Progressive disruption of hematopoietic architecture from clonal hematopoiesis to MDS.

Clonal hematopoiesis of indeterminate potential (CHIP) describes the age-related acquisition of somatic mutations in hematopoietic stem/progenitor cells (HSPC) leading to clonal blood cell expansion. Although CHIP mutations drive myeloid malignancies like myelodysplastic syndromes (MDS) it is unknown if clonal expansion is attributable to changes in cell type kinetics, or involves reorganization of the hematopoietic hierarchy. Using computational modeling we analyzed differentiation and proliferation kinetics of cultured hematopoietic stem cells (HSC) from 8 healthy individuals, 7 CHIP, and 10 MDS patients. While the standard hematopoietic hierarchy explained HSPC kinetics in healthy samples, 57% of CHIP and 70% of MDS samples were best described with alternative hierarchies. Deregulated kinetics were found at various HSPC compartments with high inter-individual heterogeneity in CHIP and MDS, while altered HSC rates were most relevant in MDS. Quantifying kinetic heterogeneity in detail, we show that reorganization of the HSPC compartment is already detectable in the premalignant CHIP state.

Choosing the source of healthy controls for studies on myeloid malignancies: all bone marrow cells are created equal, but some are more equal than others.

Bone marrow samples from discarded femoral heads are often used as healthy controls in studies investigating the in vitro characteristics of cells from patients with hematologic malignancies. Since patient samples are usually derived from iliac crest aspirates, this carries the risk that the properties of the cells from both sources might be different due to the site and method of harvesting. Comparing BM cells from iliac crest aspirates and femoral heads from age-matched healthy donors, we show that, while mesenchymal stromal cells have indistinguishable properties between both sources, hematopoietic stem and progenitor cells (HSPC) from femoral heads show a considerable proliferative advantage in vitro. These data therefore suggest that experiments comparing leukemic cells from the iliac crest to healthy HSPC obtained from femoral heads should be interpreted with caution.

Comparative analysis of extracellular vesicle isolation methods from human AML bone marrow cells and AML cell lines.

Cellular crosstalk between hematopoietic stem/progenitor cells and the bone marrow (BM) niche is vital for the development and maintenance of myeloid malignancies. These compartments can communicate via bidirectional transfer of extracellular vesicles (EVs). EV trafficking in acute myeloid leukemia (AML) plays a crucial role in shaping the BM microenvironment into a leukemia-permissive niche. Although several EV isolation methods have been developed, it remains a major challenge to define the most accurate and reliable procedure. Here, we tested the efficacy and functional assay compatibility of four different EV isolation methods in leukemia-derived EVs: (1) membrane affinity-based: exoEasy Kit alone and (2) in combination with Amicon filtration; (3) precipitation: ExoQuick-TC; and (4) ultracentrifugation (UC). Western blot analysis of EV fractions showed the highest enrichment of EV marker expression (e.g., CD63, HSP70, and TSG101) by precipitation with removal of overabundant soluble proteins [e.g., bovine serum albumin (BSA)], which were not discarded using UC. Besides the presence of damaged EVs after UC, intact EVs were successfully isolated with all methods as evidenced by highly maintained spherical- and cup-shaped vesicles in transmission electron microscopy. Nanoparticle tracking analysis of EV particle size and concentration revealed significant differences in EV isolation efficacy, with exoEasy Kit providing the highest EV yield recovery. Of note, functional assays with exoEasy Kit-isolated EVs showed significant toxicity towards treated target cells [e.g., mesenchymal stromal cells (MSCs)], which was abrogated when combining exoEasy Kit with Amicon filtration. Additionally, MSC treated with green fluorescent protein (GFP)-tagged exoEasy Kit-isolated EVs did not show any EV uptake, while EV isolation by precipitation demonstrated efficient EV internalization. Taken together, the choice of EV isolation procedure significantly impacts the yield and potential functionality of leukemia-derived EVs. The cheapest method (UC) resulted in contaminated and destructed EV fractions, while the isolation method with the highest EV yield (exoEasy Kit) appeared to be incompatible with functional assays. We identified two methods (precipitation-based ExoQuick-TC and membrane affinity-based exoEasy Kit combined with Amicon filtration) yielding pure and intact EVs, also suitable for application in functional assays. This study highlights the importance of selecting the right EV isolation method depending on the desired experimental design.

Compartment-specific mutational landscape of clonal hematopoiesis.

Clonal hematopoiesis (CH) is characterized by somatic mutations in blood cells of individuals without hematologic disease. While the mutational landscape of CH in peripheral blood (PB) has been well characterized, detailed analyses addressing its spatial and cellular distribution in the bone marrow (BM) compartment are sparse. We studied CH driver mutations in healthy individuals (n = 261) across different anatomical and cellular compartments. Variant allele frequencies were higher in BM than PB and positively correlated with the number of driver variants, yet remained stable during a median of 12 months of follow-up. In CH carriers undergoing simultaneous bilateral hip replacement, we detected ASXL1-mutant clones in one anatomical location but not the contralateral side, indicating intra-patient spatial heterogeneity. Analyses of lineage involvement in ASXL1-mutated CH showed enriched clonality in BM stem and myeloid progenitor cells, while lymphocytes were particularly involved in individuals carrying the c.1934dupG variant, indicating different ASXL1 mutations may have distinct lineage distribution patterns. Patients with overt myeloid malignancies showed higher mutation numbers and allele frequencies and a shifting mutation landscape, notably characterized by increasing prevalence of DNMT3A codon R882 variants. Collectively, our data provide novel insights into the genetics, evolution, and spatial and lineage-specific BM involvement of CH.

Comprehensive characterization of central BCL-2 family members in aberrant eosinophils and their impact on therapeutic strategies.

PURPOSE: Hypereosinophilia represents a heterogenous group of severe medical conditions characterized by elevated numbers of eosinophil granulocytes in peripheral blood, bone marrow or tissue. Treatment options for hypereosinophilia remain limited despite recent approaches including IL-5-targeted monoclonal antibodies and tyrosine kinase inhibitors. METHODS: To understand aberrant survival patterns and options for pharmacologic intervention, we characterized BCL-2-regulated apoptosis signaling by testing for BCL-2 family expression levels as well as pharmacologic inhibition using primary patient samples from diverse subtypes of hypereosinophilia (hypereosinophilic syndrome n = 18, chronic eosinophilic leukemia not otherwise specified n = 9, lymphocyte-variant hypereosinophilia n = 2, myeloproliferative neoplasm with eosinophilia n = 2, eosinophilic granulomatosis with polyangiitis n = 11, reactive eosinophilia n = 3). RESULTS: Contrary to published literature, we found no difference in the levels of the lncRNA Morrbid and its target BIM. Yet, we identified a near complete loss of expression of pro-apoptotic PUMA as well as a reduction in anti-apoptotic BCL-2. Accordingly, BCL-2 inhibition using venetoclax failed to achieve cell death induction in eosinophil granulocytes and bone marrow mononuclear cells from patients with hypereosinophilia. In contrast, MCL1 inhibition using S63845 specifically decreased the viability of bone marrow progenitor cells in patients with hypereosinophilia. In patients diagnosed with Chronic Eosinophilic Leukemia (CEL-NOS) or Myeloid and Lymphatic Neoplasia with hypereosinophilia (MLN-Eo) repression of survival was specifically powerful. CONCLUSION: Our study shows that MCL1 inhibition might be a promising therapeutic option for hypereosinophilia patients specifically for CEL-NOS and MLN-Eo.

AXL Inhibition in Macrophages Stimulates Host-versus-Leukemia Immunity and Eradicates Naïve and Treatment-Resistant Leukemia.

Acute leukemias are systemic malignancies associated with a dire outcome. Because of low immunogenicity, leukemias display a remarkable ability to evade immune control and are often resistant to checkpoint blockade. Here, we discover that leukemia cells actively establish a suppressive environment to prevent immune attacks by co-opting a signaling axis that skews macrophages toward a tumor-promoting tissue repair phenotype, namely the GAS6/AXL axis. Using aggressive leukemia models, we demonstrate that ablation of the AXL receptor specifically in macrophages, or its ligand GAS6 in the environment, stimulates antileukemic immunity and elicits effective and lasting natural killer cell- and T cell-dependent immune response against naïve and treatment-resistant leukemia. Remarkably, AXL deficiency in macrophages also enables PD-1 checkpoint blockade in PD-1-refractory leukemias. Finally, we provide proof-of-concept that a clinical-grade AXL inhibitor can be used in combination with standard-of-care therapy to cure established leukemia, regardless of AXL expression in malignant cells. SIGNIFICANCE: Alternatively primed myeloid cells predict negative outcome in leukemia. By demonstrating that leukemia cells actively evade immune control by engaging AXL receptor tyrosine kinase in macrophages and promoting their alternative priming, we identified a target which blockade, using a clinical-grade inhibitor, is vital to unleashing the therapeutic potential of myeloid-centered immunotherapy.This article is highlighted in the In This Issue feature, p. 2659.

CHIP and hips: clonal hematopoiesis is common in patients undergoing hip arthroplasty and is associated with autoimmune disease.

Clonal hematopoiesis (CH) is an age-related condition predisposing to blood cancer and cardiovascular disease (CVD). Murine models demonstrate CH-mediated altered immune function and proinflammation. Low-grade inflammation has been implicated in the pathogenesis of osteoarthritis (OA), the main indication for total hip arthroplasty (THA). THA-derived hip bones serve as a major source of healthy hematopoietic cells in experimental hematology. We prospectively investigated frequency and clinical associations of CH in 200 patients without known hematologic disease who were undergoing THA. Prevalence of CH was 50%, including 77 patients with CH of indeterminate potential (CHIP, defined as somatic variant allele frequencies [VAFs] ≥2%), and 23 patients harboring CH with lower mutation burden (VAF, 1% to 2%). Most commonly mutated genes were DNMT3A (29.5%), TET2 (15.0%), and ASXL1 (3.5%). CHIP is significantly associated with lower hemoglobin, higher mean corpuscular volume, previous or present malignant disease, and CVD. Strikingly, we observed a previously unreported association of CHIP with autoimmune diseases (AIDs; multivariable adjusted odds ratio, 6.6; 95% confidence interval, 1.7-30; P = .0081). These findings underscore the association between CH and inflammatory diseases. Our results have considerable relevance for managing patients with OA and AIDs or mild anemia and question the use of hip bone-derived cells as healthy experimental controls.

Tens of images can suffice to train neural networks for malignant leukocyte detection.

Convolutional neural networks (CNNs) excel as powerful tools for biomedical image classification. It is commonly assumed that training CNNs requires large amounts of annotated data. This is a bottleneck in many medical applications where annotation relies on expert knowledge. Here, we analyze the binary classification performance of a CNN on two independent cytomorphology datasets as a function of training set size. Specifically, we train a sequential model to discriminate non-malignant leukocytes from blast cells, whose appearance in the peripheral blood is a hallmark of leukemia. We systematically vary training set size, finding that tens of training images suffice for a binary classification with an ROC-AUC over 90%. Saliency maps and layer-wise relevance propagation visualizations suggest that the network learns to increasingly focus on nuclear structures of leukocytes as the number of training images is increased. A low dimensional tSNE representation reveals that while the two classes are separated already for a few training images, the distinction between the classes becomes clearer when more training images are used. To evaluate the performance in a multi-class problem, we annotated single-cell images from a acute lymphoblastic leukemia dataset into six different hematopoietic classes. Multi-class prediction suggests that also here few single-cell images suffice if differences between morphological classes are large enough. The incorporation of deep learning algorithms into clinical practice has the potential to reduce variability and cost, democratize usage of expertise, and allow for early detection of disease onset and relapse. Our approach evaluates the performance of a deep learning based cytology classifier with respect to size and complexity of the training data and the classification task.

Computational modeling of stem and progenitor cell kinetics identifies plausible hematopoietic lineage hierarchies.

Classically, hematopoietic stem cell (HSC) differentiation is assumed to occur via progenitor compartments of decreasing plasticity and increasing maturity in a specific, hierarchical manner. The classical hierarchy has been challenged in the past by alternative differentiation pathways. We abstracted experimental evidence into 10 differentiation hierarchies, each comprising 7 cell type compartments. By fitting ordinary differential equation models with realistic waiting time distributions to time-resolved data of differentiating HSCs from 10 healthy human donors, we identified plausible lineage hierarchies and rejected others. We found that, for most donors, the classical model of hematopoiesis is preferred. Surprisingly, multipotent lymphoid progenitor differentiation into granulocyte-monocyte progenitors is plausible in 90% of samples. An in silico analysis confirmed that, even for strong noise, the classical model can be identified robustly. Our computational approach infers differentiation hierarchies in a personalized fashion and can be used to gain insights into kinetic alterations of diseased hematopoiesis.

Bone marrow stromal cells from MDS and AML patients show increased adipogenic potential with reduced Delta-like-1 expression.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. Increasing evidence suggests that alterations in the non-hematopoietic microenvironment (bone marrow niche) can contribute to or initiate malignant transformation and promote disease progression. One of the key components of the bone marrow (BM) niche are BM stromal cells (BMSC) that give rise to osteoblasts and adipocytes. It has been shown that the balance between these two cell types plays an important role in the regulation of hematopoiesis. However, data on the number of BMSC and the regulation of their differentiation balance in the context of hematopoietic malignancies is scarce. We established a stringent flow cytometric protocol for the prospective isolation of a CD73+ CD105+ CD271+ BMSC subpopulation from uncultivated cryopreserved BM of MDS and AML patients as well as age-matched healthy donors. BMSC from MDS and AML patients showed a strongly reduced frequency of CFU-F (colony forming unit-fibroblast). Moreover, we found an altered phenotype and reduced replating efficiency upon passaging of BMSC from MDS and AML samples. Expression analysis of genes involved in adipo- and osteogenic differentiation as well as Wnt- and Notch-signalling pathways showed significantly reduced levels of DLK1, an early adipogenic cell fate inhibitor in MDS and AML BMSC. Matching this observation, functional analysis showed significantly increased in vitro adipogenic differentiation potential in BMSC from MDS and AML patients. Overall, our data show BMSC with a reduced CFU-F capacity, and an altered molecular and functional profile from MDS and AML patients in culture, indicating an increased adipogenic lineage potential that is likely to provide a disease-promoting microenvironment.