Since the establishment of functional repopulation assays in the late 1990s, accumulating studies have demonstrated the existence of cancer stem cells (CSCs) that possess self-renewal capability and the potential to generate differentiated daughter cells (1–4). Purification of a unique cell population based on the expression of specific cell surface markers enabled the prospective isolation of CSCs in various types of cancers. A prime example is acute myeloid leukemia (AML), which has been extensively studied as a model disease for the establishment of the CSC theory. Although T-cell acute lymphoblastic leukemia (T-ALL) has many similarities in pathophysiological features to AML, the CSC concept in T-ALL has not been firmly established. Leukemia-initiating cells (LICs), which can generate leukemia in a xenograft setting, have been confirmed in both human T-ALL patients and mouse models (5–12), but common stem cell markers have not been identified in this disease.

Unlike AML, which arises from the bone marrow, T-ALL clones originally emerge in the thymus, which does not provide a niche for hematopoietic stem cells (HSCs) (13–15). In many T-ALL cases, oncogenes are driven by a chromosomal translocation involving the T-cell receptor (TCR) gene locus, which is associated with somatic recombination in immature thymocytes (16). This suggests that T-ALL arises from committed T-cell precursors, but not from multi-potent HSCs. It is likely that developing thymocytes acquire stemness capability as a consequence of genetic and epigenetic abnormalities. On the other hand, recent studies have shown that early thymocytes can self-renew under certain condition (17, 18). Therefore, it is also possible that T-ALL arises from thymocytes that already possess self-renewal potential.

Acute lymphoblastic leukemia (ALL) is the most common type of childhood malignancy (19). Approximately 20% of ALL cases are classified as T-ALL. T-ALL is an aggressive malignancy characterized by the clonal proliferation of immature T-cell precursors that arise from the thymus and infiltrate into the bone marrow and peripheral blood (13–16). Enormous progress has been made in the treatment of T-ALL in the past few decades, with long-term remission observed in approximately 80% of children and 60% of adult patients (20, 21). However, a substantial fraction of T-ALL patients fail to respond to induction therapy or relapse within 2 years of diagnosis. The prognosis for this group of patients is very poor, with a 5-year survival rate of less than 25% (22).

T-ALL development requires multi-step genetic alterations of crucial oncogenes and tumor suppressors via different recurrent mechanisms, such as chromosomal translocations, intrachromosomal rearrangements, and mutations in protein-coding genes or enhancer elements, as well as epigenetic abnormalities (13–16). These alterations commonly affect genes that are required for cell growth, survival, and differentiation during normal T-cell development (14, 16). Results from recent genome-wide sequencing studies across different types of cancers indicate that ALL exhibits the fewest genomic abnormalities compared with other hematological malignancies and solid tumors (23, 24). This suggests that relatively few molecular alterations are crucial and significant enough to hijack the normal developmental program and promote malignant transformation.

The concept of CSCs originates from the observation that tumors consist of a hierarchically organized, heterogeneous population of cells with a minority of biologically distinct subsets capable of self-renewing and giving rise to clonal daughter cells (1–4). A number of studies have shown the existence of CSCs in various types of cancers. The CSC model also indicates that this rare cell population is able to tolerate therapeutic agents such as chemotherapy and radiation that eradicate the bulk of the rapidly proliferating tumor cells, thus resulting in inevitable cancer relapse in the long term (1–4, 59).

The most definitive property of stem cells lies in their self-renewal ability (1–4). Self-renewal in normal cells or CSCs gives rise either to one stem and one differentiated daughter cell via asymmetric division or to two stem cells via symmetric division. The general consensus in stem cell research is that CSCs are able to initiate and maintain clonal growth in long-term repopulation assays where the cancer cells are serially transplanted into immunodeficient recipient mice. The purification of a unique cell population based on the expression of specific cell surface markers has allowed researchers to isolate CSCs in various cancers, including AML and breast cancer (60–62). However, such populations have not been well characterized in many other cancers, including T-ALL. Hence, other terms, such as “tumor-initiating cells (TICs)” or “LICs,” have been coined to refer to the ability of transplanted cells to initiate tumor formation or leukemia in animals and are more preferentially used in experimental settings (1). Notably, the TIC/LIC concept is distinct from the “cell-of-origin” idea, as TIC/LIC strictly refers to cells in which tumorigenesis can be initiated (63), whereas the cell of origin that received the first oncogenic “hit” would progressively accumulate mutations during clonal evolution of the tumor. The acquisition of stem cell-like properties may occur at a much later stage of tumorigenesis in the evolved cells than the original cell that received only the first hit. In this regard, John Dick has proposed that TICs/LICs should be defined by their ability to (a) generate tumors in xenograft models that are representative of the parent tumors, (b) generate tumors upon serial passages in xenograft models, and, lastly, (c) give rise to daughter cells that can proliferate but might not be able to establish tumors after serial passages (1).

The presence of LICs was first reported by Dick and his colleagues in the late 1990s in studies of AML (60, 61). In a series of seminal studies, they showed that a rare subset of CD34⁺CD38⁻ cells isolated from AML patients was able to initiate the disease when transplanted into severe combined immunodeficient (SCID) mice (60). Crucially, the more differentiated CD34⁺CD38⁺ cells were unable to generate leukemia. In the initial study, secondary transplant of leukemic cells from SCID mice failed to generate leukemia. However, using a more immunocompromised non-obese diabetic (NOD)/SCID mouse model, the authors demonstrated that CD34⁺CD38⁻ cells have self-renewal properties (61). Furthermore, this group has shown that the engrafted CD34⁺CD38⁻ cells were able to give rise to more differentiated leukemic cells (61). Thus, this study demonstrated the presence of a leukemic hierarchy, with the CD34⁺CD38⁻ LICs at the top of the pyramid.

These results have also been challenged by studies utilizing more immunocompromised mouse models. For example, in the NOD/LtSz-scid IL-2Rγchain^null (NSG) mouse model, AML LICs are not only present exclusively in CD34⁺CD38⁻ cells (64). Results from this model showed that LICs can also be found in more differentiated CD34⁻ and CD38⁺ cells. The concept of LICs was also challenged by a study in which leukemic cells from Ras-induced T-cell lymphoma or an Eμ-Myc model of pre-B/B-cell lymphoma were shown to engraft in non-congenic animals regardless of the number of cells injected (65). The authors stressed the need to interpret data from serial transplantations more carefully, since failure to show engraftment could simply be due to the inability of the human cells to adapt to the microenvironment in the mouse.

The identification of LICs in ALL is even more challenging. To date, the identity and presence of LICs in human ALL has not been firmly established and is still debatable. Early studies in B-cell ALL (B-ALL) reported that the relatively immature CD34⁺CD19⁻ cells could contain LICs (66, 67). However, recent studies have found that more mature CD34⁺CD19⁺ leukemic blasts could initiate leukemia in ETV6-RUNX1- or TEL-AML1-positive B-ALL cases (68). In addition, a more recent study on MLL-AF4-positive infant ALL indicated that the LICs capable of reconstituting transplanted mice are exclusively CD19⁺ but exhibit variable CD34 expression (69). These studies highlight the heterogeneity of LICs in B-ALL cases and suggest that different cytogenetic abnormalities might play a role in determining the type of LICs present.

Similarly, the nature of LICs in human T-ALL has not been well characterized. An early study suggested that CD34⁺CD4⁻ and CD34⁺CD7⁻ cells, which make up a fraction of the leukemic cells from pediatric T-ALL patients, had leukemia-initiating properties when engrafted into NOD/SCID mice (5). A follow-up study investigating LIC activity in cortical/mature T-ALL patients reported that the CD34⁺CD7⁻ population from these patients contained normal hematopoietic cells that were able to differentiate into different lineages, while the CD34⁺CD7⁺ cells possessed LIC capability (6). Dick and Chiu et al. have also reported that the CD7⁺CD1a⁻ subset is enriched for LIC activity and exhibits glucocorticoid resistance (7).

Although the findings on LICs in primary human T-ALL are limited, several studies have been performed on transgenic animal models of T-ALL.

Another important consideration in the study of LICs is the interaction between leukemia cells and non-leukemia cells in the microenvironment. Bone marrow niche is essential for the maintenance and regulation of normal HSCs (78, 79). AML and ALL cells also home and expand in the bone marrow. Several studies have shown that signals from the bone marrow niche can dictate the survival of LICs and their responses to various types of treatment administered (80, 81).

Notably, two recent studies have elucidated the roles of bone marrow niche in T-ALL pathogenesis and implicated the CXCL12-CXCR4 signaling axis in the maintenance and progression of T-ALL (82, 83). CXCL12 is a chemokine secreted from endothelial and mesenchymal cells in the bone marrow and binds to its G protein-coupled receptor CXCR4 (79). Pitt et al. showed that in the bone marrow, T-ALL cells reside in close contact with stroma cells that secrete Cxcl12 (82). Deletion of the Cxcr4 receptor resulted in a reduction of leukemia burden and their infiltration into the bone marrow, thymus, and spleen in mouse model of T-ALL (82). Treatment of patient-derived human T-ALL cells in xenografts with a CXCR4 antagonist also produced the same result. Importantly, the authors observed a reduction in LIC activity in the absence of Cxcr4 in mice (82). Passaro et al. independently showed that depletion of CXCR4 affected T-ALL cell migration and expansion (83). Furthermore, the authors reported that calcineurin regulates CXCR4 expression in a cortactin-dependent manner (83). Those studies demonstrated the roles of the bone marrow niche in the maintenance of T-ALL.

One of the fundamental questions in LIC research is whether the LICs are derived from cells that already have self-renewal potential, such as HSCs, or whether they emerge from differentiated cells by newly acquiring stemness capability. T-ALL is derived from committed T-cell precursors in the thymus, which does not provide a niche for HSCs. The chromosomal translocation involving the TCR gene locus found in many T-ALL cases is associated with somatic recombination in immature thymocytes (16). These findings suggest that developing thymocytes likely acquire stemness capability as a consequence of genetic and epigenetic abnormalities. Tal1 and Lmo1/2 transgenic mice show an increased number of thymic progenitors that can generate leukemia, indicating that these oncogenic transcription factors are capable of inducing LIC ability in immature thymocytes.

On the other hand, recent studies have shown that normal thymocytes can self-renew in the absence of competitive precursor replacement (17, 18, 84). In general, HSCs differentiate into common lymphoid progenitor (CLP) cells in the bone marrow. CLPs migrate into the thymus and are committed to T-cell precursors that can differentiate into the DN to DP stage of thymocytes. In this well-accepted model, a continuous supply of lymphoid progenitor cells from the bone marrow is necessary to support T-cell development. Interestingly, Martins and Rodewald et al. recently reported that in Rag2^−/−γc^−/−Kit^W/Wv mice, which do not produce lymphoid progenitors from the bone marrow, a transplanted wild-type thymus sustained T-cell development for a long period of time (17). Similarly, Peaudecerf and Rocha et al. reported that in Rag2^−/−γc^−/−IL7 receptor^−/− mice engrafted with a wild-type thymus, persistent development of donor T-cells was observed (18). In this setting, host lymphoid progenitors can still migrate into the thymus and replace donor thymocytes but cannot differentiate after the DN2 stage, because IL7R signaling is required for the proliferation of early T-cell progenitors. Thus, competitive replacement by the host lymphoid progenitors is restricted to the DN1 and DN2 stages in this mouse model. This indicates that the donor thymus, which contains DN3 thymocytes, sustained T-cell development. Although this mechanism may be activated only when the competitive DN3 thymocytes are absent, these studies indicate that the thymus harbors cell populations with self-renewal potential that are capable of reconstituting the full diversity of T-cells.

Importantly, a large fraction of mice develop T-ALL in these settings (85). Tal1 and Lmo2 expression is strongly upregulated in these mouse T-ALL cells, and Notch1 mutations are also frequently found. This is consistent with observations in Tal1 and Lmo2 transgenic mice, which exhibit LICs in the DN3 subset and acquire Notch1 mutations (8). One possible mechanism is that differentiation arrest and expansion of DN3 thymocytes caused by overexpression of oncogenic transcription factors result in a loss of competitive replacement by bone marrow-derived progenitor cells, leading to activation of self-renewal machinery and malignant transformation. Alternatively, a loss of competitive replacement may result in the failure to silence the transcription factors that are normally expressed in stem and progenitor cells. Although the intrinsic mechanism of self-renewal in thymocytes is still unclear, these studies suggest that in T-ALL, LICs may arise from thymocytes that already have self-renewal potential via cellular competition.

Mouse studies have suggested that cellular competition potentially triggers the self-renewal capability of immature thymocytes, which may eventually lead to malignant transformation via the acquisition of genetic abnormalities such as Notch1 mutations. In human T-ALL, a loss of competition may be caused by overexpression of oncogenic transcription factors such as TAL1 and LMO2. Notably, these transcription factors themselves are also involved in the stem cell regulatory program during normal hematopoiesis.

In general, cellular differentiation of hematopoietic cells is associated with developmental restrictions that can be illustrated by the analogy of a “ball rolling down a hill” (86). During the differentiation process, HSCs lose their self-renewal and lineage potential. This process is regulated by an epigenetic and transcriptional network (87–89). A number of hematopoietic transcription factors are involved in this process. For example, TAL1 has been implicated as an essential regulator of hematopoiesis (33). TAL1 is expressed in normal HSCs, progenitor cells, and erythro-megakaryocytic lineages. Studies in knockout mouse models have revealed that this factor is required for hematopoietic specification and the genesis of hematopoietic cells (28, 29). In normal hematopoietic cells, TAL1 forms a large transcriptional complex with E-protein, LMO2, LDB1, and GATA (90). Several other transcription factors, including RUNX1 and the ETS family proteins, also frequently co-regulate downstream target genes (91).

Interestingly, these transcription factors co-occupy their own regulatory elements and positively regulate each other, thus forming an interconnected auto-regulatory loop (87, 88, 92). This structure is also termed a “core regulatory circuit” (CRC) and has been reported in other stem cells (93–95). For example, in embryonic stem cells, three key transcription factors that establish stem cell identity, OCT4, SOX2, and NANOG regulate each other (93, 94). This mechanism is thought to reinforce and stabilize downstream gene expression by “interlocking” the regulatory loop and is likely required for stem cell properties (92). Importantly, ectopic expression of these transcription factors can reprogram somatic cells back into stem cells, as has been established for the production of induced pluripotent stem cells (96). Similarly, recent studies have demonstrated that adult somatic fibroblasts can be reprogrammed into multi-potent hematopoietic stem progenitor cells by ectopic overexpression of TAL1, LMO2, RUNX1, GATA2, and ERG (“iHSPCs”) (97). This clearly indicates that a relatively small number of transcription factors are sufficient to control cell fate and identity.

In contrast to the regulatory circuit in HSCs, a very different type of transcriptional program is formed in developing thymocytes to regulate genes that are essential for T-cell differentiation (98). This process requires a number of transcription factors working in a cascade as well as the interactions in the microenvironment (Figure 1). Briefly, the NOTCH ligand expressed on thymic stromal cells induces expression of the transcription factors TCF7 and GATA3, which regulate other key transcription factors such as BCL11B and LEF1 (98). During this process, stem cell transcription factors such as TAL1 and LMO2 are gradually silenced, resulting in the loss of stem and progenitor cell potential. Meanwhile, E-proteins (E2A and HEB) are functionally and transcriptionally upregulated to induce RAG1, RAG2, and PTCRA, for example, which are required for somatic TCR recombination (99, 100). Such orchestrated stage-specific regulation of transcription factors mediates the T-cell differentiation process like a “ball rolling down a hill.” TAL1 and LMO2 silencing and E-protein upregulation are crucial to controlling the reciprocal switch from self-renewal to lineage-specific genetic programs. In other words, ectopic expression of TAL1 and LMO2 in developing thymocytes may rewrite the internal regulatory program.

Interestingly, TAL1 and LMO2 function as oncogenes in T-ALL cells, similar to their behavior in normal HSCs (33). TAL1 is expressed in 40–60% of T-ALL cases due to chromosomal translocation, intrachromosomal rearrangement, or mutations in non-coding elements (16, 36–38). These alterations replace an endogenous regulatory element controlling TAL1 expression with a new, potent enhancer that drives ectopic expression of this oncogene. Similarly, LMO2 or its related gene LMO1 is ectopically expressed in T-ALL cells due to chromosomal translocation or mutations in the regulatory elements (16, 101, 102). LMO1 or LMO2 is often expressed together with TAL1. In T-ALL cells, TAL1 and LMO proteins form a transcriptional complex with E-proteins and GATA3 (103, 104). Their regulatory partners in normal HSCs, RUNX1, ETS1, and MYB are also endogenously expressed in T-cells (98). We previously reported that TAL1, GATA3, RUNX1, and MYB co-occupy their own regulatory elements and positively regulate each other, forming the interconnected auto-regulatory structure (Figure 2) (105). These factors coordinately regulate downstream target genes. All these mechanisms are essentially the same as the machinery observed in normal HSCs.

At the same time, TAL1 counteracts the function of E-proteins by sequestering them, thus preventing them from transcriptionally inducing genes required for T-cell differentiation (99, 100). In this context, E-proteins act as tumor suppressors, as several groups have shown that E2a-deficient mice develop T-cell lymphoma and that this deficiency accelerates leukemia onset and progression in Tal1-transgenic mice (10, 106). Our recent study also revealed that in human T-ALL cells, TAL1 opposes the expression of E-protein target genes (105). Thus, the imbalance between the oncogenic TAL1 complex and E-protein is a primary determinant underlying the molecular pathogenesis of T-ALL (Figure 3) (107). Together, ectopic expression of TAL1 and LMO1/2 leads to the induction of HSC-like machinery and disruption of the T-cell differentiation program.

In this regard, it would be interesting to identify genes that are abnormally induced by the TAL1 complex in T-ALL cells. Recently, our group used a targeted approach to identify regulatory elements that are differentially controlled by TAL1 and E-proteins (108). From this analysis, we discovered an enhancer situated within a cluster of seven genes belonging to the GTPase of Immunity Associated Protein (GIMAP) family. This region is associated with active histone marks in T-ALL cells but not in the normal human thymus, suggesting that the GIMAP enhancer is aberrantly activated in T-ALL cells. Importantly, GIMAP genes are expressed in mouse HSCs and CD4 or CD8 SP mature T-cells, while they are downregulated in DN3-4 stage thymocytes where TAL1 is also silenced. Using an in vivo reporter system in zebrafish, we showed that the GIMAP enhancer can be activated in normal hematopoietic stem and progenitor cells but not in the thymus. In addition, a reporter assay in human T-ALL cell lines indicated that the GIMAP enhancer is activated by TAL1 and its regulatory partners (GATA3 and RUNX1) and is repressed by E-proteins (E2A and HEB). Although ectopic expression of human GIMAP genes in immature zebrafish thymocytes did not induce tumor formation, their overexpression accelerated leukemia development in the presence of the MYC oncogene. Thus, our results revealed that aberrant activation of the GIMAP enhancer contributes to T-cell leukemogenesis.

While GIMAP genes have been known to be involved in the development of mature T- and B-lymphocytes (109–112), another group has also implicated their importance in HSC survival and maintenance (113). The work of Chen et al. on Gimap5^−/− mice demonstrated that Gimap5 regulates the survival of HSCs and other early hematopoietic progenitors by stabilizing the Mcl-1 protein, which is an anti-apoptotic Bcl-2 family member (113). The HSCs in Gimap5-deficient mice exhibited defective long-term repopulation capacity, as demonstrated by their impaired engrafting ability. This study provided insights into the critical roles of GIMAP genes in the survival of HSCs and early progenitor cells. Notably, NOTCH1 was also identified as a positive regulator of the GIMAP genes in T-ALL cells (114, 115). A functional study by Chadwick et al. showed that Gimap5 mediates apoptosis protection in T-ALL cells upon its upregulation by NOTCH1 (114). Together with our findings, these studies suggest that as a consequence of TAL1/LMO overexpression and activation of the NOTCH1 pathway, the GIMAP genes could be reactivated in immature thymocytes in which they are normally repressed, possibly by E-proteins, thereby contributing to leukemogenesis.

Another gene that has been implicated in stem cells and is also aberrantly activated by the TAL1 complex in T-ALL is the ALDH1A2 gene (105, 116). Based on our ChIP-seq and gene expression data, this gene was one of the top candidate genes directly regulated by TAL1 in human T-ALL cells (105). ALDH activity has been proposed to be a universal CSC marker, as demonstrated by the tumorigenic and self-renewal properties of ALDH⁺ cells isolated from leukemia and many solid tumors (117–119). Among the 19 isoforms in the ALDH family, only a few of them, including ALDH1A2 are involved in retinoic acid signaling, which has been known to be associated with the stemness characteristics of CSCs. Another group and our recent study indicated that ALDH1A2 is induced by TAL1 via an internal enhancer in T-ALL cells (116) (and Zhang and Tan et al., unpublished data). Although the role of GIMAPs and ALDH1A2 in the self-renewal potential of malignant T-cells is yet to be elucidated, their ability to mark stem cells and T-ALL cells may be used as a signature of the aberrant transcriptional program induced by T-ALL oncogenes.

The transformation mechanism in T-ALL is very efficient. T-ALL oncogenes alter the intrinsic transcriptional regulatory program by disrupting the differentiation machinery and by introducing the stem cell-like properties into developing thymocytes. This may initiate or reactivate the self-renewal ability that potentially exists in thymocytes. This process is mediated by a relatively small number of oncogenic transcription factors and seems not require the accumulation of a large number of genetic and chromosomal abnormalities until it obtains the hallmarks of cancer.

In other words, this mechanism poses a potential severe risk hidden in the thymus. Thymocytes may always be “primed” to initiate leukemogenesis. As recently reported (17, 18, 84), the competitive replacement of thymocytes via a continuous supply of lymphoid progenitor cells from the bone marrow plays an important tumor suppressive role in homeostasis. Further investigation is necessary to elucidate the loss-of-competition mechanism in human T-ALL. In particular, it is of great interest to analyze whether T-ALL develops from a self-renewal pool prior to the TCR rearrangement or pre-leukemic clones, which harbor the TCR translocation newly acquire the self-renewal capability. Single cell sequencing analysis is ideal to detect the emergence of those clones. Another important consideration is the mechanism of self-renewal in the ETP subtype of T-ALL. ETP cases show a very different genomic landscape and gene expression signature as compared to non-ETP cases. For example, mutations of NOTCH1 are less frequently found in ETP (27), thus suggesting that different oncogenic mechanisms are involved. Establishment of proper model systems is needed to analyze LICs in this particular subtype.

The mechanisms described above can be also therapeutic targets to eliminate LICs in T-ALL. Disruption of the transcriptional complex involving TAL1 would efficiently block the formation of the CRC and revert the functional imbalance between oncogenic TAL1 complex and E-protein tumor suppressors. Rabbit and his colleagues have developed a peptide and intracellular antibody targeting LMO2 protein to disassociate the TAL1–LMO2 complex (32, 120, 121). Inhibition of transcriptional machinery by small-molecule inhibitors of CDK7 or BRD4 concurrently reduces expressions of multiple oncogenic transcription factors in T-ALL, thereby leading to cell death (122, 123). Moreover, targeting CXCR4/CXCL12 signaling is an ideal strategy to disrupt the interaction between T-ALL cells and stroma cells in the bone marrow niche, as recently reported (82, 83). Additionally, identification of specific cell surface markers associated with LIC capability in T-ALL is critical for developing better therapeutic strategy.

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.