Search

SummaryHematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay of 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 remained unknown. We mapped genome-wide chromatin contacts and TF binding profiles in HSPC subsets (HSC, CMP, GMP, MEP) and found that heptad 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. These findings suggest that specific heptad-TF combinations play critical roles in regulating hematopoietic differentiation and provide a valuable resource for development of targeted therapies to manipulate specific HSPC subsets.

Myelodysplastic syndromes with ring sideroblasts (MDS-RS) commonly originate from mutations in the splicing factorSF3B1(SF3B1mt).SF3B1mtcause RNA mis-splicing, mechanistically established as the major driver of RS development. However, little is known about RS fate and biology after their initial formation in the human bone marrow. We here achieve isolation of viable RS from patient samples, enabling the first complete investigation ofSF3B1mtdevelopment from stem cell to RS. We show that RS skew MACS-isolated CD34+data towards erythroid features not recapitulated in single-cell RNAseq. We demonstrate that RS divide, differentiate, enucleate and actively respond to mis-splicing/oxidative stress, decreasing wildtype stem cell fitness via GDF15 overproduction. We identify circulating RS as a uniform clinical feature associated with disease burden. Finally, we establish thatSF3B1mtmis-splicing intensifies during erythroid differentiation and demonstrate through combined transcriptomics/proteomics an uncoupling of RNA/protein biology in RS encompassing severe and dysfunctional mis-splicing of proapoptotic genes.Statement of significanceWe here combine a novel method for RS isolation with state-of-the-art multiomics to perform the first complete investigation ofSF3B1mtMDS-RS hematopoiesis from stem cell to RS. We identify the survival mechanisms underlyingSF3B1mterythropoiesis and establish an active role for erythroid differentiation and RS themselves inSF3B1mtMDS-RS pathogenesis.

Chin and colleagues used detailed mutational analysis of aged mice and transplantation to evaluate the mouse as a model of clonal hematopoiesis (CH). Their data suggest that while murine hematopoietic stem cells acquire mutations in CH-associated genes when aged and CH clones can expand after transplantation (as in humans), these are rare events. Nevertheless, genetically manipulated murine models mimicking human CH are feasible and may prove useful in the future.

Anucleate cells - platelets and erythrocytes - constitute over 95% of all hematopoietic stem cell (HSC) output, but the clonal dynamics of HSC contribution to these lineages remains largely unexplored. Here, we use lentiviral RNA cellular barcoding and transplantation of HSCs, combined with single-cell RNA-seq, for quantitative analysis of clonal behavior with a multi-lineage readout - for the first time including anucleate and nucleate lineages. We demonstrate that most HSCs steadily contribute to hematopoiesis, but acute platelet depletion shifts the output of multipotent HSCs to the exclusive production of platelets, with the additional emergence of new myeloid-biased clones. Our approach therefore enables comprehensive profiling of multi-lineage output and transcriptional heterogeneity of individual HSCs, giving insight into clonal dynamics in both steady state and under physiological stress.

Extensive genetic and epigenetic variegation has been demonstrated in many malignancies. Importantly, their interplay has the potential to contribute to disease progression and treatment resistance. To shed light on the complex relationships between these different sources of intra-tumour heterogeneity, we explored their relative contributions to the evolutionary dynamics of Acute Lymphoblastic Leukemia (ALL) in children with Down syndrome, which has particularly poor prognosis. We quantified the tumour propagating potential of genetically distinct sub-clones using serial transplantation assays and SNP-arrays. While most leukaemias were characterized by a single dominant subclone, others were highly heterogeneous. Importantly, we provide clear and direct evidence that genotypes and phenotypes with functional relevance to leukemic progression and treatment resistance can co-segregate within the disease. Hence, individual genetic lesions can be restricted to well-defined cell immunophenotypes, corresponding to different stages of the leukemic differentiation hierarchy and varied proliferation potentials. As a result of this difference in fitness, which can be accurately quantified via competitive transplantation assays, matching diagnostic, post-treatment, and relapse leukemias can be dominated by different genotypes, including pre-leukemic clones persisting throughout the disease progression and treatment. Intriguingly, plasticity also appears to be a temporally defined property that can segregate with genotype. These results suggest that Down Syndrome ALL should be viewed as a complex matrix of cells exhibiting genetic and epigenetic heterogeneity that foster extensive clonal evolution and competition. Therapeutic intervention reshapes this ‘eco-system’ and may provide the right conditions for the preferential expansion of selected compartments and subsequently relapse.

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.

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematopoietic stem cell (HSC) disorders characterized by aberrant HSC differentiation, cytopenia, and an increased risk of progression to leukemia. The low-risk subtype MDS with ring sideroblasts (MDS-RS) is specifically characterized by expanded and ineffective erythropoiesis, with more than 80% of patients displaying mutations in the core spliceosome component SF3B1 (SF3B1 mt). A hallmark of the MDS-RS bone marrow (BM) is the progressive accumulation of ring sideroblasts (RS), erythroblasts displaying perinuclear mitochondria loaded with aberrant ferritin-iron complexes. Whilst several in vitro and in vivo model systems exist for studying the impact of SF3B1 mt on erythropoiesis and RS development, primary SF3B1 mt erythroid biology remains largely unexplored due to the inability to purify live SF3B1 mt cells or fully replicate BM conditions in vitro. To address this issue, we developed an innovative two-step method to isolate live ring sideroblasts from SF3B1 mt MDS-RS BM aspiration material with extremely high purity (as determined through droplet digital PCR-based genotyping [Fig. 1A] and morphology-based detection through Prussian blue staining [Fig. 1B,C]). Unexpectedly, evaluation of matching peripheral blood samples showed that circulating ring sideroblasts are strikingly common in MDS-RS (Fig. 1D), with their abundance being significantly positively associated with clinically-determined BM RS frequencies and serum erythropoietin levels, as well as negatively associated with hemoglobin levels. Through high-throughput Chromium 3'-based single-cell RNA sequencing (scRNAseq) analysis of purified RS, we then showed that these cells comprise a heterogeneous population encompassing all stages of the erythroid differentiation continuum, from early progenitors to orthochromatic erythroblasts (Fig. 1E). The RS transcriptome was shown to be dynamically regulated towards the maintenance of cell survival during late terminal erythroid differentiation (exemplified through parkin 1 [PINK1] expression), with SF3B1 K700E erythroblasts employing multiple strategies to preserve homeostasis despite undergoing extreme oxidative stress. These observations were confirmed through a parallel whole-transcript RNAseq investigation comprising CD34 + and GPA +-enriched samples obtained from normal bone marrow (NBM) donors and SF3B1 K700E MDS-RS patients, as well as purified RS samples. This bulk RNAseq experiment validated the RS transcriptomic signature observed in scRNAseq (Fig. 1F) and allowed for a detailed investigation of RNA splicing. SF3B1 K700E-associated alternative splicing in CD34 + and RS was consistent with previous literature, but also highly context-dependent and with substantial changes in scope and magnitude throughout erythroid differentiation (Fig. 1G-I). Finally, we substantiated these RNAseq results through Tandem Mass Tag-based semi-quantitative proteomic analysis of purified RS and GPA-enriched cells from NBM donors and MDS-RS patients. We confirmed that ring sideroblast survival is heavily dependent on redox balance modulation and suppression of ER stress via an increased dependence on glutamine, mirroring the molecular mechanisms observed in malignancy. Additionally, our data strongly indicate that the RS population is a major modulator of the MDS-RS BM microenvironment due to expression of stress factors (with particular emphasis on GDF15, erythroferrone and IL-18). In conclusion, our integrative analysis of primary RS constitutes a unique platform for the study of MDS-RS, with special interest for the investigation of potential drivers of disease severity or treatment avenues. Figure 1 Figure 1. Disclosures Kretzschmar: Vanadis Diagnostics, a PerkinElmer company.: Current Employment.

Mutations in the RNA splicing gene SF3B1 are found in >80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS). We investigated the origin of SF3B1 mutations within the bone marrow hematopoietic stem and progenitor cell compartments in patients with MDS-RS. Screening for recurrently mutated genes in the mononuclear cell fraction revealed mutations in SF3B1 in 39 of 40 cases (97.5%), combined with TET2 and DNMT3A in 11 (28%) and 6 (15%) patients, respectively. All recurrent mutations identified in mononuclear cells could be tracked back to the phenotypically defined hematopoietic stem cell (HSC) compartment in all investigated patients and were also present in downstream myeloid and erythroid progenitor cells. While in agreement with previous studies, little or no evidence for clonal (SF3B1 mutation) involvement could be found in mature B cells, consistent involvement at the pro-B-cell progenitor stage was established, providing definitive evidence for SF3B1 mutations targeting lymphomyeloid HSCs and compatible with mutated SF3B1 negatively affecting lymphoid development. Assessment of stem cell function in vitro as well as in vivo established that only HSCs and not investigated progenitor populations could propagate the SF3B1 mutated clone. Upon transplantation into immune-deficient mice, SF3B1 mutated MDS-RS HSCs differentiated into characteristic ring sideroblasts, the hallmark of MDS-RS. Our findings provide evidence of a multipotent lymphomyeloid HSC origin of SF3B1 mutations in MDS-RS patients and provide a novel in vivo platform for mechanistically and therapeutically exploring SF3B1 mutated MDS-RS.

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.

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell-restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs.

A high proportion of patients with lower-risk del(5q) myelodysplastic syndromes will respond to treatment with lenalidomide. The median duration of transfusion-independence is 2 years with some long-lasting responses, but almost 40% of patients progress to acute leukemia by 5 years after starting treatment. The mechanisms underlying disease progression other than the well-established finding of small TP53-mutated subclones at diagnosis remain unclear. We studied a longitudinal cohort of 35 low- and intermediate-1-risk del(5q) patients treated with lenalidomide (n=22) or not (n=13) by flow cytometric surveillance of hematopoietic stem and progenitor cell subsets, targeted sequencing of mutational patterns, and changes in the bone marrow microenvironment. All 13 patients with disease progression were identified by a limited number of mutations in TP53, RUNX1, and TET2, respectively, with PTPN11 and SF3B1 occurring in one patient each. TP53 mutations were found in seven of nine patients who developed acute leukemia, and were documented to be present in the earliest sample (n=1) and acquired during lenalidomide treatment (n=6). By contrast, analysis of the microenvironment, and of hematopoietic stem and progenitor cells by flow cytometry was of limited prognostic value. Based on our data, we advocate conducting a prospective study aimed at investigating, in a larger number of cases of del(5q) myelodysplastic syndromes, whether the detection of such mutations before and after lenalidomide treatment can guide clinical decision-making.

Rare multipotent haematopoietic stem cells (HSCs) in adult bone marrow with extensive self-renewal potential can efficiently replenish all myeloid and lymphoid blood cells1, securing long-term multilineage reconstitution after physiological and clinical challenges such as chemotherapy and haematopoietic transplantations2,3,4. HSC transplantation remains the only curative treatment for many haematological malignancies, but inefficient blood-lineage replenishment remains a major cause of morbidity and mortality5,6. Single-cell transplantation has uncovered considerable heterogeneity among reconstituting HSCs7,8,9,10,11, a finding that is supported by studies of unperturbed haematopoiesis2,3,4,12 and may reflect different propensities for lineage-fate decisions by distinct myeloid-, lymphoid- and platelet-biased HSCs7,8,9,10,13. 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 multipotency11,14. 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.

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.

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.

© 2017 The Author(s) Myelodysplastic syndromes and chronic myelomonocytic leukemia are blood disorders characterized by ineffective hematopoiesis and progressive marrow failure that can transform into acute leukemia. The DNA methyltransferase inhibitor 5-azacytidine (AZA) is the most effective pharmacological option, but only ∼50% of patients respond. A response only manifests after many months of treatment and is transient. The reasons underlying AZA resistance are unknown, and few alternatives exist for non-responders. Here, we show that AZA responders have more hematopoietic progenitor cells (HPCs) in the cell cycle. Non-responder HPC quiescence is mediated by integrin α5 (ITGA5) signaling and their hematopoietic potential improved by combining AZA with an ITGA5 inhibitor. AZA response is associated with the induction of an inflammatory response in HPCs in vivo. By molecular bar coding and tracking individual clones, we found that, although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders.

Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis.

Distinct from other forms of acute lymphoblastic leukemia (ALL), infant ALL with mixed lineage leukemia (MLL) gene rearrangement, the most common leukemia occurring within the first year of life, might arise without the need for cooperating genetic lesions. Through Ig/TCR rearrangement analysis of MLL-AF4+ infant ALL at diagnosis and xenograft leukemias from mice transplanted with the same diagnostic samples, we established that MLL-AF4+ infant ALL is composed of a branching subclonal architecture already at diagnosis, frequently driven by an Ig/TCR-rearranged founder clone. Some MLL-AF4+ clones appear to be largely quiescent at diagnosis but can reactivate and dominate when serially transplanted into immunodeficient mice, whereas other dominant clones at diagnosis can become more quiescent, suggesting a dynamic competition between actively proliferating and quiescent subclones. Investigation of paired diagnostic and relapse samples suggested that relapses often occur from subclones already present but more quiescent at diagnosis. Copy-number alterations identified at relapse might contribute to the activation and expansion of previously quiescent subclones. Finally, each of the identified subclones is able to contribute to the diverse phenotypic pool of MLL-AF4+ leukemia-propagating cells. Unraveling of the subclonal architecture and dynamics in MLL+ infant ALL may provide possible explanations for the therapy resistance and frequent relapses observed in this group of poor prognosis ALL.

Background and aim: Treatment with erythropoietin stimulating agents (ESA) is first-line treatment for anemic low-risk MDS patients, but although clinical variables such as endogenous serum erythropoietin levels have been associated with response, the bone marrow (BM) cell populations targeted during ESA-induced erythroid improvement have not been identified. Initiating SF3B1 mutations in MDS-RS arise in the multipotent hematopoietic stem cell (HSC), and we previously showed that most SF3B1-mutated MDS-RS patients retain a small residual wildtype HSC population, something that we propose may have clinical relevance (Mortera-Blanco, et al, Blood 2017). In this study, we aimed to map clonal dynamics of mutant and wildtype hematopoietic stem and progenitor cells (HSPCs) during steady-state and ESA treatment. Methodology: We used advanced flow cytometry to sort and characterize HSPC populations in BM samples of SF3B1-mutated MDS-RS patients with stable anemia, before and during ESA response, and during chronic transfusion dependency. In addition to conventional HSPC markers, we added CD105 to the antibody panel as it has been reported to distinguish committed erythroid progenitors (Mori, et al, PNAS 2015). Variant allele frequency (VAF) of the known SF3B1 mutations was determined using droplet digital PCR. Results: Expression patterns of mature CD38+ progenitors; common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs) and megakaryocytic-erythroid progenitors (MEPs), mostly followed published patterns with CD105 expression as expected in a subset of MEPs. The immature CD38- HSCs, multipotent progenitors (MPPs), and lymphoid-primed MPPs (LMPPs) should be negative for the CD105 surface marker (Mori, et al, PNAS 2015) but we confirmed a very low expression ( Conclusion: Our data indicate that presence of wildtype CD34+ BM populations in anemic MDS-RS patients is associated with better erythroid output and milder anemia. Our results show poor discriminative power of SF3B1 mutational burden in total BM as routinely assessed in the clinic, whereas SF3B1 VAF in defined CD34+ HSPC populations shows a strong correlation with treatment need. Expression of the surface marker CD105 identifies an immature CD34+CD38- BM population that expands during erythroid responses to ESA treatment. Because this population also can be detected at low levels in normal BM donors, and has a lower mutation burden than other HSPC populations in MDS-RS patients, we propose that this wildtype population acts as a reserve source of erythroblasts during stress erythropoiesis, possibly bypassing the traditional hierarchy. Disclosures No relevant conflicts of interest to declare.

A rostral brainstem structure, the pedunculopontine nucleus (PPN), is severely affected by Parkinson's disease (PD) pathology and is regarded a promising target for therapeutic deep-brain stimulation (DBS). However, understanding the PPN's role in PD and assessing the potential of DBS are hampered by the lack of a suitable model of PPN degeneration. Rats were rendered Parkinsonian through a unilateral substantia nigra pars compacta (SNpc) stereotaxic injection of the proteasome inhibitor Lactacystin, to investigate whether the lesion's pathological effects spread to impact the integrity of PPN cholinergic neurons which are affected in PD. At 5 weeks post-surgery, stereological analysis revealed that the lesion caused a 48 % loss of dopaminergic SNpc neurons and a 61 % loss of PPN cholinergic neurons, accompanied by substantial somatic hypotrophy in the remaining cholinergic neurons. Magnetic resonance imaging revealed T2 signal hyper-/hypointensity in the PPN of the injected hemisphere, respectively at weeks 3 and 5 post-lesion. Moreover, isolated PPN cholinergic neurons revealed no significant alterations in key autophagy mRNA levels, suggesting that autophagy-related mechanisms fail to protect the PPN against Lactacystin-induced cellular changes. Hence, the current results suggest that the Lactacystin PD model offers a suitable model for investigating the role of the PPN in PD.

In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis. Copyright © 2013 Elsevier Inc. All rights reserved.

Abstract 1738 Since the initial description of V617F somatic mutation in patients with Philadelphia chromosome negative myeloproliferative neoplasms (MPNs), a remarkable association between alterations in the JAK2 gene and MPNs has emerged. In addition to V617F, a number of other mutations have been detected in exons 12–15 of the JAK2 gene. Furthermore, a specific JAK2 haplotype predisposes to somatic V617F mutation and MPN. However, the link between JAK mutation and MPNs is not straightforward. For example, in familial cases of MPNs, occurrence of V617F is heterogeneous and occurs as a somatic rather than germline mutation. The underlying inherited genetic abnormality in many of these cases remains unknown. Similarly, occurrence of JAK2 V617F in sporadic MPNs is also heterogeneous and is associated with variable disease characteristics. Thus, understanding of the relationship between JAK2 mutation and MPN disease phenotype remains far from complete. We herein report a family with germline V617I mutation (Figure 1), associated with mild/moderate thrombocythaemia and thrombosis. All patients had normal haematocrit, WBC and peripheral blood (PB) morphology without splenomegaly or other abnormality on physical examination. P1: Presented in 2006 at the age of 53 with a significant ischaemic cerebrovascular event and a platelet count of 750 × 109/l, with a history of longstanding thrombocythaemia (>10 years; 700–970 × 109/l). Bone marrow examination showed normal architecture with increased numbers of morphologically normal megakaryocytes and no fibrosis. She was commenced on aspirin and hydroxycarbamide resulting in good control of the platelet count. Aspirin was not tolerated due to recurrent epistaxis. Subsequent JAK2 mutation screening by pyrosequencing demonstrated an abnormal pyrogram pattern subsequently identified to be V617I by Sanger sequencing. Quantification of allelic level by a pyrosequencing assay designed to detect V617I confirmed heterozygous (≈50%) V617I in PB mononuclear cells (MNCs), CD3+ cells, CD66+ myeloid cells, buccal swab DNA and hair follicle DNA. P2: Daughter of P1. 34 years. Asymptomatic. Persistent thrombocythemia (470–604 × 109/l). Heterozygous (≈50%) V617I in PB MNCs, CD3+ T cells, CD66+ myeloid cells, buccal swab DNA and hair follicle DNA. P3: Son of P1. 36 years. Asymptomatic. Persistent thrombocythemia (606–648 × 109/l). Heterozygous (≈50%) V617I in PB MNCs, CD3+ T cells, CD66+ myeloid cells and buccal swab DNA. P4: Son of P1. 38 years. Asymptomatic. Persistent thrombocythemia (456–526 × 109/l). Heterozygous (≈50%) V617I in PB MNCs, CD3+ T cells, CD66+ myeloid cells and buccal swab DNA. P5: Daughter of P1. 40 years. Platelet count 294 × 109/l. V617I negative in all tissues. V617I has been previously reported to occur rarely in MPN (PMID: 19074595) and to be constitutively-activating in cell line models (PMID: 18326042) and molecular dynamic simulations (PMID: 19744331). Single cell intracellular quantitative pSTAT3 FACS analysis of PB cells from P1-4 demonstrated GCSF hyper-responsiveness of V617I positive PB CD33+ and CD34+ cells. For example, in comparison with normal controls (NC; n=6) V617I CD33+ myeloid cells (n=4) showed a 14-fold increased pSTAT3 mean fluorescent intensity relative to unstimulated cells in response to 15 minutes stimulation with 0.8 ng/ml GCSF (11% vs 156%; P Patients 2–4 are currently under observation and receiving low-dose aspirin only. Germline activating JAK2 mutation is a previously unreported cause of inherited thrombocythaemia (autosomal dominant) and should be considered in familial cases or when rare JAK2 mutations are detected. Importantly, V617F allele specific PCR, would not detect such mutations and, consequently, it is possible that similar cases may have been missed. Finally, the mild/moderate thrombocytosis (without evidence of fibrosis over 50 years) observed with human germline V617I provides fundamental insights into the role of JAK2 mutations in the pathophysiology of human MPNs. Disclosures: No relevant conflicts of interest to declare.

Evidence for distinct human cancer stem cells (CSCs) remains contentious and the degree to which differentcancer cells contribute to propagating malignancies in patients remains unexplored. In low- to intermediate-risk myelodysplastic syndromes (MDS), we establish the existence of rare multipotent MDS stem cells (MDS-SCs), and their hierarchical relationship to lineage-restricted MDS progenitors. All identified somatically acquired genetic lesions were backtracked to distinct MDS-SCs, establishing their distinct MDS-propagating function invivo. In isolated del(5q)-MDS, acquisition of del(5q) preceded diverse recurrent driver mutations. Sequential analysis in del(5q)-MDS revealed genetic evolution in MDS-SCs and MDS-progenitors prior to leukemic transformation. These findings provide definitive evidence for rare human MDS-SCs invivo, with extensive implications for the targeting of the cells required and sufficient for MDS-propagation. © 2014 Elsevier Inc.

The ASXL1 gene encodes a chromatin-binding protein involved in epigenetic regulation in haematopoietic cells. Loss-of-function ASXL1 mutations occur in patients with a range of myeloid malignancies and are associated with adverse outcome. We have used lentiviral-based shRNA technology to investigate the effects of ASXL1 silencing on cell proliferation, apoptosis, myeloid differentiation and global gene expression in human CD34+ cells differentiated along the myeloid lineage in vitro. ASXL1-deficient cells showed a significant decrease in the generation of CD11b+ and CD15+ cells, implicating impaired granulomonocytic differentiation. Furthermore, colony-forming assays showed a significant increase in the number of multipotent mixed lineage colony-forming unit (CFU-GEMM) colonies and a significant decrease in the numbers of granulocyte-macrophage CFU (CFU-GM) and granulocyte CFU (CFU-G) colonies in ASXL1-deficient cells. Our data suggests that ASXL1 knockdown perturbs human granulomonocytic differentiation. Gene expression profiling identified many deregulated genes in the ASXL1-deficient cells differentiated along the granulomonocytic lineage, and pathway analysis showed that the most significantly deregulated pathway was the LXR/RXR activation pathway. ASXL1 may play a key role in recruiting the polycomb repressor complex 2 (PRC2) to specific loci, and we found over-representation of PRC2 targets among the deregulated genes in ASXL1-deficient cells. These findings shed light on the functional role of ASXL1 in human myeloid differentiation. © 2013 Blackwell Publishing Ltd.

The blood system is maintained by a small pool of haematopoietic stem cells (HSCs), which are required and sufficient for replenishing all human blood cell lineages at millions of cells per second throughout life. Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood, crucial for preventing bleeding - a common and life-threatening side effect of many cancer therapies - and major efforts are focused at identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow transplantation or chemotherapy. Although it has become clear that distinct HSC subsets exist that are stably biased towards the generation of lymphoid or myeloid blood cells, we are yet to learn whether other types of lineage-biased HSC exist or understand their inter-relationships and how differently lineage-biased HSCs are generated and maintained. The functional relevance of notable phenotypic and molecular similarities between megakaryocytes and bone marrow cells with an HSC cell-surface phenotype remains unclear. Here we identify and prospectively isolate a molecularly and functionally distinct mouse HSC subset primed for platelet-specific gene expression, with enhanced propensity for short- and long-term reconstitution of platelets. Maintenance of platelet-biased HSCs crucially depends on thrombopoietin, the primary extrinsic regulator of platelet development. Platelet-primed HSCs also frequently have a long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs. These findings show that HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs at the apex. They also demonstrate that molecular and functional priming for platelet development initiates already in a distinct HSC population. The identification of a platelet-primed HSC population should enable the rational design of therapies enhancing platelet output. © 2013 Macmillan Publishers Limited. All rights reserved.

According to current models for hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs; Lin– Sca-1+ c-Kit+ CD34+ Flt3hi) and common myeloid progenitors (CMPs; Lin– Sca- 1+ c-Kit+ CD34+ CD41hi) establish an early branch point for separate lineage commitment pathways from hematopoietic stem cells, with the notable exception that both pathways are proposed to generate all myeloid innate immune cell types through the same myeloidrestricted pre-granulocyte-macrophage progenitor (pre-GM; Lin– Sca-1– c-Kit+ CD41– FcγRII/III– CD150– CD105– ). By single cell transcriptome profiling of pre-GMs we identify distinct myeloid differentiation pathways: a Gata1-expressing pathway generates mast cells, eosinophils, megakaryocytes and erythroid cells, and a Gata1-negative pathway that generates monocytes, neutrophils and lymphocytes. These results identify an early hematopoietic lineage bifurcation, separating the myeloid lineages prior to their segregation from other hematopoietic lineage potentials.

Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19(+) B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R(+)CD19(+) ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R(+) myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R(+) myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported.

Quiescent leukaemic cells account for minimal residual disease in childhood lymphoblastic leukaemia

MicroRNAs (miRs) are involved in many aspects of normal and malignant hematopoiesis, including hematopoietic stem cell (HSC) self-renewal, proliferation, and terminal differentiation. However, a role for miRs in the generation of the earliest stages of lineage committed progenitors from HSCs has not been identified. Using Dicer inactivation, we show that the miR complex is not only essential for HSC maintenance but is specifically required for their erythroid programming and subsequent generation of committed erythroid progenitors. In bipotent pre-MegEs, loss of Dicer up-regulated transcription factors preferentially expressed in megakaryocyte progenitors (Gata2 and Zfpm1) and decreased expression of the erythroid-specific Klf1 transcription factor. These results show a specific requirement for Dicer in acquisition of erythroid lineage programming and potential in HSCs and their subsequent erythroid lineage differentiation, and in particular indicate a role for the miR complex in achieving proper balance of lineage-specific transcriptional regulators necessary for HSC multilineage potential to be maintained.

Summary 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.

Natural killer (NK) cells serve as important effectors for antitumor immunity, and CD56+CD45+ NK cells can be routinely derived from human embryonic stem cells (hESCs). However, little is know about the ability of hESC-derived NK cells to mediate an effective in vivo antitumor response. Using bioluminescent imaging, we now demonstrate that H9 line hESC-derived NK cells mediate effective clearance of human tumor cells in vivo. In addition to increased in vitro killing of diverse tumor targets, the in vivo tumor clearance by H9 hESC-derived NK cells was more effective compared with NK cells derived from umbilical cord blood (UCB). Phenotypic analysis demonstrates the hESC-derived NK cells are uniformly CD94+CD117low/−, an NK-cell population characterized by potent cytolytic activity and thus more competent to mediate tumor clearance. These studies demonstrate that hESCs provide an important model to study human lymphocyte development and may serve as a novel source for antitumor immunotherapy.

Human embryonic stem cells (hESCs) provide an important means to characterize early stages of hematopoietic development. However, the in vivo potential of hESC-derived hematopoietic cells has not been well defined. We demonstrate that hESC-derived cells are capable of long-term hematopoietic engraftment when transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Human CD45(+) and CD34(+) cells are identified in the mouse bone marrow (BM) more than 3 months after injection of hESCs that were allowed to differentiate on S17 stromal cells for 7-24 days. Secondary engraftment studies further confirm long-term repopulating cells derived from hESCs. We also evaluated two mechanisms that may inhibit engraftment: host immunity and requirement for homing to BM. Treatment with anti-ASGM1 antiserum that primarily acts by depletion of natural killer cells in transplanted mice leads to improved engraftment, likely due to low levels of HLA class I expressed on hESCs and CD34(+) cells derived from hESCs. Intra-BM injection also provided stable engraftment, with hematopoietic cells identified in both the injected and contra-lateral femur. Importantly, no teratomas are evident in animals injected with differentiated hESCs. These results demonstrate that SCID-repopulating cells, a close surrogate for hematopoietic stem cells, can be derived from hESCs. Moreover, both adaptive and innate immune effector cells may be barriers to engraftment of these cells.

An increased prevalence of hyperhomocysteinemia has been observed among patients with end-stage renal disease, and numerous studies have demonstrated that kidney function is one of the most important determinants of plasma total homocysteine (tHcy) concentration. In an effort to understand the mechanism of hyperhomocysteinemia in renal disease, we chose, as our model, living kidney donors who had undergone uninephrectomy. We studied 10 living kidney donors and measured fasting plasma tHcy, plasma creatinine, folate, vitamins B(12) and B(6), and high-sensitivity C-reactive protein (hsCRP) 24 hours before nephrectomy and 2 days, 6 weeks, and 6 months after nephrectomy compared to the values 24 hours before nephrectomy. Mean fasting tHcy and creatinine concentrations were significantly higher in donors 2 days, 6 weeks and 6 months after nephrectomy they were 24 hours before nephrectomy. Both the increases in tHcy levels 2 days after nephrectomy and subsequent decreases 6 weeks and 6 months after are paralleled by the changes in plasma creatinine values, although neither returned to its presurgery value. Decreases in tHcy are significantly correlated with decreases in creatinine values. The B vitamins were unchanged, and the hsCRP level was increased 2 days after surgery but had returned to the baseline level after 6 weeks. We conclude that tHcy and creatinine levels parallel each other after uninephrectomy and that the gradual decrease in tHcy is accounted for by hypertrophy of the remaining kidney. Our results, the first to be obtained from living kidney donors, support the hypothesis that renal metabolism of tHcy is the mechanism responsible for the correlation between renal function and plasma tHcy level.

A high proportion of lower-risk del(5q) MDS patients will respond to treatment with lenalidomide. The estimated duration of transfusion-independence is 2 years including some long-lasting responses, but almost 40% of patients progress to acute leukemia by 5 years after start of treatment. As the molecular mechanisms underlying disease progression in del(5q) MDS remain to be elucidated, we do not know how to predict disease progression or how to monitor patients during lenalidomide treatment. We previously reported that small TP53 mutated subclones predict for an unfavorable outcome in del(5q) patients, and that these subclones expand with disease progression. However, whether or not other somatic mutations or factors related to the bone marrow microenvironment also contribute to disease progression has not been comprehensively assessed. We studied a longitudinal cohort of 35 low- and intermediate-1-risk del(5q) patients treated with lenalidomide (n=22), or other treatments including stem cell transplantation (n=13) by flow cytometric surveillance of hematopoietic stem and progenitor cells (HSPC) subsets, targeted sequencing of mutational patterns, and changes in the bone marrow microenvironment. In our cohort, 13 of 35 patients progressed to either higher-risk MDS (n=4) or leukemia (n=9), 12 of whom were treated with lenalidomide. Progression was associated with the detection of a restricted subset of new recurrent mutations, either alone or in combination: TP53 (n=9, p=0.0004), TET2 (n=6, p=0.006), RUNX1 (n=3, p=0.044), and PTPN11 (n=1). Regardless of whether the three mutations (TP53, TET2 and RUNX1) were present in the initial sample or whether they subsequently developed, testing positive for any of them carried a high probability (13/16, 81%) for predicting progression. For 11 out of 13 patients the new mutations were detected prior to the time point of clinical progression and the median time from detection of the mutation to clinical evidence of progression was 42 months (range 0-83.9). Thus, we were able to detect the mutation in the majority of cases well before clinical signs of disease progression. Seven of the nine patients who developed leukemia carried a TP53 mutation. Based on a median sequencing depth of 370 reads, the mutation was considered present pre-treatment in one of these patients and to have developed under treatment in the other six. Using flow cytometry for surveillance of HSPC subsets in lenalidomide-treated patients, we found that neither lenalidomide treatment nor the acquisition of additional mutations led to any uniform profound changes in the hematopoietic hierarchy unless the patient showed clinical signs of progression. Microarray analysis of mesenchymal stromal cells (MSC) exhibited an expression footprint consistent with MSC with high expression of typical MSC markers and absence of hematopoietic gene signatures. However, we observed only minor differences in gene expression between pre- treatment del(5q) and healthy MSC. In conclusion, while flow cytometric analysis of HSPC populations or analysis of the microenvironment had limited predictive value in this cohort of lower-risk del(5q) MDS, all patients who progressed to either higher-risk MDS or leukemia were identified by harboring recurrent mutations in a limited number of genes, i.e., TP53, RUNX1, TET2, and PTPN11. Based on our data, we advocate for conducting a prospective study aimed at investigating in a larger number of del(5q) MDS cases pre- and post-lenalidomide treatment, whether the detection of such mutations can guide clinical decision making, such as suggesting which patients should undergo hematopoietic cell transplantation. Disclosures No relevant conflicts of interest to declare.

The association between somatic JAK2 mutation and myeloproliferative neoplasms (MPNs) is now well established. However, because JAK2 mutations are associated with heterogeneous clinical phenotypes and often occur as secondary genetic events, some aspects of JAK2 mutation biology remain to be understood. We recently described a germline JAK2V617I mutation in a family with hereditary thrombocytosis and herein characterize the hematopoietic and signaling impact of JAK2V617I. Through targeted sequencing of MPN-associated mutations, exome sequencing, and clonality analysis, we demonstrate that JAK2V617I is likely to be the sole driver mutation in JAK2V617I-positive individuals with thrombocytosis. Phenotypic hematopoietic stem cells (HSCs) were increased in the blood and bone marrow of JAK2V617I-positive individuals and were sustained at higher levels than controls after xenotransplantation. In signaling and transcriptional assays, JAK2V617I demonstrated more activity than wild-type JAK2 but substantially less than JAK2V617F. After cytokine stimulation, JAK2V617I resulted in markedly increased downstream signaling compared with wild-type JAK2 and comparable with JAK2V617F. These findings demonstrate that JAK2V617I induces sufficient cytokine hyperresponsiveness in the absence of other molecular events to induce a homogeneous MPN-like phenotype. We also provide evidence that the JAK2V617I mutation may expand the HSC pool, providing insights into both JAK2 mutation biology and MPN disease pathogenesis.

Background: Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukaemia (CMML) are haematological disorders that develop in haematopoietic stem or progenitor cells (HSPCs) and are characterised by ineffective haematopoiesis. 5'-Azacitidine (AZA), a DNA demethylating agent, is the primary drug for the treatment of high-risk MDS and CMML and response is associated with improved survival benefits. However, only half of treated patients will ever respond to AZA and the molecular basis for poor response is currently unknown. There are few alternative therapies for the non-responders. Additionally, AZA response is rarely sustained and a substantial fraction of responders will eventually relapse. The in vivo effect of AZA therapy on dysplastic cells in responders is unclear and there are no predictive markers for impending relapse in responders. Methods: To address these fundamental questions, we enrolled 18 high-risk MDS and CMML patients on a compassionate access program for AZA in Australia. Bone marrow was collected at seven different points - before treatment; through 6 cycles of treatment; and at up to two years after initiation - and we isolated high-purity CD34+ HSPCs (Figure A). 10 patients had a complete response while 8 were poorer responders. We performed RNA-seq to query the transcriptomes (and validated by Fluidigm-based PCR) and deduced the clonal evolution in the bone marrow in response to AZA therapy (by whole exome-sequencing, followed by targeted capture resequencing, and genotyping of individual CFU colonies). Our findings were validated in an independent cohort of 57 patients. We used flow cytometry to develop a clinically relevant prognostic assay for AZA resistance and developed a novel stromal co-culture based functional drug testing platform to rationally discover combinational drug therapies to overcome AZA resistance. Results: We hypothesised that primary AZA resistance would be driven by pre-existing molecular differences between responders and non-responders. Analysis of the pre-treatment RNA-seq data strikingly revealed the differential expression of 1148 genes between responders and non-responders (Figure B). Pathway analyses of these genes indicated that cell cycle and DNA damage response pathways were relatively up-regulated in responders compared to non-responders, indicating that HSPCs of non-responders are more quiescent compared to responders (Figure C). We validated these gene expression differences in independent patient cohorts from the U.K. and Sweden (n=57; 27 responders, 30 non-responders). We then adapted a flow cytometry based assay, amenable to prospective use in a clinical diagnostic setting, to directly detect the increased quiescence of CD34+ CD38+ haematopoietic progenitors in unsorted bone marrows of non-responders across all cohorts (Figure D). Finally, to reverse the quiescence of progenitor cells of non-responders, and make them more susceptive to AZA, we leveraged our RNA-seq discoveries to target pathways that were relatively up-regulated in non-responders. Using a stromal co-culture drug testing platform that we developed, we discovered that inhibiting integrin-linked signalling combinatorially with AZA improved the functionality of dysplastic cells (Figure E). Additionally, dysplastic cells were particularly sensitive to the inhibition of the mTOR pathway. To trace the fate of dysplastic cells as patients undergo AZA therapy, and thereby understand the basis of eventual relapse in responders, we performed whole exome sequencing of all patients (Figure F). Using the mutations as "molecular barcodes", we deduced the clonal architecture in each individual and observed the clonal evolution that occurred in response to AZA treatment. Combined with genotyping of CFU colonies grown in vitro, we have discovered that clonal haematopoiesis originating from resistant multipotent cells bearing mutations persists even upon complete response and forms the basis for eventual relapse (Figure G). Conclusions: Our findings, across independent cohorts and relevant to both MDS and CMML, have immediate clinical utility not simply to prospectively identify AZA non-responders but also by suggesting combinatorial therapies that could improve response. Finally, elucidating the in vivo effects of AZA therapy lay the foundation for developing more durable treatments. Figure 1. Figure 1. Disclosures Kulasekararaj: Alexion: Consultancy. Lynch:Celgene: Employment, Equity Ownership. Campbell:14M genomics: Other: Co-founder and consultant.

Molecularly targeted therapy frequently induces remissions in cancer, but rarely achieves complete disease eradication, with resulting risk of disease relapse/progression. Chronic myeloid leukemia (CML) is a good example, with rare, propagating stem cells (SCs) that are incompletely eradicated by BCR-ABL directed tyrosine kinase inhibitor (TKI) therapy in most cases. Consequently, CML relapse usually occurs following treatment discontinuation. Further, some patients fail to achieve a satisfactory response to TKI therapy and are at risk of disease progression. Ultimately, the depth of response to TKIs is dictated by CML-SCs, however, it has proven challenging to characterise this crucial population of cells as they reside in the same compartment as their normal hematopoietic stem cell (HSC) counterparts from which they cannot be reliably distinguished throughout the disease course. Advances in single cell transcriptomics are opening up unprecedented opportunities to unravel heterogeneity in cell populations. However, to date, this technology has primarily been used to analyse normal tissues, partly reflecting the lack of sensitivity for detection of somatic mutations using current techniques. Herein, we developed a novel protocol allowing targeted amplification of BCR-ABL during single cell cDNA library generation using a modification of a published protocol (PMID:24385147). We then applied this method to analyse 1082 bone marrow SCs (defined as Lin-CD34+CD38-) from a cohort of 10 patients with CML and 3 normal controls. We first validated our method using the BCR-ABL +ve K562 cell line. Surprisingly, using standard methods, BCR-ABL was only detected in a small minority of cells. With our modified technique, we demonstrate robust detection of BCR-ABL in 100% of single K562 cells with parallel whole transcriptome amplification from the same cell. Further, using a plasmid Òspike-inÓ, we demonstrate sensitivity to detect a single molecule of BCR-ABL per cell, without false +ve BCR-ABL amplification in 142 cells tested. We then analysed 40 SCs from a patient with CML who was in a cytogenetic remission (CyR) following 3 months of TKI therapy. BCR-ABL was detected in 16 cells and was absent in 24 cells. With an average of 3.3 million mapped reads per single cell, we detected over 4600 genes per cell with good correlation of single cell ensemble data with bulk analysis. Using this approach we were uniquely able to compare BCR-ABL +ve and -ve cells and identify a large number of differentially expressed genes, which would not have been detected through bulk analysis. We selected a number of genes of biologic interest for validation by Q-PCR and 80% of them were confirmed as differentially expressed, a number of these are highlighted in the heatmap (see Figure). We then analysed 717 single SCs from 8 CML diagnostic samples. This analysis identified marked heterogeneity of BCR-ABL expression with frequencies comparable to the published literature (9% to 92%). Unsupervised clustering analysis revealed many genes that were differentially expressed between BCR-ABL +ve and -ve SCs e.g. CML-SCs showed upregulation of cell cycle signatures and decreased expression of HSC-affiliated genes. We next analysed 223 single SCs from a series of 5 patients in CyR following TKI initiation. BCR-ABL positive SCs could still be detected, albeit at a reduced frequency (0.5% to 45%). In contrast to the diagnostic samples, CML-SCs showed quiescence and HSC signatures that were comparable to normal HSCs. Furthermore, through analysis of serial samples from diagnosis to remission from the same patient, this single-cell approach uniquely allowed analysis of gene expression specifically of the CML-SCs which evade TKI therapy, facilitating the identification of a number of candidate gene-sets including cell surface markers and potentially ÒdruggableÓ targets. Finally, through analysis of a patient with early blast crisis transformation of CML during TKI therapy, we illustrate how single cell RNA sequencing might be applied to predict such early disease progression events. We herein describe a new approach for single cell RNA sequencing of CML-SCs that might be applied to fate-map persistent CML-SCs during and following treatment for discovery research and also to refine precision medicine in CML. This approach could be applied across a range of clonal disorders with potential broader relevance for cancer research. Disclosures No relevant conflicts of interest to declare.

GATA3 has been identified as a master regulator of T helper cells, as well as being important for early thymic progenitors and T-cell commitment. However, Gata3 expression initiates already at the hematopoietic stem cell (HSC) level, implicating a potential role also in the regulation of HSCs. Herein we used a conditional Gata3 knockout strategy in which Gata3 expression was completely deleted from the earliest stage of embryonic hematopoietic development after emergence of HSCs from hemogenic endothelium. Through a detailed analysis of HSCs at the phenotypic and functional level, we demonstrate that steady-state levels of HSCs are normal in Gata3fl/flVav-Cretg/+ mice. Moreover, through long-term primary and secondary transplantation experiments, we also unequivocally demonstrate that Gata3 has a redundant role in post-transplantation HSC self-renewal.

Abstract 2335 The stepwise lineage-commitment from hematopoietic stem cells (HSCs) in the bone marrow (BM) to T-lymphocyte-restricted progenitors in the thymus represents a paradigm for how distinct stages of lineage restriction from a multipotent to a lineage-restricted progenitor require different extrinsic cues. However, the commitment stage at which progenitors migrate from the BM to the thymus remains unclear. Previous studies demonstrated the existence of adult early thymic progenitors (ETPs) restricted to T lymphocyte and granulocyte-monocyte (GM) fates (Bell and Bhandoola, Nature 2008; Wada et al., Nature 2008). The challenge remains to couple this thymic T–GM restricted progenitor to candidate thymus seeding progenitors (TSPs) identified in the BM, such as common lymphoid progenitors, lymphoid-primed multipotent progenitors (LMPPs) and HSCs, which all also possess B cell potential. Using high resolution FACS and sensitive clonal assays, we have identified the most primitive ETPs in the neonatal thymus and demonstrate at the single cell level that rare Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs possess combined granulocyte-monocyte (GM), T and B lymphocyte but not megakaryocyte-erythroid (MkE) lineage potentials, identical to closely molecularly related thymus-seeding progenitors in the BM. Moreover, global molecular profiling demonstrates that neonatal Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs cluster closest to multipotent progenitors in the BM, rather than subsequent (DN1 and DN2) progenitor stages in the thymus. Finally, in support of Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs with combined T, B and GM lineage potentials, being the most multipotent progenitors in the thymus, phenotypic and functional studies demonstrate that the neonatal thymus is not seeded by HSCs or MPPs with MkE potential. These findings establish for the first time a distinct lineage commitment stage for the transition of T-lineage commitment from the BM to the remote thymus. Disclosures: No relevant conflicts of interest to declare.

SummaryThe relationships between normal and leukemic stem/progenitor cells are unclear. We show that in ∼80% of primary human CD34+ acute myeloid leukemia (AML), two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. Both populations have leukemic stem cell (LSC) activity and are hierarchically ordered; one LSC population gives rise to the other. Global gene expression profiling shows the LSC populations are molecularly distinct and resemble normal progenitors but not stem cells. The more mature LSC population most closely mirrors normal granulocyte-macrophage progenitors (GMP) and the immature LSC population a previously uncharacterized progenitor functionally similar to lymphoid-primed multipotential progenitors (LMPPs). This suggests that in most cases primary CD34+ AML is a progenitor disease where LSCs acquire abnormal self-renewal potential.

Abstract 1585 Recent studies utilising surrogate leukaemic stem cell (LSC) assays have suggested that LSCs in acute lymphoblastic leukaemias (ALLs) might be neither rare, nor phenotypically or functionally distinct. However, studies of candidate LSCs in surrogate assays might not recapitulate the full leukaemic potential of candidate LSCs in patients, and in particular their responsiveness and resistance to therapeutic targeting. Therefore, we have investigated the identity, molecular and functional properties, and persistence of different subsets of candidate LSCs in childhood ALL, at diagnosis and during the course of clinical and molecular remissions in response to chemotherapy, and their relationship to subsequent relapses. First, we investigated 6 patients diagnosed with “good prognosis” TEL-AML1+ ALL, and at diagnosis we found TEL-AML1+ leukaemic cells within the immature B cell progenitor compartment (proB: 34+38+19+), mature B-cells (34-19+), as well as in a population expressing an aberrant combination of stem cell (34+38-/lo) and B-cell (19+) cell surface markers. These stem/B (34+38-/lo19+) cells were all TEL-AML1+ and not present in age-matched normal bone marrow controls. In contrast, haematopoietic stem cells (HSC: 34+38-19-) were not part of the TEL-AML1+ leukaemic clone in any of the patients. 15 days into chemotherapy, all TEL-AML1+ mature B-cells were eliminated in all patients, and this was followed by a clearance of leukaemic proB cells by day 28 of treatment. In striking contrast, leukaemic stem/B cells were still detectable at day 28, but in all TEL-AML1 patients, at later stages all leukaemic cells including the stem/B cells were undetectable, and at the same time these patients went into complete remission with less than 1 leukaemic cell in 10e4 cells detectable. A similar pattern was observed in a case of “high risk” BCR-ABL+ ALL: BCR-ABL+ proB and B-cells were efficiently eliminated by day 90 of the course of chemotherapy, and up to 180 days into the treatment only 34+38-/lo19+ stem/B cells remained part of the BCR-ABL+ clone. In agreement with the persistence of BCR-ABL+ 34+38-/lo19+ stem/B cells, this patient relapsed 17 months after the initiation of chemotherapy. In order to understand the underlying mechanisms of the observed functional and therapeutic heterogeneity seen in leukaemic subpopulations, we performed comparative gene-expression analysis of diagnostic leukaemic stem/B and proB cells of TEL-AML1+ patients. This analysis revealed a differential gene expression pattern between leukaemic stem/B and proB cells, with positive regulators of cell cycle being the most distinctly up regulated genes in leukaemic proB cells. In agreement with this, cell cycle analysis of 3 diagnostic TEL-AML1+ cases also showed proB cells to be more actively cycling compared to the more quiescent state of the leukaemic stem/B compartment (proB: G0 42%; G1 40%; S,G2,M 18% vs. stem/B: G0 81%; G1 18%; S,G2,M 1%), providing a potential mechanistic basis for the relative therapy resistance of ALL stem/B cells. Taken together the present studies suggest that quiescent 34+38-/lo19+ stem/B cells are selectively resistant to chemotherapy, and most likely the origin of relapses when these occur in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

BACKGROUND: The in vivo clinical significance of malignant stem cells remains unclear. METHODS: Patients who have the 5q deletion (del[5q]) myelodysplastic syndrome (interstitial deletions involving the long arm of chromosome 5) have complete clinical and cytogenetic remissions in response to lenalidomide treatment, but they often have relapse. To determine whether the persistence of rare but distinct malignant stem cells accounts for such relapses, we examined bone marrow specimens obtained from seven patients with the del(5q) myelodysplastic syndrome who became transfusion-independent while receiving lenalidomide treatment and entered cytogenetic remission. RESULTS: Virtually all CD34+, CD38+ progenitor cells and stem cells that were positive for CD34 and CD90, with undetectable or low CD38 (CD38−/low), had the 5q deletion before treatment. Although lenalidomide efficiently reduced these progenitors in patients in complete remission, a larger fraction of the minor, quiescent, CD34+,CD38-/low, CD90+ del(5q) stem cells as well as functionally defined del(5q) stem cells remained distinctly resistant to lenalidomide. Over time, lenalidomide resistance developed in most of the patients in partial and complete remission, with recurrence or expansion of the del(5q) clone and clinical and cytogenetic progression. CONCLUSIONS: In these patients with the del(5q) myelodysplastic syndrome, we identified rare and phenotypically distinct del(5q) myelodysplastic syndrome stem cells that were also selectively resistant to therapeutic targeting at the time of complete clinical and cytogenetic remission. (Funded by the EuroCancerStemCell Consortium and others.)