269 research outputs found
Unravelling haematopoietic stem cell dysfunction in isolated Del(5q) myelodysplastic syndromes
No full textMyelodysplastic syndromes (MDS) represent a heterogeneous group of haematological malignancies. A subgroup of MDS patients are characterized by heterozygous deletion of the long arm of chromosome 5, Del(5q), as the only karyotypic abnormality. The commonly deleted region (CDR) on chromosome 5 contains approximately forty-two genes and haploinsufficiency of one or more of these genes is thought to be the basis for Del(5q) MDS pathogenesis.
The 5q deletion originates in the Hematopoietic Stem Cell (HSC) compartment and Del(5q) HSCs have a clonal advantage, outcompeting healthy HSCs in the bone marrow of patients. Although they have a competitive advantage in situ, Del(5q) HSCs perform poorly in functional stem cell assays in vitro and in vivo.
A mouse model of Del(5q) MDS, the Cd74-Nid67 model, carries a heterozygous deletion of eight genes located within the CDR. Cd74-Nid67 haploinsufficiency causes macrocytic anaemia and bone marrow dysplasia in mice. However, the impact of Cd74-Nid67 haploinsufficiency on HSC function has not previously been investigated.
The results presented, herein, demonstrate that haploinsufficiency of Cd74-Nid67 has a significant impact on HSC self-renewal and repopulation potential. Furthermore, two genes within this region, Rps14 and Rbm22, are identified as likely candidates responsible for Cd74-Nid67 HSC dysfunction. Finally, we demonstrate that Cd74-Nid67 HSC dysfunction is driven by a P53-dependent mechanism.
This study provides important insights into the mechanistic basis for disease development and propagation, which may facilitate the development of improved therapeutic avenues for Del(5q) MDS patients
Investigating the specific roles of the growth factor kit ligand in the regulation of murine haematopoiesis
No full textCharacterisation and targeting of stem cells in myelodysplastic syndromes
No full textUnderstanding which cells within a cancer are responsible for its initiation and propagation is vital if we are to achieve cure. If cancer stem cells are the only population able to sustain a tumour long term, designing therapeutic strategies to target this population will give medical science the best chance of long-term cure. Significant controversy remains over the existence of cancer stem cells, predominantly due to the lack of a sensitive human cancer stem cell assay. This thesis investigates whether two haematological malignancies, myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML) can only be driven by rare and distinct cancer stem cells. We have demonstrated that low and intermediate-1 risk MDS is driven solely by the stem cell (Lin- CD34+ CD38- CD90+ CD45RA-) by developing a novel genetic approach, tracing all somatic mutations and karyotypic abnormalities back to this population. Prior to this study, very little was known about the clonal architecture of CMML. By performing detailed phenotypic, functional, molecular and genetic analysis of patients with CMML, we were able to demonstrate that the most likely candidate driver cell in these patients was also the stem cell rather than any of the down-stream progenitors. Currently, effective therapeutic strategies for MDS or CMML are very limited. Allogeneic stem cell transplantation is the only potential cure and not suitable for most patients. Cancer stem cells, including MDS stem cells are known to be highly quiescent and selectively resistant to therapy. Having demonstrated that both MDS and CMML were driven by stem cells, we developed a novel therapeutic targeting strategy. Using the thrombopoietin receptor agonist, Romiplostim, we were able to activate stem cells and enhance their subsequent sensitivity to chemotherapy dramatically. This approach may facilitate improved remission rates and prevent cancer stem cell driven relapse in many diseases
The emergence and early fate decisions of stem and progenitor cells in the haematopoietic system
No full textThe alternative road map describes the separation of lympho-myeloid and myeloid-megakaryocyte-erythroid (myeloid-Mk-E) lineages as the earliest haematopoietic commitment event. However, a number of aspects of this lineage restriction process remain poorly understood. Herein this work identified a lympho-myeloid restricted progenitor in the embryo, which resembles the adult LMPP, and demonstrated that lymphoid lineage restriction is initiated prior to definitive haematopoiesis, much earlier than previously appreciated. In vivo fate mapping showed that lympho-myeloid progenitors significantly contribute to steady state myelopoiesis in the embryo. The early thymic progenitor (ETP) as most primitive cell in the thymus was characterised and demonstrated to sustain B, T and myeloid but not Mk potentials at the single cell level. The ETP therefore largely resembles the cellular properties of lympho-myeloid progenitors in bone marrow and foetal liver, which points to these cells as candidate thymus seeding progenitors (TSP). Furthermore the existence of a putative Mk progenitor was explored within the LSKCD150+CD48+Gata1pos compartment of a Gata1 reporter mouse providing the basis for a future prospective characterisation. Finally, this work evaluated the earliest lineage restriction of von Willebrand factor (Vwf)-EGFP+ and EGFP- haematopoietic stem cells (HSCs) through in vitro paired daughter fate mapping. Single Vwf+ HSCs showed heterogeneous Mk priming and more frequently sustained Mk potential after cell division. Moreover, analysis of lineage priming between daughter cells revealed the asymmetric expression of key lineage determinants and stem cell regulators, which might be employed as reporters for future fate mapping studies
The role and impact of Del(5q) and TP53 mutations on haematopoietic stem and progenitor cells during the progression of Myelodysplastic Syndrome to Acute Myeloid Leukaemia
No full textMyelodysplastic Syndromes (MDS) are a heterogeneous group of haematological
malignancies characterised by ineffective haematopoiesis and dysplastic bone
marrow changes. Patients frequently progress to Acute Myeloid Leukaemia and
this is associated with very poor clinical outcomes. This thesis focusses on a
subtype of MDS, Del(5q) MDS.
Sequencing analysis revealed that Del(5q) is frequently the initiating
lesion in low risk isolated Del(5q) MDS and tracking patients over time
demonstrated progressive disease is associated with the acquisition of new
mutations (including Tp53 mutations). Early in disease, MDS appears to be
sustained by the Lin-CD34+CD38-CD90+CD45RA- MDS stem cells. In one
patient, however, at the point of disease progression and after the acquisition of
a Tp53 mutation, self-renewal activity could be detected in a progenitor cell
population outside the stem cell compartment.
On the basis of these findings and previous evidence linking Tp53
mutations with progression of Del(5q) MDS, mouse models were used to
investigate if and how Del(5q) and mutant Tp53 collaborate to perturb
haematopoiesis. An established model of Del(5q) (heterozygous deletion of
Cd74-Nid67) was combined with a Tp53 knockout model and conditional knockin
models of Tp53 mutations found in MDS patients. This work showed Tp53
loss and mutations collaborate with Del(5q) to alter haematopoietic stem/
progenitor cell number, function and differentiation ability. Del(5q)+/-Tp53-/-
cells also uniquely develop long-term self-renewal potential in vitro which can
be associated with chromosomal instability. Preliminary evidence also suggests
a possible role for Del(5q) itself in the development of chromosomal instability.</p
Clonal dynamics of TEL-AML1+ childhood ALL: an in vivo model
No full textETV6–RUNX1 gene fusion is an early or initiating genetic lesion of Childhood Acute Lymphoblastic Leukaemia, followed, in disease progression, by a modest number of recurrent or "driver" copy number alterations. Among patients diagnosed with this particular cytogenetic subtype of ALL cure rate reaches over 80% . However, treatment protocols are extremely demanding, and the molecular mechanisms driving drug-resistance and relapse in the remaining 10% of patient are still unknown. Recently a genetic signature of subclones arranged in a complex non-linear or branching architecture has been identified in this leukaemia, pioneering similar observations in many other cancer types. At present, most reports of this intratumor diversity represent static, in-depth snapshots of clonal diversity of tumours at a given time. "Real-time" longitudinal and spatial analysis of subclonal evolutionary dynamics is required to understand the functional characteristics of individual subclones. Such approach also promises to reveal the overall clinical relevance of this phenomenon. This project therefore aims to understand whether genetically distinct subclones are also functionally different, particularly with respect to chemoresistance. A mouse model that allows for the independent exposure of the same tumour to treatment multiple times, and the tracking of clonal dynamics by mean of a selected pool of genetic markers was established. While the analysis is still ongoing, preliminary data confirm that tumours generated from the same inoculum are highly similar in their genetic makeup across recipients, that multiple clones within a tumour can survive chemotherapy, but sensitivity of clones equally represented within the bulk can vary, and unexpectedly that spatially segregated subclones can be identified in this model of liquid cancer. The complete analysis of the collected samples by mFISH as well as by single cell whole genome sequencing will provide unprecedented insight into the impact of cytotoxic treatment on intratumour genetic heterogeneity, and might reveal novel mechanisms of chemoresistance and relapse. Additionally, a lentiviral overexpression system was designed to endogenously manipulate clonal interactions through the restored expression of TEL and PAX5 in primary cells from patients, and evaluate their impact on clonal relationships and hierarchies and to validate the results obtained from the first part of the study. Lentiviral vectors for TEL and PAX5 second hit overexpression were produced and tested by Western blot and qPCR. The TEL vectors were adopted in a preliminary in vivo experiment, showing no effect on overall engraftment ability
Collaboration of Ezh2 and Runx1 inactivating mutations in malignant haematopoiesis
No full textExtensive efforts have shed light on the identity and biology of cancer stem cells, required and sufficient for the propagation of hematological malignancies and solid tumours. Much less is understood about the closely related issue as to the identity and properties of the normal stem and progenitor cells targeted by oncogenic lesions, and how the nature of the targeted cell might impact on the biology and clinical picture of the resulting cancer. To address this, we developed a mouse model allowing targeted inactivation of Ezh2 and Runx1 to different haematopoietic compartments. Inactivating mutations of EZH2 and RUNX1 frequently co-occur in haematological malignancies with markedly different phenotypes including myelodysplastic syndrome (MDS) and early thymic progenitor (ETP) leukaemia.
Inactivation of Ezh2 and Runx1 in adult haematopoietic stem cells (HSCs) resulted in perturbed haematopoiesis leading to development of an MDS-like disease. Unexpectedly, this MDS phenotype could be fully reproduced when Ezh2 and Runx1 inactivation was targeted to multipotent progenitors (MPPs) using Flt3-Cre. Furthermore, the disease was transplantable by MPPs, but not more committed progenitor populations, demonstrating that MDS tumour propagating potential is not exclusive to intrinsically self-renewing HSCs.
Targeting Ezh2 and Runx1 inactivation to early lympho-myeloid progenitors did not result in an MDS phenotype. These mice showed a marked expansion of ETPs within the thymus, combined with a block in thymocyte differentiation. These expanded ETPs displayed transcriptional features characteristic of ETP leukaemia, a treatment-resistant acute leukaemia subtype hypothesised to originate from ETPs. Combination of inactivation of Ezh2 and Runx1 in ETPs with the constitutively activating Flt3-ITD signalling mutation resulted in an aggressive lympho-myeloid acute leukaemia, which could be propagated by the expanded ETP population. These findings demonstrate the potential of lympho-myeloid progenitors such as ETPs to become leukaemia stem cells which propagate a disease retaining lympho-myeloid features.
We used this novel ETP leukaemia model to explore therapeutic targeting of Ezh2-inactivated ETP leukaemias using inhibitors of the bromodomain and extra terminal (BET) proteins. Aberrant transcription resulting from epigenetic changes induced by Ezh2 loss could be reversed by BET inhibitors, and these compounds showed therapeutic efficacy against both mouse and human ETP leukaemias in vitro and in vivo.</p
TARGET-seq genotyped single-cell RNA sequencing of hematopoietic stem cells and megakaryocyte-erythroid progenitor cells from a dual SF3B1-mutant MDS-RS patient and 3 healthy donors
No full textThis dataset consists of TARGETseq (genotype-targeted plate-based SmartSeq2) single-cell RNA sequencing data of purified hematopoietic stem cells (HSC) and megakaryocyte-erythroid progenitors (MEP) from the bone marrow of a dual SF3B1-mutant MDS-RS patient (Patient 1 in the associated publication) over 2 timepoints (39 months post-diagnosis, with dominant SF3B1-N626D, and 118 months post-diagnosis, with dominant SF3B1-K666N); and purified HSC/MEP from the bone marrow of three healthy donors. The objective of this data collection was to assess the molecular characteristics that increase fitness in SF3B1-mutant HSC as compared to normal HSC.
The dataset is approximately 187 GB and includes the file types: zip, xlsx, bam, bam.bai, rds.Denna datauppsättning består av TARGETseq (genotypriktad plattbaserad SmartSeq2) RNA-sekvenseringsdata från renade hematopoetiska stamceller (HSC) och megakaryocyt-erytroida progenitorer (MEP) från benmärgen hos en dubbel SF3B1-mutant MDS-RS patient vid två tillfällen poäng (39 månader efter diagnos och 118 månader efter diagnos); och renad benmärg HSC/MEP från tre friska donatorer. Syftet med denna datainsamling var att bedöma de molekylära egenskaperna som ökar "fitness" i SF3B1-mutant HSC jämfört med normal HSC. Se den engelska beskrivningen för mer information
Autophagy-dependent generation of free fatty acids is essential for normal neutrophil differentiation by guiding an energy-metabolic switch
No full textNeutrophils are critical and short lived mediators of innate immunity that require constant replenishment. Their differentiation in the bone marrow requires extensive cytoplasmic and nuclear remodeling, but the processes governing these energy-consuming changes are unknown. While previous studies show that autophagy is required for differentiation of other blood cell lineages, its function during granulopoiesis has remained elusive. Autophagy was described as a critical process in HSC differentiation and in memory- and regulatory T cells, where it prevents excessive glycolysis and maintains lipid metabolic homeostasis. In the myeloid lineage, an essential role for autophagy is to prevent pro-inflammatory macrophage polarization and to limit glycolytic metabolism of acute myeloid leukemia. While these studies provide robust in vivo evidence for the relevance of autophagy in the differentiation of hematopoietic and immune cells, the targets and mechanisms of autophagy remain elusive. Here, we show that metabolism and autophagy are developmentally programmed and essential for neutrophil differentiation in vivo. Atg7 -deficient neutrophil precursors had increased glycolytic activity but impaired mitochondrial respiration, decreased ATP production and accumulated lipid droplets. Inhibiting autophagy-mediated lipid degradation or fatty acid oxidation alone was sufficient to cause defective differentiation, while administration of fatty acids or pyruvate for mitochondrial respiration rescued differentiation in autophagy deficient neutrophil precursors. Together, we show that autophagy-mediated lipolysis provides free fatty acids to support a mitochondrial respiration pathway essential to neutrophil differentiation. Evidence presented here also contributed to studies on autophagy-mediated metabolic homeostasis in HSCs, Treg cells and myeloid leukemia, suggesting that this pathway may act broadly during differentiation.</p
The role of Notch and GATA3 in postnatal and adult haematopoiesis
No full textThe role of Notch in cell fate determination and lineage restriction in the bone marrow (BM) is controversial in the field. Recent studies have convincingly shown that Notch is dispensable for haematopoietic stem cell (HSC) regulation in adult haematopoiesis (Maillard et al., 2008). In contrast, Notch signaling has been proposed to be of importance in the regulation of BM megakaryocyte progenitor differentiation, based on dominant negative genetic approaches, identifying a potentially distinct role for Notch in adult BM haematopoiesis (Mercher et al., 2008). Here, I found that by selectively ablating the gene coding the transcription factor recombination signal-binding protein J kappa (RBP-Jk), to which all canonical Notch signaling converges, canonical Notch signaling does not mediate HSC maintenance, neither in steady state nor in conditions of stress. Furthermore, I propose, in contrast with previous studies (Mercher et al., 2008), that canonical Notch signaling plays no role in myeloerythropoiesis cell lineage commitment in the BM. My data also show that key Notch target genes are suppressed by RBP-Jk, as their expression is unaffected in Notch1-deficient BM progenitors, while target genes are upregulated in Rbp-Jk-deleted megakaryocyte and erythroid progenitors. This establishes for the first time in mammalian cells in vivo, that Notch target genes are kept in a suppressed state by RBP-Jk, potentially restricting T cell commitment to the thymus and not to the BM, at the expense of myeloerythropoiesis.Notch signaling and GATA3 are two master regulators in T cell commitment (Han et al., 2002; Ho et al., 2009; Pui et al., 1999; Radtke et al., 1999; Zhu et al., 2004). However, although very well established as being involved in the thymic stages of T cell restriction, there is little evidence of Notch and GATA3 being involved in the migration of a thymus settling progenitor (TSP) from the BM to the thymus or in the establishment of the earliest thymic progenitor (ETP) in the thymus. From this thesis work, I conclude that Notch signaling is essential for the emergence of ETPs in the thymus in a NOTCH1-independent manner. Moreover, I demonstrate, as supported by a very recent published study (Hosoya et al., 2009), that GATA3 is important for the development of the earliest T cell progenitor.GATA1 and GATA2 mediate haematopoietic stem cell maintenance in the BM. GATA1 is required for erythropoiesis, megakaryocytes and eosinophils while GATA2 is important for the proliferation and survival of HSCs. In contrast, a role for GATA3 in the BM has never been established. By using a Gata3-conditional knockout mouse model, I demonstrate that GATA3 is dispensable for HSC maintenance in steady state and following active haematopoietic regeneration as well as for HSC self-renewal in the BM
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