Metoclopramide treatment blocks CD93-signaling-mediated self-renewal of chronic myeloid leukemia stem cells

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组蛋白去乙酰化酶抑制剂在急性髓系白血病中的应用

急性髓系白血病（AML）是造血干/祖细胞恶性克隆性疾病,以骨髓、血液和其他组织中髓系起源的异常原始细胞增殖为特征。“3+7”诱导方案（蒽环类药物联合阿糖胞苷）一直是治疗AML的基石,但仍有部分AML患者无法耐受强化疗或完全缓解后复发,目前AML的总体疗效仍不乐观。因此,寻找新药物以提高AML患者疗效具有重要的临床意义。越来越多的研究证明,表观遗传对AML的发生、发展起重要作用。组蛋白去乙酰化酶抑制剂（HDACi）是表观遗传修饰的分子靶向药物,可抑制组蛋白去乙酰化酶（HDAC）的活性,上调组蛋白赖氨酸的乙酰化水平,目前已应用于AML临床研究中,在联合治疗中显现出良好的耐受性与治疗效果。本综述介绍了HDAC和HDACi的分类依据以及在临床上的应用,阐述了伏立诺他、贝利司他、帕比司他、戊丙酸、恩替诺特、西达本胺等6种HDACi在AML中的临床前研究结果和临床应用研究进展,讨论了HDACi与其他抗癌药物联用在AML中的作用机制,并对HDACi今后的发展提出了建议,期望为临床治疗AML提供参考。     

精原干细胞自我更新和分化的调控

精原干细胞（spermatogonial stem cells,SSCs）是体内自然状态下惟一能将遗传信息传至子代的成体干细胞,它们能通过维持自我更新和分化的稳定从而保证雄性生命过程中精子发生的持续进行。了解SSCs自我更新和分化的调节机制有助于阐明精子发生机理,并为探究其他组织中成体干细胞增殖分化的调节机制提供依据。然而目前对于SSCs自我更新和分化的调控机制所知甚少。SSCs的更新与分化遵循特定模式,受以睾丸支持细胞为主要成分的微环境及各种内分泌因素如胶质细胞源神经营养因子（GDNF）、维生素、Ets转录因子ERM/Etv5等的调控。本文评述了SSCs更新与分化的模式以及上述因素对其更新、分化的调控,探讨了其中可能涉及的信号通路,以期为本领域及其他成体干细胞相关研究提供借鉴。     

Epigenetic modifications and self-renewal regulation of mouse germline stem cells

Germline stem (GS) cells were established from gonocytes and spermatogonia of postnatal mouse testes. GS cells proliferate in the presence of several kinds of cytokines, and a small percentage of GS cells also show spermatogonial stem cell (SSC) activity, i.e., they differentiate into sperm after being transplanted into infertile mouse testes without endogenous spermatogenesis. Interestingly, in GS cell culture, we also found that pluripotent stem cells (multipotent germline stem cells (mGS cells)) could be derived and these mGS cells do not have normal androgenetic genomic imprinting marks that are shown in GS cells, e.g., H19 hypermethylation. A new culture system for fetal male germ cells (embryonic GS (eGS) cells) has also been recently developed. Although these cells exhibited SSC potential, the offspring from cultured cells showed heritable imprinting defects in their DNA methylation patterns. In an attempt to understand the self-renewal machinery in SSCs, we transfected H-Ras and cylin D2 into GS cells, and successfully reconstructed the SSC self-renewal ability without using exogenous cytokines. Although these cells showed SSC activity in germ cell transplantation assays, we also found development of seminomatous tumors, possibly induced by excessive self-renewing signal. These stem cell culture systems are useful tools not only for understanding the mechanisms of self-renewal or epigenetic reprogramming but also for clarifying the mechanism of germ cell tumor development.     

CDKIs p18INK4c and p57Kip2 are involved in quiescence of CML leukemic stem cells after treatment with TKI

Chronic Myeloid Leukemia (CML) is sustained by a small population of cells with stem cell characteristics known as Leukemic Stem Cells that are positive to BCR-ABL fusion protein, involved with several abnormalities in cell proliferation, expansion, apoptosis and cell cycle regulation. Current treatment options for CML involve the use of Tirosine Kinase Inhibitor (Imatinib, Nilotinib and Dasatinib), that efficiently reduce proliferation proliferative cells but do not kill non proliferating CML primitive cells that remain and contributes to the persistence of the disease.

In order to understand the role of Cyclin Dependent Kinase Inhibitors in CML LSC permanence after TKI treatment, in this study we analyzed cell cycle status, the levels of several CDKIs and the subcellular localization of such molecules in different CML cell lines, as well as primary CD34⁺CD38^?lin^? LSC and HSC.

Our results demonstrate that cellular location of p18^INK4c and p57^Kip2 seems to be implicated in the antiproliferative activity of Imatinib and Dasatinib in CML cells and also suggest that the permanence of quiescent stem cells after TKI treatment could be associated with a decrease in p18^INK4c and p57^Kip2 nuclear location. The differences in p18^INK4cand p57^Kip2activities in CML and normal stem cells suggest a different cell cycle regulation and provide a platform that could be considered in the development of new therapeutic options to eliminate LSC.     

Nicotinamide phosphoribosyltransferase inhibitors selectively induce apoptosis of AML stem cells by disrupting lipid homeostasis

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造血干细胞研究进展

造血干细胞（hematopoietic stem cell,HSC）是成体干细胞研究领域的范式。对造血干细胞自我更新和不对称分裂分子遗传学机制的诠释,将不仅帮助理解成体干细胞“干性”维持的发育遗传学机制,也将对白血病干细胞和其他类型肿瘤干细胞的发育起源及开发针对肿瘤干细胞的靶向治疗模式产生深远的影响。     

Mitochondrial respiration defects modulate differentiation but not proliferation of hematopoietic stem and progenitor cells

Mitochondrial energy production is involved in various cellular processes. Here we show that ATP content is significantly increased in lineage-restricted progenitor cells compared with hematopoietic stem and progenitor cells (HSPCs) or more differentiated cells. Transplantation analysis using a mouse model of mitochondrial disease revealed that mitochondrial respiration defects resulted in a significant decrease in the total number and repopulating activity of bone marrow cells, although the number of HSPCs increased. The proliferative activity of HSPCs and lineage-restricted progenitor cells was not impaired by reduction of ATP content and there seems to be no associated increase in reactive oxygen species levels and apoptosis. Our findings indicate that mitochondrial respiration defects modulate HSPC commitment/differentiation into lineage-restricted progenitor cells.     

Akt suppresses DLK for maintaining self-renewal of mouse embryonic stem cells

Mouse embryonic stem cells (ES cells) can proliferate indefinitely. To identify potential signals involved in suppression of self-renewal, we previously screened a kinase/phosphatase expression library in ES cells, and observed that inhibition of Dual Leucine zipper-bearing Kinase (DLK) increased relative cell numbers. DLK protein was detected in both the pluripotent and differentiated states of mouse ES cells while DLK kinase activity increased upon differentiation. Overexpression of DLK in mouse ES cells displayed reductions in relative cell/colony numbers and Nanog expression, suggesting a suppressive role of DLK in self-renewal. By examining protein sequences of DLK, we identified 2 putative Akt phosphorylation sites at S584 and T659. Blocking PI3K/Akt signaling with LY-294002 enhanced DLK kinase activity dramatically. We found that Akt interacts with and phosphorylates DLK. Mutations of DLK amino acid residues at putative Akt phosphorylation sites (S584A, T659A, or S584A and T659A) diminished the level of DLK phosphorylation. While the mutated DLKs (S584A, T659A, or S584A and T659A) were expressed, a further reduction in cell/colony numbers and Nanog expression appeared in mouse ES cells. In addition, these mutant DLKs (S584A, T659A, or S584A and T659A) exhibited more robust kinase activity and cell death compared to wild type DLK or green fluorescence (GFP) controls. In summary, our results show that DLK functions to suppress self-renewal of mouse ES cells and is restrained by Akt phosphorylation.     

Non-small cell lung cancer stem/progenitor cells are enriched in multiple distinct phenotypic subpopulations and exhibit plasticity

Cancer stem cells (CSCs) represent a population of cancer cells that possess unique self-renewal and differentiation characteristics required for tumorigenesis and are resistant to chemotherapy-induced apoptosis. Lung CSCs can be enriched by several markers including drug-resistant side population (SP), CD133^pos and ALDH^high. Using human non-small cell lung adenocarcinoma cell lines and patient-derived primary tumor cells, we demonstrate that SP cells represent a subpopulation distinct from other cancer stem/progenitor cell (CS/PC) populations marked by CD133^pos or ALDH^high. The non-CS/PCs and CS/PCs of each subpopulation are interconvertible. Epithelial-mesenchymal transition (EMT) promotes the formation of CD133^pos and ALDH^high CS/PC subpopulations while suppressing the SP CS/PC subpopulation. Rac1 GTPase activity is significantly increased in cells that have undergone EMT, and targeting Rac1 is effective in inhibiting the dynamic conversion of non-CS/PCs to CS/PCs, as well as the CS/PC activity. These results imply that various subpopulations of CS/PCs and non-CS/PCs may achieve a stochastic equilibrium in a defined microenvironment, and eliminating multiple subpopulations of CS/PCs and effectively blocking non-CS/PC to CS/PC transition, by an approach such as targeting Rac1, can be a more effective therapy.     

NPR-A regulates self-renewal and pluripotency of embryonic stem cells

Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.     

E35 ablates acute leukemia stem and progenitor cells in vitro and in vivo

Leukemia stem cells (LSCs) have critical functions in acute leukemia (AL) pathogenesis, participating in its initiation and relapse. Thus, identifying new molecules to eradicate LSCs represents a high priority for AL management. This work identified E35, a novel Emodin derivative, which strongly inhibited growth and enhanced apoptosis of AL stem cell lines, and primary stem and progenitor cells from AL cases, while sparing normal hematopoietic cells. Furthermore, functional assays in cultured cells and animals suggested that E35 preferentially ablated primitive leukemia cell populations without impairing their normal counterparts. Moreover, molecular studies showed that E35 remarkably downregulated drug-resistant gene and dramatically inhibited the Akt/mammalian target of rapamycin signaling pathway. Notably, the in vivo anti-LSC activity of E35 was further confirmed in murine xenotransplantation models. Collectively, these findings indicate E35 constitutes a novel therapeutic candidate for AL, potentially targeting leukemia stem and progenitor cells.     

METTL16 drives leukemogenesis and leukemia stem cell self-renewal by reprogramming BCAA metabolism

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Retinoic acid maintains self-renewal of murine embryonic stem cells via a feedback mechanism

Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass (ICM) that are able to self-renew or undergo differentiation depending on a complex interplay of extracellular signals and intracellular factors. However, the feedback regulation of differentiation-dependent ESC self-renewal is poorly understood. Retinoic acid (RA), a derivative of vitamin A, plays a critical role in ESC differentiation and embryogenesis. In the present study, we demonstrate that short-term treatment of murine (m) ESCs with RA during the early differentiation stage prevented spontaneous differentiation of mESCs. The RA-treated cells maintained self-renewal capacity and could differentiate into neuronal cells, cardiomyocytes, and visceral endoderm cells derived from three germ layers. The differentiation-inhibitory effect of RA was mimicked by conditioned medium from RA-treated ESCs and was accompanied with up-regulated expression of leukemia inhibitory factor (LIF), Wnt3a, Wnt5a, and Wnt6. Such RA-induced prevention of ESC differentiation was attenuated by a neutralizing antibody against LIF or by a specific Wnt antagonist Fz8-Fc and was totally reversed in the presence of both of them. Furthermore, knock-down of beta-catenin, a component of the Wnt signaling pathway, by small interfering RNA counteracted the effect of RA. In addition, RA treatment enhanced expression of endodermal markers GATA4 and AFP but inhibited expression of primitive ectodermal marker Fgf-5 and mesodermal marker Brachyury. These findings reveal a novel role of RA in ESC self-renewal and provide new insight into the regulatory mechanism of differentiation-dependent self-renewal of ESCs, in which Wnt proteins and LIF induced by RA have the synergistic action. The short-term treatment of ESCs with RA also offers a unique model system for study of the regulatory mechanism that controls self-renewal and specific germ-layer differentiation of ESCs.     

组蛋白去甲基化酶1在干细胞增殖与分化中的调控作用

表观遗传学在干细胞的分化与成熟过程中扮演着重要的角色。其中发现组蛋白去甲基化酶1（LSD1）可以动态地调节组蛋白的甲基化状态,进而调控基因转录的激活和抑制以及X染色体失活等过程,LSD1在肿瘤干细胞、胚胎干细胞、神经干细胞及诱导多能干细胞中均有表达,并影响这些干细胞的增殖和分化过程。就LSD1在干细胞增殖与分化中的调控作用的研究进展进行综述。     

The miR 302-367 cluster drastically affects self-renewal and infiltration properties of glioma-initiating cells through CXCR4 repression and consequent disruption of the SHH-GLI-NANOG network

Glioblastoma multiforme (GBM) is the most common form of primary brain tumor in adults, often characterized by poor survival. Glioma-initiating cells (GiCs) are defined by their extensive self-renewal, differentiation, and tumor initiation properties. GiCs are known to be involved in tumor growth and recurrence, and in resistance to conventional treatments. One strategy to efficiently target GiCs in GBM consists in suppressing their stemness and consequently their tumorigenic properties. In this study, we show that the miR-302-367 cluster is strongly induced during serum-mediated stemness suppression. Stable miR-302-367 cluster expression is sufficient to suppress the stemness signature, self-renewal, and cell infiltration within a host brain tissue, through inhibition of the CXCR4 pathway. Furthermore, inhibition of CXCR4 leads to the disruption of the sonic hedgehog (SHH)-GLI-NANOG network, which is involved in self-renewal and expression of the embryonic stem cell-like signature. In conclusion, we demonstrated that the miR-302-367 cluster is able to efficiently trigger a cascade of inhibitory events leading to the disruption of GiCs stem-like and tumorigenic properties.