Biology subjects

Mead, A. J.

Publications and source records attributed to Mead, A. J..

12 recordsLinked to original sources

bioRxiv2025

Genome editing for treatment of JAK2 V617F-driven myeloproliferative neoplasms

JAK2 V617F is a common haematological driver mutation and underlies most cases of myeloproliferative neoplasms (MPNs). Reducing variant allele frequency (VAF) is an important treatment goal, but no current treatment modalities fully and specifically inhibit mutant signalling. Thus, the consequences of V617F inactivation are unclear, including whether selective inhibition of V617F signalling will eradicate mutant cells due to oncogene addiction. Here, we describe an allele-selective CRISPR-Cas genome editing strategy that achieves selective and efficient JAK2 V617F inactivation in patient stem and progenitor cells. We show that JAK2 V617F heterozygous cells are not oncogene addicted and maintain viability and differentiation potential upon loss of their mutant allele. In contrast, homozygous mutant cells are eradicated upon deletion of both mutant alleles. Across in vitro, organoid, xenotransplantation and single-cell assays, selective deletion of V617F alleles reverts MPN hallmarks including erythroid clonogenicity, inflammatory gene expression signatures, as well as myelofibrosis and splenomegaly phenotypes in an in vivo xenograft model. Collectively, our results show that is it possible to revert heterozygous JAK2 V617F mutant cells to a normal phenotype and suggest that ex vivo genome editing of stem and progenitor cells may be a viable treatment option to achieve rapid and deep reductions in VAF.

cancer biology↗

bioRxiv2025

Hypercholesterolaemia promotes epigenetic memory in hematopoietic stem cells that persists after lipid lowering and causes systemic immunometabolic dysfunction mediated by macrophage metabolic reprogramming

In patients with atherosclerotic cardiovascular disease (ASCVD) lowering LDL cholesterol (LDL-C) reduces the risk of adverse cardiovascular events. However, these patients remain at continued risk of adverse cardiovascular events despite achieving optimal LDL-C lowering. We hypothesised that this residual risk is mediated by epigenetic programming of haematopoietic stem cells (HSC) which persists after cholesterol levels are lowered and results in sustained effects on innate immune cell metabolism, that is not resolved by cholesterol lowering. We found that exposure to high cholesterol (HC) in vivo induced long-term metabolic changes in macrophages differentiated ex vivo from bone marrow, leading to a pro-inflammatory profile; these changes persisted in vivo despite cholesterol lowering. HSC from HC mice had altered chromatin accessibility that persisted after cholesterol lowering and was also present in bone marrow monocytes and tissue resident macrophages. HC provoked RUNX1-dependent downregulation of stearoyl-CoA desaturase (SCD) which reduced mono-unsaturated fatty acid (MUFA) availability for OxPhos in murine and human monocytes and macrophages. Supplementation with MUFA restored OxPhos capacity and promoted a shift towards a less pro-inflammatory macrophage phenotype in HC-trained BMDM. Bone marrow chimera and lineage tracking studies revealed that prior exposure of HSC to HC conferred adverse systemic metabolic effects on normocholesterolemic mice, with increased adipose tissue mass and increased migration of macrophages derived from HC-exposed HSC into adipose tissue, resulting in increased adipose tissue inflammation and systemic glucose intolerance. These findings indicate that HC results in long-lasting immuno-epigenetic memory in HSC which is refractory to lipid lowering, and provide strong evidence that exposure to HC can have prolonged consequences that require new therapeutic approaches, beyond cholesterol lowering.

immunology↗

bioRxiv2025

comBO: A combined human bone and lympho-myeloid bone marrow organoid for pre-clinical modelling of haematopoietic disorders

The bone marrow supports lifelong blood and immune cell production. Current human bone marrow organoid models do not include both lymphoid and myeloid elements and lack the complexity of stromal cell types present in native haematopoietic tissues, precluding the accurate ex vivo modelling of human pathologies. Here we introduce "comBOs" (combined bone and lympho-myeloid bone marrow organoids) that include osteolineage, vascular, lymphoid and myeloid cells. comBOs are generated by the differentiation of induced pluripotent stem cells guided by physiologically-relevant oxygen and cytokine exposures within an innovative granular microgel scaffold to increase scalability and reproducibility. We demonstrate that comBOs can be used to generate "chimeroids" - incorporating healthy or aberrant cells from adult donors - and recapitulate features of diseased microenvironments. ComBOs are one of the most physiologically-relevant human organoid systems to date, and this study showcases the potential of 3D in vitro disease models for discovery science and translational studies.

cancer biology↗

bioRxiv2023

Chromothripsis orchestrates leukemic transformation in blast phase MPN through targetable amplification of DYRK1A

Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a common event in cancer. Whether chromothripsis might constitute an actionable molecular event amenable to therapeutic targeting remains an open question. We describe recurrent chromothripsis of chromosome 21 in a subset of patients in blast phase of a myeloproliferative neoplasm (BP-MPN), which alongside other structural variants leads to amplification of a region of chromosome 21 in [~]25% of patients ( chr21amp). We report that chr21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. The chr21amp event is highly clonal and present throughout the hematopoietic hierarchy. DYRK1A, a serine threonine kinase and transcription factor, is the only gene in the 2.7Mb minimally amplified region which showed both increased expression and chromatin accessibility compared to non-chr21amp BP-MPN controls. We demonstrate that DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development, including DNA repair, STAT signalling and BCL2 overexpression. DYRK1A is essential for BP-MPN cell proliferation in vitro and in vivo, and DYRK1A inhibition synergises with BCL2 targeting to induce BP-MPN cell apoptosis. Collectively, these findings define the chr21amp event as a prognostic biomarker in BP-MPN and link chromothripsis to a druggable target.

cancer biology↗

bioRxiv2023

Erythroid differentiation intensifies RNA mis-splicing in SF3B1-mutant myelodysplastic syndromes with ring sideroblasts

Myelodysplastic syndromes with ring sideroblasts (MDS-RS) commonly originate from mutations in the splicing factor SF3B1 (SF3B1mt). SF3B1mt cause RNA mis-splicing, mechanistically established as the major driver of RS development. However, little is known about RS fate and biology after their initial formation in the human bone marrow. We here achieve isolation of viable RS from patient samples, enabling the first complete investigation of SF3B1mt development from stem cell to RS. We show that RS skew MACS-isolated CD34+ data towards erythroid features not recapitulated in single-cell RNAseq. We demonstrate that RS divide, differentiate, enucleate and actively respond to mis-splicing/oxidative stress, decreasing wildtype stem cell fitness via GDF15 overproduction. We identify circulating RS as a uniform clinical feature associated with disease burden. Finally, we establish that SF3B1mt mis-splicing intensifies during erythroid differentiation and demonstrate through combined transcriptomics/proteomics an uncoupling of RNA/protein biology in RS encompassing severe and dysfunctional mis-splicing of proapoptotic genes. Statement of significanceWe here combine a novel method for RS isolation with state-of-the-art multiomics to perform the first complete investigation of SF3B1mt MDS-RS hematopoiesis from stem cell to RS. We identify the survival mechanisms underlying SF3B1mt erythropoiesis and establish an active role for erythroid differentiation and RS themselves in SF3B1mt MDS-RS pathogenesis.

cancer biology↗

bioRxiv2022

MARVEL: An integrated alternative splicing analysis platform for single-cell RNA sequencing data

Alternative splicing is an important source of heterogeneity in gene expression between individual cells but remains an understudied area due to the paucity of computational tools to analyze splicing dynamics at single-cell resolution. Here, we present MARVEL, a comprehensive R package for single-cell splicing analysis applicable to RNA sequencing generated from the plate- and droplet-based methods. We performed extensive benchmarking of MARVEL against available tools and demonstrated its utility by analyzing iPSC differentiation into endoderm cells and cardiomyocytes. MARVEL enables systematic and integrated splicing and gene expression analysis of single cells to characterize the splicing landscape and reveal biological insights.

bioinformatics↗

bioRxiv2022

Deciphering TP53 mutant Cancer Evolution with Single-Cell Multi-Omics

TP53 is the most commonly mutated gene in human cancer, typically occurring in association with complex cytogenetics and dismal outcomes. Understanding the genetic and non-genetic determinants of TP53-mutation driven clonal evolution and subsequent transformation is a crucial step towards the design of rational therapeutic strategies. Here, we carry out allelic resolution single-cell multi-omic analysis of haematopoietic stem/progenitor cells (HSPC) from patients with a myeloproliferative neoplasm who transform to TP53-mutant secondary acute myeloid leukaemia (AML), a tractable model of TP53-mutant cancer evolution. All patients showed dominant TP53 multi-hit HSPC clones at transformation, with a leukaemia stem cell transcriptional signature strongly predictive of adverse outcome in independent cohorts, across both TP53-mutant and wild-type AML. Through analysis of serial samples and antecedent TP53-heterozygous clones, we demonstrate a hitherto unrecognised effect of chronic inflammation, which supressed TP53 wild-type HSPC whilst enhancing the fitness advantage of TP53 mutant cells. Our findings will facilitate the development of risk-stratification, early detection and treatment strategies for TP53-mutant leukaemia, and are of broader relevance to other cancer types.

cancer biology↗

bioRxiv2022

Single cell genotyping of matched bone marrow and peripheral blood cells in treatment naive and AZA-treated MDS and CMML

Progressively acquired somatic mutations in hematopoietic stem cells are central to pathogenesis in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). They can lead to proliferative advantages, impaired differentiation and progressive cytopenias. MDS or CMML patients with high-risk disease are treated with hypomethylating agents including 5-azacytidine (AZA). Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. To address this, we characterised the somatic mutations of individual stem, progenitor (common myeloid progenitor, granulocyte monocyte progenitor, megakaryocyte erythroid progenitor), and matched circulating (monocyte, neutrophil, naive B cell) haematopoietic cells in treatment naive and AZA-treated MDS and CMML via high-throughput single cell genotyping. The mutational burden was similar across multiple hematopoietic cell types, and even the most mutated stem and progenitor clones maintained their capacity to differentiate to mature myeloid and, in some cases, lymphoid cell types in vivo. Our data show that even highly mutated HSPCs contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment. Key points* Highly mutated HSPCs contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment. * The mutational burden in matched bone marrow and peripheral blood cells in MDS and CMML was similar throughout myelopoiesis.

cancer biology↗

bioRxiv2021

Developmental stage- and site-specific transitions in lineage specification and gene regulatory networks in human hematopoietic stem and progenitor cells

Human hematopoiesis is a dynamic process that starts in utero 4 weeks post-conception. Understanding the site- and stage-specific variation in hematopoiesis is important if we are to understand the origin of hematological disorders, many of which occur at specific points in the human lifespan. To unravel how the hematopoietic stem/progenitor cell (HSPC) compartments change during human ontogeny and the underlying gene regulatory mechanisms, we compared 57,489 HSPCs from 5 different tissues spanning 4 developmental stages through the human lifetime. Single-cell transcriptomic analysis identified significant site- and developmental stage-specific transitions in cellular architecture and gene regulatory networks. Uncommitted stem cells showed progression from cycling to quiescence and increased inflammatory signalling during ontogeny. We demonstrate the utility of this dataset for understanding aberrant hematopoiesis through comparison to two cancers that present at distinct timepoints in postnatal life - juvenile myelomonocytic leukemia, a childhood cancer, and myelofibrosis, which classically presents in older adults.

developmental biology↗

bioRxiv2020

A novel human fetal liver-derived model reveals that MLL-AF4 drives a distinct fetal gene expression program in infant ALL

Although 90% of children with acute lymphoblastic leukemia (ALL) are now cured1, the prognosis of infant-ALL (diagnosis within the first year of life) remains dismal2. Infant-ALL is usually caused by a single genetic hit that arises in utero: rearrangement of the MLL/KMT2A gene (MLL-r). This is sufficient to give rise to a uniquely aggressive and treatment-refractory leukemia compared to older children with the same MLL-r3-5. The reasons for disparate outcomes in patients of different ages with identical driver mutations are unknown. This paper addresses the hypothesis that fetal-specific gene expression programs co-operate with MLL-AF4 to initiate and maintain infant-ALL. Using direct comparison of fetal and adult HSC and progenitor transcriptomes we identify fetal-specific gene expression programs in primary human cells. We show that MLL-AF4-driven infant-ALL, but not MLL-AF4 childhood-ALL, displays expression of fetal-specific genes. In a direct test of this observation, we find that CRISPR-Cas9 gene editing of primary human fetal liver cells to produce a t(4;11)/MLL-AF4 translocation replicates the clinical features of infant-ALL and drives infant-ALL-specific and fetal-specific gene expression programs. These data strongly support the hypothesis that fetal-specific gene expression programs co-operate with MLL-AF4 to initiate and maintain the distinct biology of infant-ALL.

cancer biology↗

bioRxiv2019

Single-cell analyses reveal aberrant pathways for megakaryocyte-biased hematopoiesis in myelofibrosis and identify mutant clone-specific targets

Myelofibrosis is a severe myeloproliferative neoplasm characterised by increased numbers of abnormal bone marrow megakaryocytes that induce progressive fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed high-throughput single-cell transcriptome profiling of 50,538 hematopoietic stem/progenitor cells (HSPCs), single-cell proteomics, genomics and functional assays. We identified an aberrant pathway for direct megakaryocyte differentiation from the earliest stages of hematopoiesis in myelofibrosis and associated aberrant molecular signatures, including surface antigens selectively expressed by JAK2-mutant HSPCs. Myelofibrosis megakaryocyte progenitors were heterogeneous, with distinct expression of fibrosis and proliferation-associated genes and putative therapy targets. We validated the immunoglobulin receptor G6B as a promising JAK2-mutant clone-specific antigen warranting further development as an immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and more broadly illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.

cancer biology↗

bioRxiv2018

Unraveling intratumoral heterogeneity through high-sensitivity single-cell mutational analysis and parallel RNA-sequencing

Single-cell RNA-sequencing has emerged as a powerful tool to resolve transcriptional heterogeneity. However, its application to study cancerous tissues is currently hampered by the lack of coverage across key mutation hotspots in the vast majority of cells, which prevents correlation of genetic and transcriptional readouts from the same single cell. To overcome this, we developed TARGET-seq, a method for the high-sensitivity detection of multiple mutations within single-cells from both genomic and coding DNA, in parallel with unbiased, high-depth whole transcriptome analysis. We demonstrate how this technique uniquely resolves transcriptional and genetic tumor heterogeneity in myeloproliferative neoplasm stem/progenitor cells, providing insights into deregulated pathways of mutant and non-mutant cells. TARGET-seq provides a powerful tool to resolve molecular signatures of genetically distinct subclones of tumor cells.

genomics↗