Integrative approaches to high-throughput data in lymphoid leukemias (on transcriptomes, the whole-genome mutational landscape, flow cytometry and gene copy-number alterations)

Within this thesis I developed a new approach for the analysis and integration of heterogeneous leukemic data sets applicable to any high-throughput analysis including basic research. All layers are stored in a semantic graph which facilitates modifications by just adding edges (relationships/attributes) and nodes (values/results) as well as calculating biological consensus and clinical correlation. The front-end is accessible through a GUI (graphical user interface) on a Java-based Semantic Web server. I used this framework to describe the genomic landscape of T-PLL (T-cell prolymphocytic leukemia), which is a rare (~0.6/million) mature T-cell malignancy with aggressive clinical course, notorious treatment resistance, and generally low overall survival. We have conducted gene expression and copy-number profiling as well as NGS (next-generation sequencing) analyses on a cohort comprising 94 T-PLL cases. TCL1A (T-cell leukemia/lymphoma 1A) overexpression and ATM (Ataxia Telangiectasia Mutated) impairment represent central hallmarks of T-PLL, predictive for patient survival, T-cell function and proper DNA damage responses. We identified new chromosomal lesions, including a gain of AGO2 (Argonaute 2, RISC Catalytic Component; 57.14% of cases), which is decisive for the chromosome 8q lesion. While we found significant enrichments of truncating mutations in ATM mut/no del (p=0.01365), as well as FAT (FAT Atypical Cadherin) domain mutations in ATM mut/del (p=0.01156), JAK3 (Janus Kinase 3) mut/ATM del cases may represent another tumor lineage. Using whole-transcriptome sequencing, we identified novel structural variants affecting chromosome 14 that lead to the expression of a TCL1A-TCR (T-cell receptor) fusion transcript and a likely degradated TCL1A protein. Two clustering approaches of normal T-cell subsets vs. leukemia gene expression profiles, as well as immunophenotyping-based agglomerative clustering and TCR repertoire reconstruction further revealed a restricted, memory-like T-cell phenotype. This is to date the most comprehensive, multi-level, integrative study on T-PLL and it led to an evolutionary disease model and a histone deacetylase-inhibiting / double strand break-inducing treatment that performs better than the current standard of chemoimmunotherapy in preclinical testing

Enhancer-associated H3K4 methylation safeguards in vitro germline competence.

Funder: Studienstiftung des Deutschen VolkesGermline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions

Polycomb proteins as organizers of 3D genome architecture in embryonic stem cells

Polycomb group proteins (PcGs) control the epigenetic and transcriptional state of developmental genes and regulatory elements during mammalian embryogenesis. Moreover, PcGs can also contribute to 3D genome organization, adding an additional layer of complexity to their regulatory functions. Understanding the mechanistic basis and the dynamics of PcG-dependent chromatin structures will help us untangle the full complexity of PcG function during development. Since most studies concerning the 3D organization of PcG-bound chromatin in mammals have been performed in embryonic stem cells (ESCs), here we will focus on this cell type characterized by its unique self-renewal and pluripotency properties. More specifically, we will highlight recent findings and discuss open questions regarding how PcG-dependent changes in 3D chromatin architecture control gene expression, cellular identity and differentiation potential in ESCs. We believe that this can serve to illustrate the diverse regulatory mechanisms by which PcG proteins control the proper execution of gene expression programs during mammalian embryogenesis.T.P. is supported by a doctoral fellowship from the DAAD (Germany). A.R.-I. is supported by the ‘Programa STAR-Santander Universidades, Campus Cantabria Internacional de la convocatoria CEI 2015 de Campus de Excelencia Internacional’ (Spain).Peer reviewe

A Critical Evaluation of Analytic Aspects of Gene Expression Profiling in Lymphoid Leukemias with Broad Applications to Cancer Genomics

In cancer research, transcriptional aberrations are often deduced from mRNA-based gene expression profiling (GEP). Although transcriptome sequencing (RNA-seq) has gained ground in the recent past, mRNA-based microarrays remain a useful asset for high-throughput experiments in many laboratories. Possible reasons are the lower per-sample costs and the opportunity to analyze obtained GEP data in association with published data sets. There are established and widely used methods for the analysis of microarray data, which increase the comparability of different GEP data sets and facilitate data-mining approaches. However, analytic pitfalls, such as batch effects and issues of sample purity, e.g. by complex tissue composition, are often not properly addressed by these standard approaches. Moreover, most of these tools do not capitalize on the full range of public data sources or do not take advantage of the analytic possibilities for functional interpretation or of comprehensive meta-analyses. We present an overview of the most critical steps in the analysis of microarray-based GEP data. We discuss software and database query solutions that may be useful for each step and for generally overcoming analytic challenges. Aside from machine-learning applications to classify and cluster samples, we describe clinical applications of GEP, including a novel exploratory algorithm to identify potential biomarkers of prognosis in small sample cohorts as demonstrated by exemplary data from lymphatic leukemias. Overall, this review and the attached source code provide guidance to both molecular biologists and bioinformaticians / biostatisticians to properly conduct GEP analyses as well as to evaluate the clinical / biological relevance of obtained results

AKT-pathway Inhibition in Chronic Lymphocytic Leukemia Reveals Response Relationships Defined by TCL1

Cell survival in chronic lymphocytic leukemia (CLL) largely depends on B-cell receptor-induced AKT activation. Gain-of-function genomic lesions of PI3K-AKT-mTOR pathway components are usually absent in CLL. We previously established that a BCR-mediated growth response in CLL is determined by the oncogene T-cell leukemia 1 (TCL1) through a sensitizer effect on AKT phospho-activation. Despite high clinical response rates following AKT-cascade inhibition in CLL, resistances in a substantial proportion of patients call for reliable pre- and post-exposure strata to better predict compound responses. Using a panel of inhibitors with differential vertical affinities in the PI3K-AKT-mTOR axis, we describe distinct patterns and determinants of sensitivities in 75 CLL samples. The compounds specifically impacted the BCR-induced physical TCL1-AKT interaction. In general, there was an efficient and tumorselective abrogation of cell survival in suspension or protective stromal-cell cultures. However, biochemical and survival responses were heterogeneous across CLL and showed only incomplete overlap across inhibitors. Sensitivity clusters could be defined by differential responses to selective pan-PI3K inhibition vs. compounds acting more down-stream. An elevated PI3K/AKT/mTOR activation state conferred sensitivity or resistance, depending on the applied inhibitor. In fact, down-stream interception by mTOR or dual mTOR/PI3K inhibition appears more efficient in cases expressing the BCR-response and poor-risk determinants of ZAP70 or TCL1. Finally, exploiting the TCL1-AKT interaction, peptide-based TCL1-interphase mimics were potent in steric AKT antagonization and in reducing CLL cell survival. Overall, this study provides informative response relationships in AKT-pathway interception that can help refining predictive models in BCR-pathway inhibition in CLL

Semi-automated cancer genome analysis using high-performance computing

Next-generation sequencing (NGS) has turned from a new and experimental technology into a standard procedure for cancer genome studies and clinical investigation. While a multitude of software packages for cancer genome data analysis have been made available, these need to be combined into efficient analytical workflows that cover multiple aspects relevant to a clinical environment and that deliver handy results within a reasonable time frame. Here, we introduce Quick-NGS Cancer as a new suite of bioinformatics pipelines that is focused on cancer genomics and significantly reduces the analytical hurdles that still limit a broader applicability of NGS technology, particularly to clinically driven research. QuickNGS Cancer allows a highly efficient analysis of a broad variety of NGS data types, specifically considering cancer-specific issues, such as biases introduced by tumor impurity and aneuploidy or the assessment of genomic variations regarding their biomedical relevance. It delivers highly reproducible analysis results ready for interpretation within only a few days after sequencing, as shown by a reanalysis of 140 tumor/normal pairs from The Cancer Genome Atlas (TCGA) in which QuickNGS Cancer detected a significant number of mutations calling pipeline. Finally, QuickNGS Cancer obtained several unexpected mutations in leukemias that could be confirmed by Sanger sequencing

The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo

Poised enhancers (PEs) represent a genetically distinct set of distal regulatory elements that control the expression of major developmental genes. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally characterized in embryonic stem cells (ESC), it is currently unknown whether PEs are functionally conserved in vivo. Here, we show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that the interactions between PEs and their target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation within specific vertebrate clades, with only a few being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential roles during the induction of major developmental genes in vivo. Poised enhancers (PEs) in embryonic stem cells have accessible chromatin, are bound by repressive Polycomb Group proteins, and interact with their targets prior to activation. However, whether this is recapitulated in vivo is unknown. Here the authors show PEs display these features in mouse embryos, are prevalent across vertebrates, and are required for developmental gene expression

A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells

The serine/threonine death-associated protein kinases (DAPK) provide pro-death signals in response to (oncogenic) cellular stresses. Lost DAPK expression due to (epi) genetic silencing is found in a broad spectrum of cancers. Within B-cell lymphomas, deficiency of the prototypic family member DAPK1 represents a predisposing or early tumorigenic lesion and high-frequency promoter methylation marks more aggressive diseases. On the basis of protein studies and meta-analyzed gene expression profiling data, we show here that within the low-level context of B-lymphocytic DAPK, particularly CLL cells have lost DAPK1 expression. To target this potential vulnerability, we conceptualized B-cell-specific cytotoxic reconstitution of the DAPK1 tumor suppressor in the format of an immunokinase. After rounds of selections for its most potent cytolytic moiety and optimal ligand part, a DK1KD-SGIII fusion protein containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv SGIII against the B-cell-exclusive endocytic glyco-receptor CD22 was created. Its high purity and large-scale recombinant production provided a stable, selectively binding, and efficiently internalizing construct with preserved robust catalytic activity. DK1KD-SGIII specifically and efficiently killed CD22-positive cells of lymphoma lines and primary CLL samples, sparing healthy donor-or CLL patient-derived non-B cells. The mode of cell death was predominantly PARP-mediated and caspase-dependent conventional apoptosis as well as triggering of an autophagic program. The notoriously high apoptotic threshold of CLL could be overcome by DK1KD-SGIII in vitro also in cases with poor prognostic features, such as therapy resistance. The manufacturing feasibility of the novel CD22-targetingDAPK immunokinase and its selective antileukemic efficiency encourage intensified studies towards specific clinical application. (C) 2016 AACR

Comprehensive Analysis of Disease-Related Genes in Chronic Lymphocytic Leukemia by Multiplex PCR-Based Next Generation Sequencing.

High resolution molecular studies have demonstrated that the clonal acquisition of gene mutations is an important mechanism that may promote rapid disease progression and drug resistance in chronic lymphocytic leukemia (CLL). Therefore, the early and sensitive detection of such mutations is an important prerequisite for future predictive CLL diagnostics in the clinical setting.Here, we describe a novel, target-specific next generation sequencing (NGS) approach, which combines multiplex PCR-based target enrichment and library generation with ultra-deep high-throughput parallel sequencing using a MiSeq platform. We designed a CLL specific target panel, covering hotspots or complete coding regions of 15 genes known to be recurrently mutated and/or related to B-cell receptor signaling.High-throughput sequencing was performed using as little as 40 ng of peripheral blood B-cell DNA from 136 CLL patients and a dilution series of two ATM- or TP53-mutated cell lines, the latter of which demonstrated a limit of mutation detection below 5%. Using a stringent functional assessment algorithm, 102 mutations in 8 genes were identified in CLL patients, including hotspot regions of TP53, SF3B1, NOTCH1, ATM, XPO1, MYD88, DDX3X and the B-cell receptor signaling regulator PTPN6. The presence of mutations was significantly associated with an advanced disease status und molecular markers of an inferior prognosis, such as an unmutated IGHV mutation status or positivity for ZAP70 by flow cytometry.In summary, targeted sequencing using an amplicon based library technology allows a resource-efficient and sensitive mutation analysis for diagnostic or exploratory purposes and facilitates molecular subtyping of patient sets with adverse prognosis