The role of GATA-1 isoforms in haematopoiesis

GATA-1 is a key haematopoietic transcription factor which plays a pivotal role in differentiation of the erythroid, megakaryocytic, eosinophilic, mast cell and dendritic cell lineages. Since its initial cloning and characterisation in 1989 a huge amount of information has been gathered on the molecular mechanisms of action of GATA-1. This knowledge has helped understanding of the processes by which cells enact differentiation programmes and suppress alternative lineage choices. GATA-1 produces at least two protein isoforms – the well characterised GATA-1 full-length (GATA-1FL) isoform and a truncated isoform – GATA-1 short (GATA-1s). GATA-1FL comprises two conserved Zinc fingers (which interact with DNA and essential co-factors), a C-terminal tail (of mostly unknown function) and an N-terminal domain (thought to confer activation properties to the molecule, but which may also be involved in transcriptional repression). GATA-1s lacks the N-terminal domain but is otherwise identical. The biological role of GATA-1s is unknown and this isoform received scant attention until the discovery that GATA-1FL mutations were linked to a rare, but highly informative, acute megakaryoblastic leukaemia seen in children with Down syndrome (constitutional trisomy 21). This discovery was particularly interesting, not only because the association between trisomy 21 and the X-linked GATA-1 mutation was extremely tight (being seen in 100% of the cases examined), but also because the GATA-1FL mutations were not randomly located, but rather clustered within the N-terminus, allowing unhindered production of the GATA-1s isoform. This finding led to interest in the pathological and physiological role of GATA-1s in haematopoiesis. Some insight has been gained into the pathological role of GATA-1s by creation of a GATA-1s knock-in transgenic mouse and by experiments looking at the ability of GATA-1s to rescue GATA-1 deficient embryonic stem (ES) cell lines. GATA-1s produces hyper-proliferation of fetal liver meg-erythroid progenitors but allows at least partial differentiation of these cells. However, a number of key questions remain. In particular what is the physiological role of GATA-1s and the reason for the tight association between trisomy 21 and GATA-1s mutations? Given this background, this thesis describes experiments designed to address the physiological role of GATA-1s, to establish whether additional GATA-1 isoforms exist, and to investigate the association between GATA-1 isoform expression and trisomy 21. Firstly a comprehensive expression analysis was performed in murine and human primary tissues and cell lines. This aimed to identify whether GATA-1s had a unique expression profile, either in particular lineages, or at distinct stages of haematological ontogeny. Reverse-transcriptase polymerase chain reaction (RT-PCR) and western blot analyses showed that the expression patterns of GATA-1s and GATA-1FL were virtually identical, with the possible exception of one human primary monocytic cell preparation which appeared to preferentially express GATA-1s. Before proceeding to further analysis of GATA-1s a search was made for additional GATA-1 isoforms using in silico analysis, RT-PCR and western blotting. This led to identification of a clone carrying a GATA-1 mutation involving the C-terminal tail, derived from a patient with chronic myeloid leukaemia. An analysis of the properties of this clone was performed, confirming its altered C-terminus and demonstrating that this conferred increased transactivation properties on the molecule as measured by luciferase assays. This observation suggests that the C-terminal tail may be an important, and previously under-recognised, functional region of the GATA-1 molecule. The discovery of this potentially hyper-functioning GATA-1 mutation led to investigation of whether GATA-1 mutations could be a widespread phenomenon in CML. However, GATA-1 mutational analysis in 21 patient samples from CML blast crisis did not reveal any additional coding mutations. To address the physiological role of GATA-1s, attempts were made to perform gene targeting in murine embryonic stem cells to produce isoform specific knock-out cells i.e. ES cells engineered so that they exclusively express the GATA-1FL isoform (a GATA-1s knock-out) or the GATA-1s isoform (a GATA-1FL knockout). These cells could then be used in in vitro haematopoietic differentiation assays and for transcriptional profiling. In this way it was hoped to establish whether GATA-1s fulfilled any unique roles in primitive or definitive haematopoiesis that could not be compensated for by the presence of the GATA-1FL isoform. Unfortunately, despite evidence of apparently successful targeting from PCR screening of ES cell clones, it was impossible to confirm the existence of endogenously targeted alleles on Southern blotting. Following exhaustive attempts at screening further clones and subclones (more than 1000 clones in total), this approach was abandoned in favour of transgenic expression of GATA-1 isoforms in cell lines. Transgenic expression studies in murine ES cells showed that whilst GATA-1FL expression led to an expansion in numbers and maturity of erythroid and non-erythroid haematopoietic colonies in vitro, GATA-1s was incapable of supporting colony formation in this assay. Studies then moved on to human cell lines. Two cell lines were identified, both capable of in vitro haematopoietic differentiation into megakaryocytic and erythroid cells, but one carrying trisomy 21 (Meg-01) and the other disomic for chromosome 21 (K562). GATA-1FL expression in these cells generally drove differentiation along the megakaryocytic or erythroid lineage as measured by DNA ploidy analysis, haemoglobinisation, upregulation of erythroid or megakaryocytic gene expression (by quantitative PCR) and suppression of alternative lineage genes (PU.1 and Ikaros) and genes associated with progenitor proliferation (cyclin D2 and c-myb). GATA-1s, in contrast, produced less evidence of differentiation with lower DNA ploidy, less up-regulation of erythroid genes and failure to repress other lineage and haematopoietic progenitor associated genes. Examination of the link with trisomy 21 confirmed that that the chromosome 21 candidate gene Erg3 was upregulated in trisomic cells and that expression of GATA-1s appeared to confer a selective advantage in the presence of trisomy 21. However, no clear mechanistic reasons for the selective advantage could be identified. Overall, these studies show widespread GATA-1s expression in haematopoietic cells, confirm the association with inadequate repression of genes associated with primitive progenitors, and suggest that the C-terminal tail of GATA-1 may be an important functional part of the molecule. Finally, these observations have generated a number of testable hypotheses which could form the basis for future work

Challenging "privileged" stereotypes - leukemic blasts and the central nervous system

No abstract available

Comment on: Successful use of nitrous oxide during lumbar punctures: a call for nitrous oxide in pediatric oncology clinics

No abstract available

The use of Ommaya reservoirs to deliver central nervous system directed chemotherapy in childhood acute lymphoblastic leukaemia

Prophylactic eradication of central nervous system (CNS) leukaemia is the current standard of care in treating childhood acute lymphoblastic leukaemia (ALL). This is conventionally achieved through regular lumbar punctures with intrathecal injections of methotrexate into the cerebrospinal fluid (CSF). Ommaya reservoirs are subcutaneous implantable devices that provide a secure route of drug delivery into the CSF via an intraventricular catheter. They are an important alternative in cases where intrathecal injection via lumbar puncture is difficult. Among UK Paediatric Principal Treatment centres for ALL we found considerable variation in methotrexate dosing when using an Ommaya reservoir. We review the current safety and theoretical considerations when using Ommaya reservoirs and evidence for methotrexate dose adjustments via this route. We conclude by summarising the pragmatic consensus decision to use 50% of the conventional intrathecal dose of methotrexate when it is administered via Ommaya reservoir in front-line ALL therapy

Reply: Methotrexate neurotoxicity due to drug interactions: an inadequate folinic acid effect

No abstract available

Drug interactions may be important risk factors for methotrexate neurotoxicity, particularly in pediatric leukemia patients

Purpose: Methotrexate administration is associated with frequent adverse neurological events during treatment for childhood acute lymphoblastic leukemia. Here, we present evidence to support the role of common drug interactions and low vitamin B12 levels in potentiating methotrexate neurotoxicity. Methods: We review the published evidence and highlight key potential drug interactions as well as present clinical evidence of severe methotrexate neurotoxicity in conjunction with nitrous oxide anesthesia and measurements of vitamin B12 levels among pediatric leukemia patients during therapy. Results: We describe a very plausible mechanism for methotrexate neurotoxicity in pediatric leukemia patients involving reduction in methionine and consequential disruption of myelin production. We provide evidence that a number of commonly prescribed drugs in pediatric leukemia management interact with the same folate biosynthetic pathways and/or reduce functional vitamin B12 levels and hence are likely to increase the toxicity of methotrexate in these patients. We also present a brief case study supporting out hypothesis that nitrous oxide contributes to methotrexate neurotoxicity and a nutritional study, showing that patients. Conclusions: Use of nitrous oxide in pediatric leukemia patients at the same time as methotrexate use should be avoided especially as many suitable alternative anesthetic agents exist. Clinicians should consider monitoring levels of vitamin B12 in patients suspected of having methotrexate- induced neurotoxic effects

High throughput sequencing in acute lymphoblastic leukemia reveals clonal architecture of central nervous system and bone marrow compartments

No abstract available

Research into cancer metabolomics: towards a clinical metamorphosis

The acknowledgement that metabolic reprogramming is a central feature of cancer has generated high expectations for major advances in both diagnosis and treatment of malignancies through addressing metabolism. These have so far only been partially fulfilled, with only a few clinical applications. However, numerous diagnostic and therapeutic compounds are currently being evaluated in either clinical trials or pre-clinical models and new discoveries of alterations in metabolic genes indicate future prognostic or other applicable relevance. Altogether, these metabolic approaches now stand alongside other available measures providing hopes for the prospects of metabolomics in the clinic. Here we present a comprehensive overview of both ongoing and emerging clinical, pre-clinical and technical strategies for exploiting unique tumour metabolic traits, highlighting the current promises and anticipations of research in the field

The impact of therapy for childhood acute lymphoblastic leukaemia on intelligence quotients; results of the risk-stratified randomized central nervous system treatment trial MRC UKALL XI

<p>Background: The MRC UKALLXI trial tested the efficacy of different central nervous system (CNS) directed therapies in childhood acute lymphoblastic leukaemia (ALL). To evaluate morbidity 555/1826 randomised children underwent prospective psychological evaluations. Full Scale, verbal and performance IQs were measured at 5 months, 3 years and 5 years. Scores were compared in; (1) all patients (n = 555) versus related controls (n = 311), (2) low-risk children (presenting white cell count (WCC) < 50 × 109/l) randomised to intrathecal methotrexate (n = 197) versus intrathecal and high-dose intravenous methotrexate (HDM) (n = 202), and (3) high-risk children (WCC ≥ 50 × 109/l, age ≥ 2 years) randomised to HDM (n = 79) versus cranial irradiation (n = 77).</p> <p>Results: There were no significant differences in IQ scores between the treatment arms in either low- or high-risk groups. Despite similar scores at baseline, results at 3 and 5 years showed a significant reduction of between 3.6 and 7.3 points in all three IQ scores in all patient groups compared to controls (P < 0.002) with a higher proportion of children with IQs < 80 in the patient groups (13% vs. 5% at 3 years p = 0.003).</p> <p>Conclusion: Children with ALL are at risk of CNS morbidity, regardless of the mode of CNS-directed therapy. Further work needs to identify individuals at high-risk of adverse CNS outcomes.</p&gt

Central nervous system involvement in childhood acute lymphoblastic leukemia: challenges and solutions

Delivery of effective anti-leukemic agents to the central nervous system (CNS) is considered essential for cure of childhood acute lymphoblastic leukemia. Current CNS-directed therapy comprises systemic therapy with good CNS-penetration accompanied by repeated intrathecal treatments up to 26 times over 2–3 years. This approach prevents most CNS relapses, but is associated with significant short and long term neurotoxicity. Despite this burdensome therapy, there have been no new drugs licensed for CNS-leukemia since the 1960s, when very limited anti-leukemic agents were available and there was no mechanistic understanding of leukemia survival in the CNS. Another major barrier to improved treatment is that we cannot accurately identify children at risk of CNS relapse, or monitor response to treatment, due to a lack of sensitive biomarkers. A paradigm shift in treating the CNS is needed. The challenges are clear – we cannot measure CNS leukemic load, trials have been unable to establish the most effective CNS treatment regimens, and non-toxic approaches for relapsed, refractory, or intolerant patients are lacking. In this review we discuss these challenges and highlight research advances aiming to provide solutions. Unlocking the potential of risk-adapted non-toxic CNS-directed therapy requires; (1) discovery of robust diagnostic, prognostic and response biomarkers for CNS-leukemia, (2) identification of novel therapeutic targets combined with associated investment in drug development and early-phase trials and (3) engineering of immunotherapies to overcome the unique challenges of the CNS microenvironment. Fortunately, research into CNS-ALL is now making progress in addressing these unmet needs: biomarkers, such as CSF-flow cytometry, are now being tested in prospective trials, novel drugs are being tested in Phase I/II trials, and immunotherapies are increasingly available to patients with CNS relapses. The future is hopeful for improved management of the CNS over the next decade