Oncogenic Kras Initiates Leukemia in Hematopoietic Stem Cells

How oncogenes modulate the self-renewal properties of cancer-initiating cells is incompletely understood. Activating KRAS and NRAS mutations are among the most common oncogenic lesions detected in human cancer, and occur in myeloproliferative disorders (MPDs) and leukemias. We investigated the effects of expressing oncogenic Kras^G12D from its endogenous locus on the proliferation and tumor-initiating properties of murine hematopoietic stem and progenitor cells. MPD could be initiated by Kras^G12D expression in a highly restricted population enriched for hematopoietic stem cells (HSCs), but not in common myeloid progenitors. Kras^G12D HSCs demonstrated a marked in vivo competitive advantage over wild-type cells. Kras^G12D expression also increased the fraction of proliferating HSCs and reduced the overall size of this compartment. Transplanted Kras^G12D HSCs efficiently initiated acute T-lineage leukemia/lymphoma, which was associated with secondary Notch1 mutations in thymocytes. We conclude that MPD-initiating activity is restricted to the HSC compartment in Kras^G12D mice, and that distinct self-renewing populations with cooperating mutations emerge during cancer progression.     

Neovascularization and Hematopoietic Stem Cells

Vasculogenesis and angiogenesis are the major forms of blood vessel formation. Angiogenesis is the process where new vessels grow from pre-existing blood vessels, and is very important in the functional recovery of pathological conditions, such as wound healing and ischemic heart diseases. The development of better animal model and imaging technologies in past decades has greatly enriched our understanding on vasculogenesis and angiogenesis processes. Hypoxia turned out to be an important driving force for angiogenesis in various ischemic conditions. It stimulates expression of many growth factors like vascular endothelial growth factor, platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor, which play critical role in induction of angiogenesis. Other cellular components like monocytes, T cells, neutrophils, and platelets also play significant role in induction and regulation of angiogenesis. Various stem/progenitor cells also being recruited to the ischemic sites play crucial role in the angiogenesis process. Pre-clinical studies showed that stem/progenitor cells with/without combination of growth factors induce neovascularization in the ischemic tissues in various animal models. In this review, we will discuss about the fundamental factors that regulate the angiogenesis process and the use of stem cells as therapeutic regime for the treatment of ischemic diseases.     

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.     

白血病干细胞的生物学特性

白血病干细胞是第一个被确认的肿瘤干细胞,在白血病的形成和病情的发展中具有重要作用。深入研究其生物学特性将为白血病的发病机制以及干细胞靶向治疗奠定理论基础。该文介绍白血病干细胞的免疫表型、自我更新机制、生存优势及多重耐药等。     

Differential Sensitivity of Telomerase from Human Hematopoietic Stem Cells and Leukemic Cell Lines to Mild Hyperthermia

We have investigated the effects of hyperthermia (HT) on cell proliferation and telomerase activity of human hematopoietic stem cells (HSCs) and compared with human leukemic cell lines (TF-1, K562 and HL-60). The cells were exposed to HT at 42 and 43 °C up to 120 min. The cells were incubated at 37 °C for 96 h. Then the cells were collected and assayed for cell proliferation, viability, telomerase activity, and terminal restriction fragment (TRF) lengths. The enzyme activity from HSCs was decreased up to 68.6 at 42 and 85.1 % at 43 °C for 120 min. This inhibition in leukemic cells was up to 28.9 and 53.6 % in TF-1; 53 and 63.9 % in K562; 45.2 and 61.1 % in HL-60 cells. The treated cells showed TRF lengths about 5.3 kb for control HL-60 cells, 5.0 kb for HL-60 cells treated at 42 and 4.5 kb at 43 °C for 120 min. In HSCs, the TRF length was about 4.5 kb for untreated cells and 4.0–4.5 kb for treated cells at 42 and 43 °C for 120 min. The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. TRF length was constant at treated two types of cells, which implies that in cells subjected to mild HT no telomere shortening was observed.     

Hck和Lyn激酶与慢性髓细胞性白血病的关系

Src家族激酶（Sre—family kinase,SFK）是一组非受体型酪氨酸蛋白激酶,其家族成员Hck （hemopoietic cell kinase）和Lyn（v-yes-1 Yanaguchi sarcoma viral related oncogene homolog）激酶在慢性髓细胞性白血病（chronic myelogenous leukemia,CML）细胞中被BCR—ABL异常激活,并作用于多种细胞内信号转导分子,影响细胞的增殖、凋亡和迁移。深入研究SFK在CML发生和发展中的作用,对进一步认识CML发病机制及其靶向性治疗有着重要的实际意义。     

A PDE Model for Imatinib-Treated Chronic Myelogenous Leukemia

We derive a model for describing the dynamics of imatinib-treated chronic myelogenous leukemia (CML). This model is a continuous extension of the agent-based CML model of Roeder et al. (Nat. Med. 12(10), 1181–1184, 2006) and of its recent formulation as a system of difference equations (Kim et al. in Bull. Math. Biol. 70(3), 728–744, 2008). The new model is formulated as a system of partial differential equations that describe various stages of differentiation and maturation of normal hematopoietic cells and of leukemic cells. An imatinib treatment is also incorporated into the model. The simulations of the new PDE model are shown to qualitatively agree with the results that were obtained with the discrete-time (difference equation and agent-based) models. At the same time, for a quantitative agreement, it is necessary to adjust the values of certain parameters, such as the rates of imatinib-induced inhibition and degradation.     

胚胎干细胞体外分化为造血干细胞

造血干细胞移植已成为治疗白血病、再生障碍性贫血、重症免疫缺陷征、地中海贫血、急性放射病、某些恶性实体瘤和淋巴瘤等造血及免疫系统功能障碍性疾病的成熟技术和重要手段,另外这一技术还被尝试用于治疗艾滋病,已取得积极的效果。但是由于移植需要配型相同的供体,并且过程复杂,使得造血干细胞移植因缺少配型相同的供体来源以及费用昂贵而不能被广泛应用。胚胎干细胞是一种能够在体外保持未分化状态并且能进行无限增殖的细胞,在适合条件下能够分化为体内各种类型的细胞,研究胚胎干细胞分化为造血干细胞,不仅可作为研究动物的早期造血发生的模型,而且可以增加造血干细胞的来源,还可以通过基因剔除、治疗性克隆等方法来解决移植排斥的问题,从而为造血干细胞移植的发展扫除了障碍,因此有着重要的研究价值和应用前景。现对胚胎干细胞体外分化为造血干细胞的诱导方法,诱导过程中的调控机制,并对胚胎干细胞分化为造血干细胞的存在问题和发展前景进行讨论。     

骨髓移植与造血干细胞研究

骨髓移植是目前治疗白血病等血液疾病最有效的方法。在经历了多年的发展并已取得巨大成就的今天,骨髓移植仍然面,临很多问题。造血干细胞研究必将解决骨髓移植中的诸多问题。重点对造骨髓移植的发展历史,所面临的问题以及造血干细胞研究的进展进行简要的概述。     

Frozen Cord Blood Hematopoietic Stem Cells Differentiate into Higher Numbers of Functional Natural Killer Cells In Vitro than Mobilized Hematopoietic Stem Cells or Freshly Isolated Cord Blood Hematopoietic Stem Cells

Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of NK cells that are cytotoxic but not exhausted. NK cell differentiation from hematopoietic stem cells (HSC) has become an alluring option for NK cell therapy, with umbilical cord blood (UCB) and mobilized peripheral blood (PBCD34⁺) being the most accessible HSC sources as collection procedures are less invasive. In this study we compared the capacity of frozen or freshly isolated UCB hematopoietic stem cells (CBCD34⁺) and frozen PBCD34⁺ to generate NK cells in vitro. By modifying a previously published protocol, we showed that frozen CBCD34⁺ cultures generated higher NK cell numbers without loss of function compared to fresh CBCD34⁺ cultures. NK cells generated from CBCD34⁺ and PBCD34⁺ expressed low levels of killer-cell immunoglobulin-like receptors but high levels of activating receptors and of the myeloid marker CD33. However, blocking studies showed that CD33 expression did not impact on the functions of the generated cells. CBCD34⁺-NK cells exhibited increased capacity to secrete IFN-γ and kill K562 in vitro and in vivo as compared to PBCD34⁺-NK cells. Moreover, K562 killing by the generated NK cells could be further enhanced by IL-12 stimulation. Our data indicate that the use of frozen CBCD34⁺ for the production of NK cells in vitro results in higher cell numbers than PBCD34⁺, without jeopardizing their functionality, rendering them suitable for NK cell immunotherapy. The results presented here provide an optimal strategy to generate NK cells in vitro for immunotherapy that exhibit enhanced effector function when compared to alternate sources of HSC.     

Acadesine Kills Chronic Myelogenous Leukemia (CML) Cells through PKC-Dependent Induction of Autophagic Cell Death

CML is an hematopoietic stem cell disease characterized by the t(9;22) (q34;q11) translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL) was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients.     

Strategic Treatment Interruptions During Imatinib Treatment of Chronic Myelogenous Leukemia

Although imatinib is an effective treatment for chronic myelogenous leukemia (CML), and nearly all patients treated with imatinib attain some form of remission, imatinib does not completely eliminate leukemia. Moreover, if the imatinib treatment is stopped, most patients eventually relapse (Cortes et al. in Clin. Cancer Res. 11:3425–3432, 2005). In Kim et al. (PLoS Comput. Biol. 4(6):e1000095, 2008), the authors presented a mathematical model for the dynamics of CML under imatinib treatment that incorporates the anti-leukemia immune response. We use the mathematical model in Kim et al. (PLoS Comput. Biol. 4(6):e1000095, 2008) to study and numerically simulate strategic treatment interruptions as a potential therapeutic strategy for CML patients. We present the results of numerous simulated treatment programs in which imatinib treatment is temporarily stopped to stimulate and leverage the anti-leukemia immune response to combat CML. The simulations presented in this paper imply that treatment programs that involve strategic treatment interruptions may prevent leukemia from relapsing and may prevent remission for significantly longer than continuous imatinib treatment. Moreover, in many cases, strategic treatment interruptions may completely eliminate leukemic cells from the body. Thus, strategic treatment interruptions may be a feasible clinical approach to enhancing the effects of imatinib treatment for CML. We study the effects of both the timing and the duration of the treatment interruption on the results of the treatment. We also present a sensitivity analysis of the results to the parameters in the mathematical model.     

Stochastic Dynamics of Hematopoietic Tumor Stem Cells

Many tumors derive from the transformation of normal stem cells into cancer stem-cells that retain their self-renewal capacity. This modern view of cancer has provided a natural explanation for the striking parallels which exist between these two different types of self-renewing cells. Here we develop a simple mathematical model to investigate the implications of this concept regarding the evolution of tumors in the hematopoietic system. Our results unequivocally demonstrate that stochastic effects related to the finite size of the active stem-cell population have a profound influence on the dynamics of cancer evolution. For input parameters compatible with both the natural history of human cancer and mouse models, our results show how stochastic dynamics alone may lead to both remission in some cases and rapid expansion in others.     

Molecular Signatures of Quiescent,Mobilized and Leukemia-Initiating Hematopoietic Stem Cells

Hematopoietic stem cells (HSC) are rare, multipotent cells capable of generating all specialized cells of the blood system. Appropriate regulation of HSC quiescence is thought to be crucial to maintain their lifelong function; however, the molecular pathways controlling stem cell quiescence remain poorly characterized. Likewise, the molecular events driving leukemogenesis remain elusive. In this study, we compare the gene expression profiles of steady-state bone marrow HSC to non-self-renewing multipotent progenitors; to HSC treated with mobilizing drugs that expand the HSC pool and induce egress from the marrow; and to leukemic HSC in a mouse model of chronic myelogenous leukemia. By intersecting the resulting lists of differentially regulated genes we identify a subset of molecules that are downregulated in all three circumstances, and thus may be particularly important for the maintenance and function of normal, quiescent HSC. These results identify potential key regulators of HSC and give insights into the clinically important processes of HSC mobilization for transplantation and leukemic development from cancer stem cells.     

ARAP3 Functions in Hematopoietic Stem Cells

ARAP3 is a GTPase-activating protein (GAP) that inactivates Arf6 and RhoA small GTPases. ARAP3 deficiency in mice causes a sprouting angiogenic defect resulting in embryonic lethality by E11. Mice with an ARAP3 R302,303A mutation (Arap3^KI/KI) that prevents activation by phosphoinositide-3-kinase (PI3K) have a similar angiogenic phenotype, although some animals survive to adulthood. Here, we report that hematopoietic stem cells (HSCs) from rare adult Arap3^KI/KI bone marrow are compromised in their ability to reconstitute recipient mice and to self-renew. To elucidate the potential cell-autonomous and non-cell-autonomous roles of ARAP3 in hematopoiesis, we conditionally deleted Arap3 in hematopoietic cells and in several cell types within the HSC niche. Excision of Arap3 in hematopoietic cells using Vav1-Cre does not alter the ability of ARAP3-deficient progenitor cells to proliferate and differentiate in vitro or ARAP3-deficient HSCs to provide multi-lineage reconstitution and to undergo self-renewal in vivo. Thus, our data suggest that ARAP3 does not play a cell-autonomous role in HSPCs. Deletion of Arap3 in osteoblasts and mesenchymal stromal cells using Prx1-Cre resulted in no discernable phenotypes in hematopoietic development or HSC homeostasis in adult mice. In contrast, deletion of Arap3 using vascular endothelial cadherin (VEC or Cdh5)-driven Cre resulted in embryonic lethality, however HSCs from surviving adult mice were largely normal. Reverse transplantations into VEC-driven Arap3 conditional knockout mice revealed no discernable difference in HSC frequencies or function in comparison to control mice. Taken together, our investigation suggests that despite a critical role for ARAP3 in embryonic vascular development, its loss in endothelial cells minimally impacts HSCs in adult bone marrow.     

Stability Analysis of a Model of Interaction Between the Immune System and Cancer Cells in Chronic Myelogenous Leukemia

We describe here a simple model for the interaction between leukemic cells and the autologous immune response in chronic phase chronic myelogenous leukemia (CML). This model is a simplified version of the model we proposed in Clapp et al. (Cancer Res 75:4053–4062, 2015). Our simplification is based on the observation that certain key characteristics of the dynamics of CML can be captured with a three-compartment model: two for the leukemic cells (stem cells and mature cells) and one for the immune response. We characterize the existence of steady states and their stability for generic forms of immunosuppressive effects of leukemic cells. We provide a complete co-dimension one bifurcation analysis. Our results show how clinical response to tyrosine kinase inhibitors treatment is compatible with the existence of a stable low disease, treatment-free steady state.     

Studies on the Longevity,Sequestration and Release of the Leukocytes in Chronic Myelogenous Leukemia

The intravascular life-span of leukocytes labelled in vitro with radioactive di-isopropylfluorophosphate was studied in 12 patients with chronic myelogenous leukemia (CML). In relapse, leukocyte specific activity (LSA) disappeared slowly; in remission, LSA curves approached normal and only a small proportion of LSA disappeared slowly. The level of maturation of the leukocytes that persisted in the blood was investigated by a leukocyte fractionation technique which excluded immature myeloid cells from leukocyte samples. The influence of extracorpuscular factors upon the pattern of disappearance of LSA was investigated by means of cross-transfusion experiments, and LSA curves obtained with in vitro and in vivo labelling were compared. The results suggest that: (1) the intravascular life-span of the mature leukemic neutrophil is prolonged in relapse and in remission; (2) intrinsically abnormal leukocytes are sequestered in an extravascular pool(s) but recycling occurs; (3) extracorpuscular factors modify the LSA curves; (4) exchange of leukocytes between intravascular and extravascular pools may not occur in relapse; and (5) the intravascular and extravascular pools constitute a self-sustaining pool(s) not replenished from a non-miscible precursor pool.     

The Mechanism of Action of Splenic Irradiation in Chronic Myelogenous Leukemia

The mechanism of action of splenic irradiation in the induction of a remission in chronic myelogenous leukemia was investigated in six patients using a leukocyte kinetic approach. The leukocytes were labelled in vitro with radioactive diisopropylfluorophosphate-32 and returned to the circulation. The effect of treatment on the rate of change of leukocyte specific activity was determined. The results suggest (1) that irradiation of the spleen damages granulopoietic cells as they cycle back and forth between the spleen, blood and other extravascular compartments; (2) that damage to exchangeable granulopoietic cells in transit through the irradiated spleen may explain the long remission often encountered after this form of therapy.