John E. Dick

A human colon cancer cell capable of initiating tumour growth in immunodeficient mice

Colon cancer is one of the best-understood neoplasms from a genetic perspective, yet it remains the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. What has yet to be established is whether every colon cancer cell possesses the potential to initiate and sustain tumour growth, or whether the tumour is hierarchically organized so that only a subset of cells—cancer stem cells—possess such potential. Here we use renal capsule transplantation in immunodeficient NOD/SCID mice to identify a human colon cancer-initiating cell (CC-IC). Purification experiments established that all CC-ICs were CD133+; the CD133- cells that comprised the majority of the tumour were unable to initiate tumour growth. We calculated by limiting dilution analysis that there was one CC-IC in 5.7 × 104 unfractionated tumour cells, whereas there was one CC-IC in 262 CD133+ cells, representing >200-fold enrichment. CC-ICs within the CD133+ population were able to maintain themselves as well as differentiate and re-establish tumour heterogeneity upon serial transplantation. The identification of colon cancer stem cells that are distinct from the bulk tumour cells provides strong support for the hierarchical organization of human colon cancer, and their existence suggests that for therapeutic strategies to be effective, they must target the cancer stem cells.

Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells.

A workshop was convened by the AACR to discuss the rapidly emerging cancer stem cell model for tumor development and progression. The meeting participants were charged with evaluating data suggesting that cancers develop from a small subset of cells with self-renewal properties analogous to organ

Targeting of CD44 eradicates human acute myeloid leukemic stem cells.

The long-term survival of patients with acute myeloid leukemia (AML) is dismally poor. A permanent cure of AML requires elimination of leukemic stem cells (LSCs), the only cell type capable of initiating and maintaining the leukemic clonal hierarchy. We report a therapeutic approach using an activating monoclonal antibody directed to the adhesion molecule CD44. In vivo administration of this antibody to nonobese diabetic-severe combined immune-deficient mice transplanted with human AML markedly reduced leukemic repopulation. Absence of leukemia in serially transplanted mice demonstrated that AML LSCs are directly targeted. Mechanisms underlying this eradication included interference with transport to stem cell–supportive microenvironmental niches and alteration of AML-LSC fate, identifying CD44 as a key regulator of AML LSCs. The finding that AML LSCs require interaction with a niche to maintain their stem cell properties provides a therapeutic strategy to eliminate quiescent AML LSCs and may be applicable to other types of cancer stem cells.

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.

Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity.

Emerging evidence suggests cancer stem cells sustain neoplasms; however, little is understood of the normal cell initially targeted and the resultant cancer stem cells. We show here, by tracking individual human leukemia stem cells (LSCs) in nonobese diabetic–severe combined immunodeficiency mice serially transplanted with acute myeloid leukemia cells, that LSCs are not functionally homogeneous but, like the normal hematopoietic stem cell (HSC) compartment, comprise distinct hierarchically arranged LSC classes. Distinct LSC fates derived from heterogeneous self-renewal potential. Some LSCs emerged only in recipients of serial transplantation, indicating they divided rarely and underwent self-renewal rather than commitment after cell division within primary recipients. Heterogeneity in LSC self-renewal potential supports the hypothesis that they derive from normal HSCs. Furthermore, normal developmental processes are not completely abolished during leukemogenesis. The existence of multiple stem cell classes shows the need for LSC-targeted therapies.

Evolution of the Cancer Stem Cell Model

Genetic analyses have shaped much of our understanding of cancer. However, it is becoming increasingly clear that cancer cells display features of normal tissue organization, where cancer stem cells (CSCs) can drive tumor growth. Although often considered as mutually exclusive models to describe tumor heterogeneity, we propose that the genetic and CSC models of cancer can be harmonized by considering the role of genetic diversity and nongenetic influences in contributing to tumor heterogeneity. We offer an approach to integrating CSCs and cancer genetic data that will guide the field in interpreting past observations and designing future studies.

Stem cell concepts renew cancer research

Although uncontrolled proliferation is a distinguishing property of a tumor as a whole, the individual cells that make up the tumor exhibit considerable variation in many properties, including morphology, proliferation kinetics, and the ability to initiate tumor growth in transplant assays. Understanding the molecular and cellular basis of this heterogeneity has important implications in the design of therapeutic strategies. The mechanistic basis of tumor heterogeneity has been uncertain; however, there is now strong evidence that cancer is a cellular hierarchy with cancer stem cells at the apex. This review provides a historical overview of the influence of hematology on the development of stem cell concepts and their linkage to cancer.

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.

A newly discovered class of human hematopoietic cells with SCID-repopulating activity

The detection of primitive hematopoietic cells based on repopulation of immune-deficient mice is a powerful tool to characterize the human stem-cell compartment. Here, we identify a newly discovered human repopulating cell, distinct from previously identified repopulating cells, that initiates multilineage hematopoiesis in NOD/SCID mice. We call such cells CD34neg-SCID repopulating cells, or CD34neg-SRC. CD34neg-SRC are restricted to a Lin–CD34–CD38– population without detectable surface markers for multiple lineages and CD38 or those previously associated with stem cells (HLA-DR, Thy-1 and CD34). In contrast to CD34+ subfractions, Lin–CD34–CD38– cells have low clonogenicity in short-and long-term in vitro assays. The number of CD34neg-SRC increased in short-term suspension cultures in conditions that did not maintain SRC derived from CD34+ populations, providing independent biological evidence of their distinctiveness. The identification of this newly discovered cell demonstrates complexity of the organization of the human stem-cell compartment and has important implications for clinical applications involving stem-cell transplantation.

Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice

We have used the random chromosomal integration sites of retrovirus vectors as unique clonal markers to analyze cell lineage relationships within the hemopoietic stem cell hierarchy. Using a high efficiency protocol for retrovirus-mediated gene transfer, anemic W/Wv mutant mice were reconstituted with bone marrow cells infected with a NEO vector. Analysis of the DNA from bone marrow, thymus, and spleen of these reconstituted W/Wv mice indicated insertion of the vector into primitive pluripotent stem cells capable of producing both myeloid and lymphoid progeny as well as into more committed stem cells apparently restricted to either the myeloid or lymphoid lineages. The neo gene was also expressed in these mice, as they contained a variety of G418 resistant in vitro colony-forming cells. These results demonstrate high-efficiency gene transfer and expression in primitive hemopoietic stem cells and provide a direct approach for analyzing the hemopoietic stem cell hierarchy.