TET1 is a tumor suppressor of hematopoietic malignancy

The methylcytosine dioxygenase TET1 (‘ten-eleven translocation 1’) is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.National Institutes of Health (U.S.) (5RO1HD045022)National Institutes of Health (U.S.) (5R37CA084198

Quantitative proteomics to characterize specific histone H2A proteolysis in chronic lymphocytic leukemia and the myeloid THP-1 cell line

Proteome studies on hematological malignancies contribute to the understanding of the disease mechanism and to the identification of new biomarker candidates. With the isobaric tag for relative and absolute quantitation (iTRAQ) method we analyzed the protein expression between B-cells of healthy people and chronic lymphocytic leukemia (CLL) B-cells. CLL is the most common lymphoid cancer of the blood and is characterized by a variable clinical course. By comparing samples of patients with an aggressive vs. indolent disease, we identified a limited list of differentially regulated proteins. The enhanced sensitivity attributed to the iTRAQ labels led to the discovery of a previously reported but still not clarified proteolytic product of histone H2A (cH2A) which we further investigated in light of the suggested functional properties of this modification. In the exploratory proteome study the Histone H2A peptide was up-regulated in CLL samples but a more specific and sensitive screening of a larger patient cohort indicated that cH2A is of myeloid origin. Our subsequent quantitative analysis led to a more profound characterization of the clipping in acute monocytic leukemia THP-1 cells subjected to induced differentiation

Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases.

We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs

Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia

Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis

Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia

Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis

Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common type of leukemia. The Cancer Genome Atlas Research Network has demonstrated the increasing genomic complexity of acute myeloid leukemia (AML). In addition, the network has facilitated our understanding of the molecular events leading to this deadly form of malignancy for which the prognosis has not improved over past decades. AML is a highly heterogeneous disease, and cytogenetics and molecular analysis of the various chromosome aberrations including deletions, duplications, aneuploidy, balanced reciprocal translocations and fusion of transcription factor genes and tyrosine kinases has led to better understanding and identification of subgroups of AML with different prognoses. Furthermore, molecular classification based on mRNA expression profiling has facilitated identification of novel subclasses and defined high-, poor-risk AML based on specific molecular signatures. However, despite increased understanding of AML genetics, the outcome for AML patients whose number is likely to rise as the population ages, has not changed significantly. Until it does, further investigation of the genomic complexity of the disease and advances in drug development are needed. In this review, leading AML clinicians and research investigators provide an up-to-date understanding of the molecular biology of the disease addressing advances in diagnosis, classification, prognostication and therapeutic strategies that may have significant promise and impact on overall patient survival

Differential splicing and allelic imbalance as pathomechanisms of recurring mutations in Acute Myeloid Leukemia

Acute myeloid leukemia is an aggressive malignancy which proves fatal if left untreated. Most patients respond to intensive chemotherapy, however refractory or relapsing disease is still a major contributor of poor patient outcome. New generation sequencing methods enabled the identification of genes harboring recurrent mutations in this disease, and they are being used to inform clinical decisions. In the studies presented in this thesis the aim was to improve our understanding of these mutations to further refine clinical decision making. The first study provided an overview of splicing factor mutations, which affect around 20% of all acute myeloid leukemia patients. It highlighted the association of splicing factor mutations with clinical and molecular parameters and further showed that splicing factor mutations are not independent prognostic markers in acute myeloid leukemia. A novel differential splice junction usage pipeline was used to quantify aberrant splicing patterns in mutated patients in two large sequencing datasets. The usage of two splice junctions was shown to identify patients with poor prognosis thereby providing an example of how our findings can be translated to clinical practice. The purpose of the second study was to examine allelic imbalance of recurrent mutations, a currently underappreciated phenomenon in acute myeloid leukemia. Using a large patient sample pool with matched DNA- and RNA-sequencing data we were able to compare variant calling pipelines between both sequencing methods to determine whether recurrent mutations are over- or underrepresented in RNA. We defined weighted allelic imbalance as a parameter for statistically comparing variant allele frequencies between DNA and RNA and identified allelic imbalance in nine out of eleven recurrently mutated genes examined in this study. Furthermore, recurrent mutations in GATA2 were also shown to exhibit preferential transcription for the mutant allele in a pooled validation cohort of three independent datasets. In summary, our studies show how customized bioinformatics pipelines can lead to an improved pathomechanistic understanding of recurrent mutations in acute myeloid leukemia and provide a foothold for further study of these mutations in high throughput sequencing experiments.Die Akute Myeloische Leukämie ist eine aggressive Krebserkrankung die unbehandelt tödlich verläuft. Die Mehrheit der Patienten spricht auf eine intensive Chemotherapie an, jedoch resultieren refraktäre Erkrankungsverläufe oder Rezidive immer noch in einer schlechten Gesamtprognose. Hochdurchsatz-Sequenzierungsverfahren erlaubten die Identifikation von Genen, die in dieser Erkrankung häufig mutiert sind. Diese Mutationen ermöglichen eine Risikostratifizierung der Patienten und fließen in Therapie-Entscheidungen ein. Das Ziel der in dieser Dissertation präsentierten Studien war es, die funktionelle Bedeutung einiger dieser Mutationen genauer zu charakterisieren. Die erste Studie charakterisierte Spliceosom-Mutationen, die bei etwa 20% aller Patienten mit einer Akuten Myeloischen Leukämie beobachtet werden. Die Assoziation von Spliceosom-Mutationen mit klinischen und molekularen Parametern wurde untersucht und zeigte, dass Spliceosom-Mutationen keine unabhängige prognostische Wertigkeit besitzen. Eine neue Analyse-Methode zur Splicing-Quantifizierung wurde zur Untersuchung von aberranten Splicing-Mustern in Patienten mit Mutationen in diesen Genen entwickelt. Diese wurde in der Folge auf zwei große Sequenzierdatensätze angewandt. Zwei der aberranten Splicing-Muster konnten genutzt werden, um Patienten mit einer schlechten Prognose zu identifizieren und stellen damit die klinische Bedeutung der Ergebnisse beispielhaft dar. Das Ziel der zweiten Studie war es, ein allelisches Ungleichgewicht von häufigen Mutationen zu untersuchen. Mittels eines großen Patientenkollektivs mit gepaarten DNA- und RNA-Sequenzierungsdaten konnten eine Über- oder Unterrepräsentation von häufigen bei AML Patienten beobachteten Mutationen auf RNA-Ebene bestimmt werden. Wir definierten die “weighted allelic imbalance” als einen Parameter für den statistischen Vergleich der Allelfrequenzen von rekurrenten Mutationen in DNA und RNA und stellten ein allelisches Ungleichgewicht in neun von elf untersuchten Gen-Mutationen fest. Weiterhin konnte die bevorzugte Transkription des mutierten Allels von GATA2 in einer Validierungskohorte, bestehend aus drei unabhängigen Datensätzen, gezeigt werden. Zusammenfassend, zeigen diese Studien wie maßgeschneiderte bioinformatische Arbeitsabläufe zu einem verbesserten pathomechanistischen Verständnis von rekurrenten Mutationen in der Akuten Myeloischen Leukämie führen können und stellen einen Baustein für die weitere Erforschung solcher Mutationen mit Hilfe von Hochdurchsatz-Experimenten dar

NON-GENETIC MECHANISMS OF CHEMORESISTANCE IN ACUTE MYELOID LEUKEMIA

Acute Myeloid Leukemia (AML) is the most common type of acute hematological malignancy in adults. 40–60% of patients relapse due to the emergence of cellular resistance to anti- leukemic drugs. Drug resistance in leukemic cells has been associated with intratumoral heterogeneity, among which quiescence, specifically, is considered a key factor for cell survival. Experimental evidence collected both from patients and model systems suggests that relapse is due to rare persistent AML cells which survive chemotherapy. Chemotherapy persistent cells are not yet biologically and molecularly defined. Open questions are whether persistent cells are drug-resistant, what are their cellular and molecular features, as well as their content in leukemic stem cells. My working hypothesis is that chemoresistance in AMLs is associated with specific phenotypic states that characterize rare cell populations found within the pool of quiescent leukemic cells. These cells are selected by chemotherapy and represent the cellular basis of the relapse. To test my hypothesis, I explored transcriptional and functional characteristics of quiescent leukemic cells and tested the effect of chemotherapy. Here, I present two newly established xeno-models of chemoresistant human AMLs that closely recapitulate clinical data. My data show that quiescent cells accumulate over time and quiescent and proliferating cells can switch from one state to another. Notably, quiescent cells are selectively spared by chemotherapy and show resistance to additional rounds of treatment. Finally, quiescent cells appear as the only carrier of tumorigenic capacity in AMLs and are, therefore, deemed essential for leukemia development and, possibly, relapse

Partner independent fusion gene detection by multiplexed CRISPR-Cas9 enrichment and long read nanopore sequencing

Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment. Fusion gene partner choice and breakpoint-position promiscuity restricts diagnostic detection, even for known and recurrent configurations. Here, we develop FUDGE (FUsion Detection from Gene Enrichment) to accurately and impartially identify fusions. FUDGE couples target-selected and strand-specific CRISPR-Cas9 activity for fusion gene driver enrichment - without prior knowledge of fusion partner or breakpoint-location - to long read nanopore sequencing with the bioinformatics pipeline NanoFG. FUDGE has flexible target-loci choices and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in one sequencing run. We observe on-average 665 fold breakpoint-site enrichment and identify nucleotide resolution fusion breakpoints within 2 days. The assay identifies cancer cell line and tumor sample fusions irrespective of partner gene or breakpoint-position. FUDGE is a rapid and versatile fusion detection assay for diagnostic pan-cancer fusion detection

Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification

Non-invasive epigenome editing is a promising strategy for engineering gene expression programs, yet potency, specificity, and persistence remain challenging. Here we show that effective epigenome editing is gated at single-base precision via 'keyhole' sites in endogenous regulatory DNA. Synthetic repressors targeting promoter keyholes can ablate gene expression in up to 99% of primary cells with single-gene specificity and can seamlessly repress multiple genes in combination. Transient exposure of primary T cells to keyhole repressors confers mitotically heritable silencing that persists to the limit of primary cultures in vitro and for at least 4 weeks in vivo, enabling manufacturing of cell products with enhanced therapeutic efficacy. DNA recognition and effector domains can be encoded as separate proteins that reassemble at keyhole sites and function with the same efficiency as single chain effectors, enabling gated control and rapid screening for novel functional domains that modulate endogenous gene expression patterns. Our results provide a powerful and exponentially flexible system for programming gene expression and therapeutic cell products