Search

Eradicating leukaemia stem cells (LSCs) and curing stem cell-derived cancers is hugely challenging. This is exemplified in chronic myeloid leukaemia (CML), where the t(9;22)(q34;q11) chromosomal translocation results in an abnormal chimeric BCR-ABL1 tyrosine kinase in a haematopoietic stem cell (HSC). Treatment of CML with tyrosine kinase inhibitors (TKIs) targeting the BCRABL1 protein is considered an exemplar of precision medicine, but unfortunately, TKIs do not cure most patients. Hence, unmet clinical needs arise in CML through the persistence and propagation of LSCs that are not eradicated by TKI treatment as they yield a source of disease recurrence. The persistence of LSCs after TKI treatment is related to tyrosine kinaseindependent mechanisms which include intrinsic properties of LSCs determined by epigenetic alterations, dysregulated transcriptional networks, and mitochondrial/metabolic changes. Importantly, both leukemic and normal HSCs share these properties along with quiescence and self-renewal ability supported in specialised haematopoietic bone marrow (BM) niches. The BM mesenchymal cell (BMSC) plays an essential niche role in HSC and LSC maintenance. Therefore, a deeper understanding of BMSC-dependent regulation of CML LSCs is required to determine how CML manipulates the haematopoietic niche and protect LSCs from TKI treatment. In this thesis, I set out to identify genes downstream of established autocrine signalling loops which drive the persistence of CML LSCs in a BCRABL1 independent manner. I examined seventeen publicly available CML RNA-seq datasets obtained from the Gene Expression Omnibus for novel gene signatures (Chapter 3). To understand the possible bone marrow niche cell interactions, dataset samples from human primary CD34⁺ enriched normal donor and CML samples (Greater Glasgow and Clyde National Health Service) cultured alongside human stromal, myeloid, and Lymphoid CML cell lines were also examined. A Fluidigm platform was used for LSC-niche co-culture samples to verify the autocrine signalling loops identified from published RNA sequencing data (Chapter 4). Collectively, the analyses inferred the expected widespread disturbances of haemopoietic networks. Gene sets enriched in CML LSCs downstream of previously reported axes included IL-6, TGF-β-BMP, TNF-α as well as PI3KBMP and NF-κB- expression pathways. During this study, significantly expressed genes from both human CML LSCs and a murine model of CML were mined for overlapping genes of novel significance. These datasets allowed us to compare normal HSCs, highly proliferative LSCs, quiescent BCR-ABL⁺ LSCs, as well as CML cell lines, and murine CML cells. Gene enrichment for Pituitary tumour-transforming gene 1 (PTTG1) and its interacting protein (PTTG1IP) were found and CML co-cultures showed increased expression of PTTG1 transcriptional targets, c- Myc, p53, FGF2, p21, prolactin and MMP2. To confirm whether the cell source of expression was the LSC, or niche cells co-culture data were re-examined. We hypothesised that the BMSC niche harbours and selectively maintains LSC persistence through cellular secretion. Our findings reveal a role for the secretory BMP/TGF-β superfamily, particularly Activin signalling, during the progression of CML. These correspond with earlier observations in CML that propose a switch from BMP to ActivinA (INHBA) signalling during treatment resistance and progressive disease. Throughout the in-silico study, we explored the role of the TGF-β superfamily and ActivinA associated genes within the CML BM niche and how they could be involved in the augmentation of a PTTG1-LSC gene network. In Chapter 4-5 we report an unanticipated finding. When CML cells were cocultured with BMSCs, macrophage chemotaxis and activation pathways were upregulated. This highlights CMLs ability to recruit immune cells to their specialised niche. We explored CML immune cell datasets focusing on macrophages from CML and normal healthy BM samples. CML exposed macrophages provided strong evidence that the niche can provide AcitivinA secretion that drives Activin signalling during CML. Thus, macrophages could pose an interesting therapeutic target along with other niche-related adjuvant approaches for TKI–resistant patients or following treatment discontinuation. To unravel the insights, we observed during our in-silico analyses, we sought to determine the in vivo effects of the niche on CML-LSCs and the potential synergistic effect of the BM towards disease progression and relapse during therapy (Chapter 2). To achieve this, we employed a CML mouse model (SCLtTA×BCR–ABL1) in which BCR–ABL1 expression is mainly targeted to the haematopoietic population. Through this approach, our data established that BCR–ABL1 expression produced a myeloproliferative disorder (with a similar temporal onset) after BM transplantation into a large cohort of mice. From this observation, we could produce varying clinical scenarios that represent significant time points during the development and transformation of the CML BM microenvironment. With these cohorts, we developed protocols for bone digestion and whole-bone mount imaging allowing for endosteal and central marrow cell visualisation, providing the basis for single-cell studies localizing niche constituents within the BM. In summary, the re-analysis of existing CML transcriptomic datasets provided insights into BM niche pathways that are potentially involved in promoting the survival of a subset of quiescent BCR-ABL1⁺ LSCs via a BCR-ABLindependent mechanism.

Summary The human myeloproliferative neoplasms are a biologically fascinating group of chronic myeloid malignancies. This perspective outlines how a post-doctoral fellowship working in Don Metcalf's unit proved a formative and immensely enjoyable experience for me and my family. It laid the foundation for a subsequent body of work over 3 decades which revealed the genetic basis of these diseases, defined how these genetic alterations subvert normal haematopoiesis, altered clinical practice and provided insights of broad biological relevance for cancer and cytokine signalling.

Comprehensive cataloguing of the acute myeloid leukaemia (AML) genome has revealed a high frequency of mutations and deletions in epigenetic factors that are frequently linked to treatment resistance and poor patient outcome. In this review, we discuss how the epigenetic mechanisms that underpin normal haematopoiesis are subverted in AML, and in particular how these processes are altered in childhood and adolescent leukaemias. We also provide a brief summary of the burgeoning field of epigenetic-based therapies, and how AML treatment might be improved through provision of better conceptual frameworks for understanding the pleiotropic molecular effects of epigenetic disruption.

The haematopoietic system manifests several age-associated phenotypes including anaemia; loss of regenerative capacity, especially in the face of insults such as infection, chemotherapy or blood loss; and increased risk of clonal haematopoiesis and blood cancers. The cellular alterations that underpin these age-related phenotypes, which typically manifest in individuals aged over 70, remain elusive. We aimed to investigate whether changes in HSC population structure with age might underlie any aspects of haematopoietic system ageing. We sequenced 3579 genomes from single-cell-derived colonies of haematopoietic stem cell/multipotent progenitors (HSC/MPPs) from 10 haematologically normal subjects aged 0-81 years. HSC/MPPs accumulated 17 somatic mutations/year after birth with no increased rate of mutation accumulation in the elderly. HSC/MPP telomere length declined by 30 bp/yr. In cord blood and adults aged To interrogate changes in HSC population structure with age, we used the pattern of unique and shared mutations between the sampled cells from each individual to reconstruct their phylogenetic relationships. The frequency of branch-points (known as coalescences) in phylogenetic trees in a neutrally evolving, well-mixed population of somatic cells is primarily determined by the product of population size and time between symmetric self-renewal cell divisions (Nt). Smaller populations and more frequent symmetric divisions both increase the density of coalescences. Specific clones can come to dominate either through neutral drift or positive selection. We found that haematopoiesis in adults aged 75 showed profoundly decreased clonal diversity. In each elderly subject, 30-60% of haematopoiesis was accounted for by 12-18 independent clones, each contributing 1-34% of blood production. Most clones had begun their expansion before age 40, but only 22% had known driver mutations. We used the ratio of non-synonymous to synonymous mutations (dN/dS) to identify any excess of non-synonymous (driver) mutations in the dataset. This genome-wide selection analysis estimated that 1/34 to 1/12 non-synonymous mutations were drivers, occurring at a constant rate throughout life, such that the set of 300 - 400 HSC/MPPs sampled from each adult individual harboured around 100 driver mutations, over 10-fold higher than the number of known drivers we could identify. Novel drivers affected a wider pool of genes than identified in blood cancers. The genes DNMT3A, ZNF318 and HIST2H3D were identified as being under significant positive selection in HSC/MPPs, despite ZNF318 and HIST2H3D not being enriched in the setting of myeloid malignancies. Loss of Y chromosome conferred selective benefits on HSC/MPPs in males. Simulations from a simple model of haematopoiesis, with constant HSC population size and constant acquisition of driver mutations conferring moderate fitness benefits, entirely explained the abrupt change in clonal structure observed in the elderly, which could not be explained by neutral models incorporating drift alone. Our data supports the view that dramatically decreased clonal diversity is a universal feature of haematopoiesis in aged humans, underpinned by pervasive positive selection acting on many more genes than currently known. By old age the majority of HSCs harbour at least one driver mutation. With such ubiquity of driver mutations, selected purely for their competitive advantage within the stem cell compartment, and with the wholesale rewiring of cellular pathways they induce, it is feasible that they may contribute to age-related phenotypes beyond the increased risk of blood cancer. Disclosures Spencer: Wugen, Inc.: Consultancy, Other: Stock Options. Vassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy. Kent: STRM.bio: Research Funding. Campbell: Mu Genomics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Myeloid leukaemia of Down syndrome (ML‐DS) is an acute megakaryoblastic/erythroid leukaemia uniquely found in children with Down syndrome (constitutive trisomy 21). It has a unique clinical course, being preceded by a pre‐leukaemic condition known as transient abnormal myelopoiesis (TAM), and provides an excellent model to study multistep leukaemogenesis. Both TAM and ML‐DS blasts carry acquired N‐terminal truncating mutations in the erythro‐megakaryocytic transcription factor GATA1. These result in exclusive production of a shorter isoform (GATA1s). The majority of TAM cases resolve spontaneously without the need for treatment; however, around 10% acquire additional cooperating mutations and transform to leukaemia, with differentiation block and clinically significant cytopenias. Transformation is driven by the acquisition of additional mutation(s), which cooperate with GATA1s to perturb normal haematopoiesis.

As one of the most highly consumed amino acids in cultured cancer cells, glutamine is an attractive target for anti-cancer therapy, and glutaminase inhibitors are currently in clinical trials. In this issue, Ni et al. show that blocking this pathway by targeting the glutamine importer ASCT2 (SLC1A5) decreases tumorigenesis in mouse leukaemia models while largely sparing normal haematopoiesis.

PURPOSE OF REVIEW Recent work has shown that the Musashi 2 (Msi2) gene plays important roles in normal and malignant hematopoiesis. Here, we give an overview on the role of Msi2 in the regulation and function of primitive hematopoietic cells as well as in leukaemic progression. We also discuss the molecular pathways in which Msi2 acts in both normal and leukaemic blood cells. RECENT FINDINGS Msi2 gain and loss of function experiments have shown that it plays an important role in regulating the heamatopoietic stem cell pool. Msi2 has also been found to be overexpressed in human myeloid leukaemias correlating with poor prognosis, therefore Msi2 may be considered as a prognostic marker for acute myeloid leukaemia. SUMMARY Further studies into the molecular pathways through which Msi2 modulates primitive progenitor function will provide insight into the regulation of normal haematopoiesis and a better understanding of the mechanisms governing the leukaemic transformation process. This will be crucial for the development of effective therapies.

For many years there was a widely accepted picture of how a haematopoietic stem cell (HSC) gives rise to the multiple types of blood and immune cells. This described the general nature of stem and progenitor cells and the pathways of cell development. Recent years have seen many attempts to re-draw the map of haematopoiesis. These have become increasingly complex, and they often envisage multiples routes to some cell types. The ‘established’ view that self-renewal in haematopoiesis only occurs in HSCs has been challenged by the recognition of self-renewing HSC-derived progenitor cells that display at least some fate restriction. This evolution of how normal haematopoiesis is viewed has inevitable implications for understanding the origins, disease progression and classification of the leukaemias. In essence, some progenitor cells are now seen as possessing a larger repertoire of routes to end-fates than was previously thought. This leads one to ask whether leukaemia stem cells are equally or less versatile than their normal counterparts?

Bone marrow samples were collected from the ribs of 20 healthy adult male Iranian camels (Camelus dromedarius). The bone marrow smears were stained using Wright's stain. Blood samples were collected at the same time for routine haematological examination. The development and morphology of the blood cells in the bone marrow of the camels were similar to those of other domestic species. The mean myeloid/erythroid ratio was 1.21, the mean erythroid percentage was 42.7% and the mean myeloid percentage was 52.0%. The mean percentage of other cells was 5.3%. The mean percentage of eosinophils in the myeloid series was higher than in other domestic species. Iron stores, estimated from Perl's stain, ranged from scant (1+) to moderate (3+) but most samples had 2+ iron content. All peripheral blood results were within reference ranges.

Multiple myeloma (mm) is characterized by plasma cell infiltration in the bone marrow, suppression of normal haematopoiesis, destruction of bone, renal failure, and recurrent infections. The extent of organ involvement, severity of complications, drug sensitivity and therefore the clinical course differ widely among patients. Specific and reliable laboratory examinations and especially morphology are required to evaluate its aggressivity and extent and for recognition of complications. In this review of MM and its variants we emphasize the impact of morphology in both bone marrow biopsy and aspiration on the diagnostic and prognostic approach to MM, and on the planning of appropriate therapy.

Successful chemotherapy of acute leukaemias (AL) depends not only on the extent to which leukaemic cells are destroyed, but also on the state of normal haematopoiesis and its capacity for repair.

Written by eleven authors under the editorial guidance of Owen P. Smith and Ian M. Hann, this book of 240 pages aims to inform physicians in training for paediatric haematology, with special emphasis on the progress due to the advances in molecular diagnosis of diseases. Starting with an overview on normal haematopoiesis, leading the reader through the jungle of the cytokine networks and normal coagulation, the chapter on molecular biology and genetic manipulation introduces the main molecular techniques currently used for diagnosis and predicting prognosis of the main malignant haematological disorders. The management of infections is presented in a concise manner and the indications for blood and blood component transfusion are discussed. A chapter on anaemia, with reference to different age groups, is followed by information of the main neoplastic diseases encountered. One may regret a certain lack of coloured illustrations, but summarising tables and figures are concise and informative. A valuable up-date in paediatric haematology. Eur J Pediatr (2003) 162: 733 DOI 10.1007/s00431-003-1244-5

Children with Down syndrome (DS) have an increased risk of developing leukaemias — particularly acute megakaryoblastic leukaemia (AMKL) — but the link between DS and cancer is not well understood. In the September issue of Nature Genetics, Wechsler et al. report that people with DS who develop AMKL all carry a somatic mutation in a gene encoding a transcription factor that is required for normal haematopoiesis.

SUMMARY The lymphoproliferative disorders and the myelodysplastic syndromes are thought to result from the clonal expansion of a single abnormal precursor cell. Seven patients who were found to have concurrent myelodysplasia and lymphoproliferation are described. In all cases myelodysplasia and lymphoproliferation were diagnosed simultaneously or myelodysplasia preceded overt lymphoproliferation by a brief interval. Thus, myelodysplasia in these patients was not related to treatment given for a lymphoproliferative disorder. Stem cell theory is reviewed with special reference to evidence for an adult human stem cell common to both haemopoietic and lymphoid cell lines (CFU-L-M). The presence of concurrent myelodysplasia and lymphoproliferation in these seven patients may be the result of the clonal expansion of an abnormal pluripotential stem cell. It may be possible in some cases to achieve remission of the lymphoproliferative disorder with return of normal haematopoiesis if residual normal stem cells are present and repopulate the marrow following cytotoxic therapy.

A review is given about the normal kinetics of granulopoiesis and the possible effects of drugs on the normal haematopoiesis. Subsequently some properties of acute leukaemia are described, and finally some remarks are made about management policies of acute leukaemia and the effects of cytostatic drugs on normal and leukaemic haematopoiesis.

NORMAL haematopoiesis is initiated and maintained by haematopoietic stem cells. These cells perform two functions: (1) they differentiate into mature haemic elements, and (2) they maintain and renew the population of stem cells. It is these stem cells which seed, differentiate and reproduce, thus effecting successful haematopoietic transplants.