Biology subjects

Wilkinson, A. C.

Publications and source records attributed to Wilkinson, A. C..

13 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

The ERCC6L2-MRI-KU complex coordinates NHEJ at staggered DNA double-strand breaks.

ERCC6L2 disease is a recessive bone marrow failure (BMF) syndrome caused by mutations in the SNF2-like putative DNA helicase ERCC6L2. While implicated in DNA replication, double strand break (DSB) repair via non-homologous end joining (NHEJ), and interstrand crosslink (ICL) repair, how ERCC6L2 supports haematopoietic longevity remains unclear. Investigating this in vivo, we find that an Ercc6l2-deficient haematopoietic stem and progenitor cell (HSPC) compartment in mice is unexpectedly resilient. Ercc6l2 loss was also tolerated in mice co-deficient for endogenous formaldehyde detoxification, which precipitates early-onset BMF in models of Fanconi anaemia. Instead, Ercc6l2-deficient mice display a mild immunodeficiency, arising from defects in immunoglobulin class-switch recombination (CSR), that synergise with shieldin-deficiency, implicating ERCC6L2 and shieldin in distinct repair mechanisms. Furthermore, we demonstrate that ERRC6L2 stimulates chromosome fusions in the context of staggered, but not blunt dysfunctional telomeres. We reconcile ERCC6L2s NHEJ function through proteomic elucidation of its endogenous interactome and AlphaFold structural modelling to reveal a complex formed of ERCC6L2 and KU that is bridged by the NHEJ accessory factor MRI/CYREN. Consequently, ERCC6L2-MRI inter-dependence characterises CSR. Together, our findings implicate the ERCC6L2-MRI complex as a KU-regulatory DNA translocase coordinating classical-NHEJ at staggered-end DSBs. We suggest that similar staggered-end breaks represent the pathological substrates driving haematopoietic failure in ERCC6L2 disease.

molecular biology↗

bioRxiv2025

Polymer-based culture system enables expansion of human haematopoietic stem and progenitor cells while preserving ageing-associated transcriptional programs

The global rise in ageing populations is driving an increase in age-associated diseases, including haematological malignancies such as myelodysplastic syndromes and acute myeloid leukaemia. A major challenge in addressing this burden is the lack of experimental systems that enable mechanistic studies and therapeutic screening in ageing haematopoiesis. Here, we evaluate a polymer-based ex vivo culture platform for studying human haematopoietic stem and progenitor cells (HSPCs) in the context of ageing. Using CD34+ cells from cord blood, bone marrow and peripheral blood from different age groups, we show that this culture system enables robust expansion of HSPCs while preserving age-associated transcriptional programs. Single-cell RNA sequencing revealed enrichment of key progenitor populations with consistent transcriptional identities across donors. Importantly, expanded HSPCs from older individuals retained ageing-associated signatures, such as upregulation of inflammatory pathways. These findings demonstrate that our polymer-based expansion system enables robust modelling of ageing haematopoiesis and provide a single cell transcriptional landscape of this ageing model. We envision that this system could provide a versatile platform for mechanistic studies and pharmacological testing, addressing a critical gap in experimental frameworks for age-related haematological disease research.

cell biology↗

bioRxiv2025

Inhibition of MLLT1 limits growth of MLL-AF4 leukaemias without killing healthy haematopoietic stem cells

A major challenge in cancer therapeutics has been the identification of targets that are selectively toxic to cancer cells while displaying limited effects on healthy counterparts. Toxicities related to blood production from haematopoietic stem and progenitor cells (HSPCs) can be particularly problematic and result in patient morbidity and mortality. Within haematological malignancies, therapy response rates and patient survival vary widely between cancer subtypes, with leukaemias driven by the MLL-AF4 fusion protein associated with poor prognosis. MLLT1 has been recently identified as a key potential target in acute myeloid leukaemia. Here we evaluated a panel of leukaemia cell lines and healthy HSPCs for their sensitivity to the MLLT1 inhibitor SGC-iMLLT. We found that SGC-iMLLT strongly inhibited MLL-AF4-driven leukaemia growth in vitro and in vivo. By contrast, SGC-iMLLT did not alter in vitro colony forming potential of human HSPCs or affect long-term in vivo function of mouse HSPCs. These results suggest that SGC-iMLLT may have a promising therapeutic window in the treatment of MLL-AF4-driven leukaemias, and that further clinical development is warranted.

cancer biology↗

bioRxiv2025

Development of iPSC-Derived T Cells Targeting EGFR Neoantigens in Non-Small Cell Lung Cancer

A long-sought goal of cancer immunotherapy is to mass-produce T cells that specifically target tumor neoantigens. One decisive challenge is the identification of neoantigens derived from cancer driver genes. Here, we identify T cells that recognize the NSCLC-associated EGFR C797S mutation, which confers resistance to current inhibitors and is linked to poor prognosis. To overcome limitations in T cell availability, we reprogrammed EGFR C797S-specific T cells into induced pluripotent stem cells (iPSCs) and re-differentiated them into CD8 T cells. These iPSC-derived T cells specifically recognized the EGFR C797S mutation and effectively killed cancer cells expressing this mutation. Our findings underscore the potential of targeting driver mutation-derived neoantigens for immunotherapy and demonstrate that iPSC-derived T cells can mediate antitumor effects. Collectively, this approach combining neoantigen identification with T cell reprogramming may offer a promising strategy for targeting drug-resistant tumors.

immunology↗

bioRxiv2024

Reducing oxidative stress improves ex vivo polymer-based human haematopoietic stem and progenitor cell culture and gene editing

Self-renewing multipotent haematopoietic stem cells (HSCs) have the unique capacity to stably regenerate the entire blood and immune systems following transplantation. HSCs are used clinically to reconstitute a healthy blood system in patients suffering from a range of haematological diseases. However, HSCs are very rare and have been challenging to grow ex vivo, which has hampered efforts to collect large numbers of HSCs for both basic research and clinical therapies. Polymer-based culture conditions have recently been developed to support expansion of mouse and human haematopoietic stem and progenitor cells (HSPCs). While mouse HSPCs expanded rapidly in polymer-based cultures, growth speeds for human HSPCs in polymer-based cultures was limited to [~]70-fold over 4-weeks. Here we have found that reducing oxidative stress improves human HSPC growth in these conditions. We describe an optimised culture condition that improves growth to 250-1400-fold over 4-weeks through reducing oxygen tension and the addition of antioxidants. These conditions also enable efficient gene editing in these polymer-based cultures. We envision these improved culture conditions will support a range of research into human HSPC biology and provide a platform for clinical-scale HSPC expansion and gene editing.

cell biology↗

bioRxiv2024

Hypoxia-Inducible Factor-1 activated by PIM1 assembles a non-canonical transcription complex and resultant regulon that drives progression of JAK2V617F myeloproliferative neoplasms

Hypoxia-inducible factors (HIFs) are master transcriptional regulators, central to cellular survival under limited oxygen (hypoxia) and frequently activated within malignancy. Malignant context affects the role of HIFs within oncogenesis; however, the mechanisms regulating HIF context-specificities are not well characterised. Applying the JAK2V617F (JVF) model of myeloproliferative neoplasms (MPNs), in which HIF-1 is active in normoxia (20% O2), we sought to determine whether the modality of HIF-1 activation directs its function. We identify that HIF-1 is stabilised in JVF cells downstream of STAT1/5 signalling and upregulation of PIM1: PIM1 mediates phosphorylation of HIF-1 (Thr498/Ser500) in JVF cells that inhibits proteasomal degradation. PIM1 inhibition eradicates HIF-1 from JVF cells. Applying a single-input dual-omics output chromatin interactome methodology (DOCIA), we define JVF-specific transcription cofactors and genomic redistribution of HIF-1, and a JVF-HIF-1 regulon in primary haematopoietic stem/progenitor cells. In a cohort of 172 JVF-MPN patients, we observe significant association of the JVF-HIF-1 regulon (but strikingly, not canonical HIF-1 genes) with disease severity, progression, and patient survival. Finally, we identify a core set of JVF-HIF-1 targets significantly associated with spontaneous transformation of MPNs to AML. Our findings identify that HIF-1 activation by the JVF-PIM1 axis substantially alters its function, and that this reprogramming drives MPN disease progression, restoring the potential for targeted therapies that delineate HIF-1 activity co-opted by malignancy from essential roles within physiological oxygen homeostasis. Key PointsO_LIHIF-1 activation via PIM1 in JAK2V617F-MPNs drives non-canonical transcription complex formation/function. C_LIO_LIThe JAK2V617F-HIF-1 regulon drives MPN disease progression, transformation to AML and worse patient outcomes. C_LI

cancer biology↗

bioRxiv2022

Large-scale in vivo CRISPR screens identify SAGA complex members as a key regulators of HSC lineage commitment and aging

The biological mechanisms that sustain the vast blood production required for healthy life remain incompletely understood. To search for novel regulators of hematopoiesis, we performed genome-wide in vivo hematopoietic stem cell (HSC)-based CRISPR knockout screens for regulators of hematopoiesis. We discovered SAGA complex members, including Tada2b and Taf5l, as key regulators of hematopoiesis. Loss of Tada2b or Taf5l strongly inhibited hematopoiesis in vivo, led to a buildup of immature hematopoietic cells in the bone marrow, and was associated with upregulation of interferon pathway genes. Loss of these factors also enhanced the cell outgrowth and the interferon pathway in an in vivo human myelodysplastic syndrome model, suggesting that loss of SAGA complex activity could contribute to hematological disease progression. In summary, this study has identified the SAGA complex as an important regulator of hematopoiesis.

genetics↗

bioRxiv2022

A Single Cell Cloning Platform for Gene Edited Functional Murine Hematopoietic Stem Cells

Gene editing using engineered nucleases frequently produces on- and off-target indels in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain genetically heterogenous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin- population of pre-cultured HSCs. Using the Prkdcscid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo-manipulation platform establishes a novel paradigm to control genetic heterogeneity in HSC gene editing and therapy.

cell biology↗

bioRxiv2021

A highly efficient reporter system for identifying and characterizing in vitro expanded hematopoietic stem cells

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved more than 200-fold expansion of functional HSCs, but their molecular characterization has not been possible due to the substantial majority of cells being non-HSCs and single cell-initiated cultures displaying substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. Linking single clone functional transplantation data with single clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to actively cycling fetal liver HSCs and shares a gene expression signature with functional HSCs from all sources, including Prdm16, Fstl1 and Palld. This new tool can now be applied to a wide-range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

cell biology↗

bioRxiv2020

Streamlined and quantitative detection of chimerism in mouse tissue using digital PCR

Animal chimeras are widely used for biomedical discoveries, from developmental biology to cancer research. However, the accurate quantitation of mixed cell types in chimeric and mosaic tissues has challenges. Here, we have developed and characterized a droplet digital PCR single-nucleotide discrimination assay to detect chimerism among albino and non-albino mouse strains. In addition, we have validated that this assay is compatible with crude lysate from most organs, drastically streamlining sample preparation. This chimerism detection assay has many additional advantages over existing methods including its robust nature, minimal technical bias, and ability to report the total number of cells in a prepared sample. Importantly, the concepts developed and discussed here are readily adapted to other genomic loci to accurately measure mixed cell populations in any tissue.

genetics↗

bioRxiv2020

Cas9-AAV6 Gene Correction of Beta-Globin in Autologous HSCs Improves Sickle Cell Disease Erythropoiesis in Mice

CRISPR/Cas9-mediated beta-globin (HBB) gene correction of Sickle Cell Disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a novel paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated. Here, we used a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observed a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo RBC features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.

developmental biology↗

bioRxiv2017

Establishment In Culture Of Expanded Potential Stem Cells

Mouse embryonic stem cells are derived from in vitro explantation of blastocyst epiblasts1,2 and contribute to both the somatic lineage and germline when returned to the blastocyst3 but are normally excluded from the trophoblast lineage and primitive endoderm4-6. Here, we report that cultures of expanded potential stem cells (EPSCs) can be established from individual blastomeres, by direct conversion of mouse embryonic stem cells (ESCs) and by genetically reprogramming somatic cells. Remarkably, a single EPSC contributes to the embryo proper and placenta trophoblasts in chimeras. Critically, culturing EPSCs in a trophoblast stem cell (TSC) culture condition permits direct establishment of TSC lines without genetic modification. Molecular analyses including single cell RNA-seq reveal that EPSCs share cardinal pluripotency features with ESCs but have an enriched blastomere transcriptomic signature and a dynamic DNA methylome. These proof-of-concept results open up the possibility of establishing cultures of similar stem cells in other mammalian species.

developmental biology↗