Biology subjects

Psaila, B.

Publications and source records attributed to Psaila, B..

14 recordsLinked to original sources

bioRxiv2026

CAR T cell therapy selectively depletes disease-driving mutant calreticulin cells in xenotransplants and human organoid models of myelofibrosis

Targeted immunotherapies have revolutionized outcomes for lymphoid malignancies, but success in myeloid neoplasms is limited by the lack of amenable targets and immunologically hostile tumor microenvironment (TME). Myeloproliferative neoplasms are chronic myeloid blood cancers, a third of which are driven by mutations in calreticulin (mutCALR). This yields a common neoepitope that binds to, and activates, the thrombopoietin receptor and results in display of the oncoprotein on the extracellular membrane of disease-driving cells, exposing a therapeutic vulnerability. Here, we present a first-in-class chimeric antigen receptor (CAR) T-cell therapy that specifically targets mutCALR+ cells, both in vitro and in vivo. The CAR T-cell therapy selectively depleted mutCALR+ stem cells from patients with myelofibrosis while sparing healthy stem cells, and improved survival in mutCALR leukemia xenografts. To mimic myelofibrotic marrow, we developed a bespoke human chimeroid model and showed no decrease in the potency of CAR T cell-mediated target cell killing even in a fibrotic tumor microenvironment. We also devised a method to boost cell surface expression of mutCALR in CD34+ cells isolated from patients with accelerated/blast phase MPN (defined as >10 % blasts in peripheral blood or bone marrow), enhancing CAR T targeting. This study presents a therapeutic with potential to eradicate mutCALR-driven malignancies and highlights an innovative strategy to evaluate blood cancer-targeting immunotherapies in a relevant TME. One Sentence SummaryA first-in-class CAR T-cell therapy targeting mutant calreticulin selectively depletes malignant stem cells in vivo and in fibrotic human organoids.

cancer biology↗

bioRxiv2025

Redefining the topology of the human bone marrow using augmented spatial transcriptomic analysis

The bone marrow (BM) is the main site of haematopoiesis in adult life. Our understanding of the pathogenesis of BM-derived blood cancers is limited by lack of spatial contextualisation. While emerging spatial transcriptomic (ST) platforms offer unprecedented opportunities for spatially-resolved cellular phenotyping, we recognise and incorporate the power of AI-based tissue feature detection to enhance ST workflows. We perform ST analysis to define the topology of the normal bone marrow (BM) and BM in myeloproliferative neoplasms (MPNs), profiling 5,104,452 cells across 30 human BM samples. Following rigorous histology-based QC, we identify spatially-restricted trajectories of haematopoiesis and extend our understanding of the haematopoietic stem cell (HSC) niche. We find that BM fibrosis in MPN is associated with expansion of distinct immune and stromal co-enriched cell neighbourhoods. We then present a machine learning (ML)-based model trained on ST data that quantifies BM microenvironmental deviation, identifying heretofore unrecognised inter- and intra-individual sample heterogeneity in MPN. Our study demonstrates the potential for AI-based augmented ST analysis, and redefines our understanding of human BM topology.

cancer biology↗

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

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↗

bioRxiv2024

The fetal specific gene LIN28B is essential for human fetal B-lymphopoiesis and initiation of KMT2A::AFF1 infant leukemia

Infant ALL (iALL) is initiated in utero, most often by rearrangement of the KMT2A gene (KMT2Ar). It carries a very poor prognosis despite a lack of additional oncogenic driver mutations common in childhood ALL. Here, we aimed to identify specific properties of human fetal hematopoietic stem/progenitor cells (HSPC) that promote leukemic transformation in KMT2Ar iALL using molecular, functional and in vivo assays. First, by comparing transcriptomes of human fetal HSPC to adult HSPC we derived a fetal-specific gene signature and identified the fetal oncogene LIN28B and its downstream effectors among the top hits. These genes were also expressed in iALL. Functional assays revealed that LIN28B was essential in human fetal liver (FL) CD34+ cells to maintain proliferation and stemness, and support B- and NK-lymphopoiesis. To interrogate the role of LIN28B in iALL, we utilised a human FL-derived CRISPR-Cas9 KMT2A::AFF1 model. In this model, LIN28B-expressing leukemias were more proliferative in vitro and in vivo, with this advantage being lost upon LIN28B knockdown. Mechanistic studies showed that LIN28B acts by stabilizing key early B-lymphoid genes, epigenetic regulators, and cell cycle and anti-apoptotic genes. Finally, In the absence of LIN28B, human FL CD34+ cells fail to transform upon induction of KMT2A::AFF1 translocation. Thus, LIN28B has an essential role in normal human fetal B-lymphopoiesis, and is necessary for the initiation of KMT2A::AFF1 iALL in fetal cells in the absence of co-operating mutations. It has a role in making leukemias more aggressive, suggesting it is a potential target in LIN28B-expressing leukemias.

cancer 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↗

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

A pro-inflammatory stem cell niche drives myelofibrosis through a targetable galectin 1 axis

Myeloproliferative neoplasms are stem cell-driven cancers associated with a large burden of morbidity and mortality. The majority of patients present with early-stage disease, but a substantial proportion progress to myelofibrosis and/or secondary leukemia, advanced cancers with a poor prognosis and high symptom burden. Currently, it remains difficult to predict progression, and we lack therapies that reliably prevent or reverse fibrosis development. A major bottleneck to the discovery of disease-modifying therapies has been an incomplete understanding of the interplay between perturbed cellular and molecular states. Several cell types have individually been implicated, but a comprehensive analysis of myelofibrotic bone marrow is lacking. We therefore mapped the crosstalk between bone marrow cell types in myelofibrotic bone marrow. We found that inflammation and fibrosis are orchestrated by a quartet of immune and stromal cell lineages - with basophils and mast cells creating a TNF signaling hub, communicating with megakaryocytes, mesenchymal stromal cells and pro-inflammatory fibroblasts. We identified the y-galactoside binding protein galectin 1 as a striking biomarker of progression to myelofibrosis and poor survival in multiple patient cohorts, and as a promising therapeutic target, with reduced myeloproliferation and fibrosis in vitro and in vivo and improved survival following galectin 1 inhibition. In human bone marrow organoids, TNF increased galectin 1 expression, suggesting a feedback loop wherein the pro-inflammatory MPN clone creates a self-reinforcing niche, fueling progression to advanced disease. This study provides a valuable resource for studying hematopoietic cell-niche interactions, with broad relevance for cancer-associated inflammation and disorders of tissue fibrosis.

cancer biology↗

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

Human bone marrow organoids for disease modelling, discovery and validation of therapeutic targets in hematological malignancies

A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from iPSCs committed to mesenchymal, endothelial and hematopoietic lineages. These 3-dimensional structures capture key features of human bone marrow - stroma, lumen-forming sinusoidal vessels and myeloid cells including pro-platelet forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGF{beta} stimulation and engraftment with myelofibrosis but not healthy donor-derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow-like milieu. This enabling technology is likely to accelerate discovery and prioritization of novel targets for bone marrow disorders and blood cancers. Significance StatementWe present a 3D, vascularised human bone marrow organoid that supports growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment, and provides a much-needed, ex vivo tool for prioritization of new therapeutics.

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↗

bioRxiv2019

Improving the diagnosis and classification of Ph-negative myeloproliferative neoplasms through deep phenotyping

Myeloproliferative neoplasms (MPNs) are clonal disorders characterized by excessive proliferation of myeloid lineages. Accurate classification and appropriate management of MPNs requires integration of clinical, morphological and genetic findings. Despite major advances in understanding the molecular and genetic basis, morphological assessment of the bone marrow trephine (BMT) remains paramount in differentiating between MPN subtypes and reactive conditions. However, morphological assessment is heavily constrained by a reliance on subjective, qualitative and poorly reproducible criteria. To address this, we have developed a machine-learning strategy for the automated identification and quantitative analysis of megakaryocyte morphology using clinical BMT samples. Using a sample cohort of recently diagnosed or established ET (n = 48) and reactive control cases (n = 42) we demonstrated a high predictive accuracy (AUC = 0.95) of automated tissue ET diagnosis based upon these specific megakaryocyte phenotypes. These separate morphological phenotypes showed evidence of specific genotype associations, which offers promise that an automated cell phenotyping approach may be of clinical diagnostic utility as an adjunct to standard genetic and molecular tests. This has great potential to assist in the routine assessment of newly diagnosed or suspected MPN patients and those undergoing treatment / clinical follow-up. The extraction of quantitative morphological data from BMT sections will also have value in the assessment of new therapeutic strategies directed towards the bone marrow microenvironment and can provide clinicians and researchers with objective, quantitative data without significant demands upon current routine specimen workflows.

pathology↗

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↗