Investigating the molecular drivers of CNS disease in a murine model of infant leukaemia

Infant leukaemia is a rare, aggressive entity with poor outcomes. Infant leukaemia is defined by the age of the patient, less than one year, and is commonly associated with rearrangements of the MLL gene. There are many unique features that distinguishMLLrearranged infant leukaemia from other paediatric leukaemias, including a high rate of central nervous system (CNS) involvement. CNS involvement is typically a leukaemic infiltrate of themeninges, a niche that is very different fromthe primary site of disease, the bone marrow (BM), in terms of nutrient abundance and cellular composition. While our understanding of how leukaemia cells survive and propagate in the CNS has progressed, there are many unanswered questions about infant leukaemia-specific features and the contribution of the niche. This thesis explores the cellular dynamics of leukaemia in the CNS niche, the differential regulation of immune cell interactions and growth factor pathways through transplantation assays, bulk RNA sequencing and functional experiments. In previous studies, two microRNAs (miR-128a and miR-130b) were identified as being upregulated in MLL-AF4+ infant patient samples. Overexpression of these microRNAs individually in mouse fetal liver haematopoietic stem and progenitor cells resulted in microRNA-dependent lineage-specific acute leukaemias in the context of Mll- AF4. Terminal leukaemia development in these immunocompetent models of infant leukaemia was associated with CNS involvement in a leptomeningeal distribution representative of human disease. These mouse models form the foundation of the investigation of CNS niche-specific features in infant leukaemia in this thesis. The functional properties of leukaemia propagating cells (LPC) in both niches, in either model, were explored. Data are presented that show different LPCs are very similar, are able to give rise to one another and are all represented in the CNS niche. Further transplantation assays show a lasting functional impact on LPCs and their ability to repopulate leukaemia following exposure to the CNS niche in both model systems. The thesis goes on to strengthen these findings by describing the transcriptomic differiv ences between these LPCs. The RNA sequencing data generated are also used to validate the miR-128a overexpression Mll-AF4, or pro-B infant acute lymphoblastic leukaemia, model. The next section of the thesis outlines transcriptomic differences between LPCs from the CNS and BM niches in both models. The discussion focuses on comparisons to existing datasets and on two novel differentially regulated processes; interaction with immune cells and growth factor signalling. Subsequent sections present functional followup experiments exploring these two novel themes. The data shown describe nichespecific differences in macrophage, T cell and NK cell dynamics and, of particular interest, imply suppression of anti-leukaemia macrophage and T cell responses in the CNS niche. In the final section, increased activation of the PI3K pathway in CNS-derived compared with BM-derived leukaemia cells is explored through downregulation of the inhibitory regulator PTEN.MiR-93, which targets PTENand CDKN1A, a downstream target of the PI3K pathway, is proposed as a global regulator of this cell-intrinsic niche-specific pathway activation. miR-93 is shown to be upregulated in CNS-derived leukaemia cells in the miR-128a overexpressionMll-AF4 model and three independent infant leukaemia patient-derived xenograft models. To conclude this section, suppression of miR-93 activity is shown to impair leukaemia cell engraftment to the CNS niche. Overall, this thesis will put forward new insights into cell-intrinsic and cell-extrinsic mechanisms of leukaemia cell survival and propagation within the CNS niche in infant leukaemia murine models

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

Responses to environmental enrichment differ with sex and genotype in a transgenic mouse model of Huntington's disease.

BACKGROUND: Environmental enrichment (EE) in laboratory animals improves neurological function and motor/cognitive performance, and is proposed as a strategy for treating neurodegenerative diseases. EE has been investigated in the R6/2 mouse model of Huntington's disease (HD), where increased social interaction, sensory stimulation, exploration, and physical activity improved survival. We have also shown previously that HD patients and R6/2 mice have disrupted circadian rhythms, treatment of which may improve cognition, general health, and survival. METHODOLOGY/PRINCIPAL FINDINGS: We examined the effects of EE on the behavioral phenotype and circadian activity of R6/2 mice. Our mice are typically housed in an "enriched" environment, so the EE that the mice received was in addition to these enhanced housing conditions. Mice were either kept in their home cages or exposed daily to the EE (a large playground box containing running wheels and other toys). The "home cage" and "playground" groups were subdivided into "handling" (stimulated throughout the experimental period) and "no-handling" groups. All mice were assessed for survival, body weight, and cognitive performance in the Morris water maze (MWM). Mice in the playground groups were more active throughout the enrichment period than home cage mice. Furthermore, R6/2 mice in the EE/no-handling groups had better survival than those in the home cage/no-handling groups. Sex differences were seen in response to EE. Handling was detrimental to R6/2 female mice, but EE increased the body weight of male R6/2 and WT mice in the handling group. EE combined with handling significantly improved MWM performance in female, but not male, R6/2 mice. CONCLUSIONS/SIGNIFICANCE: We show that even when mice are living in an enriched home cage, further EE had beneficial effects. However, the improvements in cognition and survival vary with sex and genotype. These results indicate that EE may improve the quality of life of HD patients, but we suggest that EE as a therapy should be tailored to individuals

Tissue-specific immunopathology in fatal COVID-19

Funding: Inflammation in COVID-19: Exploration of Critical Aspects of Pathogenesis (ICECAP) receives funding and support from the Chief Scientist Office (RapidResearch in COVID-19 programme [RARC-19] funding call, “Inflammation in Covid-19: Exploration of Critical Aspects of Pathogenesis; COV/EDI/20/10” to D.A.D., C.D.L., C.D.R., J.K.B., and D.J.H.), LifeArc (through the University of Edinburgh STOPCOVID funding award to K.D., D.A.D., and C.D.L.), UK Research and Innovation (UKRI) (Coronavirus Disease [COVID-19] Rapid Response Initiative; MR/V028790/1 to C.D.L., D.A.D., and J.A.H.), and Medical Research Scotland (CVG-1722-2020 to D.A.D., C.D.L., C.D.R., J.K.B., and D.J.H.). C.D.L. is funded by a Wellcome Trust Clinical Career Development Fellowship(206566/Z/17/Z). J.K.B. and C.D.R. are supported by the Medical Research Council (grant MC_PC_19059) as part of the International Severe AcuteRespiratory Infection Consortium Coronavirus Clinical Characterisation Consortium (ISARIC-4C). D.J.H., I.H.U., and M.E. are supported by the Industrial Centre for Artificial Intelligence Research in Digital Diagnostics. S.P. is supported by Kidney Research UK, and G.T. is supported by the Melville Trust for the Cure and Care of Cancer. Identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and sequencing work was supported by theU.S. Food and Drug Administration grant HHSF223201510104C (“Ebola Virus Disease: correlates of protection, determinants of outcome and clinicalmanagement”; amended to incorporate urgent COVID-19 studies) and contract 75F40120C00085 (“Characterization of severe coronavirus infection inhumans and model systems for medical countermeasure development and evaluation”; awarded to J.A.H.). J.A.H. is also funded by the Centre of Excellence in Infectious Diseases Research and the Alder Hey Charity. R.P.-R. is directly supported by the Medical Research Council Discovery Medicine North Doctoral Training Partnership. The group of J.A.H. is supported by the National Institute for Health Research Health Protection Research Unit in Emerging and Zoonotic Infections at the University of Liverpool in partnership with Public Health England and in collaboration with Liverpool School of Tropical Medicine and the University of Oxford.Rationale: In life-threatening Covid-19, corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of SARS-CoV-2 or an independent immunopathologic process is unknown. Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses, and the relationships between viral presence, inflammation, and organ injury. Methods: Tissue was acquired from eleven detailed post-mortem examinations. SARS-CoV-2 organotropism was mapped by multiplex PCR and sequencing, with cellular resolution achieved by in situ viral spike protein detection. Histological evidence of inflammation was quantified from 37 anatomical sites, and the pulmonary immune response characterized by multiplex immunofluorescence. Measurements and main results: Multiple aberrant immune responses in fatal Covid-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein, both between and within tissues. An arteritis was identified in the lung, which was further characterised as a monocyte/myeloid-rich vasculitis, and occurred along with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticulo-endothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues. Conclusions: Tissue-specific immunopathology occurs in Covid-19, implicating a significant component of immune-mediated, virus-independent immunopathology as a primary mechanism in severe disease. Our data highlight novel immunopathological mechanisms, and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma cell responses as well as promoting pathogen tolerance in Covid-19.Publisher PDFPeer reviewe

Responses to environmental enrichment differ with sex and genotype in a transgenic mouse model of Huntington's disease.

BACKGROUND: Environmental enrichment (EE) in laboratory animals improves neurological function and motor/cognitive performance, and is proposed as a strategy for treating neurodegenerative diseases. EE has been investigated in the R6/2 mouse model of Huntington's disease (HD), where increased social interaction, sensory stimulation, exploration, and physical activity improved survival. We have also shown previously that HD patients and R6/2 mice have disrupted circadian rhythms, treatment of which may improve cognition, general health, and survival. METHODOLOGY/PRINCIPAL FINDINGS: We examined the effects of EE on the behavioral phenotype and circadian activity of R6/2 mice. Our mice are typically housed in an "enriched" environment, so the EE that the mice received was in addition to these enhanced housing conditions. Mice were either kept in their home cages or exposed daily to the EE (a large playground box containing running wheels and other toys). The "home cage" and "playground" groups were subdivided into "handling" (stimulated throughout the experimental period) and "no-handling" groups. All mice were assessed for survival, body weight, and cognitive performance in the Morris water maze (MWM). Mice in the playground groups were more active throughout the enrichment period than home cage mice. Furthermore, R6/2 mice in the EE/no-handling groups had better survival than those in the home cage/no-handling groups. Sex differences were seen in response to EE. Handling was detrimental to R6/2 female mice, but EE increased the body weight of male R6/2 and WT mice in the handling group. EE combined with handling significantly improved MWM performance in female, but not male, R6/2 mice. CONCLUSIONS/SIGNIFICANCE: We show that even when mice are living in an enriched home cage, further EE had beneficial effects. However, the improvements in cognition and survival vary with sex and genotype. These results indicate that EE may improve the quality of life of HD patients, but we suggest that EE as a therapy should be tailored to individuals

Tissue-specific tolerance in fatal Covid-19

ABSTRACT Background Tissue inflammation is associated with organ dysfunction and death in Covid-19. The efficacy of dexamethasone in preventing mortality in critical Covid-19 suggests that inflammation has a causal role in death. Whether this deleterious inflammation is a direct response to the presence of SARS-CoV-2, or an independent immuno-pathologic process, is unknown. Methods Tissue was acquired from detailed post-mortem examinations conducted on 11 well characterised hospitalised patients with fatal Covid-19. SARS-CoV-2 organotropism was mapped at an organ level by multiplex PCR and sequencing, with cellular resolution achieved by in situ viral spike (S) protein detection. Histological evidence of inflammation and organ injury was systematically examined, and the pulmonary immune response characterized with multiplex immunofluorescence. Findings SARS-CoV-2 was detected across a wide variety of organs, most frequently in the respiratory tract but also in numerous extra-pulmonary sites. Minimal histological evidence of inflammation was identified in non-pulmonary organs despite frequent detection of viral RNA and protein. At a cellular level, viral protein was identified without adjacent inflammation in the intestine, liver and kidney. Severe inflammatory change was restricted to the lung and reticulo-endothelial system. Diffuse alveolar damage, pulmonary thrombi and a monocyte/myeloid-predominant vasculitis were the predominant pulmonary findings, though there was not a consistent association between viral presence and either the presence or nature of the inflammatory response within the lung. Immunophenotyping revealed an influx of macrophages, monocytes and T cells into pulmonary parenchyma. Bone marrow examination revealed plasmacytosis, erythroid dysplasia and iron-laden macrophages. Plasma cell excess was also present in lymph node, spleen and lung. These stereotyped reticulo-endothelial responses occurred largely independently of the presence of virus in lymphoid tissues. Conclusions Tissue inflammation and organ dysfunction in fatal Covid-19 do not map to the tissue and cellular distribution of SARS-CoV-2, demonstrating tissue-specific tolerance. We conclude that death in Covid-19 is primarily a consequence of immune-mediated, rather than pathogen-mediated, organ inflammation and injury. Funding The Chief Scientist Office, LifeArc, Medical Research Scotland, UKRI (MRC)