Jessica McLeod

Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia

In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3Amut) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3Amut-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3Amut arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.

Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma

We used high-resolution SNP genotyping to identify regions of genomic gain and loss in the genomes of 212 medulloblastomas, malignant pediatric brain tumors. We found focal amplifications of 15 known oncogenes and focal deletions of 20 known tumor suppressor genes (TSG), most not previously implicated in medulloblastoma. Notably, we identified previously unknown amplifications and homozygous deletions, including recurrent, mutually exclusive, highly focal genetic events in genes targeting histone lysine methylation, particularly that of histone 3, lysine 9 (H3K9). Post-translational modification of histone proteins is critical for regulation of gene expression, can participate in determination of stem cell fates and has been implicated in carcinogenesis. Consistent with our genetic data, restoration of expression of genes controlling H3K9 methylation greatly diminishes proliferation of medulloblastoma in vitro. Copy number aberrations of genes with critical roles in writing, reading, removing and blocking the state of histone lysine methylation, particularly at H3K9, suggest that defective control of the histone code contributes to the pathogenesis of medulloblastoma.

Distinct routes of lineage development reshape the human blood hierarchy across ontogeny

Adjusting hematopoietic hierarchy In adults, more than 300 billion blood cells are replenished daily. This output arises from a cellular hierarchy where stem cells differentiate into a series of multilineage progenitors, culminating in unilineage progenitors that generate over 10 different mature blood cell types. Notta et al. mapped the lineage potential of nearly 3000 single cells from 33 different cell populations of stem and progenitor cells from fetal liver, cord blood, and adult bone marrow (see the Perspective by Cabezas-Wallscheid and Trumpp). Prenatally, stem cell and progenitor populations were multilineage with few unilineage progenitors. In adults, multilineage cell potential was only seen in stem cell populations. Science, this issue p. 10.1126/science.aab2116; see also p. 126 As humans age, progenitor cells take over from stem cells the task of producing a steady supply of blood cells. [Also see Perspective by Cabezas-Wallscheid and Trumpp] INTRODUCTION The hematopoietic road map is a compilation of the various lineage differentiation routes that a stem cell takes to make blood. This program produces greater than 10 blood cell fates and is responsible for generating more than 300 billion cells daily. On several occasions over the past six decades, the murine road map has been reconceived due to new information overturning dogma. However, the human road map has changed little. In the human model, blood differentiation initiates at the level of multipotent stem cells and passes through a series of increasingly lineage-restricted oligopotent and, finally, unipotent progenitor intermediates. One critical oligopotent intermediate is the common myeloid progenitor (CMP), believed to be the origin of all myeloid (My), erythroid (Er), and megakaryocyte (Mk) cells. Although murine studies challenge the existence of oligopotent progenitors, a comprehensive analysis of human My-Er-Mk differentiation is lacking. Moreover, whether the pool of oligopotent intermediates is fixed across human development (fetal to adult) is unknown. RATIONALE The differentiation road map taken by human hematopoietic stem cells (HSCs) is fundamental to our understanding of blood homeostasis, hematopoietic malignancies, and regenerative medicine. RESULTS We mapped the cellular origins of My, Er, and Mk lineages across three time points in human blood development: fetal liver (FL), neonatal cord blood (CB), and adult bone marrow (BM). Using a cell-sorting scheme based on markers linked to Er and Mk lineage specification (CD71 and CD110), we found that previously described populations of multipotent progenitors (MPPs), CMPs, and megakaryocyte-erythroid progenitors (MEPs) were heterogeneous and could be further purified. Nearly 3000 single cells from 11 cellular subsets from the CD34+ compartment of FL, CB, and BM (33 subsets in total) were evaluated for their My, Er, and Mk lineage potential using an optimized single-cell assay. In FL, the ratio of cells with multilineage versus unilineage potential remained constant in both the stem cell (CD34+CD38–) and progenitor cell (CD34+CD38+) enriched compartments. By contrast, in BM, nearly all multipotent cells were restricted to the stem cell compartment, whereas unilineage progenitors dominated the progenitor cell compartment. Oligopotent progenitors were only a negligible component of the human blood hierarchy in BM, leading to the inference that multipotent cells differentiate into unipotent cells directly by adulthood. Mk/Er activity predominantly originated from the stem cell compartment at all developmental time points. In CB and BM, most Mks emerged as part of mixed clones from HSCs/MPPs, indicating that Mks directly branch from a multipotent cell and not from oligopotent progenitors like CMP. In FL, an almost pure Mk/Er progenitor was identified in the stem cell compartment, although less potent Mk/Er progenitors were also present in the progenitor compartment. In a hematological condition of HSC loss (aplastic anemia), Mk/Er but not My progenitors were more severely depleted, pinpointing a close physiological connection between HSC and the Mk/Er lineage. CONCLUSION Our data indicate that there are distinct road maps of blood differentiation across human development. Prenatally, Mk/Er lineage branching occurs throughout the cellular hierarchy. By adulthood, both Mk/Er activity and multipotency are restricted to the stem cell compartment, whereas the progenitor compartment is composed of unilineage progenitors forming a “two-tier” system, with few intervening oligopotent intermediates. Roadmaps of human blood stem cell differentiation. The classical model envisions that oligopotent progenitors such as CMP are an essential intermediate stage from which My/Er/Mk differentiation originates. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation. In a classical view of hematopoiesis, the various blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34+ cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er, and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead, only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult “two-tier” hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.

A 17-gene stemness score for rapid determination of risk in acute leukaemia.

Refractoriness to induction chemotherapy and relapse after achievement of remission are the main obstacles to cure in acute myeloid leukaemia (AML). After standard induction chemotherapy, patients are assigned to different post-remission strategies on the basis of cytogenetic and molecular abnormalities that broadly define adverse, intermediate and favourable risk categories. However, some patients do not respond to induction therapy and another subset will eventually relapse despite the lack of adverse risk factors. There is an urgent need for better biomarkers to identify these high-risk patients before starting induction chemotherapy, to enable testing of alternative induction strategies in clinical trials. The high rate of relapse in AML has been attributed to the persistence of leukaemia stem cells (LSCs), which possess a number of stem cell properties, including quiescence, that are linked to therapy resistance. Here, to develop predictive and/or prognostic biomarkers related to stemness, we generated a list of genes that are differentially expressed between 138 LSC+ and 89 LSC− cell fractions from 78 AML patients validated by xenotransplantation. To extract the core transcriptional components of stemness relevant to clinical outcomes, we performed sparse regression analysis of LSC gene expression against survival in a large training cohort, generating a 17-gene LSC score (LSC17). The LSC17 score was highly prognostic in five independent cohorts comprising patients of diverse AML subtypes (n = 908) and contributed greatly to accurate prediction of initial therapy resistance. Patients with high LSC17 scores had poor outcomes with current treatments including allogeneic stem cell transplantation. The LSC17 score provides clinicians with a rapid and powerful tool to identify AML patients who do not benefit from standard therapy and who should be enrolled in trials evaluating novel upfront or post-remission strategies.

Tracing the origins of relapse in acute myeloid leukaemia to stem cells

In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving remission. Historically, the failure of therapy has been thought to be due to mutations that produce drug resistance, possibly arising as a consequence of the mutagenic properties of chemotherapy drugs. However, other lines of evidence have pointed to the pre-existence of drug-resistant cells. For example, deep sequencing of paired diagnosis and relapse acute myeloid leukaemia samples has provided direct evidence that relapse in some cases is generated from minor genetic subclones present at diagnosis that survive chemotherapy, suggesting that resistant cells are generated by evolutionary processes before treatment and are selected by therapy. Nevertheless, the mechanisms of therapy failure and capacity for leukaemic regeneration remain obscure, as sequence analysis alone does not provide insight into the cell types that are fated to drive relapse. Although leukaemia stem cells have been linked to relapse owing to their dormancy and self-renewal properties, and leukaemia stem cell gene expression signatures are highly predictive of therapy failure, experimental studies have been primarily correlative and a role for leukaemia stem cells in acute myeloid leukaemia relapse has not been directly proved. Here, through combined genetic and functional analysis of purified subpopulations and xenografts from paired diagnosis/relapse samples, we identify therapy-resistant cells already present at diagnosis and two major patterns of relapse. In some cases, relapse originated from rare leukaemia stem cells with a haematopoietic stem/progenitor cell phenotype, while in other instances relapse developed from larger subclones of immunophenotypically committed leukaemia cells that retained strong stemness transcriptional signatures. The identification of distinct patterns of relapse should lead to improved methods for disease management and monitoring in acute myeloid leukaemia. Moreover, the shared functional and transcriptional stemness properties that underlie both cellular origins of relapse emphasize the importance of developing new therapeutic approaches that target stemness to prevent relapse.

An Integrated Analysis of Heterogeneous Drug Responses in Acute Myeloid Leukemia That Enables the Discovery of Predictive Biomarkers

Many promising new cancer drugs proceed through preclinical testing and early-phase trials only to fail in late-stage clinical testing. Thus, improved models that better predict survival outcomes and enable the development of biomarkers are needed to identify patients most likely to respond to and benefit from therapy. Here, we describe a comprehensive approach in which we incorporated biobanking, xenografting, and multiplexed phospho-flow (PF) cytometric profiling to study drug response and identify predictive biomarkers in acute myeloid leukemia (AML) patients. To test the efficacy of our approach, we evaluated the investigational JAK2 inhibitor fedratinib (FED) in 64 patient samples. FED robustly reduced leukemia in mouse xenograft models in 59% of cases and was also effective in limiting the protumorigenic activity of leukemia stem cells as shown by serial transplantation assays. In parallel, PF profiling identified FED-mediated reduction in phospho-STAT5 (pSTAT5) levels as a predictive biomarker of in vivo drug response with high specificity (92%) and strong positive predictive value (93%). Unexpectedly, another JAK inhibitor, ruxolitinib (RUX), was ineffective in 8 of 10 FED-responsive samples. Notably, this outcome could be predicted by the status of pSTAT5 signaling, which was unaffected by RUX treatment. Consistent with this observed discrepancy, PF analysis revealed that FED exerted its effects through multiple JAK2-independent mechanisms. Collectively, this work establishes an integrated approach for testing novel anticancer agents that captures the inherent variability of response caused by disease heterogeneity and in parallel, facilitates the identification of predictive biomarkers that can help stratify patients into appropriate clinical trials.

Corrigendum: Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia

This corrects the article DOI: 10.1038/nature13038

Erratum: Corrigendum: Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia

This corrects the article DOI: 10.1038/nature13038

Erratum: Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia (Nature (2014) 506 (328-333) DOI: 10.1038/nature13038)

This corrects the article DOI: 10.1038/nature13038