Based on the fact that the unlimited self-renewal capacity in normal hematopoiesis is limited to HSCs and lost in progenitor cells and the discovery that AMLSCs belong to the CD34+CD38-cell population, AMLSCs were hypothesized to originate from HSCs. This hypothesis was strongly supported by the observation that CD34+CD38- AML cells could transfer disease to SCID mice, whereas CD34+CD38+ AML cells could not . In AML, the cell of origin has been largely studied based on the delicate discrimination of leukemic cell populations harboring AML-specific genetic mutations by multi-color flow cytometric analysis and identification of cell populations with LSC capacity validated by long-term culture-initiating cell assay
A previous study using a powerful leukemic driver
In rare cases, a single gene fusion of
Through these studies, the concept of AMLSC has differentiated into two developmental stages: pre-LSCs and LSC, which is also called leukemia-initiating cells. NGS studies have found that mutations in epigenetic/chromatin regulator genes, such as DTA (
CD34 expression is universally accepted as a basic marker for AMLSCs, and CD38 null or low expression of CD38 has been considered a reliable marker of AMLSCs. Many studies identifying putative LSC markers in AML have proposed candidate biomarkers for AMLSCs as those enriched in the CD34+CD38- cell fraction. T cell immunoglobulin mucin-3 (TIM-3) [27, 28], CD25, CD32 , CD96 , and C-type lectin-like molecule-1 (CLL-1)  have been suggested as LSC markers based on this old belief. However,
Ng and Dick performed an important study to investigate true LSC gene signatures. They sorted four fractions of primary AML cells using two surface markers, CD34 and CD38, and then split them for microarray and mouse transplant experiments. They analyzed the difference in gene expression between cells capable of repopulating NSG (NOD/SCID gamma) mice and cells incapable of repopulating
Furthermore, several studies have suggested that LSCs reside within the CD34- cell population in certain AML cases. For example, some fractions of CD34- leukemic cells with
Recent advances in genomics have revealed an unexpectedly remarkable heterogeneity of genetic aberrations in AML. The Cancer Genome Atlas (TCGA) project opens mutational landscapes of AML, which shows a huge complexity in view of the numbers and combinations of recurrent genetic aberrations [14, 15]. According to these reports, the median number of mutated genes usually exceeds 3 in most AML cases, and the number of recurrently mutated genes is more than 30. As a result, the combination of these gene mutations causes remarkable heterogeneity in AML in view of the co-occurrence of gene mutations. When we consider combinations of triple gene mutations, the frequency of the most common combination of
Current standard induction chemotherapy with cytarabine and idarubicin effectively induces complete morphological remission in approximately 70% of patients with newly diagnosed AML . However, a significant proportion of patients treated with intensive chemotherapy and subsequent allogeneic hematopoietic stem cell transplantation experience relapse or refractoriness. A treatment strategy with chemotherapy regimens similar to that in the initial treatment is usually ineffective because leukemic clones resistant to the initial treatment expand through clonal evolution or surviving leukemic clones gain new mutations . Shlush
Another study revealed that LSC frequencies are remarkably increased (up to 90 folds) at the time of relapse in AML. However, the LSC marker frequencies do not change accordingly . In this study, among the various markers, including CD32, CD33, CD45RA, CD47, CD96, CD97, CD99, CD123, HLA-DR, interleukin-1 receptor accessory protein (IL-1RAP), and TIM-3, only TIM-3 and CD96 showed increasing tendencies without statistical significance. TIM-3 expression levels were increased in patients with AML with failed chemotherapy . In addition, there is evidence of plasticity of LSC surface markers in the same patient over time. For example, CD25+ LSC were shown to give rise to progeny of CD25- LSC capable of leukemic engraftment in serial transplantation assays in patient-derived xenograft models .
In summary, AMLSCs can change during treatment through clonal selection, plasticity of surface markers, and modification of the stemness transcriptional programs, even without increased LSC marker expression.
Although there has been a lot of evidence of heterogeneity from various standpoints of immunophenotype, cell cycle, metabolism, and stemness, there is still hope for an effective targeting strategy of a universal AMLSC biomarker. The Dick JE group identified 17 significant AMLSC genes through gene expression profiling selected based on results from microarray analysis and engrafting capability by xenotrans-plantation assay using samples from 78 patients with AML . 17-LSC signature genes also include genes expressed in normal HSCs, such as CD34 and GPR56. The 17-gene LSC score is also well correlated with the European LeukemiaNet risk classification widely used for the risk stratification of patients with AML in current clinical practice . Jung and Majeti investigated the methylated gene profiles in LSCs capable of being engrafted into NSG mice compared to those in leukemic blast cells  because stemness in HSCs and LSCs is known to be regulated by epigenetic mechanisms . They found that AMLSCs are hypomethylated compared to blasts, and the AMLSC epigenetic signature is largely mutation-independent and uniquely characterized by hypomethylated and upregulated HOXA cluster genes
The stemness program that is strongly expressed in AMLSCs is determined by both cell-intrinsic and cell-extrinsic mechanisms. Cell-extrinsic factors are derived from the microenvironment or niche in which the cell resides. LSCs share cellular niche components with healthy HSCs in the BM, including osteoblasts, endothelial cells, mesenchymal stromal cells (MSCs), adipocytes, and sympathetic neurons. Several factors such as stem cell factor (SCF), C-X-C motif chemokine ligand 12 (CXCL12), Notch ligands, and transforming growth factor-β are produced by these niche cells and are involved in the maintenance of HSCs . AML cells not only share a niche with HSCs for their survival but also affect HSC niche conditions in various ways. Adipocytes abundant in normal BM are important cell components of the HSC niche; however, their role is disrupted in an AML xenograft model , MSCs are also important for HSC maintenance through the secretion of various factors such as SCF and CXCL12. In an
Leukemic cells attach to the stromal ligands vascular cell adhesion molecule (VCAM-1), fibronectin, and intracellular adhesion molecule 1 (ICAM-1) of the niche via the interaction of the β-1 integrin receptor family members very late antigen-4 (VLA-4) and VLA-5, and the β-2 integrin LFA-1 . The homing receptor CXCR4 guides LSCs to CXCL12 in the BM niche. The therapeutic targeting of the CXCL12-CXCR4 interaction has shown efficacy as an adjunctive therapy to eradicate AMLSCs . Bromodomain and extra-terminal domain-containing (BET-containing) protein (BETPs) inhibitors degrade BETPs, downregulate CXCR4 and CD44 expression, and decrease the LSC population in a patient-derived xenotransplantation model . CD44, an extensively alternatively spliced adhesion molecule, mediates adhesive cell-cell and cell-extracellular matrix interactions by binding to its ligand hyaluronan in the endosteal region or osteopontin, fibronectin, and selectin. Targeting CD44 by monoclonal antibody showed promising results in effectively targeting LSCs and reversing the AML differentiation block .
Hypoxia is a well-known microenvironmental factor in HSCs/LSCs . Mantel and Broxmeyer
The BM contains a large pool of adipocytes, and leukemic cells use fatty acids as important fuels. Therefore, adipocyte targeting is being actively investigated. Restoring normal adipocyte maturation using PPARγ agonists and inhibiting fatty acid transfer into cells has been explored as future therapies .
In addition to the niche factors described above, many adhesion molecules and related pathways, including integrins/CD98, CD44/E-selectin, Eph, cadherin, GPR56, and JAM-C, are being investigated as possible future LSC niche-directed therapies (Fig. 2).
AMLSCs originating from HSPCs are the source of a more differentiated leukemic bulk and the cause of relapse and refractoriness despite the current standard treatment, which includes intensive chemotherapy and allogeneic hematopoietic cell transplantation. The discovery of the CD34+ CD38- immunophenotype as an LSC marker introduced the first model of AMLSC as a conceptual pathologic counterpart of normal HSPC. Studies in mice have shown that AMLSCs originate from hematopoietic stem cells to progenitor cells. Methodologies of multicolor flow cytometry and NGS have made deep investigations of small volumes of primary AML samples possible, which introduced the concept of pre-LSCs prior to the development of leukemia-initiating stem cells. However, the current concept is not perfect, and huge heterogeneities in terms of mutational and immunophenotypic profiles among and within patients have been revealed. Genetic aberrations detected in AML at the initial diagnosis change at the time of relapse through clonal evolution by selective survival pressure induced by cytotoxic chemotherapy. AMLSC frequencies increase during treatment over time, and the stemness program of AMLSCs becomes shallower, making patients with relapsed AML more therapy-resistant. AMLSCs have relatively common epigenetic and genetic signatures despite their huge heterogeneity. Many putative AMLSC markers have been proposed, and efforts to target AMLSCs have been made. Recent approaches to target AMLSCs and their niche have shed light on promising future treatment strategies to cure AML.
I would like to thank Gyurim Shin for her help in drawing some of the diagrams shown in
No potential conflicts of interest relevant to this article were reported.