Faiyaz Notta

The genetic basis of early T-cell precursor acute lymphoblastic leukaemia.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment

Proteins are identified that underlie the early commitment steps of human hematopoietic stem cell differentiation. Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs) to perpetually replenish mature cells via a series of lineage-restricted intermediates. Investigating the molecular state of HSCs is contingent on the ability to purify HSCs away from transiently engrafting cells. We demonstrated that human HSCs remain infrequent, using current purification strategies based on Thy1 (CD90) expression. By tracking the expression of several adhesion molecules in HSC-enriched subsets, we revealed CD49f as a specific HSC marker. Single CD49f+ cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting multipotent progenitors (MPPs). The demarcation of human HSCs and MPPs will enable the investigation of the molecular determinants of HSCs, with a goal of developing stem cell–based therapeutics.

Evolution of human BCR–ABL1 lymphoblastic leukaemia-initiating cells

Many tumours are composed of genetically diverse cells; however, little is known about how diversity evolves or the impact that diversity has on functional properties. Here, using xenografting and DNA copy number alteration (CNA) profiling of human BCR–ABL1 lymphoblastic leukaemia, we demonstrate that genetic diversity occurs in functionally defined leukaemia-initiating cells and that many diagnostic patient samples contain multiple genetically distinct leukaemia-initiating cell subclones. Reconstructing the subclonal genetic ancestry of several samples by CNA profiling demonstrated a branching multi-clonal evolution model of leukaemogenesis, rather than linear succession. For some patient samples, the predominant diagnostic clone repopulated xenografts, whereas in others it was outcompeted by minor subclones. Reconstitution with the predominant diagnosis clone was associated with more aggressive growth properties in xenografts, deletion of CDKN2A and CDKN2B, and a trend towards poorer patient outcome. Our findings link clonal diversity with leukaemia-initiating-cell function and underscore the importance of developing therapies that eradicate all intratumoral subclones.

Hematopoiesis: A Human Perspective

Despite its complexity, blood is probably the best understood developmental system, largely due to seminal experimentation in the mouse. Clinically, hematopoietic stem cell (HSC) transplantation represents the most widely deployed regenerative therapy, but human HSCs have only been characterized relatively recently. The discovery that immune-deficient mice could be engrafted with human cells provided a powerful approach for studying HSCs. We highlight 2 decades of studies focusing on isolation and molecular regulation of human HSCs, therapeutic applications, and early lineage commitment steps, and compare mouse and humanized models to identify both conserved and species-specific mechanisms that will aid future preclinical research.

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 Distinctive DNA Damage Response in Human Hematopoietic Stem Cells Reveals an Apoptosis-Independent Role for p53 in Self-Renewal

Highly regenerative tissues such as blood must possess effective DNA damage responses (DDR) that balance long-term regeneration with protection from leukemogenesis. Hematopoietic stem cells (HSCs) sustain life-long blood production, yet their response to DNA damage remains largely unexplored. We report that human HSCs exhibit delayed DNA double-strand break rejoining, persistent gammaH2AX foci, and enhanced p53- and ASPP1-dependent apoptosis after gamma-radiation compared to progenitors. p53 inactivation or Bcl-2 overexpression reduced radiation-induced apoptosis and preserved in vivo repopulating HSC function. Despite similar protection from irradiation-induced apoptosis, only Bcl-2-overexpressing HSCs showed higher self-renewal capacity, establishing that intact p53 positively regulates self-renewal independently from apoptosis. The reduced self-renewal of HSCs with inactivated p53 was associated with increased spontaneous gammaH2AX foci in secondary transplants of HSCs. Our data reveal distinct physiological roles of p53 that together ensure optimal HSC function: apoptosis regulation and prevention of gammaH2AX foci accumulation upon HSC self-renewal.

A renewed model of pancreatic cancer evolution based on genomic rearrangement patterns.

Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours.

Engraftment of human hematopoietic stem cells is more efficient in female NOD/SCID/IL-2Rgc-null recipients.

Repopulation of immunodeficient mice remains the primary method to assay human hematopoietic stem cells (HSCs). Here we report that female NOD/SCID/IL-2Rg(c)-null mice are far superior in detecting human HSCs (Lin(-)CD34(+)CD38(-)CD90(+)CD45RA(-)) compared with male recipients. When multiple HSCs were transplanted, female recipients displayed a trend (1.4-fold) toward higher levels of human chimerism (female vs male: injected femur, 44.4 +/- 9.3 vs 32.2 +/- 6.2; n = 12 females, n = 24 males; P = .1). Strikingly, this effect was dramatically amplified at limiting cell doses where female recipients had an approximately 11-fold higher chimerism from single HSCs (female vs male: injected femur, 8.1 +/- 2.7 vs 0.7 +/- 0.7; n = 28 females, n = 20 males; P < .001). Secondary transplantations from primary recipients indicate that females more efficiently support the self-renewal of human HSCs. Therefore, sex-associated factors play a pivotal role in the survival, proliferation, and self-renewal of human HSCs in the xenograft model, and recipient sex must be carefully monitored in the future design of experiments requiring human HSC assays.

PLZF is a regulator of homeostatic and cytokine-induced myeloid development

A major question in hematopoiesis is how the system maintains long-term homeostasis whereby the generation of large numbers of differentiated cells is balanced with the requirement for maintenance of progenitor pools, while remaining sufficiently flexible to respond to periods of perturbed cellular output during infection or stress. We focused on the development of the myeloid lineage and present evidence that promyelocytic leukemia zinc finger (PLZF) provides a novel function that is critical for both normal and stress-induced myelopoiesis. During homeostasis, PLZF restricts proliferation and differentiation of human cord blood-derived myeloid progenitors to maintain a balance between the progenitor and mature cell compartments. Analysis of PLZF promoter-binding sites revealed that it represses transcription factors involved in normal myeloid differentiation, including GFI-1, C/EBPalpha, and LEF-1, and induces negative regulators DUSP6 and ID2. Loss of ID2 relieves PLZF-mediated repression of differentiation identifying it as a functional target of PLZF in myelopoiesis. Furthermore, induction of ERK1/2 by myeloid cytokines, reflective of a stress response, leads to nuclear export and inactivation of PLZF, which augments mature cell production. Thus, negative regulators of differentiation can serve to maintain developmental systems in a primed state, so that their inactivation by extrinsic signals can induce proliferation and differentiation to rapidly satisfy increased demand for mature cells.

Efficacy of Retinoids in IKZF1-Mutated BCR-ABL1 Acute Lymphoblastic Leukemia

Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.