Alternative platelet differentiation pathways initiated by nonhierarchically related hematopoietic stem cells.

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.

Identification and surveillance of rare relapse-initiating stem cells during complete remission after transplantation.

ABSTRACT: Relapse after complete remission (CR) remains the main cause of mortality after allogeneic stem cell transplantation for hematological malignancies and, therefore, improved biomarkers for early prediction of relapse remains a critical goal toward development and assessment of preemptive relapse treatment. Because the significance of cancer stem cells as a source of relapses remains unclear, we investigated whether mutational screening for persistence of rare cancer stem cells would enhance measurable residual disease (MRD) and early relapse prediction after transplantation. In a retrospective study of patients who relapsed and patients who achieved continuous-CR with myelodysplastic syndromes and related myeloid malignancies, combined flow cytometric cell sorting and mutational screening for persistence of rare relapse-initiating stem cells was performed in the bone marrow at multiple CR time points after transplantation. In 25 CR samples from 15 patients that later relapsed, only 9 samples were MRD-positive in mononuclear cells (MNCs) whereas flowcytometric-sorted hematopoietic stem and progenitor cells (HSPCs) were MRD-positive in all samples, and always with a higher variant allele frequency than in MNCs (mean, 97-fold). MRD-positivity in HSPCs preceded MNCs in multiple sequential samples, in some cases preceding relapse by >2 years. In contrast, in 13 patients in long-term continuous-CR, HSPCs remained MRD-negative. Enhanced MRD sensitivity was also observed in total CD34+ cells, but HSPCs were always more clonally involved (mean, 8-fold). In conclusion, identification of relapse-initiating cancer stem cells and mutational MRD screening for their persistence consistently enhances MRD sensitivity and earlier prediction of relapse after allogeneic stem cell transplantation.

Erythroid Differentiation Enhances RNA Mis-Splicing in SF3B1-Mutant Myelodysplastic Syndromes with Ring Sideroblasts.

Myelodysplastic syndromes with ring sideroblasts (MDS-RS) commonly develop from hematopoietic stem cells (HSC) bearing mutations in the splicing factor SF3B1 (SF3B1mt). Direct studies into MDS-RS pathobiology have been limited by a lack of model systems that fully recapitulate erythroid biology and RS development and the inability to isolate viable human RS. Here, we combined successful direct RS isolation from patient samples, high-throughput multiomics analysis of cells encompassing the SF3B1mt stem-erythroid continuum, and functional assays to investigate the impact of SF3B1mt on erythropoiesis and RS accumulation. The isolated RS differentiated, egressed into the blood, escaped traditional nonsense-mediated decay (NMD) mechanisms, and leveraged stress-survival pathways that hinder wild-type hematopoiesis through pathogenic GDF15 overexpression. Importantly, RS constituted a contaminant of magnetically enriched CD34+ cells, skewing bulk transcriptomic data. Mis-splicing in SF3B1mt cells was intensified by erythroid differentiation through accelerated RNA splicing and decreased NMD activity, and SF3B1mt led to truncations in several MDS-implicated genes. Finally, RNA mis-splicing induced an uncoupling of RNA and protein expression, leading to critical abnormalities in proapoptotic p53 pathway genes. Overall, this characterization of erythropoiesis in SF3B1mt RS provides a resource for studying MDS-RS and uncovers insights into the unexpectedly active biology of the "dead-end" RS. SIGNIFICANCE: Ring sideroblast isolation combined with state-of-the-art multiomics identifies survival mechanisms underlying SF3B1-mutant erythropoiesis and establishes an active role for erythroid differentiation and ring sideroblasts themselves in SF3B1-mutant myelodysplastic syndrome pathogenesis.

A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo.

Acute myeloid leukemia (AML), the most frequent leukemia in adults, is driven by recurrent somatically acquired genetic lesions in a restricted number of genes. Treatment with tyrosine kinase inhibitors has demonstrated that targeting of prevalent FMS-related receptor tyrosine kinase 3 (FLT3) gain-of-function mutations can provide significant survival benefits for patients, although the efficacy of FLT3 inhibitors in eliminating FLT3-mutated clones is variable. We identified a T cell receptor (TCR) reactive to the recurrent D835Y driver mutation in the FLT3 tyrosine kinase domain (TCRFLT3D/Y). TCRFLT3D/Y-redirected T cells selectively eliminated primary human AML cells harboring the FLT3D835Y mutation in vitro and in vivo. TCRFLT3D/Y cells rejected both CD34+ and CD34- AML in mice engrafted with primary leukemia from patients, reaching minimal residual disease-negative levels, and eliminated primary CD34+ AML leukemia-propagating cells in vivo. Thus, T cells targeting a single shared mutation can provide efficient immunotherapy toward selective elimination of clonally involved primary AML cells in vivo.

Genome-wide transcription factor-binding maps reveal cell-specific changes in the regulatory architecture of human HSPCs.

Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay among transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs (FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2) bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remains unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac, HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (heptad, PU.1, CTCF, STAG2) in HSPC subsets (stem/multipotent progenitors plus common myeloid, granulocyte macrophage, and megakaryocyte erythrocyte progenitors) and found TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs, including PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of TF complexes was at least partially regulated by features encoded in DNA sequence motifs. Taken together, this study comprehensively characterizes the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying data sets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analyzing aberrant regulatory networks in leukemic cells.

Loss of endothelial membrane KIT ligand affects systemic KIT ligand levels but not bone marrow hematopoietic stem cells.

A critical regulatory role of hematopoietic stem cell (HSC) vascular niches in the bone marrow has been implicated to occur through endothelial niche cell expression of KIT ligand. However, endothelial-derived KIT ligand is expressed in both a soluble and membrane-bound form and not unique to bone marrow niches, and it is also systemically distributed through the circulatory system. Here, we confirm that upon deletion of both the soluble and membrane-bound forms of endothelial-derived KIT ligand, HSCs are reduced in mouse bone marrow. However, the deletion of endothelial-derived KIT ligand was also accompanied by reduced soluble KIT ligand levels in the blood, precluding any conclusion as to whether the reduction in HSC numbers reflects reduced endothelial expression of KIT ligand within HSC niches, elsewhere in the bone marrow, and/or systemic soluble KIT ligand produced by endothelial cells outside of the bone marrow. Notably, endothelial deletion, specifically of the membrane-bound form of KIT ligand, also reduced systemic levels of soluble KIT ligand, although with no effect on stem cell numbers, implicating an HSC regulatory role primarily of soluble rather than membrane KIT ligand expression in endothelial cells. In support of a role of systemic rather than local niche expression of soluble KIT ligand, HSCs were unaffected in KIT ligand deleted bones implanted into mice with normal systemic levels of soluble KIT ligand. Our findings highlight the need for more specific tools to unravel niche-specific roles of regulatory cues expressed in hematopoietic niche cells in the bone marrow.

Integrated Genomic and Transcriptomic Analysis Improves Disease Classification and Risk Stratification of MDS with Ring Sideroblasts.

PURPOSE: Ring sideroblasts (RS) define the low-risk myelodysplastic neoplasm (MDS) subgroup with RS but may also reflect erythroid dysplasia in higher risk myeloid neoplasm. The benign behavior of MDS with RS (MDSRS+) is limited to SF3B1-mutated cases without additional high-risk genetic events, but one third of MDSRS+ carry no SF3B1 mutation, suggesting that different molecular mechanisms may underlie RS formation. We integrated genomic and transcriptomic analyses to evaluate whether transcriptome profiles may improve current risk stratification. EXPERIMENTAL DESIGN: We studied a prospective cohort of MDSRS+ patients irrespective of World Health Organization (WHO) class with regard to somatic mutations, copy-number alterations, and bone marrow CD34+ cell transcriptomes to assess whether transcriptome profiles add to prognostication and provide input on disease classification. RESULTS: SF3B1, SRSF2, or TP53 multihit mutations were found in 89% of MDSRS+ cases, and each mutation category was associated with distinct clinical outcome, gene expression, and alternative splicing profiles. Unsupervised clustering analysis identified three clusters with distinct hemopoietic stem and progenitor (HSPC) composition, which only partially overlapped with mutation groups. IPSS-M and the transcriptome-defined proportion of megakaryocyte/erythroid progenitors (MEP) independently predicted survival in multivariable analysis. CONCLUSIONS: These results provide essential input on the molecular basis of SF3B1-unmutated MDSRS+ and propose HSPC quantification as a prognostic marker in myeloid neoplasms with RS.

Panhematopoietic RNA barcoding enables kinetic measurements of nucleate and anucleate lineages and the activation of myeloid clones following acute platelet depletion.

BACKGROUND: Platelets and erythrocytes constitute over 95% of all hematopoietic stem cell output. However, the clonal dynamics of HSC contribution to these lineages remains largely unexplored. RESULTS: We use lentiviral genetic labeling of mouse hematopoietic stem cells to quantify output from all lineages, nucleate, and anucleate, simultaneously linking these with stem and progenitor cell transcriptomic phenotypes using single-cell RNA-sequencing. We observe dynamic shifts of clonal behaviors through time in same-animal peripheral blood and demonstrate that acute platelet depletion shifts the output of multipotent hematopoietic stem cells to the exclusive production of platelets. Additionally, we observe the emergence of new myeloid-biased clones, which support short- and long-term production of blood cells. CONCLUSIONS: Our approach enables kinetic studies of multi-lineage output in the peripheral blood and transcriptional heterogeneity of individual hematopoietic stem cells. Our results give a unique insight into hematopoietic stem cell reactivation upon platelet depletion and of clonal dynamics in both steady state and under stress.

Targeting stem cells in myelodysplastic syndromes and acute myeloid leukemia.

The genetic architecture of cancer has been delineated through advances in high-throughput next-generation sequencing, where the sequential acquisition of recurrent driver mutations initially targeted towards normal cells ultimately leads to malignant transformation. Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are hematologic malignancies frequently initiated by mutations in the normal hematopoietic stem cell compartment leading to the establishment of leukemic stem cells. Although the genetic characterization of MDS and AML has led to identification of new therapeutic targets and development of new promising therapeutic strategies, disease progression, relapse, and treatment-related mortality remain a major challenge in MDS and AML. The selective persistence of rare leukemic stem cells following therapy-induced remission implies unique resistance mechanisms of leukemic stem cells towards conventional therapeutic strategies and that leukemic stem cells represent the cellular origin of relapse. Therefore, targeted surveillance of leukemic stem cells following therapy should, in the future, allow better prediction of relapse and disease progression, but is currently challenged by our restricted ability to distinguish leukemic stem cells from other leukemic cells and residual normal cells. To advance current and new clinical strategies for the treatment of MDS and AML, there is a need to improve our understanding and characterization of MDS and AML stem cells at the cellular, molecular, and genetic levels. Such work has already led to the identification of promising new candidate leukemic stem cell molecular targets that can now be exploited in preclinical and clinical therapeutic strategies, towards more efficient and specific elimination of leukemic stem cells.

FOXO Dictates Initiation of B Cell Development and Myeloid Restriction in Common Lymphoid Progenitors.

The development of B cells relies on an intricate network of transcription factors critical for developmental progression and lineage commitment. In the B cell developmental trajectory, a temporal switch from predominant Foxo3 to Foxo1 expression occurs at the CLP stage. Utilizing VAV-iCre mediated conditional deletion, we found that the loss of FOXO3 impaired B cell development from LMPP down to B cell precursors, while the loss of FOXO1 impaired B cell commitment and resulted in a complete developmental block at the CD25 negative proB cell stage. Strikingly, the combined loss of FOXO1 and FOXO3 resulted in the failure to restrict the myeloid potential of CLPs and the complete loss of the B cell lineage. This is underpinned by the failure to enforce the early B-lineage gene regulatory circuitry upon a predominantly pre-established open chromatin landscape. Altogether, this demonstrates that FOXO3 and FOXO1 cooperatively govern early lineage restriction and initiation of B-lineage commitment in CLPs.

T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes.

Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that T cells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific T cells. TCR-modified T cells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes.

A three-dimensional in vitro model of erythropoiesis recapitulates erythroid failure in myelodysplastic syndromes.

Established cell culture systems have failed to accurately recapitulate key features of terminal erythroid maturation, hampering our ability to in vitro model and treat diseases with impaired erythropoiesis such as myelodysplastic syndromes with ring sideroblasts (MDS-RS). We developed an efficient and robust three-dimensional (3D) scaffold culture model supporting terminal erythroid differentiation from both mononuclear (MNC) or CD34+-enriched primary bone marrow cells from healthy donors and MDS-RS patients. While CD34+ cells did not proliferate beyond two weeks in 2D suspension cultures, the 3D scaffolds supported CD34+ and MNC erythroid proliferation over four weeks demonstrating the importance of the 3D environment. CD34+ cells cultured in 3D facilitated the highest expansion and maturation of erythroid cells, including generation of erythroblastic islands and enucleated erythrocytes, while MNCs supported multi-lineage hemopoietic differentiation and cytokine secretion relevant for MDS-RS. Importantly, MDS-RS 3D-cultures supported de novo generation of ring sideroblasts and maintenance of the mutated clone. The 3D cultures effectively model a clonal disease characterized by terminal erythroid failure and can be used to assess therapeutic compounds.

The concept of leukaemic stem cells in acute myeloid leukaemia 25 years on: hitting a moving target.

The concept of leukaemic stem cells (LSCs) was experimentally suggested 25 years ago through seminal data from John Dick's group, who showed that a small fraction of cells from acute myeloid leukaemia (AML) patients were able to be adoptively transferred into immunodeficient mice. The initial estimation of the frequency was 1:250 000 leukaemic cells, clearly indicating the difficulties ahead in translating knowledge on LSCs to the clinical setting. However, the field has steadily grown in interest, expanse and importance, concomitantly with the realisation of the molecular background for AML culminating in the sequencing of hundreds of AML genomes. The literature is now ripe with contributions describing how different molecular aberrations are more or less specific for LSCs, as well as reports showing selectivity in targeting LSCs in comparison to normal haematopoietic stem and progenitor cells. However, we argue here that these important data have not yet been fully realised within the clinical setting. In this clinically focused review, we outline the difficulties in identifying and defining LSCs at the individual patient level, with special emphasis on intraclonal heterogeneity. In addition, we suggest areas of future focus in order to realise the concept as real-time benefit for AML patients.

In Vivo Labeling by CD73 Marks Multipotent Stromal Cells and Highlights Endothelial Heterogeneity in the Bone Marrow Niche.

Despite much work studying ex vivo multipotent stromal cells (MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address these questions and found EGFP+ MSCs in various organs. In vivo, EGFP+ mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP+ cells exhibited a perivascular distribution pattern. EGFP+ cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP+ specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP+ population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium.

Mapping the CLEC12A expression on myeloid progenitors in normal bone marrow; implications for understanding CLEC12A-related cancer stem cell biology.

The C-type lectin domain family 12, member A (CLEC12A) receptor has emerged as a leukaemia-associated and cancer stem cell marker in myeloid malignancies. However, a detailed delineation of its expression in normal haematopoiesis is lacking. Here, we have characterized the expression pattern of CLEC12A on the earliest stem- and myeloid progenitor subsets in normal bone marrow. We demonstrate distinct CLEC12A expression in the classically defined myeloid progenitors, where on average 39.1% (95% CI [32.5;45.7]) of the common myeloid progenitors (CMPs) expressed CLEC12A, while for granulocyte-macrophage progenitors and megakaryocyte-erythroid progenitors (MEPs), the average percentages were 81.0% (95% CI [76.0;85.9]) and 11.9% (95% CI [9.3;14.6]), respectively. In line with the reduced CLEC12A expression on MEPs, functional assessment of purified CLEC12A+/- CMPs and MEPs in the colony-forming unit assay demonstrated CLEC12A+ subsets to favour non-erythroid colony growth. In conclusion, we provide evidence that the earliest CLEC12A+ cell in the haematopoietic tree is the classically defined CMP. Furthermore, we show that CLEC12A-expressing CMPs and MEPs are functionally different than their negative counterparts. Importantly, these data can help determine which cells will be spared during CLEC12A-targeted therapy, and we propose CLEC12A to be included in future studies of myeloid cancer stem cell biology.

Canonical Notch signaling is dispensable for adult steady-state and stress myelo-erythropoiesis.

Although an essential role for canonical Notch signaling in generation of hematopoietic stem cells in the embryo and in thymic T-cell development is well established, its role in adult bone marrow (BM) myelopoiesis remains unclear. Some studies, analyzing myeloid progenitors in adult mice with inhibited Notch signaling, implicated distinct roles of canonical Notch signaling in regulation of progenitors for the megakaryocyte, erythroid, and granulocyte-macrophage cell lineages. However, these studies might also have targeted other pathways. Therefore, we specifically deleted, in adult BM, the transcription factor recombination signal-binding protein J κ (Rbpj), through which canonical signaling from all Notch receptors converges. Notably, detailed progenitor staging established that canonical Notch signaling is fully dispensable for all investigated stages of megakaryocyte, erythroid, and myeloid progenitors in steady state unperturbed hematopoiesis, after competitive BM transplantation, and in stress-induced erythropoiesis. Moreover, expression of key regulators of these hematopoietic lineages and Notch target genes were unaffected by Rbpj deficiency in BM progenitor cells.

Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.

Rare multipotent haematopoietic stem cells (HSCs) in adult bone marrow with extensive self-renewal potential can efficiently replenish all myeloid and lymphoid blood cells, securing long-term multilineage reconstitution after physiological and clinical challenges such as chemotherapy and haematopoietic transplantations. HSC transplantation remains the only curative treatment for many haematological malignancies, but inefficient blood-lineage replenishment remains a major cause of morbidity and mortality. Single-cell transplantation has uncovered considerable heterogeneity among reconstituting HSCs, a finding that is supported by studies of unperturbed haematopoiesis and may reflect different propensities for lineage-fate decisions by distinct myeloid-, lymphoid- and platelet-biased HSCs. Other studies suggested that such lineage bias might reflect generation of unipotent or oligopotent self-renewing progenitors within the phenotypic HSC compartment, and implicated uncoupling of the defining HSC properties of self-renewal and multipotency. Here we use highly sensitive tracking of progenitors and mature cells of the megakaryocyte/platelet, erythroid, myeloid and B and T cell lineages, produced from singly transplanted HSCs, to reveal a highly organized, predictable and stable framework for lineage-restricted fates of long-term self-renewing HSCs. Most notably, a distinct class of HSCs adopts a fate towards effective and stable replenishment of a megakaryocyte/platelet-lineage tree but not of other blood cell lineages, despite sustained multipotency. No HSCs contribute exclusively to any other single blood-cell lineage. Single multipotent HSCs can also fully restrict towards simultaneous replenishment of megakaryocyte, erythroid and myeloid lineages without executing their sustained lymphoid lineage potential. Genetic lineage-tracing analysis also provides evidence for an important role of platelet-biased HSCs in unperturbed adult haematopoiesis. These findings uncover a limited repertoire of distinct HSC subsets, defined by a predictable and hierarchical propensity to adopt a fate towards replenishment of a restricted set of blood lineages, before loss of self-renewal and multipotency.