Interactions between megakaryocytes and tumour cells in the bone marrow vascular stem cell niche promote tumour growth and metastasis

Specialized bone marrow microenvironments (vascular and osteoblastic 'niches') regulate normal haematopoietic stem/progenitor cells. Recently, the vascular niche has also been implicated as an area for preferential engraftment of malignant cells. The cellular and molecular factors that regulate the vascular niche and, in particular, the role of megakaryocytes are poorly understood. The aim of my work was to investigate the role of megakaryocytes in homing and engraftment of malignant cells to the bone marrow vascular niche using mouse models. C57Bl/6 wild-type and megakaryocyte-deficient, thrombopoietin (TPO)-/- mice were injected with B16 melanoma or EL4 lymphoma cell lines and the megakaryocyte-vascular niche investigated by immunohistochemistry, confocal microscopy, in vitro culture, co-cultures and gene expression by RT-PCR. In wild-type mice injected with B16 melanoma, platelet size and megakaryocyte numbers significantly increased (P<0.02). B16 tumour cells were found to produce the thrombopoietic factors VEGF, SCF and IL11. Bone marrow sinusoids were almost universally surrounded by one of more megakaryocytes tightly abutting the vascular endothelium, forming the megakaryocyte-vascular niche. Metastatic B16 cells were observed in close association with megakaryocytes in the vascular niche, consistent with this being a port of entry to the bone marrow. In TPO-/- mice, tumour growth and metastasis was markedly retarded and no tumour cells were seen in the bone marrow, suggesting that megakaryocytes play a functional role in metastasis. In TPO-/- bone marrow, vessels were more tortuous and larger in diameter (P=0.01); and expression of PF4, TSP1, VEGF and TGFβ was 70%- 90% lower, suggesting that a major proportion of angiogenic regulatory factors is producted by megakaryocytes in the bone marrow in wild-type mice. Furthermore, in wild-type mice, expression of VEGF and TGFβ significantly increased during tumour growth and metastasis while PF4 expression decreased (P<0.05). Megakaryocyte-conditioned medium (MCM) enhanced the proliferation rate of B16 cells (P<0.001) and also was highly chemotactic for B16 cells (P<0.001), an effect mediated by pertussis toxin-sensitive Gi-protein receptors and reduced in the absence of TSP1. Co-culture with B16 cells increased megakaryocyte expression of VEGF, TGFβ and TSP1 and decreased PF4, consistent with the in vivo observations, while cocultured B16 cells displayed increased expression of VEGF and TGFβ and adhesion integrins. Moreover, pretreating B16 cells with MCM prior to tail vein injection enhanced metastatic engraftment. To investigate the role of megakaryocytes in human malignancy, trephine bone marrow biopsies from patients with metastatic carcinoma were examined. Increased megakaryocyte numbers and abnormal megakaryocyte clustering were observed in the majority of patients, suggesting that megakaryocyte-tumour interactions may also occur in the setting of human metastatic disease. In conclusion, my findings suggest that megakaryocytes contribute to the integrity and function of the bone marrow vascular niche and that cellular/molecular cross talk between megakaryocytes and tumour cells may promote metastasis. Targeting these interactions may be useful as adjunctive therapy in metastatic disease

Processing single-cell RNA-seq datasets using SingCellaR

Single-cell RNA sequencing has led to unprecedented levels of data complexity. Although several computational platforms are available, performing data analyses for multiple datasets remains a significant challenge. Here, we provide a comprehensive analytical protocol to interrogate multiple datasets on SingCellaR, an analysis package in R. This tool can be applied to general single-cell transcriptome analyses. We demonstrate steps for data analyses and visualization using bespoke pipelines, in conjunction with existing analysis tools to study human hematopoietic stem and progenitor cells. For complete details on the use and execution of this protocol, please refer to Roy et al. (2021)

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

Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic 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 induced pluripotent stem cells committed to mesenchymal, endothelial, and hematopoietic lineages. These 3D structures capture key features of human bone marrow— stroma, lumen-forming sinusoids, and myeloid cells including proplatelet-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 TGFβ 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 the discovery and prioritization of novel targets for bone marrow disorders and blood cancers. SIGNIFICANCE: We present a human bone marrow organoid that supports the 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 the prioritization of new therapeutics.</p

Single-cell multi-omics identifies chronic inflammation as a driver of TP53-mutant leukemic evolution

Understanding the genetic and nongenetic determinants of tumor protein 53 (TP53)-mutation-driven clonal evolution and subsequent transformation is a crucial step toward the design of rational therapeutic strategies. Here we carry out allelic resolution single-cell multi-omic analysis of hematopoietic stem/progenitor cells (HSPCs) from patients with a myeloproliferative neoplasm who transform to TP53-mutant secondary acute myeloid leukemia (sAML). All patients showed dominant TP53 ‘multihit’ HSPC clones at transformation, with a leukemia stem cell transcriptional signature strongly predictive of adverse outcomes in independent cohorts, across both TP53-mutant and wild-type (WT) AML. Through analysis of serial samples, antecedent TP53-heterozygous clones and in vivo perturbations, we demonstrate a hitherto unrecognized effect of chronic inflammation, which suppressed TP53 WT HSPCs while enhancing the fitness advantage of TP53-mutant cells and promoted genetic evolution. Our findings will facilitate the development of risk-stratification, early detection and treatment strategies for TP53-mutant leukemia, and are of broad relevance to other cancer types

A proinflammatory 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. Most patients present with early-stage disease, but a substantial proportion progress to myelofibrosis or secondary leukemia, advanced cancers with a poor prognosis and high symptom burden. Currently, it remains difficult to predict progression, and therapies that reliably prevent or reverse fibrosis are lacking. 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 cross-talk 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 proinflammatory fibroblasts. We identified the β-galactoside-binding protein galectin-1 as a 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 after galectin-1 inhibition. In human bone marrow organoids, TNF increased galectin-1 expression, suggesting a feedback loop wherein the proinflammatory myeloproliferative neoplasm clone creates a self-reinforcing niche, fueling progression to advanced disease. This study provides a resource for studying hematopoietic cell-niche interactions, with relevance for cancer-associated inflammation and disorders of tissue fibrosis

A blood atlas of COVID-19 defines hallmarks of disease severity and specificity.

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism, and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design, and personalized medicine approaches for COVID-19