Cancer stem cells: The adventurous journey from hematopoietic to leukemic stem cells

This year’s Gairdner Foundation Award for Biomedical Research is awarded to John Dick for the discovery of leukemic stem cells and the hierarchical organization of acute myeloid leukemias. His work laid the foundation for the cancer stem cell model with numerous clinical implications for hematopoietic malignancies and solid tumors. This year’s Gairdner Foundation Award for Biomedical Research is awarded to John Dick for the discovery of leukemic stem cells and the hierarchical organization of acute myeloid leukemias. His work laid the foundation for the cancer stem cell model with numerous clinical implications for hematopoietic malignancies and solid tumors. The cellular regeneration of many tissues is driven by small populations of tissue-specific adult stem cells. A prime example for the regenerative capacity of somatic stem cells are hematopoietic stem cells (HSCs), which reside in the bone marrow of adult mammals. Notably, HSCs are capable of self-renewal and are responsible for the lifelong production and regeneration of blood and immune cells. The hematopoietic system is hierarchically organized with HSCs at the apex, giving rise to gradually more specialized progenitor populations that ultimately differentiate into functional mature blood and immune cells. A variety of hematological cancers, such as leukemias, are derived from hematopoietic stem and progenitor cells (HSPCs) by a stepwise acquisition of genetic and epigenetic aberrations that reprogram and subsequently transform the healthy hematopoietic system. The malignant transformation typically causes a block in differentiation of HSPCs, resulting in the accumulation of dysfunctional leukemic progenitors in the bone marrow or other hematopoietic organs. However, in contrast to the classical view that all leukemic cells are similar, it has become apparent that leukemias, like many other cancers, display a tremendous amount of intra-tumor heterogeneity. Most importantly, similar to the hierarchical organization of the healthy hematopoietic system, leukemias retain a hierarchical structure, with mostly few leukemic stem cells (LSCs) at the apex exhibiting unlimited self-renewal capacity fueling the bulk of the leukemia. Since LSCs, like normal HSCs, have the capacity to enter a reversible state of quiescence/dormancy, they are thought to resist standard chemotherapy and form the cellular reservoir that drives relapse by re-initiating the disease after remission. Over the past 35 years, the pioneering research by John Dick and colleagues has paved the way for our detailed understanding of how human HSCs and LSCs control the respective hierarchies of the healthy hematopoietic and malignant leukemic systems. These findings have laid the foundation for the more broadly formulated cancer stem cell (CSC) concept. It states that cancers are functionally heterogeneous often containing only a few stem cell-like cancer cells with unlimited self-renewal potential, termed CSCs, capable of clonally regenerating the tumor after a seemingly successful therapy (Reya et al., 2001Reya T. Morrison S.J. Clarke M.F. Weissman I.L. Stem cells, cancer, and cancer stem cells.Nature. 2001; 414: 105-111Google Scholar). The CSC concept has been further developed with far-reaching implications for the understanding and treatment of hierarchically organized cancers such as leukemias and numerous solid cancers (Batlle and Clevers, 2017Batlle E. Clevers H. Cancer stem cells revisited.Nat. Med. 2017; 23: 1124-1134Google Scholar). In the 1960s, the pioneering work by James Till and Ernest McCulloch marked the inception of the field of modern HSC biology. In murine transplantation models, the scientists showed that the transfer of bone marrow into irradiated mice resulted in the formation of clonal colonies in the spleen, consisting of multiple mature blood and immune cell lineages that were initiated by rare, self-renewing multipotent progenitor cells. In the following decades, murine HSCs were characterized in great detail by taking advantage of the new possibilities of fluorescence-activated cell sorting (FACS) combined with reconstitution assays in lethally irradiated mice. Spearheaded by the Weismann, Nakauchi, Morrison, and Eaves groups among others, this led to the identification of cell-surface marker profiles to isolate pure mouse HSCs that can reconstitute and maintain the hematopoietic system even after transplantation of a single HSC. However, the study of human HSCs and their role in leukemias remained much more challenging, particularly due to a lack of adequate in vivo models and tools that enabled the discrimination and prospective purification of distinct HSPC populations. In particular, the rejection of human HSCs by the murine immune system and a potential lack of cross-reactivity of endogenous murine cytokines that are required to support hematopoiesis in mice were major roadblocks in the development of xenograft models for the human hematopoietic system. In 1988, John Dick and colleagues successfully introduced xenograft mouse models that overcame these hurdles by transplanting healthy and malignant human bone marrow into severely immunodeficient mice (Kamel-Reid and Dick, 1988Kamel-Reid S. Dick J.E. Engraftment of immune-deficient mice with human hematopoietic stem cells.Science. 1988; 242: 1706-1709Google Scholar). Similar assays were introduced at the same time by the Weissman and Wilson groups. These xenograft assays enabled, for the first time, the study of human HSC function in vivo, recapitulated the pathophysiology of human disease, and provided a measure for the precise enumeration of human HSC activity (Bonnet and Dick, 1997Bonnet D. Dick J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat. Med. 1997; 3: 730-737Google Scholar; Kamel-Reid and Dick, 1988Kamel-Reid S. Dick J.E. Engraftment of immune-deficient mice with human hematopoietic stem cells.Science. 1988; 242: 1706-1709Google Scholar). Repopulation assays as introduced by Dick and colleagues have become the gold standard in the field for measuring human stem cell activity and have inspired the development of a variety of xenograft models for hematological malignancies as well as other cancers, opening the possibility for evaluating the effects of drugs on human cancer cells in a pre-clinical setting in vivo. With the advent of modern multi-parameter FACS, the identification of surface molecules and introduction of new flow cytometric gating strategies in combination with more sensitive xenograft assays permitted the identification and functional characterization of highly purified human HSPC population including the isolation of a single human HSC that is capable of long-term engraftment (Doulatov et al., 2010Doulatov S. Notta F. Eppert K. Nguyen L.T. Ohashi P.S. Dick J.E. Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development.Nat. Immunol. 2010; 11: 585-593Google Scholar; Notta et al., 2011Notta F. Doulatov S. Laurenti E. Poeppl A. Jurisica I. Dick J.E. Isolation of single human hematopoietic stem cells capable of long-term Multilineage engraftment.Science. 2011; 333: 218-221Google Scholar). Flow cytometric gating and purification schemes for HSCs and progenitors as introduced by the Dick group have been highly influential and have served as a foundation for experimental designs in the community until today. Together, the introduction of highly sensitive xenograft models and new approaches in purifying HSPC subpopulations have been instrumental for laying the grounds for our detailed understanding of healthy and malignant hematopoiesis and the validation of the CSC model as will be discussed in more detail in the next sections. More than 300 billion blood and immune cells are generated every day, orchestrated by a complex cellular differentiation program starting from a small pool of HSCs. In recent years the advent of single-cell genomic technologies has shed light on the cellular heterogeneity of such complex biological systems. However, decades ago, John Dick already recognized the importance of delineating the complex cellular relationships in the human hematopoietic system for understanding hematopoiesis. First, by combining clonal barcoding with xenograft transplantation models, his group revealed that distinct classes of HSCs that differ in their proliferative and self-renewal potential reside at the top of the hematopoietic hierarchy. Subsequently, John Dick introduced novel FACS gating schemes that enabled the prospective isolation of highly potent HSCs with long-term self-renewal capacity and pioneered the delineation of the developmental differentiation routes individual HSCs pass through while undergoing differentiation into blood and immune cells (Notta et al., 2011Notta F. Doulatov S. Laurenti E. Poeppl A. Jurisica I. Dick J.E. Isolation of single human hematopoietic stem cells capable of long-term Multilineage engraftment.Science. 2011; 333: 218-221Google Scholar, Notta et al., 2016Notta F. Zandi S. Takayama N. Dobson S. Gan O.I. Wilson G. Kaufmann K.B. McLeod J. Laurenti E. Dunant C.F. et al.Distinct routes of lineage development reshape the human blood hierarchy across ontogeny.Science. 2016; 351: aab2116Google Scholar). The molecular, cellular and functional characterization of highly purified HSPC populations and single HSPCs resulted in the identification of lineage demarcation points and revealed key insights into lineage commitment pathways of the macrophage, dendritic cell, lymphoid, erythroid, and megakaryocytic lineages (Doulatov et al., 2010Doulatov S. Notta F. Eppert K. Nguyen L.T. Ohashi P.S. Dick J.E. Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development.Nat. Immunol. 2010; 11: 585-593Google Scholar; Notta et al., 2016Notta F. Zandi S. Takayama N. Dobson S. Gan O.I. Wilson G. Kaufmann K.B. McLeod J. Laurenti E. Dunant C.F. et al.Distinct routes of lineage development reshape the human blood hierarchy across ontogeny.Science. 2016; 351: aab2116Google Scholar). These studies shaped how we perceive human blood formation, contributed to several paradigm shifts in hematopoiesis, and helped in establishing a refined model of HSC lineage commitment that is consistent with most recent models of murine hematopoiesis (Figure 1). Tracing the cellular origin of human cancers remains one of the most challenging and controversial topics in cancer research. Due to the highly overlapping features of undifferentiated leukemic cells and HSCs, John Dick and colleagues postulated already in the 1990s that a primitive stem or early progenitor cell, and not a committed cell, likely constitutes the cell of origin for malignant transformation in AML (Bonnet and Dick, 1997Bonnet D. Dick J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat. Med. 1997; 3: 730-737Google Scholar; Lapidot et al., 1994Lapidot T. Sirard C. Vormoor J. Murdoch B. Hoang T. Caceres-Cortes J. Minden M. Paterson B. Caligiuri M.A. Dick J.E. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.Nature. 1994; 367: 645-648Google Scholar). The identification of BCR-ABL translocations in normal hematopoietic progenitor cells and AML1-ETO transcripts in non-leukemic stem cells provided the first genetic evidence that healthy HSCs can be the target for transformation in human chronic myeloid leukemia and AML. Subsequently, using single-cell assays, the sequential acquisition of driver mutations starting from HSCs demonstrated the presence of pre-leukemic cells in patients (Jan et al., 2012Jan M. Snyder T.M. Corces-Zimmerman M.R. Vyas P. Weissman I.L. Quake S.R. Majeti R. Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia.Sci. Transl. Med. 2012; 4: 149ra118Google Scholar). Further proof for the hypothesis that AMLs have their initial origin in HSCs came from the finding that mutations acquired early in the disease course (e.g. DNMT3A, TET2) occurred both in multipotent HSCs and T cells, whereas mutations acquired later in clonal evolution were exclusively found in AML blasts (e.g. NPM1, FLT3-ITD) (Figure 1) (Shlush et al., 2014Shlush L.I. Zandi S. Mitchell A. Chen W.C. Brandwein J.M. Gupta V. Kennedy J.A. Schimmer A.D. Schuh A.C. Yee K.W. et al.Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia.Nature. 2014; 506: 328-333Google Scholar). This suggested that a multipotent cell, namely the HSC, must be the cell of origin in this case. Strikingly, such pre-leukemic DNMT3A mutant HSCs were shown to survive chemotherapy and displayed a proliferative advantage when compared to healthy HSCs and thus can serve as a source for relapse if fully transformed by additional mutations (Figure 1). In 2014, the concept of a long-lived, multipotent pre-leukemic stem cell pool, which derives from healthy HSCs by the acquisition of pre-leukemic mutations received unexpected support from two landmark studies that performed DNA sequencing of tens of thousands of blood samples from healthy donors. These analyses uncovered the phenomenon of clonal hematopoiesis, a clonal expansion of blood cells occurring in a considerable number of healthy aged individuals, marked by mutations in DNMT3A or other genes also recurrently mutated in hematological cancers (Köhnke and Majeti, 2021Köhnke T. Majeti R. Clonal hematopoiesis: from Mechanisms to clinical Intervention.Cancer Discov. 2021; 11: 2987-2997Google Scholar). In line with the concept of pre-leukemic HSCs, clonal hematopoiesis of indeterminate potential (CHIP) is generated by a single mutated HSC. It initiates a lifelong maintained genetically identical blood cell clone with each cell carrying the same mutation (Figure 1). The presence of CHIP is associated with increased risks for the development of hematological cancers and cardiovascular diseases, the latter potentially caused by a malfunctioning of mature mutant cells, such as macrophages (Köhnke and Majeti, 2021Köhnke T. Majeti R. Clonal hematopoiesis: from Mechanisms to clinical Intervention.Cancer Discov. 2021; 11: 2987-2997Google Scholar). Collectively, these findings provide key evidence for the role of healthy human HSCs as the cell of origin in leukemias and highlight the clinical relevance of the identification of pre-leukemic HSCs as the source of clonal hematopoiesis and precursors for AML and other hematological cancers. Throughout the past 50 years, it has become apparent that cancer cells and tissue stem cells share a multitude of common features, including high proliferative capacities, long-term self-renewal potential, and the activity of molecular pathways regulating stemness. John Dick’s seminal work in leukemias and solid cancers was instrumental for understanding the relationship between stem cells and cancer. While evidence for a hierarchical relationship among leukemic cells has already been generated throughout the 1970 and 1980s, the landmark studies by John Dick together with his colleagues Tsvee Lapidot, Dominque Bonnet, and others provided first direct experimental evidence for the existence of so-called LSCs, which sit on top of a hierarchically structured leukemia (Bonnet and Dick, 1997Bonnet D. Dick J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat. Med. 1997; 3: 730-737Google Scholar; Lapidot et al., 1994Lapidot T. Sirard C. Vormoor J. Murdoch B. Hoang T. Caceres-Cortes J. Minden M. Paterson B. Caligiuri M.A. Dick J.E. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.Nature. 1994; 367: 645-648Google Scholar). These studies demonstrated that the majority of leukemic cells in acute myeloid leukemias (AMLs) have limited proliferative and self-renewal capacity, whereas a distinct, rare population that share the immunophenotype with healthy HSCs and display a high self-renewal potential is exclusively able to re-initiate the leukemia in immunodeficient mice (Bonnet and Dick, 1997Bonnet D. Dick J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat. Med. 1997; 3: 730-737Google Scholar; Lapidot et al., 1994Lapidot T. Sirard C. Vormoor J. Murdoch B. Hoang T. Caceres-Cortes J. Minden M. Paterson B. Caligiuri M.A. Dick J.E. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.Nature. 1994; 367: 645-648Google Scholar). This discovery marked the first functional proof of a CSC, a concept that was further expanded to solid tumors and refined a few years later by Irving Weissman and colleagues (Reya et al., 2001Reya T. Morrison S.J. Clarke M.F. Weissman I.L. Stem cells, cancer, and cancer stem cells.Nature. 2001; 414: 105-111Google Scholar). Importantly, LSCs on top of the leukemic hierarchy are thought to fuel the leukemia similar to their healthy HSC counterpart. LSCs were found to share various features with HSCs, including long-term self-renewal capacity, capability to give rise to more differentiated cells, an overlapping immunophenotype, capacity to reversibly enter a quiescent cell-cycle state, and gene regulatory networks governing stemness-associated gene expression programs (Bonnet and Dick, 1997Bonnet D. Dick J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat. Med. 1997; 3: 730-737Google Scholar; Eppert et al., 2011Eppert K. Takenaka K. Lechman E.R. Waldron L. Nilsson B. Van Galen P. Metzeler K.H. Poeppl A. Ling V. Beyene J. et al.Stem cell gene expression programs influence clinical outcome in human leukemia.Nat. Med. 2011; 17: 1086-1094Google Scholar; Lapidot et al., 1994Lapidot T. Sirard C. Vormoor J. Murdoch B. Hoang T. Caceres-Cortes J. Minden M. Paterson B. Caligiuri M.A. Dick J.E. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.Nature. 1994; 367: 645-648Google Scholar). Strikingly, a high expression of stemness-associated gene signatures derived from functionally engrafting LSC subpopulations in mice were strong predictors for a poor outcome in AML patients (Eppert et al., 2011Eppert K. Takenaka K. Lechman E.R. Waldron L. Nilsson B. Van Galen P. Metzeler K.H. Poeppl A. Ling V. Beyene J. et al.Stem cell gene expression programs influence clinical outcome in human leukemia.Nat. Med. 2011; 17: 1086-1094Google Scholar; Ng et al., 2016Ng S.W.K. Mitchell A. Kennedy J.A. Chen W.C. McLeod J. Ibrahimova N. Arruda A. Popescu A. Gupta V. Schimmer A.D. et al.A 17-gene stemness score for rapid determination of risk in acute leukaemia.Nature. 2016; 540: 433-437Google Scholar). In fact, a 17-gene comprising LSC signature was derived that is highly prognostic and predicts therapy response (Ng et al., 2016Ng S.W.K. Mitchell A. Kennedy J.A. Chen W.C. McLeod J. Ibrahimova N. Arruda A. Popescu A. Gupta V. Schimmer A.D. et al.A 17-gene stemness score for rapid determination of risk in acute leukaemia.Nature. 2016; 540: 433-437Google Scholar). These studies not only impressively demonstrated the clinical relevance of LSCs in AMLs but also suggested that leukemias should be treated distinctively depending on their stemness profile. Based on these findings, a standardized laboratory test is currently under development that aims at enabling personalized treatment decisions taking the stemness features of AMLs into considerations. The findings that cancer cells can differ in their stemness characteristics and may be hierarchically organized stimulated the endeavor of finding CSCs in solid tumors from the year 2000 onwards. While not all cancers are believed to be strictly hierarchically organized, a number of human CSCs have been identified and characterized in solid tumors using reconstitution assays in xenograft systems (Kreso and Dick, 2014Kreso A. Dick J.E. Evolution of the cancer stem cell model.Cell Stem Cell. 2014; 14: 275-291Google Scholar). In 2007, John Dick discovered and deeply characterized CSC in colon cancer, demonstrating the hierarchical organization of this cancer. The findings on the hierarchical organization of cancers sparked today’s well-recognized notion of the complex heterogeneity of individual cancer cells in tumors. Importantly, this heterogeneity is not restricted to genetic mutations but includes non-genetic traits such as transcriptional programs, metabolic phenotypes, functional properties, and influence of the (micro)environment. In this context, John Dick’s work suggests that non-genetic features like stemness properties and proliferative capacities of individual cells act in concert with the genetic heterogeneity acquired through clonal evolution, reconciling the CSC concept with genetic evolution models (Kreso and Dick, 2014Kreso A. Dick J.E. Evolution of the cancer stem cell model.Cell Stem Cell. 2014; 14: 275-291Google Scholar) (Figure 1). Notably, not all cancers are believed to be strictly hierarchically organized. Lineage-tracing approaches suggest that in some tumors, CSC features are not a hard-wired phenotype but underlie a certain plasticity (Batlle and Clevers, 2017Batlle E. Clevers H. Cancer stem cells revisited.Nat. Med. 2017; 23: 1124-1134Google Scholar). Therefore, refined concepts of the CSC model consider cellular plasticity as a common feature of CSCs. Similar to normal somatic stem cells that respond to environmental cues such as infection, inflammation, or toxic substances with regenerative activities, CSCs are able to adapt their phenotype and behavior to external stimuli including therapeutic pressure. Examples of the latter include epithelial-to-mesenchymal transition (EMT), neuroendocrine differentiation, metabolic plasticity, or reversible quiescence and senescence. All these features contribute to therapy resistance of CSCs. For example, the BCL-2 antagonist Venetoclax specifically targets AML-LSCs and is among the few approved drugs that have been shown to target CSCs in a clinical setting. To escape this therapeutic pressure, LSCs can take advantage of their metabolic plasticity and upregulate their fatty acid metabolism to survive therapy (Stevens et al., 2020Stevens B.M. Jones C.L. Pollyea D.A. Culp-Hill R. D’Alessandro A. Winters A. Krug A. Abbott D. Goosman M. Pei S. et al.Fatty acid metabolism underlies venetoclax resistance in acute myeloid leukemia stem cells.Nat. Cancer. 2020; 1: 1176-1187Google Scholar). Moreover, under therapeutic and evolutionary pressure, an increase in plasticity might be associated with a switch from non-CSC to CSC-like states, facilitating escape strategies through gradual de-differentiation. Consideration of stem cell plasticity as a key CSC component may explain why it has been difficult to develop effective strategies to combat CSCs in a clinical setting. The concept of CSC plasticity adds a third, dynamic layer to the model of genetic and epigenetic heterogeneity of cancer systems developed by John Dick and others (Figure 1). Inhibition and exploitation of CSC plasticity by the application of epigenetic drugs or the elimination of CSC by immunotherapies could become an important component of future strategies to combat CSCs and prevent cancer relapse. The scientific contributions made by John Dick over the past decades are significant, both in their magnitude and diversity. The technological and conceptional advances introduced by him and his group have been highly influential and instrumental for numerous groundbreaking discoveries in the fields of hematology and oncology. While it is nowadays well recognized that individual cancer cells from the same tumor display substantial heterogeneity with regards to transcriptional programs, mutations, metabolic traits, and functional properties, the seminal discoveries made and concepts introduced by John Dick were of great importance for shaping our understanding of intra-tumor heterogeneity and for the identification of LSCs. In this context, John Dick’s pioneering work pinpoints to the importance of eradicating the pool of therapy-resistant LSCs on top of the hierarchy for preventing leukemia relapse. Finally, consideration of cellular plasticity as an additional dynamic layer of cancer heterogeneity mediating stemness, i.e., in response to therapeutic pressure, further refines the CSC concept for leukemias and solid tumors (Figure 1). The CSC concept has not only broad implications for our understanding of cancer but also for designing more efficient future treatment schemes.