Osteopenia and osteoporosis are common long-term complications of the cytotoxic conditioning regimen for hematopoietic stem cell transplantation (HSCT). We examined mesenchymal stem and progenitor cells (MSPCs) that include skeletal progenitors from mice undergoing HSCT. Such MSPCs showed reduced CFU-F frequency, increased DNA damage and enhanced occurrence of cellular senescence, while there was a reduced bone volume in animals that underwent HSCT. This reduced MSPC function correlated with elevated activation of the small RhoGTPAse Cdc42, disorganized F-actin distribution, mitochondrial abnormalities and impaired mitophagy in MSPCs. Changes and defects similar to those in mice were also observed in MSPCs from humans undergoing HSCT. A pharmacological treatment that attenuated the elevated activation of CDC42 restored F-actin fiber alignment, mitochondrial function, and mitophagy in MSPCs in vitro. Finally, targeting CDC42 activity in vivo in animals undergoing transplants improved MSPC quality to increase both bone volume and trabecular bone thickness. Our study shows that attenuation of CDC42 activity is sufficient to attenuate reduced function of MSPCs in a BM transplant setting.

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

Remodeling of the bone marrow microenvironment in chronic inflammation and in aging reduces hematopoietic stem cell (HSC) function. To assess the mechanisms of this functional decline of HSC and find strategies to counteract it, we established a model in which the Sfrp1 gene was deleted in Osterix+ osteolineage cells (OS1Δ/Δ mice). HSC from these mice showed severely diminished repopulating activity with associated DNA damage, enriched expression of the reactive oxygen species pathway and reduced single-cell proliferation. Interestingly, not only was the protein level of Catenin beta-1 (bcatenin) elevated, but so was its association with the phosphorylated co-activator p300 in the nucleus. Since these two proteins play a key role in promotion of differentiation and senescence, we inhibited in vivo phosphorylation of p300 through PP2A-PR72/130 by administration of IQ-1 in OS1Δ/Δ mice. This treatment not only reduced the b-catenin/phosphop300 association, but also decreased nuclear p300. More importantly, in vivo IQ-1 treatment fully restored HSC repopulating activity of the OS1Δ/Δ mice. Our findings show that the osteoprogenitor Sfrp1 is essential for maintaining HSC function. Furthermore, pharmacological downregulation of the nuclear b-catenin/phospho-p300 association is a new strategy to restore poor HSC function.

Supplementary Tables 1-2, Figures Legends 1-4 from Stromal Niche Cells Protect Early Leukemic FLT3-ITD+ Progenitor Cells against First-Generation FLT3 Tyrosine Kinase Inhibitors

Bone marrow transplantation (BMT) is a widely used treatment for a variety of hematological diseases. However, prior to BMT, recipients are conditioned with total body irradiation (TBI) and/or chemotherapy to eliminate their hematopoietic system, which can also damage the niche, including mesenchymal stem and progenitor cells (MSPCs). The niche is an important microenvironment for the maintenance and regulation of stem cell function, including hematopoietic stem cells. The aim of our study was to investigate the effects of BMT on MSPCs and their subsequent recovery. We found that 10 months after BMT, MSPCs exhibited long-term damage, including decreased frequency (CFU-F), accumulation of DNA damage and an increase in senescent cells. In addition, we observed over-activation of CDC42 and damage to the actin cytoskeleton in BMT MSPCs, resulting in abnormal mitochondrial control and cytoskeletal reorganisation. These abnormalities lead to impaired differentiation and regenerative capacity of MSPCs, resulting in a delay in tissue recovery and an increased risk of long-term complications. To investigate this, we tested the effect of CDC42 inhibition in BMT mice during the first week after transplantation. Attenuation of CDC42 overactivation prevented cytoskeletal changes, improved bone volume and reduced osteoporotic changes, resulting in improved osteogenesis after BMT. This finding suggests that inhibition of CDC42 may be a potential strategy to improve long-term tissue recovery after transplantation in aging transplant recipients. In summary, our study shows that MSPCs from the conditioned niche are damaged, leading to long-term defects in tissue regenerative capacity. Our results shed light on the mechanisms underlying age-related factors that impede BMT and highlight potential strategies to improve long-term tissue recovery after transplantation. Bone marrow transplantation (BMT) is a widely used treatment for a variety of hematological diseases. However, prior to BMT, recipients are conditioned with total body irradiation (TBI) and/or chemotherapy to eliminate their hematopoietic system, which can also damage the niche, including mesenchymal stem and progenitor cells (MSPCs). The niche is an important microenvironment for the maintenance and regulation of stem cell function, including hematopoietic stem cells. The aim of our study was to investigate the effects of BMT on MSPCs and their subsequent recovery. We found that 10 months after BMT, MSPCs exhibited long-term damage, including decreased frequency (CFU-F), accumulation of DNA damage and an increase in senescent cells. In addition, we observed over-activation of CDC42 and damage to the actin cytoskeleton in BMT MSPCs, resulting in abnormal mitochondrial control and cytoskeletal reorganisation. These abnormalities lead to impaired differentiation and regenerative capacity of MSPCs, resulting in a delay in tissue recovery and an increased risk of long-term complications. To investigate this, we tested the effect of CDC42 inhibition in BMT mice during the first week after transplantation. Attenuation of CDC42 overactivation prevented cytoskeletal changes, improved bone volume and reduced osteoporotic changes, resulting in improved osteogenesis after BMT. This finding suggests that inhibition of CDC42 may be a potential strategy to improve long-term tissue recovery after transplantation in aging transplant recipients. In summary, our study shows that MSPCs from the conditioned niche are damaged, leading to long-term defects in tissue regenerative capacity. Our results shed light on the mechanisms underlying age-related factors that impede BMT and highlight potential strategies to improve long-term tissue recovery after transplantation.

Supplementary Figures 1-4 from Stromal Niche Cells Protect Early Leukemic FLT3-ITD+ Progenitor Cells against First-Generation FLT3 Tyrosine Kinase Inhibitors

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.

Platelet homeostasis is essential for vascular integrity and immune defense. While the process of platelet formation by fragmenting megakaryocytes (thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of megakaryocytes by their progenitor cells (megakaryopoiesis) remains unclear. Here we use intravital 2 photon microscopy to track individual megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as crucial bone marrow niche cells that regulate megakaryopoiesis. pDCs monitor the bone marrow for platelet-producing megakaryocytes and deliver IFN-α to the megakaryocytic niche to trigger local on-demand proliferation of megakaryocyte progenitors. This fine-tuned coordination between thrombopoiesis and megakaryopoiesis is crucial for megakaryocyte and platelet homeostasis in steady state and stress. However, uncontrolled pDC function within the megakaryocytic niche is detrimental. Accordingly, we show that pDCs activated by SARS-CoV2 drive inappropriate megakaryopoiesis associated with thrombotic complications. Together, we uncover a hitherto unknown megakaryocytic bone marrow niche maintained by the constitutive delivery of pDC-derived IFN-α.

Plasmacytoid and conventional dendritic cells (pDC and cDC) are generated from progenitor cells in the bone marrow and commitment to pDCs or cDC subtypes may occur in earlier and later progenitor stages. Cells within the CD11c+MHCII-/loSiglec-H+CCR9lo DC precursor fraction of the mouse bone marrow generate both pDCs and cDCs. Here we investigate the heterogeneity and commitment of subsets in this compartment by single-cell transcriptomics and high-dimensional flow cytometry combined with cell fate analysis: Within the CD11c+MHCII-/loSiglec-H+CCR9lo DC precursor pool cells expressing high levels of Ly6D and lacking expression of transcription factor Zbtb46 contain CCR9loB220hi immediate pDC precursors and CCR9loB220lo (lo-lo) cells which still generate pDCs and cDCs in vitro and in vivo under steady state conditions. cDC-primed cells within the Ly6DhiZbtb46- lo-lo precursors rapidly upregulate Zbtb46 and pass through a Zbtb46+Ly6D+ intermediate stage before acquiring cDC phenotype after cell division. Type I IFN stimulation limits cDC and promotes pDC output from this precursor fraction by arresting cDC-primed cells in the Zbtb46+Ly6D+ stage preventing their expansion and differentiation into cDCs. Modulation of pDC versus cDC output from precursors by external factors may allow for adaptation of DC subset composition at later differentiation stages.

Stromal cells interact with immune cells during initiation and resolution of immune responses, though the precise underlying mechanisms remain to be resolved. Lessons learned from stromal cell-based therapies indicate that environmental signals instruct their immunomodulatory action contributing to immune response control. Here, to the best of our knowledge, we show a novel function for the guanine-exchange factor DOCK2 in regulating immunosuppressive function in three human stromal cell models and by siRNA-mediated DOCK2 knockdown. To identify immune function-related stromal cell molecular signatures, we first reprogrammed mesenchymal stem/progenitor cells (MSPCs) into induced pluripotent stem cells (iPSCs) before differentiating these iPSCs in a back-loop into MSPCs. The iPSCs and immature iPS-MSPCs lacked immunosuppressive potential. Successive maturation facilitated immunomodulation, while maintaining clonogenicity, comparable to their parental MSPCs. Sequential transcriptomics and methylomics displayed time-dependent immune-related gene expression trajectories, including DOCK2, eventually resembling parental MSPCs. Severe combined immunodeficiency (SCID) patient-derived fibroblasts harboring bi-allelic DOCK2 mutations showed significantly reduced immunomodulatory capacity compared to non-mutated fibroblasts. Conditional DOCK2 siRNA knockdown in iPS-MSPCs and fibroblasts also immediately reduced immunomodulatory capacity. Conclusively, CRISPR/Cas9-mediated DOCK2 knockout in iPS-MSPCs also resulted in significantly reduced immunomodulation, reduced CDC42 Rho family GTPase activation and blunted filopodia formation. These data identify G protein signaling as key element devising stromal cell immunomodulation.

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.

GATA2 zinc-finger (ZF) mutations are associated with distinct entities of myeloid malignancies. The specific distribution of these mutations points toward different mechanisms of leukemogenesis depending on the ZF domain affected. In this study, we compared recurring somatic mutations in ZF1 and ZF2. All tested ZF mutants disrupted DNA binding in vitro. In transcription assays, co-expression of FOG1 counteracted GATA2-dependent transcriptional activation, while a variable response to FOG1-mediated repression was observed for individual GATA2 mutants. In primary murine bone marrow cells, GATA2 wild-type (WT) expression inhibited colony formation, while this effect was reduced for both mutants A318T (ZF1) and L359V (ZF2) with a shift toward granulopoiesis. In primary human CD34+ bone marrow cells and in the myeloid cell line K562, ectopic expression of GATA2 L359V, but not A318T or G320D, caused a block of erythroid differentiation accompanied by downregulation of GATA1, STAT5B, and PLCG1. Our findings may explain the role of GATA2 L359V during the progression of chronic myeloid leukemia and the collaboration of GATA2 ZF1 alterations with CEBPA double mutations in erythroleukemia.

In 2016, the European Hematology Association (EHA) published the EHA Roadmap for European Hematology Research(1) aiming to highlight achievements in the diagnostics and treatment of blood disorders, and to better inform European policy makers and other stakeholders about the urgent clinical and scientific needs and priorities in the field of hematology. Each section was coordinated by one to two section editors who were leading international experts in the field. In the five years that have followed, advances in the field of hematology have been plentiful. As such, EHA is pleased to present an updated Research Roadmap, now including eleven sections, each of which will be published separately. The updated EHA Research Roadmap identifies the most urgent priorities in hematology research and clinical science, therefore supporting a more informed, focused, and ideally a more funded future for European hematology research. The eleven EHA Research Roadmap sections include Normal Hematopoiesis; Malignant Lymphoid Diseases; Malignant Myeloid Diseases; Anemias and Related Diseases; Platelet Disorders; Blood Coagulation and Hemostatic Disorders; Transfusion Medicine; Infections in Hematology; Hematopoietic Stem Cell Transplantation; CAR-T and Other Cell-based Immune Therapies; and Gene Therapy.

SUMOylation is a post-translational modification of proteins that regulates these proteins' localization, turnover or function. Aberrant SUMOylation is frequently found in cancers but its origin remains elusive. Using a genome-wide transposon mutagenesis screen in a MYC-driven B-cell lymphoma model, we here identify the SUMO isopeptidase (or deconjugase) SENP6 as a tumor suppressor that links unrestricted SUMOylation to tumor development and progression. Notably, SENP6 is recurrently deleted in human lymphomas and SENP6 deficiency results in unrestricted SUMOylation. Mechanistically, SENP6 loss triggers release of DNA repair- and genome maintenance-associated protein complexes from chromatin thereby impairing DNA repair in response to DNA damages and ultimately promoting genomic instability. In line with this hypothesis, SENP6 deficiency drives synthetic lethality to Poly-ADP-Ribose-Polymerase (PARP) inhibition. Together, our results link SENP6 loss to defective genome maintenance and reveal the potential therapeutic application of PARP inhibitors in B-cell lymphoma.