Normal Hematopoietic Stem Cells within the AML Bone Marrow Have a Distinct and Higher ALDH Activity Level than Co-Existing Leukemic Stem Cells

Persistence of leukemic stem cells (LSC) after chemotherapy is thought to be responsible for relapse and prevents the curative treatment of acute myeloid leukemia (AML) patients. LSC and normal hematopoietic stem cells (HSC) share many characteristics and co-exist in the bone marrow of AML patients. For the development of successful LSC-targeted therapy, enabling eradication of LSC while sparing HSC, the identification of differences between LSC and HSC residing within the AML bone marrow is crucial. For identification of these LSC targets, as well as for AML LSC characterization, discrimination between LSC and HSC within the AML bone marrow is imperative. Here we show that normal CD34+CD38– HSC present in AML bone marrow, identified by their lack of aberrant immunophenotypic and molecular marker expression and low scatter properties, are a distinct sub-population of cells with high ALDH activity (ALDH^bright). The ALDH^bright compartment contains, besides normal HSC, more differentiated, normal CD34+CD38+ progenitors. Furthermore, we show that in CD34-negative AML, containing solely normal CD34+ cells, LSC are CD34– and ALDH^low. In CD34-positive AML, LSC are also ALDH^low but can be either CD34+ or CD34–. In conclusion, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that LSC have lower ALDH activity than the CD34+CD38– HSC that co-exist with these LSC in the AML bone marrow. Our findings form the basis for combined functionally and immunophenotypically based identification and purification of LSC and HSC within the AML bone marrow, aiming at development of highly specific anti-LSC therapy.     

Bortezomib suppresses self-renewal and leukemogenesis of leukemia stem cell by NF-ĸB-dependent inhibition of CDK6 in MLL-rearranged myeloid leukemia

Acute myeloid leukaemia (AML) with chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukaemia (MLL) is an aggressive subtype with low overall survival. Bortezomib (Bort) is first applied in multiple myeloma. However, whether bort possesses anti-self-renewal and leukemogenesis of leukaemia stem cell (LSC) in AML with MLL rearrangements is still unclear. Here, we found that bort suppressed cell proliferation and decreased colony formation in human and murine leukaemic blasts. Besides, bort reduced the frequency and function of LSC, inhibited the progression, and extended the overall survival in MLL-AF9 (MF9) -transformed leukaemic mice. Furthermore, bort decreased the percentage of human LSC (CD34⁺CD38^-) cells and extended the overall survival in AML blasts-xenografted NOD/SCID-IL2Rγ (NSG) mice. Mechanistically, cyclin dependent kinase 6 (CDK6) was identified as a bort target by RNA sequencing. Bort reduced the expressions of CDK6 by inhibiting NF ĸB recruitment to the promoter of CDK6, leading to the abolishment of NF ĸB DNA-binding activity for CDK6 promoter. Overexpression of CDK6 partially rescued bort-induced anti-leukemogenesis. Most importantly, bort had little side-effect against the normal haematological stem and progenitor cell (HSPC) and did not affect CDK6 expression in normal HSPC. In conclusion, our results suggest that bort selectively targets LSC in MLL rearrangements. Bort might be a prospective drug for AML patients bearing MLL rearrangements.     

Cell surface marker phenotypes and gene expression profiles of murine radiation-induced acute myeloid leukemia stem cells are similar to those of common myeloid progenitors

Radiation exposure induces acute myeloid leukemia (AML) in humans and mice. Recent studies postulated that AML stem cells of spontaneous human AML arise from hematopoietic stem cells. However, other studies support the possibility that short-lived committed progenitors transform into AML stem cells, accompanied by a particular gene mutation. It remains unclear whether AML stem cells are present in radiation-induced AML, and information regarding AML-initiating cells is lacking. In this study, we identified and analyzed AML stem cells of mice with radiation-induced AML. The AML stem cells were identified by transplanting 100 bone marrow cells from mice with radiation-induced AML. We injected 100 cells of each of seven cell populations corresponding to different stages of hematopoietic cell differentiation and compared the latencies of AMLs induced in recipient mice. The identified radiation-induced AML stem cells frequently displayed similarities in both CD antigen and gene expression profiles with normal common myeloid progenitors. The number of common myeloid progenitor-like AML stem cells was significantly increased in mice with radiation-induced AML, but the progeny of common myeloid progenitors was decreased. In addition, analysis of radiation effects on the hematopoietic system showed that common myeloid progenitor cells were extremely radiosensitive and that their numbers remained at low levels for more than 2?months after radiation exposure. Our results suggest that murine radiation-induced AML stem cells arise from radiosensitive cells at a common myeloid progenitor stage.     

Proteomic Profiling Identifies Distinct Protein Patterns in Acute Myelogenous Leukemia CD34+CD38- Stem-Like Cells

Acute myeloid leukemia (AML) is believed to arise from leukemic stem-like cells (LSC) making understanding the biological differences between LSC and normal stem cells (HSC) or common myeloid progenitors (CMP) crucial to understanding AML biology. To determine if protein expression patterns were different in LSC compared to other AML and CD34+ populations, we measured the expression of 121 proteins by Reverse Phase Protein Arrays (RPPA) in 5 purified fractions from AML marrow and blood samples: Bulk (CD3/CD19 depleted), CD34-, CD34+(CMP), CD34+CD38+ and CD34+CD38-(LSC). LSC protein expression differed markedly from Bulk (n=31 cases, 93/121 proteins) and CD34+ cells (n= 30 cases, 88/121 proteins) with 54 proteins being significantly different (31 higher, 23 lower) in LSC than in either Bulk or CD34+ cells. Sixty-seven proteins differed significantly between CD34+ and Bulk blasts (n=69 cases). Protein expression patterns in LSC and CD34+ differed markedly from normal CD34+ cells. LSC were distinct from CD34+ and Bulk cells by principal component and by protein signaling network analysis which confirmed individual protein analysis. Potential targetable submodules in LSC included the proteins PU.1(SP1), P27, Mcl1, HIF1α, cMET, P53, Yap, and phospho-Stats 1, 5 and 6. Protein expression and activation in LSC differs markedly from other blast populations suggesting that studies of AML biology should be performed in LSC.     

Identification of leukemia stem cells in acute myeloid leukemia and their clinical relevance

Acute myeloid leukemia (AML) is considered to be a disease of stem cells. A rare defective stem cell population is purported to drive tumor growth. Similarly to their normal counterparts, leukemic stem cells (LSC) divide extreme slowly. This may explain the ineffectiveness of conventional chemotherapy in combatting this disease. Novel treatment strategies aimed at disrupting the binding of LSC to stem cell niches within the bone marrow might render the LSC vulnerable to chemotherapy and thus improving treatment outcome. This review focuses on the detection of LSC, our current knowledge about their cellular and molecular biology, and LSC interaction with the niche. Finally, we discuss the clinical relevance of LSC and prospective targeted treatment strategies for patients with AML.     

Identification of CD166 as a Surface Marker for Enriching Prostate Stem/Progenitor and Cancer Initiating Cells

New therapies for late stage and castration resistant prostate cancer (CRPC) depend on defining unique properties and pathways of cell sub-populations capable of sustaining the net growth of the cancer. One of the best enrichment schemes for isolating the putative stem/progenitor cell from the murine prostate gland is Lin(-);Sca1(+);CD49f(hi) (LSC(hi)), which results in a more than 10-fold enrichment for in vitro sphere-forming activity. We have shown previously that the LSC(hi) subpopulation is both necessary and sufficient for cancer initiation in the Pten-null prostate cancer model. To further improve this enrichment scheme, we searched for cell surface molecules upregulated upon castration of murine prostate and identified CD166 as a candidate gene. CD166 encodes a cell surface molecule that can further enrich sphere-forming activity of WT LSC(hi) and Pten null LSC(hi). Importantly, CD166 could enrich sphere-forming ability of benign primary human prostate cells in vitro and induce the formation of tubule-like structures in vivo. CD166 expression is upregulated in human prostate cancers, especially CRPC samples. Although genetic deletion of murine CD166 in the Pten null prostate cancer model does not interfere with sphere formation or block prostate cancer progression and CRPC development, the presence of CD166 on prostate stem/progenitors and castration resistant sub-populations suggest that it is a cell surface molecule with the potential for targeted delivery of human prostate cancer therapeutics.     

The clathrin assembly protein PICALM is required for erythroid maturation and transferrin internalization in mice

Phosphatidylinositol binding clathrin assembly protein (PICALM), also known as clathrin assembly lymphoid myeloid leukemia protein (CALM), was originally isolated as part of the fusion gene CALM/AF10, which results from the chromosomal translocation t(10;11)(p13;q14). CALM is sufficient to drive clathrin assembly in vitro on lipid monolayers and regulates clathrin-coated budding and the size and shape of the vesicles at the plasma membrane. However, the physiological role of CALM has yet to be elucidated. Here, the role of CALM in vivo was investigated using CALM-deficient mice. CALM-deficient mice exhibited retarded growth in utero and were dwarfed throughout their shortened life-spans. Moreover, CALM-deficient mice suffered from severe anemia, and the maturation and iron content in erythroid precursors were severely impaired. CALM-deficient erythroid cells and embryonic fibroblasts exhibited impaired clathrin-mediated endocytosis of transferrin. These results indicate that CALM is required for erythroid maturation and transferrin internalization in mice.     

Functional Characterization of D9, a Novel Deazaneplanocin A (DZNep) Analog,in Targeting Acute Myeloid Leukemia (AML)

Aberrant epigenetic events contribute to tumorigenesis of all human cancers. Significant efforts are underway in developing new generation of epigenetic cancer therapeutics. Although clinical trials for agents targeting DNA hypermethylation and histone deacetylation have yielded promising results, developing agents that target histone methylation remains to be in the early stage. We and others have previously reported that 3-Deazaneplanocin A (DZNep) is a histone methylation inhibitor that has a wide range of anticancer effects in various human cancers. Here, focusing on acute myeloid leukemia (AML) as a model, we reported a less toxic analog of DZNep, named D9, which is shown to be efficacious in AML cell lines and patient-derived samples in vitro, as well as AML tumorigenesis in vivo. Gene expression analysis in a panel of AML cell lines treated with D9 identified a set of genes that is associated with D9 sensitivity and implicated in multiple oncogenic signaling pathways. Moreover, we show that D9 is able to deplete the leukemia stem cells (LSC) and abolish chemotherapy-induced LSC enrichment, leading to dramatic elimination of AML cell survival. Thus, D9 appears to be a robust epigenetic compound that may constitute a potential for AML therapy.     

T‐lymphoid progenitors – we know what they are,but know not what they may be

An improved understanding of the biology underlying leukemogenesis, including the determination of the cells of leukemia origin, is of great importance as it can have immediate implications on patient treatment and management. The article by Riemke et al ( 2016 ) provides further evidence that a subgroup of acute myeloid leukemia (AML), the most common acute leukemia in adults, might arise from T‐lymphoid progenitor cells. This study not only supports that the lymphoid fate of early T‐cell progenitors is not yet fully stabilized but also shows that under oncogenic conditions, this multilineage plasticity potential of T‐lymphoid progenitors can lead to transdifferentiation into myeloid leukemia. While gene expression profiles suggest that approximately 5% of all AML cases originate from T‐lymphoid progenitors, novel treatment strategies targeting JAK2/STAT3 signaling might open new avenues for this AML cohort.     

Identification and targeting leukemia stem cells: The path to the cure for acute myeloid leukemia

Accumulating evidence support the notion that acute myeloid leukemia(AML) is organized in a hierarchical system, originating from a special proportion of leukemia stem cells(LSC). Similar to their normal counterpart, hematopoietic stem cells(HSC), LSC possess selfrenewal capacity and are responsible for the continued growth and proliferation of the bulk of leukemia cells in the blood and bone marrow. It is believed that LSC are also the root cause for the treatment failure and relapse of AML because LSC are often resistant to chemotherapy. In the past decade, we have made significant advancement in identification and understanding the molecular biology of LSC, but it remains a daunting task to specifically targeting LSC, while sparing normalHSC. In this review, we will first provide a historical overview of the discovery of LSC, followed by a summary of identification and separation of LSC by either cell surface markers or functional assays. Next, the review will focus on the current, various strategies for eradicating LSC. Finally, we will highlight future directions and challenges ahead of our ultimate goal for the cure of AML by targeting LSC.     

Mobilization of human lymphoid progenitors after treatment with granulocyte colony-stimulating factor

Hemopoietic stem and progenitor cells ordinarily residing within bone marrow are released into the circulation following G-CSF administration. Such mobilization has a great clinical impact on hemopoietic stem cell transplantation. Underlying mechanisms are incompletely understood, but may involve G-CSF-induced modulation of chemokines, adhesion molecules, and proteolytic enzymes. We studied G-CSF-induced mobilization of CD34+ CD10+ CD19- Lin- and CD34+ CD10+ CD19+ Lin- cells (early B and pro-B cells, respectively). These mobilized lymphoid populations could differentiate only into B/NK cells or B cells equivalent to their marrow counterparts. Mobilized lymphoid progenitors expressed lymphoid- but not myeloid-related genes including the G-CSF receptor gene, and displayed the same pattern of Ig rearrangement status as their bone marrow counterparts. Decreased expression of VLA-4 and CXCR-4 on mobilized lymphoid progenitors as well as multipotent and myeloid progenitors indicated lineage-independent involvement of these molecules in G-CSF-induced mobilization. The results suggest that by acting through multiple trans-acting signals, G-CSF can mobilize not only myeloid-committed populations but a variety of resident marrow cell populations including lymphoid progenitors.     

Decoupling of tumor-initiating activity from stable immunophenotype in HoxA9-Meis1-driven AML

Increasing evidence suggests tumors are maintained by cancer stem cells; however, their nature remains controversial. In a HoxA9-Meis1 (H9M) model of acute myeloid leukemia (AML), we found that tumor-initiating activity existed in three, immunophenotypically distinct compartments, corresponding to disparate lineages on the normal hematopoietic hierarchy--stem/progenitor cells (Lin(-)kit(+)) and committed progenitors of the myeloid (Gr1(+)kit(+)) and lymphoid lineages (Lym(+)kit(+)). These distinct tumor-initiating cells (TICs) clonally recapitulated the immunophenotypic spectrum of the original tumor in vivo (including cells with a less-differentiated immunophenotype) and shared signaling networks, such that in vivo pharmacologic targeting of conserved TIC survival pathways (DNA methyltransferase and MEK phosphorylation) significantly increased survival. Collectively, H9M AML is organized as an atypical hierarchy that defies the strict lineage marker boundaries and unidirectional differentiation of normal hematopoiesis. Moreover, this suggests that in certain malignancies tumor-initiation activity (or "cancer stemness") can represent a cellular state that exists independently of distinct immunophenotypic definition.     

Human mesenchymal and murine stromal cells support human lympho-myeloid progenitor expansion but not maintenance of multipotent haematopoietic stem and progenitor cells

A major goal in haematopoietic stem cell (HSC) research is to define conditions for the expansion of HSCs or multipotent progenitor cells (MPPs). Since human HSCs/MPPs cannot be isolated, NOD/SCID repopulating cell (SRC) assays emerged as the standard for the quantification of very primitive haematopoietic cell. However, in addition to HSCs/MPPs, lympho-myeloid primed progenitors (LMPPs) were recently found to contain SRC activities, challenging this assay as clear HSC/MPP readout. Because our revised model of human haematopoiesis predicts that HSCs/MPPs can be identified as CD133⁺CD34⁺ cells containing erythroid potentials, we investigated the potential of human mesenchymal and conventional murine stromal cells to support expansion of HSCs/MPPs. Even though all stromal cells supported expansion of CD133⁺CD34⁺ progenitors with long-term myeloid and long-term lymphoid potentials, erythroid potentials were exclusively found within erythro-myeloid CD133^lowCD34⁺ cell fractions. Thus, our data demonstrate that against the prevailing assumption co-cultures on human mesenchymal and murine stromal cells neither promote expansion nor maintenance of HSCs and MPPs.     

Stem cell factor consistently improves thymopoiesis after experimental transplantation of murine or human hematopoietic stem cells in immunodeficient mice

Deficient thymopoiesis is a pivotal determinant of impaired immune competence following hematopoietic stem cell transplantation (HSCT). Stem cell factor (SCF) is essentially involved in early thymopoiesis. We evaluated whether SCF administration would improve recovery of thymopoiesis following HSCT in immunodeficient mice receiving: 1) bone marrow (BM) transplantation of congenic mice; or 2) human fetal liver HSCT in the human immune system mouse model. Following murine BM transplantation, SCF significantly enhanced thymopoiesis and peripheral T cell recovery in lymph nodes and spleen. SCF did not affect BM lymphoid progenitor recovery and/or expansion. Median thymic cellularity increased from 0.9 in PBS- to 266 × 10(4)/thymus in SCF-treated mice (p = 0.05). Following human HSCT in human immune system mice, higher thymic cellularity was observed in SCF-treated mice. Double-negative and early double-positive thymocyte subsets increased, but especially late double-positive, CD4 single-positive, and CD8 single-positive thymocyte subsets were significantly enhanced (p < 0.05). These results show that exogenous supply of SCF may significantly improve murine and human posttransplant thymopoiesis, for which the effect is probably exerted by directly promoting T cell development intrathymically rather than by enhanced entry of prethymically expanded lymphoid progenitors.     

Human Flt3 is expressed at the hematopoietic stem cell and the granulocyte/macrophage progenitor stages to maintain cell survival

FLT3/FLK2, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis, and the constitutively active form of the FLT3 mutation is one of the most common genetic abnormalities in acute myelogenous leukemia. In murine hematopoiesis, Flt3 is not expressed in self-renewing hematopoietic stem cells, but its expression is restricted to the multipotent and the lymphoid progenitor stages at which cells are incapable of self-renewal. We extensively analyzed the expression of Flt3 in human (h) hematopoiesis. Strikingly, in both the bone marrow and the cord blood, the human hematopoietic stem cell population capable of long-term reconstitution in xenogeneic hosts uniformly expressed Flt3. Furthermore, human Flt3 is expressed not only in early lymphoid progenitors, but also in progenitors continuously along the granulocyte/macrophage pathway, including the common myeloid progenitor and the granulocyte/macrophage progenitor. We further found that human Flt3 signaling prevents stem and progenitors from spontaneous apoptotic cell death at least through up-regulating Mcl-1, an indispensable survival factor for hematopoiesis. Thus, the distribution of Flt3 expression is considerably different in human and mouse hematopoiesis, and human FLT3 signaling might play an important role in cell survival, especially at stem and progenitor cells that are critical cellular targets for acute myelogenous leukemia transformation.     

Murine models based on acute myeloid leukemia-initiating stem cells xenografting

Acute myeloid leukemia(AML) is an aggressive malignant disease defined by abnormal expansion of myeloid blasts. Despite recent advances in understanding AML pathogenesis and identifying their molecular subtypes based on somatic mutations, AML is still characterized by poor outcomes, with a 5-year survival rate of only 30%-40%, the majority of the patients dying due to AML relapse. Leukemia stem cells(LSC) are considered to be at the root of chemotherapeutic resistance and AML relapse. Although numerous studies have tried to better characterize LSCs in terms of surface and molecular markers, a specific marker of LSC has not been found, and still the most universally accepted phenotypic signature remains the surface antigens CD34+CD38- that is shared with normal hematopoietic stem cells. Animal models provides the means to investigate the factors responsible for leukemic transformation, the intrinsic differences between secondary post-myeloproliferative neoplasm AML and de novo AML, especially the signaling pathways involved in inflammation and hematopoiesis. However, AML proved to be one of the hematological malignancies that is difficult to engraft even in the most immunodeficient mice strains, and numerous ongoing attempts are focused to develop "humanized mice" that can support the engraftment of LSC. This present review is aiming to in-troduce the field of AML pathogenesis and the concept of LSC, to present the current knowledge on leukemic blasts surface markers and recent attempts to develop best AML animal models.     

Targeting miR‐193a‐AML1‐ETO‐β‐catenin axis by melatonin suppresses the self‐renewal of leukaemia stem cells in leukaemia with t (8;21) translocation

AML1‐ETO, the most common fusion oncoprotein by t (8;21) in acute myeloid leukaemia (AML), enhances hematopoietic self‐renewal and leukemogenesis. However, currently no specific therapies have been reported for t (8;21) AML patients as AML1‐ETO is still intractable as a pharmacological target. For this purpose, leukaemia cells and AML1‐ETO‐induced murine leukaemia model were used to investigate the degradation of AML1‐ETO by melatonin (MLT), synthesized and secreted by the pineal gland. MLT remarkedly decreased AML1‐ETO protein in leukemic cells. Meanwhile, MLT induced apoptosis, decreased proliferation and reduced colony formation. Furthermore, MLT reduced the expansion of human leukemic cells and extended the overall survival in U937T‐AML1‐ETO‐xenografted NSG mice. Most importantly, MLT reduced the infiltration of leukaemia blasts, decreased the frequency of leukaemia stem cells (LSCs) and prolonged the overall survival in AML1‐ETO‐induced murine leukaemia. Mechanistically, MLT increased the expression of miR‐193a, which inhibited AML1‐ETO expression via targeting its putative binding sites. Furthermore, MLT decreased the expression of β‐catenin, which is required for the self‐renewal of LSC and is the downstream of AML1‐ETO. Thus, MLT presents anti‐self‐renewal of LSC through miR‐193a‐AML1‐ETO‐β‐catenin axis. In conclusion, MLT might be a potential treatment for t (8;21) leukaemia by targeting AML1‐ETO oncoprotein.     

Primitive AML progenitors from most CD34(+) patients lack CD33 expression but progenitors from many CD34(-) AML patients express CD33

BACKGROUND: AML blast populations are heterogeneous in their phenotype and functional properties, and contain a small subset of cells that regenerate leukemia in immunocompromised mice or produce clonogenic progeny in long-term cultures. This suggests the existence of a hierarchy of AML progenitor cells. CD33 is a myeloid marker absent on normal hematopoietic stem cells but expressed in about 75% of AML patients, and has been used for BM purging strategies and Ab-targeted therapies. These CD33 Ab therapies benefit only a minority of AML patients, suggesting that AML stem cells are heterogeneous in their CD33 expression. METHODS: In order to evaluate this question, we determined expression levels of CD34 and CD33 on AML progenitors with long-term in vitro proliferative ability and NOD/SCID engrafting ability. RESULTS: The CD34(+) CD33(-) subfraction contained the majority of progenitors detected in vitro and most often engrafted the mice. This proliferation was leukemic from the CD34(+) AML patients, however from the CD34(-) AML patients only normal progenitors were detected in this fraction in some cases. DISCUSSION: These data suggest that most leukemic progenitors of CD34(+) patients do not express CD33. In contrast, CD34(-) AML primitive leukemic progenitors may be CD33(+). CD34(-) AML patients could potentially benefit most from CD33-targeted therapies or purging.