Functional Impact of Preleukemic Mutations on the Human Hematopoietic Stem and Progenitor Cell Compartment

Dissecting functional consequences of gene mutations contributes to understanding disease development and identifying potential treatment strategies. Mutations in epigenetic regulators (especially in DNMT3A, TET2, and ASXL1, termed DTA mutations) have been identified in acute myeloid leukemia patients, as well as individuals without history of hematologic malignancies. Mutations in these genes are hypothesized to confer a clone a competitive advantage, leading to preferential expansion of this specific clone. DTA mutations are proposed to be preleukemic events that are acquired before leukemia onset and are thought to elevate the risk for acquisition of additional mutations that in turn drive malignant transformation. Clones harboring these mutations are able to survive chemotherapy, potentially increasing the risk for disease recurrence. In this thesis, I investigated the impact of DTA mutations in the human hematopoietic stem and progenitor cell compartment, as well as the role of preleukemic events in patients with AML t(8;21). Using CRISPR/Cas9, site-specific mutations were introduced in CD34+ progenitor cells freshly isolated from umbilical cord blood samples. By delivering Cas9 nuclease together with an in vitro transcribed sgRNA as ribonucleoprotein, I was able to achieve editing efficiencies ranging from 40% for the sgRNA targeting exon 13 of ASXL1 to over 90% for the sgRNA targeting exon 6 of TET2. A known hotspot mutation in DNMT3A R882H was introduced with an efficiency of up to 50% in the cell bulk. After successful genetic modification, cells were subjected to in vitro assays to assess phenotype, self-renewal properties, differentiation capacities, and long-term culture-initiating potential. Clonal composition and expansion were assessed via deep sequencing and subsequent analysis of introduced specific insertions and deletions. To investigate the role and importance of preleukemic mutations in AML leukemogenesis, a comprehensive genetic in-depth characterization of AML t(8;21) was performed. To this aim, DNA from the time of diagnosis was analyzed via next generation sequencing with a targeted gene panel including exons and coding regions of 66 genes recurrently mutated in hematologic malignancies. From 56 patients, samples from clinical remission were analyzed for the previously identified mutations by targeted re-sequencing of amplicons spanning the target region. In this thesis, I was able to demonstrate that mutations in DNMT3A, TET2, and ASXL1 influence the behavior of hematopoietic stem and progenitor cells (HSPCs) in a gene-specific fashion. Mutations in ASXL1 had only little effect on HSPCs with no observed changes in expression of differentiation markers after culture for three weeks and no enhanced proliferation in long-term culture. The only hint that ASXL1 mutations have an impact on the fitness of HSPCs was increased colony forming ability after long-term culture, meaning that although progenitor cells did not proliferate during the long-term culture period, they were able to sustain and generate colonies later. DNMT3A mutations led to a longer retention of CD34 marker expression in differentiation culture experiments, but not enhanced serial replating capacity. TET2-mutated cells, in contrast, showed delayed myeloid marker expression in short-term culture and increased self-renewal of committed progenitor cells. Both, DNMT3A and TET2 mutations led to clonal expansion of distinct cell clones during long-term culture with a competitive advantage over wild type cells. The clonal composition of TET2- and DNMT3A-mutated samples differed, with TET2mut samples being more diverse. Collectively my data indicate that DNMT3A mutations preferentially influence primitive HSC clones, whereas TET2 mutations enhance the fitness of committed progenitor cell clones. ASXL1 mutations might need additional events to drive clonal expansion. Analysis of patient samples revealed that mutations in DNMT3A and TET2 are early events in AML t(8;21) and potentially acquired before the onset of the disease. ASXL1 mutations are neither early nor late events in these patients but are potentially cooperating events in leukemia transformation. In this thesis, I was able to demonstrate that DTA mutations enhance the fitness of distinct human hematopoietic stem or progenitor cell clones, in terms of self-renewal and proliferative capacity, leading to clonal expansion. The results obtained here highlight the importance of monitoring specific preleukemic events to prevent disease recurrence as well as development of severe hematologic conditions.Um die Entwicklung von Krankheiten aufzudecken und etwaige zielgerichtete Behandlungsstrategien zu entwickeln, ist es essenziell den funktionellen Einfluss bestimmter Genmutationen zu untersuchen. Mutationen in epigenetischen Regulatoren wie DNMT3A, TET2 und ASXL1 (auch DTA-Mutationen genannt) wurden sowohl in Patienten mit akuter myeloischer Leukämie als auch in Patienten ohne hämatologische Erkrankung gefunden. Diese Mutationen führen zu einem Vorteil bestimmter Zellklone, die daraufhin expandieren. DTA-Mutationen werden auch als prä-leukämische Mutationen bezeichnet, die vor dem Ausbruch der Leukämie bereits in Stamm- und Vorläuferzellen vorkommen und ein erhöhtes Risiko für das Auftreten weiterer Mutationen bedingen. Zusätzliche Mutationen in einem prä-leukämischen Klon können wiederum zur malignen Transformation des Zellklons führen. Des Weiteren können Zellen mit prä-leukämischen Mutationen resistent gegen Chemotherapie sein und somit das Risiko eines Rezidivs erhöhen. In dieser Arbeit habe ich den Einfluss von Mutationen in DNMT3A, TET2 und ASXL1 auf humane hämatopoetische Stamm- und Vorläuferzellen untersucht, sowie die Rolle von prä-leukämischen Mutationen in Patienten mit AML t(8;21) erforscht. Mit Hilfe von CRISPR/Cas9 wurden spezifische Mutationen in CD34-positiven Vorläuferzellen eingeführt, die frisch aus Nabelschnurblut isoliert wurden. Die Cas9-Nuklease wurde zusammen mit in vitro-transkribierter sgRNA in Form von Ribonukleoproteinen in die Zellen gebracht. In Exon 13 von ASXL1 wurden Insertionen und Deletionen in mindestens 40% der Zellen generiert und in Exon 6 des Gens TET2 konnten in mehr als 90% der Zellen Insertionen und Deletionen entdeckt werden. Die spezifische Mutation DNMT3A R882H wurde in bis zu 50% der transfizierten Zellen eingeführt. Die erfolgreich mutierten Zellen wurden anschließend in in vitro-Experimenten untersucht in Hinsicht auf Veränderungen im Phänotyp, der Fähigkeit zur Selbsterneuerung, Differenzierungsfähigkeit und der Fähigkeit sich in Langzeitkulturen zu vermehren. Die klonale Zusammensetzung, sowie die Expansion bestimmter Zellklone wurden mit Hilfe von Deep Sequencing und anschließender Analyse der generierten Insertionen und Deletionen untersucht. Patienten mit AML t(8;21) wurden auf prä-leukämische Mutationen untersucht, indem DNA vom Diagnosezeitpunkt via Next-Generation Sequenzierung analysiert wurde. Kodierende Regionen von 66 Genen, die wiederholt in malignen hämatologischen Erkrankungen auftreten, wurden in die Analyse eingeschlossen. Von 56 Patienten wurden DNA-Proben vom Zeitpunkt der klinischen Remission erneut auf die bereits identifizierten Mutationen untersucht. In dieser Arbeit konnte ich zeigen, dass Mutationen in DNMT3A, TET2 und ASXL1 unterschiedliche Effekte auf hämatopoetische Stamm- und Vorläuferzellen haben. Mutationen in ASXL1 hatten den mildesten Effekt. Weder Unterschiede in der Expression von Differenzierungsmarkern noch erhöhte Replatierungsfähigkeit in Vergleich zu Wildtypzellen wurden beobachtet. Des Weiteren zeigten die Zellen kein vermehrtes Wachstum in der Langzeitkultur. Sie waren jedoch in der Lage Kolonien nach der Langzeitkultur zu bilden, was bedeuten könnte, dass diese Zellen trotz des unveränderten Proliferationsverhaltens einen Vorteil gegenüber Wildtypzellen haben und somit in der Lage sind die Langzeitkultur zu überdauern. Zellen mit DNMT3A Mutationen exprimierten den Vorläuferzellmarker CD34 länger als Kontrollzellen. Mutationen in TET2 führten zu einer verspäteten Expression von myeloischen Differenzierungsmarkern und einem erhöhten Selbsterneuerungspotential von Vorläuferzellen. Sowohl in Zellen mit DNMT3A- als auch mit TET2-Mutationen konnte ich die Expansion bestimmter mutierter Zellklone nachweisen. Die Proben mit eingeführten Mutationen in TET2 hatten jedoch eine größere Anzahl verschiedener Insertionen und Deletionen als Proben mit DNMT3A-Veränderungen. Die Daten dieser Arbeit zeigen, dass Mutationen in DNMT3A dazu führen, dass primitive Zellklone einen evolutionären Vorteil erhalten, wohingegen reifere Vorläuferzellen von Mutationen in TET2 profitieren. Mutationen in ASXL1 führen möglicherwiese nur in Anwesenheit weiterer genetischer oder zellulärer Veränderungen zu klonaler Expansion. In Patienten mit AML t(8;21) treten Mutationen in DNMT3A und TET2 wahrscheinlich früher auf als andere Mutationen und kommen möglicherweise in Vorläuferzellen vor, bevor sich die eigentliche Erkrankung entwickelt. Mutationen in ASXL1 scheinen weder frühe noch späte Ereignisse zu sein, sondern treten irgendwann dazwischen auf. Sie kooperieren möglicherweise mit anderen Mutationen, um Zellen zu transformieren. In dieser Arbeit konnte ich zeigen, dass DTA-Mutationen die Fitness von bestimmten Stamm- und Vorläuferzellen im menschlichen Stammzellkompartiment erhöhen und so zu einer Expansion der mutierten Zellklone führen. Die Ergebnisse dieser Arbeit bestätigen, dass die Untersuchung und Beobachtung prä-leukämischer Klone dazu beitragen könnte, ein Rezidiv oder sogar die Entstehung schwerer hämatologische Erkrankungen zu verhindern

Modeling clonal hematopoiesis in umbilical cord blood cells by CRISPR/Cas9

To investigate clonal hematopoiesis associated gene mutations in vitro and to unravel the direct impact on the human stem and progenitor cell (HSPC) compartment, we targeted healthy, young hematopoietic progenitor cells, derived from umbilical cord blood samples, with CRISPR/Cas9 technology. Site-specific mutations were introduced in defined regions of DNMT3A, TET2, and ASXL1 in CD34(+) progenitor cells that were subsequently analyzed in short-term as well as long-term in vitro culture assays to assess self-renewal and differentiation capacities. Colony-forming unit (CFU) assays revealed enhanced self-renewal of TET2 mutated (TET2(mut)) cells, whereas ASXL1(mut) as well as DNMT3A(mut) cells did not reveal significant changes in short-term culture. Strikingly, enhanced colony formation could be detected in long-term culture experiments in all mutants, indicating increased self-renewal capacities. While we could also demonstrate preferential clonal expansion of distinct cell clones for all mutants, the clonal composition after long-term culture revealed a mutation-specific impact on HSPCs. Thus, by using primary umbilical cord blood cells, we were able to investigate epigenetic driver mutations without confounding factors like age or a complex mutational landscape, and our findings provide evidence for a direct impact of clonal hematopoiesis-associated mutations on self-renewal and clonal composition of human stem and progenitor cells

Virtual supersampling as post-processing step preserves the trabecular bone morphometry in human peripheral quantitative computed tomography scans

In the clinical field of diagnosis and monitoring of bone diseases, high-resolution peripheral quantitative computed tomography (HR-pQCT) is an important imaging modality. It provides a resolution where quantitative bone morphometry can be extracted in vivo on patients. It is known that HR-pQCT provides slight differences in morphometric indices compared to the current standard approach micro-computed tomography (micro-CT). The most obvious reason for this is the restriction of the radiation dose and with this a lower image resolution. With advances in micro-CT evaluation techniques such as patient-specific remodeling simulations or dynamic bone morphometry, a higher image resolution would potentially also allow the application of such novel evaluation techniques to clinical HR-pQCT measurements. Virtual supersampling as post-processing step was considered to increase the image resolution of HR-pQCT scans. The hypothesis was that this technique preserves the structural bone morphometry. Supersampling from 82 μm to virtual 41 μm by trilinear interpolation of the grayscale values of 42 human cadaveric forearms resulted in strong correlations of structural parameters (R2: 0.96–1.00). BV/TV was slightly overestimated (4.3%, R2: 1.00) compared to the HR-pQCT resolution. Tb.N was overestimated (7.47%; R2: 0.99) and Tb.Th was slightly underestimated (-4.20%; R2: 0.98). The technique was reproducible with PE%CV between 1.96% (SMI) and 7.88% (Conn.D). In a clinical setting with 205 human forearms with or without fracture measured at 82 μm resolution HR-pQCT, the technique was sensitive to changes between groups in all parameters (p < 0.05) except trabecular thickness. In conclusion, we demonstrated that supersampling preserves the bone morphometry from HR-pQCT scans and is reproducible and sensitive to changes between groups. Supersampling can be used to investigate on the resolution dependency of HR-pQCT images and gain more insight into this imaging modality.ISSN:1932-620

Virtual supersampling as post-processing step preserves the trabecular bone morphometry in human peripheral quantitative computed tomography scans.

In the clinical field of diagnosis and monitoring of bone diseases, high-resolution peripheral quantitative computed tomography (HR-pQCT) is an important imaging modality. It provides a resolution where quantitative bone morphometry can be extracted in vivo on patients. It is known that HR-pQCT provides slight differences in morphometric indices compared to the current standard approach micro-computed tomography (micro-CT). The most obvious reason for this is the restriction of the radiation dose and with this a lower image resolution. With advances in micro-CT evaluation techniques such as patient-specific remodeling simulations or dynamic bone morphometry, a higher image resolution would potentially also allow the application of such novel evaluation techniques to clinical HR-pQCT measurements. Virtual supersampling as post-processing step was considered to increase the image resolution of HR-pQCT scans. The hypothesis was that this technique preserves the structural bone morphometry. Supersampling from 82 μm to virtual 41 μm by trilinear interpolation of the grayscale values of 42 human cadaveric forearms resulted in strong correlations of structural parameters (R2: 0.96-1.00). BV/TV was slightly overestimated (4.3%, R2: 1.00) compared to the HR-pQCT resolution. Tb.N was overestimated (7.47%; R2: 0.99) and Tb.Th was slightly underestimated (-4.20%; R2: 0.98). The technique was reproducible with PE%CV between 1.96% (SMI) and 7.88% (Conn.D). In a clinical setting with 205 human forearms with or without fracture measured at 82 μm resolution HR-pQCT, the technique was sensitive to changes between groups in all parameters (p < 0.05) except trabecular thickness. In conclusion, we demonstrated that supersampling preserves the bone morphometry from HR-pQCT scans and is reproducible and sensitive to changes between groups. Supersampling can be used to investigate on the resolution dependency of HR-pQCT images and gain more insight into this imaging modality

Risse im Fundament, Flammen im Gebälk

Bone is able to react to changing mechanical demands by adapting its internal microstructure through bone forming and resorbing cells. This process is called bone modeling and remodeling. It is evident that changes in mechanical demands at the organ level must be interpreted at the tissue level where bone (re)modeling takes place. Although assumed for a long time, the relationship between the locations of bone formation and resorption and the local mechanical environment is still under debate. The lack of suitable imaging modalities for measuring bone formation and resorption in vivo has made it difficult to assess the mechanoregulation of bone three-dimensionally by experiment. Using in vivo micro-computed tomography and high resolution finite element analysis in living mice, we show that bone formation most likely occurs at sites of high local mechanical strain (p<0.0001) and resorption at sites of low local mechanical strain (p<0.0001). Furthermore, the probability of bone resorption decreases exponentially with increasing mechanical stimulus (R(2) = 0.99) whereas the probability of bone formation follows an exponential growth function to a maximum value (R(2) = 0.99). Moreover, resorption is more strictly controlled than formation in loaded animals, and ovariectomy increases the amount of non-targeted resorption. Our experimental assessment of mechanoregulation at the tissue level does not show any evidence of a lazy zone and suggests that around 80% of all (re)modeling can be linked to the mechanical micro-environment. These findings disclose how mechanical stimuli at the tissue level contribute to the regulation of bone adaptation at the organ level

Mechanisms of Targeting the MDM2-p53-FOXM1 Axis in Well-Differentiated Intestinal Neuroendocrine Tumors

Background/Aims: The tumor suppressor p53 is rarely mutated in gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) but they frequently show a strong expression of negative regulators of p53, rendering these tumors excellent targets for a p53 recovery therapy. Therefore, we analyzed the mechanisms of a p53 recovery therapy on intestinal neuroendocrine tumors in vitro and in vivo.Methods: By Western blot and immunohistochemistry, we found that in GEP-NEN biopsy material overexpression of MDM2 was present in intestinal NEN. Therefore, we analyzed the effect of a small-molecule inhibitor, nutlin-3a, in p53 wild-type and mutant GEP-NEN cell lines by proliferation assay, flow cytometry, immunofluorescence, Western blot, and by multiplex gene expression analysis. Finally, we analyzed the antitumor effect of nutlin-3a in a xenograft mouse model in vivo. During the study, the tumor volume was determined. Results: The midgut wild-type cell line KRJ-I responded to the treatment with cell cycle arrest and apoptosis. By gene expression analysis, we could demonstrate that nutlins reactivated an antiproliferative p53 response. KRJ-I-derived xenograft tumors showed a significantly decreased tumor growth upon treatment with nutlin-3a in vivo. Furthermore, our data suggest that MDM2 also influences the expression of the oncogene FOXM1 in a p53-independent manner. Subsequently, a combined treatment of nutlin-3a and cisplatin (as chemoresistance model) resulted in synergistically enhanced antiproliferative effects. Conclusion: In summary, MDM2 overexpression is a frequent event in p53 wild-type intestinal neuroendocrine neoplasms and therefore recovery of a p53 response might be a novel personalized treatment approach in these tumors.Peer Reviewe

Modeling clonal hematopoiesis in umbilical cord blood cells by CRISPR/Cas9

To investigate clonal hematopoiesis associated gene mutations in vitro and to unravel the direct impact on the human stem and progenitor cell (HSPC) compartment, we targeted healthy, young hematopoietic progenitor cells, derived from umbilical cord blood samples, with CRISPR/Cas9 technology. Site-specific mutations were introduced in defined regions of DNMT3A, TET2, and ASXL1 in CD3

Conditional (re)modeling probabilities connecting the mechanical environment (SED) with the (re)modeling events.

<p>The mechanical regulation of bone (re)modeling is characterized by probability functions describing the so-called (re)modeling rules. When computing the (re)modeling rules, it is assumed that each (re)modeling event has the same probability of occurring to rule out the dependence on the time interval or the imbalance between bone formation and bone resorption (which may be due to bone growth, loading or OVX as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062172#pone-0062172-g003" target="_blank">Figure 3</a>). The normalized SED is truncated at 40% due to the very small number of voxels above this threshold (less than 1% of the total surface voxels). The plots show the data points (mean ± standard deviation) as well as the exponential fitting functions for bone formation, resorption and quiescence in all four experimental groups: (A) Mechanical loading group, (B) control group, (C) ovariectomy group, (D) sham-operated group.</p

Coefficients of the (re)modeling curves for formation, resorption and the net bone response.

<p>Fitting functions and coefficients of the (re)modeling curves for formation, resorption and the net bone response in all animal groups.</p