitro by polyoma and SV40 tumour viruses, chemical carcinogens and X-rays, chromosomes and the reversibility of cancer, surface membrane changes in malignant cells and suppression of malignancy by inducing differentiation. The cell culture system he established in the early 1960s for the clonal development of normal haematopoietic cells made it possible to analyse the molecular basis of normal haematopoiesis, and discover the proteins that regulate viability, proliferation and differentiation of different blood cell lineages and the changes that drive leukaemia. His studies established significant general concepts, including: the differential responsiveness of cancer cells to normal regulators of development; suppression of myeloid leukaemia by inducing differentiation, bypassing malignancy-driving genetic defects; identification of chromosomes that control tumour suppression; discovering apoptosis as a major mechanism by which WT-p53 suppresses malignancy; and the ability of haematopoietic cytokines to suppress apoptosis in both normal and leukaemic cells. Leo was fortunate to witness his pioneering discoveries and ideas move from the basic science stage to effective clinical applications, using amniocentesis for prenatal detection of genetic abnormalities, augmenting normal haematopoiesis in patients with various haematopoietic deficiencies and suppressing malignancy by inducing differentiation and apoptosis.